Molecular Biology
Structure of human β2-adrenergic
receptor highlighting the cholesterolbinding site in the middle of the
receptor. Work done in the laboratory
of Raymond Stevens, Ph.D., professor.
Pick-Wei Lau, Graduate Student, and
Ian MacRae, Ph.D., Assistant Professor
MOLECULAR BIOLOGY
DEPAR TMENT OF
MOLECULAR BIOLOGY
S TA F F
Peter E. Wright, Ph.D.*
Professor and Chairman
Cecil H. and Ida M. Green
Investigator in Medical
Research
Ruben Abagyan, Ph.D.
Professor
Carlos F. Barbas III, Ph.D.**
Professor
Janet and W. Keith Kellogg II
Chair, Molecular Biology
Rajesh Belani, Ph.D.
Adjunct Assistant Professor
aTyr Pharma
La Jolla, California
Michael N. Boddy, Ph.D.
Assistant Professor
Charles L. Brooks III,
Ph.D.***
University of Michigan
Ann Arbor, Michigan
David A. Case, Ph.D.***
Rutgers University
Piscataway, New Jersey
Geoffrey Chang, Ph.D.*
Associate Professor
Eli Chapman, Ph.D.
Assistant Professor of
Molecular Biology
Jerold Chun, M.D., Ph.D.
Professor
Lluis Ribas De Pouplana,
Ph.D.
Adjunct Assistant Professor
Omnia Molecular
Barcelona, Spain
Ashok Deniz, Ph.D.
Associate Professor
H. Jane Dyson, Ph.D.
Professor
James Arthur Fee, Ph.D.
Professor of Research
2008
Elizabeth D. Getzoff,
Ph.D.****
Professor
THE SCRIPPS RESEARCH INSTITUTE
203
Ian MacRae, Ph.D.
Assistant Professor
Robyn L. Stanfield, Ph.D.
Assistant Professor
Clare McGowan, Ph.D.*****
Associate Professor
Raymond C. Stevens, Ph.D.†
Professor
David S. Goodsell, Jr., Ph.D.
Associate Professor of
Molecular Biology
Duncan E. McRee, Ph.D.
Adjunct Associate Professor
Sorrento Technology
San Diego, California
Charles D. Stout, Ph.D.
Associate Professor
Joel M. Gottesfeld, Ph.D.
Professor
David P. Millar, Ph.D.
Professor
Jennifer Harris, Ph.D.
Assistant Professor of
Biochemistry
Louis Noodleman, Ph.D.
Associate Professor
David B. Goodin, Ph.D.
Associate Professor
Christian A. Hassig, Ph.D.
Adjunct Assistant Professor
Kalypsis, Inc.
San Diego, California
Peter B. Hedlund, M.D.,
Ph.D.
Assistant Professor of
Molecular Biology
John E. Johnson, Ph.D.
Professor
Gerald F. Joyce, M.D., Ph.D.**
Professor
Dean, Faculty
Ehud Keinan, Ph.D.
Adjunct Professor
Technion-Israel Institute of
Technology
Haifa, Israel
Richard A. Lerner, M.D.,
Ph.D.**
President, The Scripps
Research Institute
Lita Annenberg Hazen
Professor of
Immunochemistry
Cecil H. and Ida M. Green
Chair in Chemistry
Scott Lesley, Ph.D.
Assistant Professor of
Biochemistry
Tianwei Lin, Ph.D.***
Assistant Professor
University of Hong Kong
Hong Kong, China
Arthur J. Olson, Ph.D.
Professor
Takanori Otomo, Ph.D.
Assistant Professor
Vijay Reddy, Ph.D.
Associate Professor
Steven I. Reed, Ph.D.*****
Professor
Paul Russell, Ph.D.*****
Professor
Michel Sanner, Ph.D.
Associate Professor
Harold Scheraga, Ph.D.
Adjunct Professor
Cornell University
Ithaca, New York
Paul R. Schimmel, Ph.D.**
Ernest and Jean Hahn
Professor of Molecular
Biology and Chemistry
Anette Schneemann, Ph.D.
Associate Professor
Subhash C. Sinha, Ph.D.
Associate Professor of
Medicinal Chemistry
Gary Siuzdak, Ph.D.
Adjunct Professor
Director, Center for Mass
Spectrometry
Vaughn V. Smider, Ph.D.
Assistant Professor of
Molecular Biology
Peiqing Sun, Ph.D.
Associate Professor
J. Gregor Sutcliffe, Ph.D.
Professor
John A. Tainer, Ph.D.*
Professor
Fujie Tanaka, Ph.D.
Associate Professor
Elizabeth Anne Thomas,
Ph.D.
Assistant Professor
James R. Williamson,
Ph.D.**
Professor
Dean, Kellogg School of
Science and Technology
Ian A. Wilson, D.Phil.*
Professor
Curt Wittenberg, Ph.D.*****
Professor
Kurt Wüthrich, Ph.D.**
Cecil H. and Ida M. Green
Professor of Structural
Biology
Xiang-Lei Yang, Ph.D.*
Associate Professor of
Molecular Biology
Todd O. Yeates, Ph.D.
Adjunct Professor
University of California
Los Angeles, California
Qinghai Zhang, Ph.D.
Assistant Professor
SERVICE FACILITIES
Gerard Kroon
Manager, Nuclear Magnetic
Resonance Facilities
204 MOLECULAR BIOLOGY
Michael E. Pique
Director, Computer Graphics
Development
2008
THE SCRIPPS RESEARCH INSTITUTE
Kelly Lee, Ph.D.
Michael Baksh, Ph.D.
Gerald Edward Dodson, Ph.D.
Brian Paegel, Ph.D.
Manidipa Banerjee, Ph.D.
Claire Louise Dovey, Ph.D.
Jefferson Perry, Ph.D.
Christine Beuck, Ph.D.
Richard R. Rivera, Ph.D.
David Boehr, Ph.D.
Jerome Dupuy, Ph.D.***
Université Mediterranée
Marseille, France
David S. Shin, Ph.D.
Yannick Bomble, Ph.D.***
National Renewable Energy
Laboratory
Golden, Colorado
Michelle Duquette-Huber,
Ph.D.
Giovanni Bottegoni, Ph.D.***
Instituto Italiano de
Tecnologia
Genova, Italy
Susanna V. Ekholm-Reed,
Ph.D.
S E N I O R S TA F F
SCIENTISTS
Marc Elsliger, Ph.D.
Quansheng Zhou, Ph.D.
S TA F F S C I E N T I S T S
Philip Arno Venter, Ph.D.
Xiaoqin Ye, M.D., Ph.D.***
University of Georgia
Athens, Georgia
Svitlana Berezhna, Ph.D.
Stephen Edgcomb, Ph.D.
Venkata Reddy Erigala, Ph.D.
Vadim Cherezov, Ph.D.
R E S E A R C H A S S O C I AT E S
Reto Horst, Ph.D.
Tommy Bui, Ph.D.
Maria Martinez-Yamout, Ph.D.
Melanie Ann Adams, Ph.D.***
University of Toronto
Toronto, Ontario, Canada
Rosa Maria Cardoso, Ph.D.††
Allan Chris Merrera Ferreon,
Ph.D.
Garrett M. Morris, Ph.D.
Fabio Agnelli, Ph.D.
Andrew Barry Carmel, Ph.D.
Josephine Chu Ferreon, Ph.D.
Chiaki Nishimura, Ph.D.
Hanna-Stina Martinsson
Ahlzén, Ph.D.
Juan Jovel Castillo, Ph.D.
Stefano Forli, Ph.D.
Amarnath Chatterjee, Ph.D.
Chi-Yu Fu, Ph.D.
Susana Chaves, Ph.D. ††
Zara Fulton, Ph.D.
Yen-Ju Chen, Ph.D.***
National Synchrotron
Radiation Research Center
Hsinchu, Taiwan
Yann Gambin, Ph.D.
Zhiyong Chen, Ph.D.***
Trius Therapeutics
San Diego, California
Joshua Gill, Ph.D.
Srinivas Chittaboina, Ph.D.††
Rajib Kumar Goswami, Ph.D.
Ji Woong Choi, Ph.D.
Arsen Grigoryan, Ph.D.
Chung Jen Chou, Ph.D.
Bettina Groschel, Ph.D.***
Ardea Biosciences, Inc.
San Diego, California
Daniel Felitsky, Ph.D.
Jeffrey Speir, Ph.D.
Reetesh Raj Akhouri, Ph.D.
Mutsuo Yamaguchi, Ph.D.
Xueyong Zhu, Ph.D.
SENIOR RESEARCH
Alexander Ivanov Alexandrov,
Ph.D.***
Takeda
San Diego, California
A S S O C I AT E S
Stephen G. Aller, Ph.D.
Ryan Burnett, Ph.D.***
Tocagen, Inc.
San Diego, California
Beatriz Gonzalez Alonso,
Ph.D.
Adrienne Elizabeth Dubin,
Ph.D.
Li Fan, Ph.D.
Maria Alejandra GamezAbascal, Ph.D.***
Universidad Autonoma de
Madrid
Madrid, Spain
Juliano Alves, Ph.D.***
Genomics Institute of the
Novartis Research
Foundation
San Diego, California
William Anderson, Ph.D.
Andrew James Annalora,
Ph.D.
Munehito Arai, Ph.D.
Li-Chiou Chuang, Ph.D. ††
Linda Maria Columbus,
Ph.D.***
University of Virginia
Charlottesville, Virginia
Julia Gavrilyuk, Ph.D.
Charles Gersbach, Ph.D.
Edith Caroline Glazer, Ph.D.
Hai-Ming Guo, Ph.D.***
Hanan Normal University
Hanan, China
Min Guo, Ph.D.
Elsa Garcin, Ph.D.***
University of Maryland
Baltimore, Maryland
Guillermo Asmar-Rovira,
Ph.D.
Julio Kovacs, Ph.D.***
SeaSpace Corporation
Poway, California
Wojciech Augustyniak,
Ph.D.***
Max-Planck Institute
Leipzig, Germany
Sandro Cosconati, Ph.D.***
University of Naples
Naples, Italy
Sung-Hun Bae, Ph.D.
Robert De Bruin, Ph.D.
Byung Woo Han, Ph.D.
Krishna Mohan Bajjuri, Ph.D.
Pritilekha Deka, Ph.D.
Rodney Harris, Ph.D.
Brent Krueger, Ph.D.***
Hope College
Holland, Michigan
Stephen Connelly, Ph.D.
Adam Corper, Ph.D.
Rey-Ting Guo, Ph.D.
Mahender Gurram, Ph.D.***
AMRI
Albany, New York
Peter Haberz, Ph.D.
MOLECULAR BIOLOGY
2008
David M. Herman, Ph.D.
Edward Lemke, Ph.D.
Deron Herr, Ph.D.
Vasco Liberal, Ph.D.***
Alert Life Sciences
Computing
Villa Novade Gaia, Portugal
Kenichi Hitomi, Ph.D.
Minsun Hong, Ph.D.
Wen-Xu Hong, Ph.D.
Fang Hu, Ph.D.
William M. Lindstrom,
Ph.D.***
Acelot, Inc.
Santa Barbara, California
Veli-Pekka Jaakola, Ph.D.
Bin Liu, Ph.D.
Thamara Janaratne, Ph.D.
Hsiao-Wei Liu, Ph.D.
Christine Jespersen, Ph.D.
Wei Liu, Ph.D.
Margaret Alice Johnson, Ph.D.
Kunheng Luo, Ph.D.
Steven Johnson, Ph.D.
Susanna Juraja, Ph.D.
Sachin Kale, Ph.D.
Tse Siang Kang, Ph.D.
Darly Joseph Manayani,
Ph.D.***
Pax Vax
San Diego, California
Mili Kapoor, Ph.D.
Santiago Cavero Martinez,
Ph.D. ††
Ananta Karmakar, Ph.D.
Tsutomu Matsui, Ph.D.
Andrey Aleksandrovich
Karyakin, Ph.D.
Derrick Meinhold, Ph.D.
THE SCRIPPS RESEARCH INSTITUTE
Wataru Nomura, Ph.D.***
Tokyo Medical and Dental
University
Tokyo, Japan
Amy Odegard, Ph.D.
Akira Onoda, Ph.D.***
Osaka University
Osaka, Japan
Bill Francesco Pedrini,
Ph.D.***
ETH Zurich
Zürich, Switzerland
Jennifer S. Scorah, Ph.D.***
CovX
La Jolla, California
Alexander Perryman, Ph.D.
Pedro Serrano-Navarro, Ph.D.
Suzanne Peterson, Ph.D.
Zahra Shajani, Ph.D.
David A. Shore, Ph.D.
Andrew Mercer, Ph.D.
Eda Koculi, Ph.D. ††
Jonathan Mikolosko, Ph.D.
Ron Piran, Ph.D.
Bethany Koehntop, Ph.D.
Mauro Mileni, Ph.D.
Irina Kufareva, Ph.D.
Sharon Kwan, Ph.D.
Maki Minakawa, Ph.D.***
Riken, Wako Institute
Saitama, Japan
William Placzek, Ph.D.***
Burnham Institute for
Medical Research
La Jolla, California
Bianca Lam, Ph.D.
Satoshi Mizuta, Ph.D.
Goran Pljevaljc̆ić, Ph.D.
Stephanie Pond, Ph.D.***
Prognosys Biosciences, Inc.
La Jolla, California
John Prudden, Ph.D.
Joachim Latzer, Ph.D.
Tetsuji Mutoh, Ph.D.
Chang-Wook Lee, Ph.D.
Mir Hussain Nawaz, Ph.D.
Chul Won Lee, Ph.D.
Tuan Nguyen, Ph.D.
Young-Tae Lee, Ph.D.
George Nicola, Ph.D.
Sophie Lefebvre, Ph.D. ††
Kyoko Noguchi, M.D., Ph.D.
Andre Schiefner, Ph.D.
Vladimir Pelmenschikov,
Ph.D.***
Vrije Universiteit
Amsterdam, the Netherlands
Dae Hee Kim, Ph.D.
Samrat Mukhopadhyay, Ph.D.
Gor Sarkisyan, Ph.D.
Lauren J. Schwimmer,
Ph.D. ††
Elena Menichelli, Ph.D.
Jason Lanman, Ph.D.
Mariana Santa-Marta, Ph.D.
Robert Pejchal, Ph.D.
Reza Khayat, Ph.D.
Davide Moiani, Ph.D.
Manuel Rueda, Ph.D.
Riturparna Sinha Roy,
Ph.D.***
Brigham and Women’s
Hospital
Boston, Massachusetts
Jessica Petrillo, Ph.D.***
Office of Chemical and
Biological Weapons, Threat
Reduction
Washington, D.C.
Emma Langley, Ph.D. ††
Christopher Roth, Ph.D.
Sung-Jean Park, Ph.D.
Donald Keidel, Ph.D.
Biswaranjan Mohanty, Ph.D.
Rae Robertson, Ph.D.
Alim Seit-Nebi, Ph.D. ††
Eva Mejia Ramirez De
Arellano, Ph.D.
Petra Langerak, Ph.D.
Gabriela Ring, Ph.D.
So-Jung Park, Ph.D.
Stephanie Pebernard, Ph.D.††
205
Kimberly A. Reynolds,
Ph.D.***
University of Texas
Dallas, Texas
Jin-Kyu Rhee, Ph.D.***
Department of Chemistry,
Scripps Research
Errin Rider, Ph.D.
Daniela Andrea Slavin,
Ph.D.***
Ligand Pharmaceuticals
San Diego, California
Elisabetta Soragni, Ph.D.
Surya Venkata Sripada,
Ph.D.***
Syngene International, Ltd.
Bangalore, India
Pawel Stanczak, Ph.D.
Thomas Steinbrecher, Ph.D.
Pal Stenmark, Ph.D.***
Karolinska Institutet
Stockholm, Sweden
Shih-Che Su, Ph.D.
Sebastian Sudek, Ph.D.
Salahuddin Syed, Ph.D.
Michael T. Sykes, Ph.D.
Blair R. Szymczyna, Ph.D.
206 MOLECULAR BIOLOGY
2008
Bin Tang, Ph.D.
Sung-Il Yoon, Ph.D.
Rebecca E. Taurog, Ph.D.
Naoto Yoshizuka, M.D., Ph.D.
Ewan Richardson Taylor,
Ph.D.
Haile Zhang, Ph.D.
Leonardo Teixeira, Ph.D.
Mauricio Carrillo Tripp, Ph.D.
Oleg Trott, Ph.D
Ulrich Tschulena, Ph.D.***
German Cancer Research
Center
Heidelberg, Germany
Julie L. Tubbs, Ph.D.
Qing Zhang, Ph.D.***
Glaxo Smith Kline
Shanghai, China
Wei Zhang, Ph.D.***
aTyr Pharma
La Jolla, California
Qiang Zhao, Ph.D.
Yong Zhao, Ph.D.***
Cambridge Soft
Cambridge, Massachusetts
THE SCRIPPS RESEARCH INSTITUTE
Tammy Dwyer, Ph.D.
San Diego State University
San Diego, California
Wayne A. Fenton, Ph.D.
Yale University
New Haven, Connecticut
Astrid Graslund, Ph.D.
Stockholm University
Stockholm, Sweden
Arne Holmgren, M.D., Ph.D.
Karolinska Institutet
Stockholm, Sweden
Barry Honig, Ph.D.
Columbia University
New York, New York
Hisatoshi Uehara, Ph.D.
Naoto Utsumi, Ph.D.***
Otsuka Pharmaceutical Co.,
Ltd.
Tokushima, Japan
S C I E N T I F I C A S S O C I AT E S
Ajay Vashisht, Ph.D.***
University of California
Los Angeles, California
Dennis Carlton, B.S.
Sangita Venkataraman, Ph.D.
Xiaoping Dai, Ph.D.
Petra Verdino, Ph.D.
Marc Deller, D.Phil.
My Vo, Ph.D.
Gye Won Han, Ph.D.
Jun Wang, Ph.D.
Wenge Han, Ph.D.
Jessica Williams, Ph.D.
Michael Allen Hanson, Ph.D.
Robert Scott Williams, Ph.D.
Vance Wong, Ph.D.
Hope Johnson, Ph.D.***
California State University
Fullerton, California
Ulrich Wuellner, Ph.D.
Teresa Jones, Ph.D.
Wei Xie, Ph.D.***
Salk Institute for Biological
Studies
La Jolla, California
Lin Wang, Ph.D.***
MedImmune, L.L.C.
Gaithersburg, Maryland
Chunping Xu, Ph.D.
V I S I T I N G I N V E S T I G AT O R S
Rui Xu, Ph.D.
Yoshiki Yamada,
Ph.D. ††
Tohru Yamagaki, Ph.D.***
Suntory Institute for
Bioorganic Research
Osaka, Japan
Kye Sook Yi, Ph.D.
Enrique Abola, Ph.D.
Andrew S. Arvai, M.S.
Ellen Yu-Lin Tsai Chien, Ph.D.
Stephen J. Benkovic, Ph.D.
Pennsylvania State University
University Park, Pennsylvania
Tobias Cramer, Ph.D.
CINECA Supercomputer
Center HPC Europe
Bologna, Italy
Arthur Horwich, M.D.
Yale University
New Haven, Connecticut
Tai-Huang Huang, Ph.D.
Academica Sinica
Taipei, Taiwan
Michael Johnson, Ph.D.
University of Chicago
Chicago, Illinois
Joseph David Ng, Ph.D.
University of Alabama
Huntsville, Alabama
Mary Jo Ondrechen, Ph.D.
Northeastern University
Boston, Massachusetts
Victoria A. Roberts, Ph.D.
University of California
San Diego, California
Robert D. Rosenstein, Ph.D.
Lawrence Berkeley National
Laboratory
Berkeley, California
* Joint appointment in The
Skaggs Institute for Chemical
Biology
** Joint appointments in the
Department of Chemistry and
The Skaggs Institute for
Chemical Biology
*** Appointment completed; new
location shown
**** Joint appointments in the
Department of Immunology
and The Skaggs Institute for
Chemical Biology
***** Joint appointment in the
Department of Cell Biology
†
††
Joint appointment in the
Department of Chemistry
Appointment completed
MOLECULAR BIOLOGY
2008
Chairman’s Overview
olecular biology forms the cornerstone of biological and biomedical research. Research in
the Department of Molecular Biology encompasses a broad range of
disciplines, extending from
structural and computational biology at one
extreme to molecular
genetics at the other.
Our scientists continue
to make rapid progress
toward a deeper understanding of the fundamental molecular events
that underlie the processes of life. Major
Peter E. Wright, Ph.D.
advances have been
made in elucidating the
structural biology of signal transduction, receptor recognition, and viral assembly; understanding mechanisms
of viral infectivity; determining the structures of membrane proteins and multidrug transporters; understanding the molecular basis of nucleic acid recognition and
DNA repair; and determining the mechanisms of protein
folding and ribosome assembly.
Progress has been made in elucidating the molecular
events involved in regulation of the cell cycle, tumor
development, induction of sleep, the molecular origins
of neuronal development and of CNS disorders, the
regulation of transcription, and decoding of genetic information in translation. Finally, new advances have been
made in the design of novel low molecular weight compounds that can specifically regulate genes and in biomolecular engineering, building novel functions into viruses,
antibodies, zinc finger proteins, RNA, and DNA. Progress in these and other areas is described in detail on
the following pages, and only a few highlights are mentioned here.
In a landmark achievement, ranked as 1 of the top 10
breakthroughs of 2007 by Science magazine, Raymond
Stevens and his collaborators determined the 3- dimensional structure of the β2-adrenergic receptor. This structure, which represents the culmination of 15 years of
research in the Stevens laboratory, promises to revolutionize research on G protein–coupled receptors (GPCRs)
and have a major impact on drug discovery. More than
800 GPCRs have been identified, making them the largest
M
THE SCRIPPS RESEARCH INSTITUTE
207
family of membrane protein receptors, yet previously, the
structure of only 1 GPCR was available, for the light
receptor rhodopsin. The structure of the β 2 -adrenergic
receptor provides the first insights into the large class
of GPCRs that regulate signal transduction processes by
binding diffusible ligands. The GPCRs control a broad
spectrum of physiologic responses, activating intracellular
signaling pathways in response to stimuli from outside
the cell. GPCRs regulate cell growth and differentiation;
control cardiovascular function, metabolism, and the
immune response; and play a key role in neurotransmission. Approximately half of currently used drugs function
by binding to and regulating receptors from the GPCR
family. The structure of the β2-adrenergic receptor provides unprecedented insights into the mechanism by which
GPCRs interact with their natural ligands and with drugs
and paves the way to design of new and more effective
pharmaceutical agents with fewer side effects.
Research in the laboratory of John Tainer has led
to a detailed understanding of the mechanism by which
mutations in a critical enzyme involved in DNA repair
lead to 3 distinct disease phenotypes. Dr. Tainer and
coworkers determined the structure of the XPD helicase,
which is absolutely required for nucleotide excision repair
of damaged DNA, and measured the effects of diseasecausing mutations on the enzymatic activity of the helicase. Mutations associated with xeroderma pigmentosum
impair the DNA helicase activity of XPD and greatly predispose patients to skin cancer. Mutations associated
with Cockayne syndrome also reduce helicase activity
and, in addition, cause the XPD enzyme to become stuck
on the DNA that is undergoing repair. Finally, trichothiodystrophy mutants cause framework defects that
disrupt the integrity of the repair machinery. Related
structural work in the Tainer laboratory provided important insights into the mechanism of base excision repair
of damaged DNA. A molecular level knowledge of mechanisms of DNA repair is of central importance to understanding cancer, developmental diseases, and aging, and
pathogen-specific DNA repair enzymes are potential targets for novel antibacterial and antifungal agents.
A collaboration between Curt Wittenberg and Paul
Russell of our department and John Yates, Department
of Cell Biology, has led to novel insights into the molecular mechanism by which cells respond to errors in DNA
replication. During normal cell division, a protein named
Nrm1 binds to DNA and represses the expression of key
genes during the G1 phase of the cell cycle. Under conditions of stress, replication stalls, and repression of
208 MOLECULAR BIOLOGY
2008
these G 1-phase genes by Nrm1 is blocked, resulting in
expression of proteins needed to correct the problem that
caused the stall. Understanding the molecular mechanisms responsible for checkpoint control during the cell
cycle is critical for understanding oncogenesis and may
eventually facilitate development of novel cancer therapeutics that target the replication checkpoints.
Recent work by Paul Schimmel and members of his
group has revealed the mechanism by which alanyl-tRNA
synthetase edits mischarged tRNAAla to correct errors of
protein synthesis. Mistranslation, which occurs when an
incorrect amino acid binds to tRNA and becomes incorporated into a protein, leads to synthesis of proteins
containing errors. Such errors of protein synthesis are
associated with numerous diseases. The research by
Dr. Schimmel and coworkers provides novel insights into
the checkpoints that guard against misincorporation of
amino acids and greatly extends our understanding of
how cells avoid errors during protein synthesis.
Research by Nick Boddy, Clare McGowan, John Tainer,
and their coworkers has led to the identification of a
previously unknown and completely unexpected family
of ubiquitin ligases that mediate cross talk between the
sumoylation and ubiquitination pathways. These SUMOtargeted ubiquitin ligases specifically target SUMOmodified proteins for ubiquitination and subsequent
proteasomal degradation. The ligases bind specifically
to sumoylated proteins and catalyze ubiquitination of the
proteins, thereby playing a central role in regulation of
sumoylation pathways and in the homeostasis of SUMOmodified proteins. SUMO-targeted ubiquitin ligases are
involved in the regulation of genome stability, and evidence exists that they play a central role in controlling
cancer metastasis, making them potential targets for novel
therapeutics designed to inhibit cancer growth.
Molecular biology remains a field of enormous opportunity and excitement. The scientists in this department
are taking full advantage of powerful new technologies
to advance our understanding of fundamental biological
processes at the molecular level. Their discoveries will
ultimately be translated into new advances in biotechnology and medicine.
THE SCRIPPS RESEARCH INSTITUTE
MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
209
Investigators’ Reports
Structural Biology of Viral
Proteins, Molecular Assemblies,
and the Immune System
I.A. Wilson, R.L. Stanfield, J. Stevens, X. Zhu, M.A. Adams,
C.H. Bell, R.M.F. Cardoso, J. Carlson, P.J. Carney, S. Connelly,
A.L. Corper, T. Cross, X. Dai, E.W. Debler, W.L. Densley, B.J.
Droese, D.C. Ekiert, M.-A. Elsliger, S. Ferguson, Z. Fulton, B.W.
Han, G.W. Han, M. Hong, M.J. Jimenez-Dalmaroni, R.N.
Kirchdoerfer, J.R. Mikolosko, R. Pejchal, G.P. Porter,
A. Schiefner, D.A. Shore, R.S. Stefanko, J.A. Vanhnasy,
P. Verdino, R. Xu, X. Xu, S.I. Yoon
o understand the immune response to invading
pathogens, such as bacteria and viruses, we
focus on the structure-function relationships of
immune molecules and their microbial targets. These
structural results are especially useful in the design of
drugs and vaccines that target the pathogens and protect the host.
T
T H E I N N AT E I M M U N E S Y S T E M
To enhance our understanding of the molecular biology of innate immune receptors, we are investigating
the activation requirements of γδ T cells. In collaboration
with W. Havran, Department of Immunology and Microbial Science, we determined the structure of junctional
adhesion molecule-like (JAML), the γδ T cell–specific
costimulatory molecule, in complex with coxsackievirusadenovirus receptor, its endogenous ligand (Fig. 1).
The structure revealed an unusually hydrophilic complex
interface that suggests potential mechanisms for receptor triggering. Upon JAML engagement, different kinases
are then recruited at the JAML intracellular domain to
activate kinase signaling cascades, production of cytokines and chemokines, and, ultimately, proliferation of
γδ T cells.
Toll-like receptors are cell-surface receptors that
detect invading microbes by recognizing a variety of
pathogen-associated molecular patterns, including bacterial cell walls and viral nucleic acids. To reveal structural mechanisms involved in activation and regulation
of these receptors, we have expressed the extracellular
domain of human Toll-like receptor 4 with myeloid differentiation protein-2 for structural studies. This immune
complex binds bacterial lipopolysaccharides, ultimately
leading to sepsis.
F i g . 1 . Crystal structure of JAML in complex with coxsackievirus-
adenovirus receptor (CAR). JAML and the receptor interact with their
membrane-distal D1 immunoglobulin domains in a face-to-face
β-sheet interaction.
Among the pattern-recognition molecules, the intracellular Nod-like receptors also act as key mediators of
innate immunity and of the inflammatory response to
microbial infections. Mutations in the genes for these
receptors are associated with chronic inflammatory barrier diseases, such as Crohn’s disease and bronchial
asthma. We have expressed and purified Nod1 and Nod2
proteins for crystallization. The studies on the Toll- and
Nod-like receptors are collaborations with R. Ulevitch,
Department of Immunology and Microbial Science, and
B. Beutler, Department of Genetics.
N O N M A M M A L I A N I N N AT E A N D A D A P T I V E I M M U N I T Y
Variable lymphocyte receptors (VLRs) play a key role
in recognition of antigens in the adaptive immune response
of jawless vertebrates. In collaboration with M.D. Cooper,
Emory University School of Medicine, Atlanta, Georgia,
we determined the crystal structure of the lamprey
210 MOLECULAR BIOLOGY
2008
VLR2913 ectodomain to 2.1-Å resolution. The VLR
folds into a horseshoe-shaped, solenoidal assembly of
5 leucine-rich repeats. Although the antigen for VLR2913
is unknown, the highly similar VLR4 interacts with the
anthrax antigen, bacillus collagen-like protein of anthracis
(BclA). We have modeled VLR4 with Modeller, on the
basis of the VLR2913 structure, and then used Autodock
4.0 software to dock the VLR4 model to BclA, in collaboration with A.J. Olson and G.M. Morris, Department
of Molecular Biology (Fig. 2). The docking results suggest that the concave surface of VLR4 is the recognition site for BclA.
THE SCRIPPS RESEARCH INSTITUTE
ion hole at P9 that is present only in the diabetes-associated MHC I-Ag7. We isolated a TCR hybridoma (21.30)
F i g . 3 . Crystals of the MHC class I–peptide–CD8αβ single-
chain complex.
F i g . 2 . Model of the complex formed by lamprey VLR4 and BclA.
The VLR4 was modeled by using the computer program Modeller,
with the lamprey VLR2913 crystal structure as a template. The VLR4
model was then tested for the lowest energy docking orientation with
BclA by using Autodock software.
CLASSICAL AND NONCLASSICAL MHC AND T-CELL
RECEPTOR SIGNALING
T-cell receptors (TCRs) recognize peptide antigen
displayed on the surface of antigen-presenting cells by
MHC molecules. Coreceptor molecules, such as CD8αβ
and CD4, provide costimulatory signals that are required
for full T-cell activation. To ascertain the structural basis
for the coreceptor function of CD8αβ and understand
the mechanisms that underlie T-cell activation, we used
a single-chain MHC-CD8αβ construct to determine the
structure of the CD8αβ-MHC-peptide complex (Fig. 3).
To explore potential mechanisms of diabetogenesis,
we are investigating whether TCRs recognize the oxyan-
that is sensitive to the presence or absence of a negatively charged P9 peptide residue and determined the
crystal structure to 3.5-Å resolution of TCR 21.30–
I-Ag7–HEL9-27, where P9 is a glycine. Surprisingly, the
structure revealed that TCR 21.30 does not directly contact the I-Ag7 P9 pocket. Experiments are under way to
ascertain the TCR sensitivity to this residue position.
MHC molecules play a critical role in initiating cellmediated immunity by presenting both foreign antigens
and self-antigens. Efficient loading of peptide antigens
on MHC class I molecules requires proteins in the endoplasmic reticulum collectively known as the peptideloading complex. The complex consists of the transporter
associated with antigen processing, tapasin, calreticulin, calnexin, ERp57, and an MHC class I molecule.
Currently, we are focusing on the structural and biochemical characterization of the transporter, tapasin,
and the peptide-free form of MHC class I molecules.
Recombinant expression of tapasin from several species, as well as recombinant expression of the transporter, have provided valuable tools for analyzing the
structure, function, and assembly of the peptide-loading complex. Biochemical and biophysical techniques
are being used to provide structural models for MHC
class Ia folding and antigen presentation. The MHC
and TCR studies are a collaboration with L. Teyton,
Department of Immunology and Microbial Science.
CD1 molecules are MHC class I antigen-presenting
molecules that present lipids, glycolipids, and lipopeptides to effector T cells. CD1 molecules are involved in
host defense and also have immunoregulatory functions.
Glycolipids presented by CD1d stimulate natural killer
MOLECULAR BIOLOGY
2008
T cells, which are of clinical interest because they rapidly
secrete cytokines that either promote or suppress different immune responses. On the basis of our structural
studies, C.-H. Wong and his group, Department of Chemistry, synthesized a series of glycolipids that are more
potent than other glycolipids tested previously and have
increased efficacy in T-cell assays. Structures for 2 of
the most stimulating glycolipids in complex with CD1d
have revealed that loading and anchoring of the lipids
are the key determinants for effective lipid presentation
and subsequent T-cell stimulation.
INFLUENZA VIRUS
To aid in design of vaccines and drugs to prevent
future influenza pandemics, we are studying proteins
from different influenza strains as part of a consortium
funded by the National Institute of Allergy and Infectious
Diseases. The 1918 flu pandemic was the most devastating epidemic in recorded world history, and efforts are
ongoing to target the neuraminidase of the 1918 influenza virus in structure-based drug design. We have
determined crystal structures for the 1918 N1 neuraminidase from crystals with an unusual defect called a
“lattice translocation.” Although the successful use of
twinned data for structure determination has become
relatively routine in recent years, structure determination
with lattice-translocation defects has only been previously reported for 5 structures. In addition, structures
of the 1918 neuraminidase in complex with zanamivir
(Relenza) and oseltamivir (Tamiflu) have revealed new
cavities for drug binding (Fig. 4) and how the presence
F i g . 4 . Molecular surface of the 1918 influenza virus N1 neuraminidase active site. Zanamivir (ball-and-stick model) has been
docked into the unliganded neuraminidase structure on the basis of
the drug’s position in the zanamivir-neuraminidase complex. This
superposition reveals a large, unoccupied cavity close to the zanamivir
binding site that may be an excellent target for the design of inhibitors.
THE SCRIPPS RESEARCH INSTITUTE
211
or absence of different ions can affect the overall assembly of the neuraminidase tetramer.
The H5N1 avian influenza viruses currently circulating in Asia, Europe, and Africa are extremely virulent in humans, causing severe disease and often death.
H5N1 viruses are not readily transmitted among humans,
possibly because of differences in the receptor specificity of the hemagglutinin viral fusion protein. To investigate structural changes critical for receptor switching
from avian to human specificity, we have developed a
baculovirus display platform that will enable us to test
large libraries of hemagglutinin mutants for binding to
immobilized glycans or human bronchial cell monolayers.
The receptor specificity of the selected variants will be
determined by using glycan arrays, and structural changes
associated with receptor switching will be characterized by using x-ray crystallography. In collaboration with
G.J. Tobin, Biological Mimetics, Inc., Frederick, Maryland, the full-length hemagglutinin from an outbreak
of influenza in Wyoming and HA2 hemagglutinin fragments from outbreaks in South Carolina in 1918 (H1H1)
and Vietnam in 2003 (H5N1) are being expressed for
structural studies of conformations before and after
fusion. In addition, many collaborations are ongoing to
investigate the neutralization of H1N1 and H5N1
viruses by monoclonal antibodies.
H I V T Y P E 1 VA C C I N E
The need for an effective HIV vaccine is greater than
ever as the virus continues to devastate areas of the
world such as sub-Saharan Africa. As a part of our
vaccine development efforts, we are studying the viral
envelope “spikes” composed of heterotrimeric complexes
of gp120 and gp41. Upon binding receptors CD4 and
CXCR4/CCR5, the trimer undergoes as yet uncharacterized conformational changes that lead to fusion of
the viral membrane with the target cell, initiating infection. Crystallization of the trimer in the prefusion state
will enable a detailed understanding of its antibody
epitope landscape and reveal how neutralizing antibodies can recognize this evolutionarily moving target.
We recently determined the crystal structure of a
human monoclonal antibody, F425-B4e8 (B4e8), that
cross-reacts with the gp120 V3 region of primary viral
isolates from subtypes B, C, and D. The B4e8 Fab in
complex with the 24mer V3 peptide RP142 showed that
the antibody recognizes a novel V3 loop conformation
with a 5-residue α-turn around the conserved GPGRA
apex of the β-hairpin loop (Fig. 5). The Fab interacts
primarily through main-chain interactions with major
212 MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
found that the prolonged luminescence is due to a
charge-transfer excited complex of an anionic stilbene
and a cationic, parallel π-stacked tryptophan. Upon
charge recombination, this complex generates exceptionally bright blue light. Formation of the complex is
supported by a deep ligand-binding pocket, which in
turn is due to a noncanonical interface between the 2
variable antibody subunits. These studies are collaborations with R.A. Lerner, K.D. Janda, and P.G. Schultz,
Department of Chemistry; D.P. Millar, Department of
Molecular Biology; and H.B. Gray, California Institute
of Technology, Pasadena, California.
HISTONE DEACETYLASES
F i g . 5 . Structure of F425-B4e8 Fab in complex with a peptide
(red) representing the V3 region of HIV type 1 gp120. B4e8 is
unusually broad in its neutralization of different HIV type 1 viral
isolates, and the peptide conformation recognized has an unusual
α-turn around the tip of the loop.
contacts to only 2 V3 peptide side chains, explaining
how B4e8 can accommodate sequence variation within
V3 and hence can neutralize different isolates of HIV
type 1.
Our research on HIV is done in collaboration with
D.R. Burton, Department of Immunology and Microbial
Science; P.E. Dawson, Department of Cell Biology;
C.-H. Wong, Department of Chemistry; J.K. Scott, Simon
Fraser University, Burnaby, British Columbia; J. Moore,
Weill Medical College of Cornell University, New York,
New York; H. Katinger, R. Kunert, and G. Stiegler, University für Bodenkultur, Vienna, Austria; R. Wyatt and
P. Kwong, Vaccine Research Center, National Institutes
of Health, Bethesda, Maryland; W. Olson, and K. Kang,
Progenics Pharmaceuticals, Inc., Tarrytown, New York;
the National Institutes of Health, Bethesda, Maryland;
and the Neutralizing Antibody Consortium of the International AIDS Vaccine Initiative, New York, New York.
BLUE FLUORESCENT ANTIBODIES
EP2-19G2, an antibody to a trans-stilbene, has
bright blue luminescence and has been used as a biosensor in various applications. By extensive biophysical
characterization of the stilbene-antibody complex, we
Histone deacetylases catalyze removal of the acetyl
group from amino-terminal lysine residues in histones,
resulting in chromatin condensation and transcriptional
repression. Inhibitors of histone deacetylases are a widely
used treatment for many types of cancer. In recent years,
these compounds have been emerging as a potential
therapy for neurodegenerative disorders, such as Friedreich
ataxia, an inherited disease that affects the nervous
system and results in muscle weakness, heart disease,
and speech difficulties. In collaboration with J.M.
Gottesfeld, Department of Molecular Biology, and with
support from the Friedreich’s Ataxia Research Alliance,
Springfield, Virginia, we are expressing several histone
deacetylases for determinations of crystal structures of
the enzymes in complex with inhibitors.
JOINT CENTER FOR STRUCTURAL GENOMICS
The Joint Center for Structural Genomics is a large
consortium of scientists from Scripps Research; the
Stanford Synchrotron Radiation Laboratory; the University of California, San Diego; the Burnham Institute
for Medical Research; and the Genomics Institute of
the Novartis Research Foundation. The center is funded
by the Protein Structure Initiative of the National Institute of General Medical Sciences. Its purpose is highthroughput structure determination of large families of
proteins with no or limited structural representatives,
biologically important targets that are conserved as the
central machinery of life, the complete proteome from
Thermotoga maritima, metagenomic and human microbiome targets, and other targets suggested by the community. To date, the members of the consortium have
pioneered many novel high-throughput methods and
technologies applicable to structural biology and have
determined more than 665 unique structures, including more than 200 novel structures in the past year.
MOLECULAR BIOLOGY
2008
PUBLICATIONS
Astronomo, R.D., Lee, H.K., Scanlan, C.N., Pantophlet, R., Huang, C.Y., Wilson,
I.A., Blixt, O., Dwek, R.A., Wong, C.-H., Burton, D.R. A glycoconjugate antigen
based on the recognition motif of a broadly neutralizing human immunodeficiency
virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the
self glycans of gp120. J. Virol. 82:6359, 2008.
Bell, C.H., Pantophlet, R., Schiefner, A., Cavacini, L.A., Stanfield, R.L., Burton,
D.R., Wilson, I.A. Structure of antibody F425-B4e8 in complex with a V3 peptide
reveals a new binding mode for HIV-1 neutralization. J. Mol. Biol. 375:969, 2008.
Burley, S.K., Joachimiak, A., Montelione, G.T., Wilson, I.A. Contributions to the
NIH-NIGMS Protein Structure Initiative from the PSI Production Centers. Structure
16:5, 2008.
Burton, D.R., Wilson, I.A. Immunology: square-dancing antibodies. Science
317:1507, 2007.
Debler, E.W., Müller, R., Hilvert, D., Wilson, I.A. Conformational isomerism can
limit antibody catalysis. J. Biol. Chem. 283:16554, 2008.
Debler, E.W., Kaufmann, G.F., Meijler, M.M., Heine, A., Mee, J.M., Pljevaljcic,
G., Di Bilio, A.J., Schultz, P.G., Millar, D.P., Janda, K.D., Wilson, I.A., Gray, H.B.,
Lerner, R.A. Deeply inverted electron-hole recombination in a luminescent antibody-stilbene complex. Science 319:1232, 2008.
Huang, C.C., Lam, S.N., Acharya, P., Tang, M., Xiang, S.H., Hussan, S.S., Stanfield, R.L., Robinson, J., Sodroski, J., Wilson, I.A., Wyatt, R., Bewley, C.A.,
Kwong, P.D. Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody
with HIV-1 gp120 and CD4. Science 317:1930, 2007.
Huang, S., Romanchuk, G., Pattridge, K., Lesley, S.A., Wilson, I.A., Matthews, R.G.,
Ludwig, M. Reactivation of methionine synthase from Thermotoga maritima (TM0268)
requires the downstream gene product TM0269. Protein Sci. 16:1588, 2007.
Johnson, S.M., Connelly, S., Wilson, I.A., Kelly, J.W. Biochemical and structural
evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J. Med. Chem. 51:260, 2008.
Kozbial, P., Xu, Q., Chiu, H.J., et al. Crystal structures of MW1337R and lin2004:
representatives of a novel protein family that adopt a four-helical bundle fold. Proteins 71:1589, 2008.
Krishna, S.S., Tautz, L., Xu, Q., et al. Crystal structure of NMA1982 from Neisseria meningitidis at 1.5 Å resolution provides a structural scaffold for nonclassical,
eukaryotic-like phosphatases. Proteins 69:415, 2007.
Mathews, I.I., McMullan, D., Miller, M.D., et al. Crystal structure of 2-keto-3deoxygluconate kinase (TM0067) from Thermotoga maritima at 2.05 Å resolution.
Proteins 70:603, 2008.
Menendez, A., Calarese, D.A., Stanfield, R.L., Chow, K.C., Scanlan, C.N.,
Kunert, R., Katinger, H., Burton, D.R., Wilson, I.A., Scott, J.K. A peptide inhibitor
of HIV-1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120. FASEB J. 22:1380, 2008.
Premkumar, L., Rife, C.L., Sri Krishna, S., et al. Crystal structure of TM1030
from Thermotoga maritima at 2.3 Å resolution reveals molecular details of its transcription repressor function. Proteins 68:418, 2007.
Sanguineti, S., Centeno Crowley, J.M., Lodeiro Merlo, M.F., Cerutti, M.L., Wilson,
I.A., Goldbaum, F.A., Stanfield, R.L., de Prat-Gay, G. Specific recognition of a
DNA immunogen by its elicited antibody. J. Mol. Biol. 370:183, 2007.
Saphire, E.O., Montero, M., Menendez, A., van Houten, N.E., Irving, M.B., Pantophlet, R., Zwick, M.B., Parren, P.W., Burton, D.R., Scott, J.K., Wilson, I.A.
Structure of a high-affinity “mimotope” peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies. J. Mol.
Biol. 369:696, 2007.
Slabinski, L., Jaroszewski, L., Rodrigues, A.P., Rychlewski, L., Wilson, I.A., Lesley, S.A., Godzik, A. The challenge of protein structure determination—lessons
from structural genomics. Protein Sci. 16:2472, 2007.
Slabinski, L., Jaroszewski, L., Rychlewski, L., Wilson, I.A., Lesley, S.A., Godzik,
A. XtalPred: a web server for prediction of protein crystallizability. Bioinformatics
23:3403, 2007.
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213
Stoll, R., Lee, B.M., Debler, E.W., Laity, J.H., Wilson, I.A., Dyson, H.J., Wright,
P.E. Structure of the Wilms tumor suppressor protein zinc finger domain bound to
DNA. J. Mol. Biol. 372:1227, 2007.
Structural Genomics Consortium; China Structural Genomics Consortium; Northeast Structural Genomics Consortium; Gräslund, S., Nordlund, P., Weigelt, J., et
al. Protein production and purification. Nat. Methods 5:135, 2008.
Wei, C.J., Xu, L., Kong, W.P., Shi, W., Canis, K., Stevens, J., Yang, Z.Y., Dell, A.,
Haslam, S.M., Wilson, I.A., Nabel, G.J. Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza
virus. J. Virol. 82:6200, 2008.
Xu, Q., Kozbial, P., McMullan, D., et al. Crystal structure of an ADP-ribosylated
protein with a cytidine deaminase-like fold, but unknown function (TM1506), from
Thermotoga maritima at 2.70 Å resolution. Proteins 71:1546, 2008.
Xu, Q., Saikatendu, K.S., Krishna, S.S., et al. Crystal structure of MtnX phosphatase
from Bacillus subtilis at 2.0 Å resolution provides a structural basis for bipartite
phosphomonoester hydrolysis of 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate.
Proteins 69:433, 2007.
Zajonc, D.M., Savage, P.B., Bendelac, A., Wilson, I.A., Teyton, L. Crystal structures
of mouse CD1d-iGb3 complex and its cognate Vα14 T cell receptor suggest a model
for dual recognition of foreign and self glycolipids. J. Mol. Biol. 377:1104, 2008.
Zajonc, D.M., Wilson, I.A. Architecture of CD1 proteins. Curr. Top. Microbiol.
Immunol. 314:27, 2007.
Zubieta, C., Joseph, R., Krishna, S.S., et al. Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA. Proteins
69:234, 2007.
Zubieta, C., Krishna, S.S., Kapoor, M., et al. Crystal structures of two novel dyedecolorizing peroxidases reveal a β-barrel fold with a conserved heme-binding
motif. Proteins 69:223, 2007.
Zubieta, C., Krishna, S.S., McMullan, D., et al. Crystal structure of homoserine Osuccinyltransferase from Bacillus cereus at 2.4 Å resolution. Proteins 68:999, 2007.
Protein Structures, Activities,
and Regulation: The Functioning
of Molecular Machines
E.D. Getzoff, A.S. Arvai, E.D. Garcin, C. Hitomi, K. Hitomi,
M.D. Kroeger, M.E. Pique, A.J. Pratt, D.S. Shin
o understand how proteins function as molecular
machines, we use structural molecular biology to
characterize and control proteins. We apply the
tools of structural, molecular, and computational biology
to analyze proteins of biological and biomedical interest, especially proteins that work synergistically with
coupled chromophores, metal ions, or other cofactors.
T
NITRIC OXIDE SYNTHASE AND A NEW APPROACH
TO DESIGN OF SELECTIVE INHIBITORS
Nitric oxide synthase (NOS) enzymes synthesize
nitric oxide, a signal for vasodilation and neurotransmission at low levels and a defensive cytotoxin at higher
levels. Synthesis of nitric oxide by NOS requires calmodulin-coordinated interactions between the catalytic, oxy-
214 MOLECULAR BIOLOGY
2008
genase, and electron-supplying reductase modules of
the enzyme. NOS uses heme, zinc, tetrahydrobiopterin,
calcium, NADPH, FMN, and FAD cofactors.
The goals of our structure-based drug design projects are to selectively inhibit inducible NOS, to prevent
inflammatory disorders, or neuronal NOS, to prevent
migraines, while maintaining blood pressure regulation
by endothelial NOS. Our x-ray crystallographic structures of wild-type and mutant NOS oxygenase dimers
with substrate, intermediate, inhibitors, cofactors, and
cofactor analogs, determined in collaboration with
J.A. Tainer, Department of Molecular Biology, and
D. Stuehr, the Cleveland Clinic, Cleveland, Ohio, provide
insights into both catalytic mechanism and inhibitor
selectivity. The nearly complete sequence and structural
conservation in the active sites of the 3 NOS isozymes
is a significant challenge in the design of isozyme-specific inhibitors. Nevertheless, our latest results indicate
that plasticity of distant isozyme-specific residues modulates conformational changes of invariant residues in
the substrate-binding site. We applied these results to
develop the anchored plasticity approach for the structure-based design of selective inhibitors (Fig. 1). In this
approach, conserved binding sites are used to anchor
the core of an inhibitor, while distant sequence differences are exploited to provide selectivity.
Our structure of the neuronal NOS reductase reveals
new insights into the complex regulatory mechanisms of
F i g . 1 . Diagram illustrating the anchored-plasticity approach to
selective drug design.
THE SCRIPPS RESEARCH INSTITUTE
this enzyme family. We integrated biochemical data with
our structures of NOS oxygenase, NOS reductase, and
calmodulin in complex with NOS peptides to propose
assembly and mechanistic hypotheses for the holoenzyme. We obtained promising results in support of these
hypotheses by using solution small-angle x-ray scattering, which can provide molecular envelopes for macromolecules and macromolecular complexes in solution.
We have proposed a moving-domain mechanism
for controlling the rate-limiting flow of electrons from
the flavin cofactors of NOS reductase to the catalytic
NOS oxygenase heme. Our assembly and mechanistic
hypotheses also explain the kinetics of regulatory sitespecific phosphorylation and dephosphorylation events
that both activate and inactivate synthesis of nitric
oxide in vivo. In ongoing research, we are using complementary biochemical, biophysical, and computational methods to define, describe, and understand
how NOS chemistry, structure, assembly, dynamics,
and protein-protein interactions regulate production of
nitric oxide in vivo.
PHOTOACTIVE PROTEINS AND CIRCADIAN CLOCKS
To understand in atomic detail how chromophorebound proteins translate sunlight into defined conformational changes for biological functions, we are exploring
the reaction mechanisms of the blue-light receptors photoactive yellow protein (PYP), photolyase, and cryptochrome. PYP is the prototype for the Per-Arnt-Sim
domain proteins of circadian clocks, whereas FAD-containing proteins of the photolyase/cryptochrome family
catalyze DNA repair or act in circadian clocks. To understand the photocycle of PYP and to propose a common
mechanism for signaling by Per-Arnt-Sim domains, we
combined ultra-high-resolution and time-resolved crystallographic structures of the PYP dark state and 2 photocycle intermediates with site-directed mutagenesis;
spectroscopy; deuterium-hydrogen exchange mass spectrometry, in collaboration with V. Woods, University of
California, San Diego; and quantum mechanical and
electrostatic computational methods, in combination
with L. Noodleman, Department of Molecular Biology.
Cryptochrome flavoproteins function as blue-light
receptors in plants and as components of circadian
clocks in animals. We determined the first crystallographic structure of a cryptochrome; the structure
revealed commonalities with the homologous photolyases in DNA binding and redox-dependent function
but showed differences in active-site and interactionsurface features. We found that this cryptochrome binds
MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
215
the same antenna cofactor found in a photolyase homolog but uses different amino acid residues to form the
cofactor-binding site. Our new structures and spectroscopy, obtained in collaboration with S. Weber, Universität Freiburg in Germany, of cryptochromes and of
photolyases from 2 other branches of the photolyase/
cryptochrome family that repair cyclobutane pyrimidine
dimers and (6-4) photoproducts help us decipher the
cryptic structure, function, and evolutionary relationships
of these fascinating redox-active proteins. Furthermore,
the (6-4) photolyase enzyme provides an excellent model
for human cryptochrome.
A simple, but functional, circadian clock can be
reconstituted in vitro from the 3 cyanobacterial proteins
KaiA, KaiB, and KaiC alone. Yet, the structure and
dynamics of the functional assembly are not understood.
Our crystallographic, dynamical light scattering and smallangle x-ray scattering studies revealed that KaiB selfassembles into a tetramer. We also study clock proteins
with PYP-like Per-Arnt-Sim domains that bind to mammalian cryptochromes. Our goal is to determine the detailed
chemistry and atomic structure of these proteins, define
their mechanisms of action and interaction, and use our
results to understand and regulate biological function.
Roberts, B.R., Tainer, J.A., Getzoff, E.D., Malencik, D.A., Anderson, S.R.,
Bomben, V.C., Meyers, K.R., Karplus, P.A., Beckman, J.S. Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS. J.
Mol. Biol. 373:877, 2007.
S U P E R O X I D E D I S M U TA S E
S M A L L - A N G L E X - R AY S C AT T E R I N G I N S O L U T I O N
The superoxide radical is a central player in the biology of reactive oxygen and nitrogen intermediates, which
mediate signaling and oxidative damage, a key factor in
aging and cancer, in vivo. Mutations in human copper
zinc superoxide dismutase (SOD), the enzyme that converts superoxide to molecular oxygen and hydrogen peroxide, cause the fatal neurodegenerative disease familial
amyotrophic lateral sclerosis, or Lou Gehrig disease. We
are analyzing the structural chemistry of SOD to help
bridge the gap from protein structures to enzyme stability and activities in vivo. We designed a zinc-free variant
of human SOD to help test the role of zinc binding and
loss in disease. Our results, obtained in collaboration
with J.A. Tainer, support the importance of the stable
SOD core structure in preventing amyloid formation and
toxic effects. For comparison, we determined the structure and stability of the SOD from the most extreme
eukaryotic thermophile known: the deep-sea hydrothermal vent worm Alvinella pompejana.
PUBLICATIONS
Garcin, E.D., Arvai, A.S., Rosenfeld, R.J., Kroeger, M.D., Crane, B.R., Andersson,
G., Andrews, G., Hamley, P.J., Mallinder, P.R., Nicholls, D.J., St-Gallay, S.A., Tinker, A.C., Gensmantel, N.P., Mete, A., Cheshire, D.R., Connolly, S., Stuehr, D.J.,
Åberg, A., Wallace, A.V., Tainer, J.A., Getzoff, E.D. Anchored plasticity opens doors
for selective inhibitor design in nitric oxide synthase. Nat. Chem. Biol., in press.
Yamamoto, J., Tanaka, Y., Hitomi, K., Getzoff, E.D., Iwai, S. Spectroscopic studies
on a novel intramolecular hydrogen bond within the (6-4) photoproduct. Nucleic
Acids Symp. Ser. (Oxf). Issue 51:79, 2007.
Structural Biology of Molecular
Interactions and Design
J.A. Tainer, A.S. Arvai, B.R. Chapados, L. Fan, E. Garcin,
G. Guenther, C. Hitomi, K. Hitomi, M.D. Kroeger, J.J. Perry,
M.E. Pique, D.S. Shin, J.L. Tubbs, R.S. Williams
e are developing new technologies and systems
to close the gaps from proteins to pathways
and from interaction networks to biological
outcomes in cells. We focus on molecular mechanisms
and relationships for proteins that control DNA damage
responses, reactive oxygen species, protein modifications, and pathogenesis. Our results have relevance for
improved understanding and therapeutic approaches
for cancer, aging, and degenerative diseases and for
bacterial pathogens.
W
C O M B I N E D W I T H C R Y S TA L L O G R A P H Y A N D
C O M P U TAT I O N
Aided by our synchrotron facility SIBLYS, we are
developing and applying technologies to develop accurate structures of protein conformation, assembly, and
interactions in solution by combining x-ray scattering
with x-ray crystallography and computation. We recently
developed methods for high-throughput analyses via
small-angle x-ray scattering. Small-angle x-ray scattering,
crystallography, and computation together allow multiscale modeling and fundamental insights to allosteric
mechanisms, self-assemblies, and dynamic molecular
machines acting as master keys to cell biology.
P R O T E I N M O D I F I C AT I O N S A N D F U N C T I O N
The ubiquitin-like protein family of posttranslational
modifications consists primarily of ubiquitin and the
small ubiquitin modifier SUMO. In collaboration with
M.N. Boddy, Department of Molecular Biology, we have
helped discover an intriguing family of proteins, SUMOtargeted ubiquitin ligases (STUbLs), that directly connect
the ubiquitination and sumoylation pathways. Uniquely,
STUbLs use SUMO interaction motifs to recognize their
sumoylated targets. STUbLs act as global regulators of
protein sumoylation levels, and cells lacking STUbLs
216 MOLECULAR BIOLOGY
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THE SCRIPPS RESEARCH INSTITUTE
have associated genomic instability and hypersensitivity to genotoxic stress; the human STUbL RNF4 is
implicated in cancer.
REACTIVE OXYGEN CONTROL ENZYMES
Superoxide dismutases (SODs) and nitric oxide synthases are master regulators for reactive oxygen species
involved in injury, pathogenesis, aging, and degenerative diseases. We tested if Alvinella pompejana, a deepsea hydrothermal vent worm, could be a eukaryotic
source for thermostable and humanlike proteins and
whether information on SOD mechanism and stability
could be obtained by examining A pompejana SOD.
We discovered that the worm SOD has a remarkably
high sequence identity with other mammalian SOD
enzymes but is substantially more stable than human
SOD. Moreover, crystals from initial conditions diffracted
just beyond 1-Å resolution. These results extend knowledge of SOD stability and catalysis and also suggest that
A pompejana may be a unique resource of macromolecules of enhanced stability for science and technology.
For human copper, zinc SOD, we are examining
single-site mutations that cause the neurodegeneration
in Lou Gehrig disease or familial amyotrophic lateral
sclerosis. Our structures show a key role for the zinc
ion in the defects associated with the disease.
For the nitric oxide synthases, our combined solution
scattering and crystallographic methods are revealing
regulatory mechanisms responsible for controlling nitric
oxide levels, which act as an important signal and as a
cytotoxin, with implications for inflammatory and neurodegenerative diseases. Our structures of the synthases,
determined in collaboration with E.D. Getzoff, Department of Molecular Biology, are enabling us to design
new inhibitors to directly control nitric oxide levels for
the treatment of human diseases.
DNA REPAIR AND GENETIC EVOLUTION
Structural knowledge allows possible selective inhibition of certain DNA repair pathways for new cancer
therapies. Endonuclease IV is an archetype for an endonuclease superfamily critical for DNA-base excision repair.
Our structures of endonuclease IV revealed a mechanism
for binding to and incising areas of DNA damage that
involves 3 metal ions and explained how the chemistry
avoids the release of toxic and mutagenic repair intermediates (Fig. 1).
Mutations in XPD helicase cause 3 distinct phenotypes: cancer-prone xeroderma pigmentosum and the
aging disorders Cockayne syndrome and trichothiodystrophy. To clarify molecular differences that underlie
F i g . 1 . Endonuclease IV E261Q DNA substrate–bound and DNA-
free x-ray structures. A, Apurinic-apyrimidinic (AP)-DNA complex
stereo shows the 3-metal-ion active site (green spheres); residues
R37, Y72, and Q261 (pink); and bound DNA substrate with both
the AP site sugar and phosphate moieties and the cognate nucleotide
(orange) flipped out from normal duplex DNA. The 2Fo-Fc electron
density map is contoured at 1 σ (blue mesh). B, DNA-free structure
with active-site phosphate and 3 zinc ions coordination. Omit map is
contoured at 2 (light blue) and 4 (dark blue) σ for the bound phosphate group. C, DNA substrate complex binding to active-site metal
ions. High-quality omit map (contoured at 2 σ, pink mesh) shows
the intact phosphodiester bond (black arrow) that constrains the Zn3
to Cyt6 O3′ distance to 2.7 Å. Based on Garcin, E.D., Hosfield, D.J.,
Desai, S.A., et al., DNA apurinic-apyrimidinic site binding and excision by endonuclease IV. Nat. Struct. Mol. Biol. 15:515, 2008.
these diseases, we determined crystal structures of the
XPD helicase catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Mutations
associated with xeroderma pigmentosum map along the
ATP-binding edge and DNA-binding channel and impair
helicase activity essential for nucleotide excision repair.
Mutations associated with xeroderma pigmentosum and
Cockayne syndrome both impair helicase activity and
likely affect functional movement. Mutations association with trichothiodystrophy lose or retain helicase
activity but map to sites in all 4 domains expected to
MOLECULAR BIOLOGY
2008
cause framework defects affecting the integrity of transcription factor IIH.
These new results broaden our understanding of
how structural changes in the XPD helicase might affect
cancer risks or result in developmental or aging phenotypes (Fig. 2). In general, the structural biology of pro-
THE SCRIPPS RESEARCH INSTITUTE
217
sion to host cells, and natural transformation. Because
they are prominently exposed on bacterial surfaces, pili
are attractive targets for the host immune response
and for vaccines and therapeutic reagents.
Our studies are providing an integrated understanding of the assembly and disassembly of type IV pili.
This understanding suggests new approaches to drug
and vaccine design for bacterial pathogens, including
Francisella tularensis, a highly virulent microorganism
that causes tularemia. Because of its high infectivity
and potential airborne transmission, F tularensis is
designated a category A bioterrorism agent.
PUBLICATIONS
Chrencik, J.E., Brooun, A., Zhang, H., Mathews, I.I., Hura, G.L., Foster, S.,
Perry, J.J.P., Streiff, M., Ramage, P., Widmer, H., Bokoch, G.M., Tainer, J.A.,
Weckbecker, G., Kuhn, P. Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1. J. Mol. Biol. 380:828, 2008.
Fan, L., Fuss, J.O., Cheng, Q.J., Arvai, A.S., Hammel M., Roberts, V.A., Cooper,
P.K., Tainer, J.A. XPD helicase structures and activities: insights into the cancer
and aging phenotypes from XPD mutations. Cell 133:789, 2008.
Garcin, E.D., Hosfield, D.J., Desai, S.A., Haas, B.J., Björas, M., Cunningham,
R.P., Tainer, J.A. DNA apurinic-apyrimidinic site binding and excision by endonuclease IV. Nat. Struct. Mol. Biol. 15:515, 2008.
Perry, J.J., Tainer, J.A., Boddy, M.N. A SIM-ultaneous role for SUMO and ubiquitin. Trends Biochem. Sci. 33:201, 2008.
Perry, J.J.P., Tainer, J.A. Structural biology of Cockayne syndrome proteins, their
interactions and insights into DNA repair mechanisms. In: Molecular Mechanisms of
Cockayne Syndrome. Ahmad, S.I. (Ed.). Landes Bioscences, Austin, TX, in press.
F i g . 2 . Structural placement of disease-causing mutations in XPD
helicase. Mapping the 3 classes of mutations onto the SaXPD structure reveals patterns associated with each disease defect. A, Stereo
pair mapping the distribution of disease-causing mutations on a XPD
C α trace. Disease-causing mutation sites (Cα colored sphere): red
(XP), greenish yellow (XP/CS), and purple (TTD). Residue F136 is
also shown (cyan). B, XPDcc fold and domain architecture (ribbons)
with labeled disease-causing mutation sites as spheres colored as
in A. C, XP mutations affect DNA- and ATP-binding regions. D, XP/CS
mutations affect HD1-HD2 conformational changes. E, TTD mutations
affect the overall framework stability. Reprinted from Fan, L., Fuss,
J.O., Cheng, Q.J., Arvai, A.S., Hammel M., Roberts, V.A., Cooper, P.K.,
Tainer, J.A. XPD helicase structures and activities: insights into the
cancer and aging phenotypes from XPD mutations. Cell 133:789,
2008. Copyright 2008, with permission from Elsevier.
teins such as XPD, which control reactive oxygen species
and DNA repair, may provide master keys to brain abnormalities, cancer, and aging.
Putnam, C.D., Hammel, M., Hura, G.L., Tainer, J.A. X-ray solution scattering
(SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q. Rev. Biophys.
40:191, 2007.
Prudden, J., Pebernard, S., Raffa, G., Slavin, D.A., Perry, J.J., Tainer, J.A.,
McGowan, C.H., Boddy, M.N. SUMO-targeted ubiquitin ligases in genome stability.
EMBO J. 26:4089, 2007.
Roberts, B.R., Tainer, J.A., Getzoff, E.D., Malencik, D.A., Anderson, S.R.,
Bomben, V.C., Meyers, K.R., Karplus, P.A., Beckman, J.S. Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS. J.
Mol. Biol. 373:877, 2007
Tubbs, J.L., Pegg, A.E., Tainer, J.A. DNA binding, nucleotide flipping, and the
helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and
its implications for cancer chemotherapy. DNA Repair (Amst.) 6:1100, 2007.
Structural Biology of Integral
Membrane Proteins
G. Chang, S. Aller, X. He, A. Karyakin, S. Lieu, P. Szewczk,
BACTERIAL PILI AND INFECTIOUS DISEASES
Type IV pili are essential virulence factors for many
bacterial pathogens and therefore act in many important infectious diseases. Functions of type IV pili include
motility, formation of microcolonies and biofilms, adhe-
T. Tuan, A. Ward, J. Yu
S
tudy of the structure of membrane proteins is
important for understanding their function. We
are interested in 4 areas: (1) the molecular struc-
218 MOLECULAR BIOLOGY
2008
tural basis for the transport of lipids and drugs across
the cell membrane by multidrug resistance (MDR)
transporters, (2) the crystallography of mammalian
MDR transporters and the structural basis of their inhibition, (3) the discovery and rational design of potent
MDR reversal agents, and (4) the development and
application of a cell-free system capable of producing
large quantities of integral membrane proteins. We use
several experimental methods, and we collaborate with
scientists in other laboratories to achieve our goals.
MDR in the treatment of cancer and infectious disease is often caused by an upregulation of drug efflux
pumps imbedded in the cell membrane. The molecular
basis of multispecificity and drug efflux by these transporters is not well understood. Through our structural
studies, we are elucidating the mechanisms for the transport of amphipathic substrate across the cell membrane
in several families of transporters: ATP-binding cassette,
small multidrug resistance, major facilitator superfamily,
and multiple antimicrobial extrusion. In collaboration
with M.G. Finn, Department of Chemistry, and Q. Zhang,
Department of Molecular Biology, we are discovering and
designing potent inhibitors to be used synergistically
with established antibiotics and cancer chemotherapeutics. In collaboration with R.A. Milligan, Department of
Cell Biology, we are using electron cryomicroscopy to
visualize transporter structures.
We have determined 4 x-ray structures of the ATPbinding cassette transporter MsbA trapped in different
conformations: 2 with nucleotide bound and 2 with no
nucleotide. Comparisons of the nucleotide-free conformations of MsbA revealed a flexible hinge formed by
extracellular loops 2 and 3. The hinge allows the nucleotide-binding domains to disassociate while the ATPbinding half sites remain facing each other. The binding
of nucleotide causes a packing rearrangement of the
transmembrane helices and changes the accessibility
of the transporter from cytoplasmic (inward) facing to
extracellular (outward) facing. The inward and outward
openings are mediated by 2 different sets of transmembrane helix interactions. Altogether, the conformational
changes between these structures suggest that large
ranges of motion may be required for substrate transport.
EmrE, an MDR transporter from the small multidrug
resistance family, functions as a homodimer of a small
4-transmembrane protein. The membrane insertion
topology of the 2 monomers is controversial. EmrE was
reported to have a unique orientation in the membrane.
Models based on electron microscopy and on several
THE SCRIPPS RESEARCH INSTITUTE
biochemical studies posit an antiparallel dimer. The
structures of EmrE in complex with a transport substrate are highly similar to the electron microscopy
structure. The first 3 transmembrane helices from each
monomer surround the substrate-binding chamber,
whereas the fourth helix participates only in dimer formation. Selenomethionine markers clearly indicate an
antiparallel orientation for the monomers, supporting
a “dual topology” model.
EmrD, an MDR transporter from the major facilitator superfamily, expels hydrophobic compounds across
the inner membrane. The x-ray structure reveals an
interior composed of hydrophobic residues, a finding
consistent with the role of EmrD in transporting amphipathic molecules that uncouple the proton gradient
across the cell membrane. Two long loops extend into
the inner leaflet side of the cell membrane and may
recognize and bind substrate directly from the lipid
bilayer. On the basis of the structure, we propose that
multisubstrate specificity, binding, and transport are
facilitated by these loop regions and the internal cavity.
Structure and Function of
Membrane-Bound Enzymes
C.D. Stout, M. Yamaguchi, R. Akhouri, V.M.M. Luna,
A. Annalora
e focus on the structure and function of membrane-bound enzymes and the development
of methods for crystallizing membrane proteins. We study the mechanism of transhydrogenase, a
mitochondrial respiratory enzyme complex that couples
proton translocation with hydride transfer. This enzyme
is essential for maintaining NADPH levels in mitochondria, and it plays a critical role in insulin secretion in
beta cells of the pancreas. We use x-ray crystallography, biochemical and spectroscopic methods, electron
microscopy in collaboration with M. Yeager, Department
of Cell Biology, and nuclear magnetic resonance in collaboration with H.J. Dyson, Department of Molecular
Biology. Currently, further progress in understanding the
structure and function of transhydrogenase is stymied
by lack of a 3-dimensional structure; therefore, our
primary effort now is to obtain diffraction-quality crystals of the enzyme in its membrane-bound configuration.
Crystallization experiments with transhydrogenase,
cytochrome ba 3 oxidase, and cytochrome P450s are
W
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2008
further enabled by use of novel detergents synthesized
by Q. Zhang, Department of Molecular Biology.
In collaboration with J.A. Fee, Department of Molecular Biology, we are studying the mechanism of cytochrome ba3 oxidase, a homolog of the terminal enzyme
of respiration in mitochondria. We are using crystal
structure analysis, mutagenesis, and spectroscopy to
visualize intermediates in the reduction of oxygen to
water, to define the pathways for protons and oxygen into
the active site, and to understand the coupling of reduction potential to proton translocation across the membrane. We have also used protein engineering to improve
the crystallization behavior of this membrane protein.
In collaboration with E.F. Johnson, Department of
Molecular Biology; J.R. Halpert, University of California, San Diego; and I. Pikuleva, University of Texas
Medical Branch, Galveston, Texas, we are characterizing the structure and function of mammalian microsomal cytochrome P450s. These membrane-associated
enzymes specifically metabolize a wide diversity of exogenous compounds and drugs. High-resolution structures
have been determined for the principal drug-metabolizing
microsomal P450s in the liver and lung in humans: 1A2,
2A6, 2A13, 2C5, 2C8, 2C9, 2C19, 3A4, and 2B4.
For 2B4, 5 structures of the enzyme in markedly different conformations provide insight to substrate binding
and membrane insertion. A structure of the brain-specific, cholesterol-metabolizing P450 CYP46 has been
determined, and recently the first structure of the mitochondrial P450 CYP24A1 has been determined at 2.0-Å
resolution (Fig. 1). This class of P450s is involved in
the metabolism of lipophilic hormones.
In collaboration with P.E. Dawson, Department of
Cell Biology, we are developing the assembly of synthetic
peptides and phospholipids into discs that contain lipid
bilayers. Nanodiscs composed of human apolipoprotein
A-I and phospholipids self-assemble into discrete, watersoluble, bilayer-containing particles. Integral membrane
proteins incorporated into these particles retain their
enzymatic activity, are amenable to biochemical assays,
and may have superior properties for crystallization in
the absence of detergents.
A major effort to determine the basis of HIV resistance to antiviral drugs is ongoing in collaboration with
A.J. Olson and J.E. Elder, Department of Molecular Biology; B.E. Torbett, Department of Molecular and Experimental Medicine; M.G. Finn, Department of Chemistry;
and D.E. McRee, ActiveSight, San Diego, California.
One aspect of this project entails determining the crystal
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219
F i g . 1 . Electron density for mitochondrial cytochrome P450
CYP24A1, the enzyme responsible for stereospecific, 6-step oxidation of vitamin D3. The active form of vitamin D3 regulates calcium
and phosphate homeostasis and stimulates cellular differentiation
but inhibits proliferation, making CYP24A1 an attractive target for
development of cancer therapeutics.
structure of mutant proteases from drug-resistant HIV
in complex with broad-spectrum inhibitors. Currently,
220 MOLECULAR BIOLOGY
2008
we are using fragment-based screening to discover new
classes of inhibitors as lead compounds for drug discovery. In these experiments, we use high-throughput
structural genomics technologies to acquire hundreds
of high-resolution data sets to discover specific binding of druglike small molecules.
PUBLICATIONS
Liu, B., Luna, V.M., Chen, Y., Stout, C.D., Fee, J.A. An unexpected outcome of
surface engineering an integral membrane protein: improved crystallization of cytochrome ba3 from Thermus thermophilus. Acta Crystallogr. Sect. F Struct. Biol.
Cryst. Commun. 63:1029, 2007.
Luna, V.M., Chen, Y., Fee, J.A., Stout, C.D. Crystallographic studies of Xe and Kr
binding within the large internal cavity of cytochrome ba3 from Thermus thermophilus: structural analysis and role of oxygen transport channels in the heme-Cu
oxidases. Biochemistry 47:4657, 2008.
Mast, N., White, M.A., Bjorkhem, I., Johnson, E.F., Stout, C.D., Pikuleva, I.A.
Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1,
the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. U. S. A.
105:9546, 2008.
THE SCRIPPS RESEARCH INSTITUTE
pathways whereby oxygen arrives at the heme a 3–CuB
center, which is deeply buried within the transmembrane domain of these enzymes.
Using xenon- and krypton-pressurized crystals of
cytochrome ba 3 , we identified the hydrophobic cavities in the oxidase. The location and characteristics of
this channel led us to believe that it is a ligand-transport channel. To gain further insight into transport
channels in cytochrome c oxidases, we have also analyzed hydrophobic channels in cytochrome c oxidases
of known structure.
The locations of the xenon-binding sites indicate a
continuous, bifurcated channel that opens from 2 points
on the protein exterior and leads directly to the dinuclear center (Fig. 1A). Opening into the bilayer (Fig. 1B)
Sansen, S., Hsu, M.-H., Stout, C.D., Johnson, E.F. Structural insight into the
altered substrate specificity of human cytochrome P450 2A6 mutants. Arch.
Biochem. Biophys. 464:197, 2007.
Schoch, G.A., Yano, J.K., Sansen, S., Dansette, P.M., Stout, C.D., Johnson, E.F.
Determinants of cytochrome P450 2C8 substrate binding: structures of complexes
with montelukast, troglitazone, felodipine, and 9-cis-retinoic acid. J. Biol. Chem.
283:17227, 2008.
White, M.A., Mast, N., Bjorkhem, I., Johnson, E.F., Stout, C.D., Pikuleva, I.A. Use
of complementary cation and anion heavy-atom salt derivatives to solve the structure
of cytochrome P450 46A1. Acta Crystallogr. D Biol. Crystallogr. 64:487, 2008.
Zhao, Y., Sun, L., Muralidhara, B.K., Kumar, S., White, M.A., Stout, C.D., Halpert,
J.R. Structural and thermodynamic consequences of 1-(4-chlorophenyl)imidazole
binding to cytochrome P450 2B4. Biochemistry 46:11559, 2007.
Structural Analysis of Oxygen
Transport Channels in Thermus
thermophilus Cytochrome ba3
Oxidase
J.A. Fee, V.M. Luna, B. Liu, Y. Chen, C.D. Stout
ytochrome c oxidase is the principal terminal oxidase in the aerobic metabolism of all animals,
plants, and yeasts and in some bacteria. In the
thermophillic bacterium Thermus thermophilus, cytochrome ba3 oxidase is the preferred respiratory enzyme
that catalyzes the flow of electrons from reduced cytochrome c to dioxygen, forming water concomitant with the
formation of a proton gradient under low oxygen tensions.
Dioxygen reduction occurs at the high-spin, heme
a 3–CuB dinuclear center. Although enzymatic turnover
has been studied extensively, little is known about the
C
F i g . 1 . Location and characteristics of the oxygen channel in cyto-
chrome ba 3. For both panels, subunit I is pale green, subunit II is
magenta, and subunit IIa is turquoise. A, Top view of the oxidase
shows the computationally derived shape of the oxygen channel
(yellow surface). Xenon and krypton molecules completely line the
hydrophobic channel from the dinuclear center to the boundary of
the protein. The CuA domain of subunit II was removed to illustrate
the unique Y shape of the channel. B, Transmembrane view of the
oxidase shows the oxygen channel opening into the lipid bilayer.
MOLECULAR BIOLOGY
2008
allows the channel to use the higher oxygen concentration in lipid bilayers. Compared with the other cytochrome c oxidases of known structure, the structure of
cytochrome ba 3 with its Y-shaped channel is unique.
The channel has 2 openings and allows unimpeded
access to the enzymatic center of the protein.
On the basis of its location and relative occupancy
(highest), the xenon 1 site (Fig. 2) serves as the portal
THE SCRIPPS RESEARCH INSTITUTE
221
by a hydrophilic path. We hypothesize that the newly
formed water molecules are repelled by the hydrophobic surface of the oxygen channel but attracted to the
hydrophilic area around the heme a3 propionates, where
they exit the dinuclear center in a hydrophilic “vent”
(Fig. 2). This spatially separated vectorial transfer from
substrate (oxygen) to product (water) cavities during
turnover would increase the overall rate of the reaction.
PUBLICATIONS
Liu, B., Luna, V.M., Chen, Y., Stout, C.D., Fee, J.A. An unexpected outcome of
surface engineering an integral membrane protein: improved crystallization of cytochrome ba3 from Thermus thermophilus. Acta Crystallogr. Sect. F Struct. Biol.
Cryst. Commun. 63:1029, 2007.
Luna, V.M., Chen, Y., Fee, J.A., Stout, C.D. Crystallographic studies of Xe and Kr
binding within the large internal cavity of cytochrome ba3 from Thermus thermophilus: structural analysis and role of oxygen transport channels in the heme-Cu
oxidases. Biochemistry 47:4657, 2008.
Chemistry and Membrane
Protein Structural Biology
Q. Zhang, M.M. Baksh, W.-X. Hong, Y. Weng
he structural characterization of integral membrane proteins has been an important challenge
for decades, and the structures of only a tiny fraction of these proteins have been solved to date. An
integral membrane protein with built-in detergents can
be regarded as the equivalent of a soluble protein, and
the many challenges in the study of membrane proteins
are associated with the use of the spherical micelleforming detergents. Currently, a pressing need exists for
new types of cell-membrane mimicking reagents to
advance the field of membrane protein structural biology.
We designed a new class of steroid-based molecules
with facial amphiphilicity instead of the end polarity
of the conventional head-to-tail detergents. The facial
amphiphiles should provide a better membrane-mimicking environment than the spherical micelle-forming detergents do and thus shield the transmembrane hydrophobic
surface of integral membrane proteins. The results of
using these amphiphiles to stabilize several integral
membrane proteins in functional states and in the protein structural characterization as well have been promising. In collaborative studies with C.D. Stout and
D.B. Goodin, Department of Molecular Biology, use of
our facial amphiphiles led to the successful crystallization of the mitochondrial cytochrome P450 CYP24A1
at 2.0-Å resolution. In collaboration with Dr. Stout,
T
F i g . 2 . Environment around the dinuclear center of cytochrome
ba 3. Surface renditions of the pocket encompassing the dinuclear
center are primarily hydrophobic (salmon). No direct access exists
from the xenon 1 (Xe1) site (slate; top) to the CuB (blue; bottom)
of the dinuclear center (sky-blue arrow). We hypothesize that after
the conversion of oxygen to water, the water exits the dinuclear center
through a hydrophilic area (surface representation in pale yellow)
surrounding the propionates of heme a3 (marine-blue arrow). Although
not present in our structures, ordered waters found at the heme a 3
propionates in a higher resolution structure of cytochrome ba 3 are
shown as orange spheres.
into the dinuclear center for oxygen. Oxygen initially binds
to CuB before being transferred to heme a 3, but there
is no straight-line access from the xenon 1 site to CuB.
Although oxygen favors the hydrophobic environment
surrounding the dinuclear center, during enzymatic
turnover, the water produced does not and must exit
222 MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
R.C. Stevens, and G. Chang, Department of Molecular
Biology, and M. Yeager, Department of Cell Biology, we
are attempting to crystallize a variety of other targets.
Meanwhile, we are combining different strategies
to overcome the many challenges in membrane protein structural biology. Systematic screening of a large
number of detergents is desirable in many circumstances, because selection of detergents is still empirical. In collaboration with K. Wüthrich, Department of
Molecular Biology, we have developed a strategy for
efficient screening of a large detergent library for the
reconstitution of integral membrane proteins. Using
this approach, we identified several new detergents
that yielded high-quality nuclear magnetic resonance
spectra of the membrane proteins OmpX and OmpW.
PUBLICATIONS
Zhang, Q., Horst, R., Geralt, M., Ma, X., Hong, W.-X., Finn, M.G., Stevens, R.C.,
Wüthrich, K. Microscale NMR screening of new detergents for membrane protein
structural biology. J. Am. Chem. Soc. 130:7357, 2008.
Zhang, Q., Ma, X., Ward, A., Hong, W.-X., Jaakola, V.-P., Stevens, R.C., Finn,
M.G., Chang, G. Designing facial amphiphiles for the stabilization of integral membrane proteins. Angew. Chem. Int. Ed. 46:7023, 2007.
G Protein–Coupled Receptors
R.C. Stevens, E.E. Abola, A.I. Alexandrov, L.K. Allin,
G.A. Asmar-Rovira, K.A. Baker, R.R. Benoit, M.H. Bracey,
Q. Chai, V.G. Cherezov, E.Y.T. Chien, E. Chun, S. Daudenarde,
J. Dupuy, A. Gámez, J. Gatchalian, M.T. Griffith,
M.A. Hanson, V.-P. Jaakola, J.S. Joseph, T.S. Kang,
J. Liu, K. Masuda, M. Michino, M. Mileni, C.B. Roth,
K.S. Saikatendu, I. Slaymaker, P. Stenmark, T. Trinh,
J. Velasquez, L. Wang, B. Wu, Q. Zhao
protein–coupled receptors (GPCRs) are the
largest family of proteins in the human genome,
with more 1000 receptors. These receptors are
a key signaling component throughout the human body
and the target for more than 50% of all drugs. In 2007,
we reported for the first time the 3-dimensional structure of a human GPCR, the β2-adrenergic receptor
(Fig. 1). Since then, we have made rapid progress on
additional cocrystal structures with different drug molecules bound and variants to probe receptor function.
In parallel with the structural studies, we are also conducting biochemical and biological experiments to further understand how the receptors transmit signals
across the cell membrane and influence so many different events within the cell (Fig. 2).
G
F i g . 1 . Structure of human β2-adrenergic receptor highlighting
the cholesterol-binding site in the middle of the receptor.
Recently, we found that cholesterol, a molecule
typically associated with membrane fluidity and curvature, is a key stabilizing molecule for GPCRs that bind
in the “cholesterol consensus motif.” The discovery of
this unique binding site provides a potential new avenue
for modulating receptor function and for showing, for
the first time, the direct binding interaction between
cholesterol and a membrane protein. Almost certainly,
this discovery is a more general event in biology. To
further compliment our studies, in collaboration with
K. Wüthrich, Department of Molecular Biology, we are
using nuclear magnetic resonance to understand the
dynamics of the receptor and the influence of ligands.
Last, we are working to decipher the protein-protein
interactions in GPCR systems between the receptors
and G proteins, arrestin, and G protein receptor kinases.
STRUCTURAL PROTEOMICS OF GPCRS
Perhaps as exciting as the first human GPCR structure is our development of technologies and a pipeline
that enable us to solve multiple GPCR structures more
rapidly and to tackle representative members of the
entire GPCR family (Fig. 3). Our development of a
MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
223
F i g . 3 . Family tree of class A human GPCRs. To date, only rho-
dopsin and one member of the amine family have been structurally
characterized.
F i g . 2 . Analysis of the increase in helical packing and thermal stability of the β 2 -adrenergic receptor due to cholesterol binding. A,
Receptor is colored by normalized occluded surface area. Red thick
lines indicate the compact areas of the receptor; blue thin lines are
the least compact. Helix IV has the lowest packing of the 7 helices
in the tertiary structure, particularly on the cytoplasmic end. Cholesterol binding stabilizes the receptor by increasing packing constraints,
especially in the vicinity of the cytoplasmic end of helix IV. From 10%
to 70% of the total available surface area is involved in packing interactions. B, Differences in the normalized occluded surface area of
the receptor due to cholesterol binding. The increase in packing of
available surface area due to cholesterol binding is 0% to 15%; the
most significant increases are for residues on helices II and IV. C,
Molecular surface representation of the receptor and cholesterol. Green
corresponds to atoms on both cholesterol and the receptor that are
within 4 Å of each other. Blue corresponds to atoms on the receptor
that are 4 and 5 Å from the cholesterol molecules. On the right, the
cholesterol molecules have been lifted out of the binding groove to
better show the interactions and the groove. D, Isothermal denaturation curves with 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin dye were used to determine the half-life of the β2-adrenergic recepter in the presence of 1 M guanidine hydrochloride with
and without both chloesterol hemisuccinate (CHS) and timolol (Tim).
The thickness of the line represents the 95% confidence interval over
3 replicates; the fitted half-lives are indicated next to the respective
curves. Both timolol and cholesterol cause an approximate 5-fold
increase in half-life under these conditions. In combination, the effect
is almost 16-fold relative to apo. Reprinted from Hanson, M.A.,
Cherezov, V., Griffith, M.T., et al. A specific cholesterol binding site is
established by the 2.8 Å structure of the human β2-adrenergic receptor. Structure 16:897, 2008, with permission from Elsevier.
structural proteomics approach to understanding entire
protein families came out of frustration with the pace
at which information on structural biology became available. Although we are now focusing on the biological
aspects of GPCRs, during the past 10 years, we have
developed new tools to change the field of structural
biology by accelerating the rate of determination of
protein structures, an endeavor that includes pioneering microliter expression/purification for structural studies,
nanovolume crystallization, and automated image collection. These technologies were initially tested at the
Joint Center for Structural Genomics (http://www.jcsg.org),
in collaboration with I.A. Wilson, Department of Molecular Biology, where the power of the new tools was
demonstrated.
Although the Joint Center for Structural Genomics 2
has continued as a successful production for the second
phase of the Protein Structure Initiative of the National
Institute of General Medical Sciences, in collaboration
with P. Kuhn, Department of Cell Biology, we have
created 2 new centers funded by the National Institutes
of Health that focus on technologic innovations in structural biology. The first center is the Joint Center for Membrane Protein Technologies (http://jcimpt.scripps.edu).
Here, in collaboration with K. Wüthrich, Q. Zhang, and
G. Chang, Department of Molecular Biology; M.G. Finn,
Department of Chemistry; and P. Kuhn and M. Yeager,
Department of Cell Biology, we do research exclusively
on membrane proteins, including GPCRs. The second
center is the Accelerated Technologies Center for Gene
to 3D Structure (http://www.atcg3d.org). Here we are
collaborating with Dr. Kuhn and with researchers from
deCODE biostructures, Bainbridge Island, Washington;
224 MOLECULAR BIOLOGY
2008
Lyncean Technologies, Palo Alto, California; and the
University of Chicago, Chicago, Illinois, on novel crystallization and x-ray methods.
THE SCRIPPS RESEARCH INSTITUTE
Serrano, P., Johnson, M.A., Almeida, M.S., Horst, R., Hermann, T., Joseph, J.S.,
Neuman, B.W., Subramanian, V., Saikatendu, K.S., Buchmeier, M.J., Stevens,
R.C., Kuhn, P., Wüthrich, K. Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome
coronavirus. J. Virol. 81:12049, 2007.
STRUCTURE-BASED DRUG DISCOVERY
Over the years, we have been involved in the basic
science and development of several therapeutic agents to
treat neurologically related disorders (e.g., phenylketonuria, pain, dystonias). With the structure determination
of a GPCR now in hand and the basic mechanistic
research in this area progressing, we will turn our attention to developing improved drugs to treat human disease.
Slaymaker, I.M., Bracey, M., Mileni, M., Garfunkle, J., Cravatt, B.F., Boger, D.L.,
Stevens, R.C. Correlation of inhibitor effects on enzyme activity and thermal stability for the integral membrane protein fatty acid amide hydrolase. Bioorg. Med.
Chem. Lett., in press.
Stevens, R.C. Generation of protein structures for the 21st century. Structure
15:1517, 2007.
Structural Genomics Consortium; China Structural Genomics Consortium; Northeast
Structural Genomics Consortium, Gräslund, S., Nordlund, P., Weigelt, J., et al. Protein production and purification. Nat. Methods 5:135, 2008.
PUBLICATIONS
Alexandrov, A.I., Mileni, M., Chien, E.Y.T., Hanson, M.A., Stevens, R.C. Microscale
fluorescent thermal stability assay for membrane proteins. Structure 16:351, 2008.
Wang, L., Gámez, A., Archer, H., Abola, E.E., Sarkissian, C.N., Fitzpartick, P.,
Wendt, D., Zhang, Y., Vellard, M., Bliesath, J., Bell, S.M., Lemontt, J.F., Scriver,
C.R., Stevens, R.C. Structural and biochemical characterization of the therapeutic
Anabaena variabilis phenylalanine ammonia lyase. J. Mol. Biol. 380:623, 2008.
Asmar-Rovira, G.A., Asseo-García, A.M., Quesada, O., Hanson, M.A., Nogueras,
C., Lasalde-Dominicci, J.A., Stevens, R.C. Biophysical and ion channel functional
characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments. J. Membr. Biol. 223:13, 2008.
Zhang, Q., Horst, R., Geralt, M., Ma, X., Hong, W.X., Finn, M.G., Stevens, R.C.,
Wüthrich, K. Microscale NMR screening of new detergents for membrane protein
structural biology. J. Am. Chem. Soc. 130:7357, 2008.
Cherezov, V., Rosenbaum, D.M., Hanson, M.A., Rasmussen, S.G.F., Thian, F.S.,
Kobilka, T.S., Choi, H.-J., Kuhn, P., Weis, W.I., Kobilka, B.K., Stevens, R.C.
High-resolution crystal structure of an engineered human β2-adrenergic G proteincoupled receptor. Science 318:1258, 2007.
Gámez, A., Wang, L., Sarkissian, C.N., Wendt, D., Fitzpatrick, P., Lemontt, J.F.,
Scriver, C.R., Stevens, R.C. Structure-based epitope and PEGylation sites mapping
of phenylalanine ammonia-lyase for enzyme substitution treatment of phenylketonuria. Mol. Genet. Metab. 91:325, 2007.
Zhang, Q., Ma, X., Ward, A., Hong, W.X., Jaakola, V.P., Stevens, R.C., Finn,
M.G., Chang, G. Designing facial amphiphiles for the stabilization of integral membrane proteins. Angew. Chem. Int. Ed. 46:7023, 2007.
Zurflüh, M.R., Zschocke, J., Linder, M., Feillet, F., Chery, C., Burlina, A., Stevens,
R.C., Thony, B., Blau, N. Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. Hum. Mutat. 29:1079, 2008.
Hanson, M.A., Brooun, A., Baker, K.A., Jaakola, V.P., Roth, C., Chien, E., Alexandrov, A., Velasquez, J., Davis, L., Griffith, M., Moy, K., Ganser-Pornillos, B.,
Kuhn, P., Ellis, S., Yeager, M., Stevens, R.C. Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale
parallel expression. Protein Expr. Purif. 56:85, 2007.
High-Throughput Approaches to
Protein Structure and Function
Hanson, M.A., Cherezov, V., Griffith, M.T., Roth, C.B., Jaakola, V.-P., Chien, E.Y.T.,
Velasquez, J., Kuhn, P., Stevens, R.C. A specific cholesterol binding site is established
by the 2.8 Å structure of the human β2-adrenergic receptor. Structure 16:897, 2008.
S.A. Lesley, H. Johnson, S. Sudek, T. Janaratne, S. Kale,
M. Deller, D. Carlton, T. Clayton, A. Grzechnik, C. Farr
Jauch, R., Ng, C.K., Saikatendu, K.S., Stevens, R.C., Kolatkar, P.R. Crystal structure and DNA binding of the homeodomain of the stem cell transcription factor
Nanog. J. Mol. Biol. 376:758, 2008.
Mathews, I.I., McMullan, D., Miller, M.D., et al. Crystal structure of 2-keto-3deoxygluconate kinase (TM0067) from Thermatoga maritima at 2.05 Å resolution.
Proteins 70:603, 2008.
Neuman, B.W., Joseph, J.S., Saikatendu, K.S., Serrano, P., Chatterjee, A., Johnson, M.A., Liao, L., Klaus, J.P., Yates, J.R. III, Wüthrich, K., Stevens, R.C.,
Buchmeier, M.J., Kuhn, P. Proteomics analysis unravels the functional repertoire of
coronavirus nonstructural protein 3. J. Virol. 82:5279, 2008.
Ng, J.D., Clark, P.J., Stevens, R.C., Kuhn, P. In situ x-ray analysis of protein crystals in low-birefringent and x-ray transmissive plastic microchannels. Acta Crystallogr. D Biol. Crystallogr. 64:189, 2008.
Ng, J.D., Stevens, R.C., Kuhn, P. Protein crystallization in restricted geometry:
advancing old ideas for modern times in structural proteomics. Methods Mol. Biol.
426:363, 2008.
Rosenbaum, D.M., Cherezov, V., Hanson, M.A., Rasmussen, S.G.F., Thian, F.S.,
Kobilka, T.S., Choi, H.-J., Yao, X.-J., Weis, W.I., Stevens, R.C., Kobilka, B.K.
GPCR engineering provides high-resolution structural insights into β2-adrenergic
receptor function. Science 318:1266, 2007.
Roth, C.B., Hanson, M.A., Stevens, R.C. Stabilization of the human β2-adrenergic
receptor TM4-TM3-TM5 helix interface by mutagenesis of Glu-1223.41, a critical
residue in GPCR structure. J. Mol. Biol. 376:1305, 2008.
valuating protein structure and function is of primary importance for understanding the basic
biology of the cell and is a challenge because of
the constantly expanding wealth of genomic information.
To address this challenge, we have established highthroughput approaches for evaluating structural and
functional diversity of proteins as part of a structural
genomics effort with the Joint Center for Structural
Genomics. We use these same tools to characterize
the molecular basis of the specificity of enzyme substrates and to probe the roles the enzymes play in cellular and metabolic pathways.
The goal of the Joint Center for Structural Genomics
is to develop a high-throughput and cost-effective structure pipeline and to use the pipeline to determine
novel protein folds and explore protein structure-function relationships. We have used this approach in an
extensive study of the thermophilic bacterium Thermotoga maritima and for targets from mouse and more
E
MOLECULAR BIOLOGY
2008
than 100 other bacterial genomes. Our technologies
have enabled us to perform comprehensive structural
studies of these proteomes. To date, these efforts have
resulted in more than 700 novel protein structures
from the center. We continue to develop technology in
membrane protein crystallization and protein complexes.
We are also using this approach to study commensal bacteria in human health and disease. Awareness
is increasing that both normal and disease states are
influenced by the composition and byproducts of the
microbial communities in our bodies. We are using highthroughput discovery methods to screen for new roles
and influences of these microbes in disease and metabolism. Combining metagenomic studies with protein
characterization and cell-based assays, we are screening large panels of secreted proteins from the human
gut in a number of basic pathways involved in inflamation, cell death, and drug metabolism.
Understanding how genome sequences are related
to the biology of an organism requires correct annotation of gene function. Many computational approaches
are available for assigning a putative gene function on
the basis of similarity to known activities. However, as
the evolutionary distances extend and the number of
putative homologs increases, the reliability of such unvalidated predictions becomes suspect. We use direct experimentation to test putative predictions and focused
ligand screens to identify new activities.
Structural Biology and Structural
Genomics With Nuclear
Magnetic Resonance
Spectroscopy
K. Wüthrich, W. Augustyniak, A. Chatterjee, M. Geralt,
R. Horst, M. Johnson, B. Pedrini, W.J. Placzek, J.K. Rhee,
P. Serrano, P. Stanczak
e are developing methods to improve the efficiency and reliability of nuclear magnetic
resonance (NMR) structure determination of
proteins in solution and are applying the methods to target proteins selected within the framework of the Joint
Center for Structural Genomics. In addition, as part of
the research of the Center for Functional and Structural
Proteomics of SARS-CoV (FSPS; http://visp.scripps.edu/
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THE SCRIPPS RESEARCH INSTITUTE
225
SARS/default.aspx), we are characterizing the proteome
of the coronavirus (SARS-CoV) that causes severe acute
respiratory syndrome (SARS).
SARS-COV STRUCTURAL GENOMICS
Although a 2003 SARS pandemic was contained by
public health measures, no vaccine or effective treatment for SARS is available, and the basic mechanisms
of coronavirus infections are not yet understood. SARSCoV contains a 29-kb positive-stranded RNA genome.
About two-thirds of the genome is devoted to encoding
a replicase polyprotein, which is cleaved by 2 viral proteases to release the mature nonstructural proteins.
These proteins are responsible for the enzymatic functions that allow the virus to replicate in infected cells
and therefore are potential targets for drug development.
The FSPS project was started with the expectation that
structure-based functional studies will reveal new functional features that are not detectable when only the
amino acid sequence is known.
S T R U C T U R E D E T E R M I N AT I O N O F T H E
NONSTRUCTURAL PROTEIN 3c
The region of the SARS-CoV nonstructural protein 3
(nsp3) that spans residues 366–722 is a functional
domain termed the SARS-unique domain (SUD) because
it is not present in other known coronaviruses. Expression in Escherichia coli indicated that SUD does not
form a single globular structure, and NMR studies showed
that it consists of at least 3 distinct structural domains,
which may provide for multiple functions (Fig. 1). A
central globular domain, SUD-M (M stands for middle),
spans residues 527–651. The NMR structure of this
protein shows a macrodomain fold with similarity to that
of proteins that bind the important regulatory molecule
ADP-ribose in eukaryotic cells (Fig. 2).
Structure-based attempts to determine the function of
SUD-M started with a search of the Protein Data Bank for
structural homologs of SUD-M. The closest 3-dimensional
structural homolog was the protein nsp3b, which is
located immediately N-terminal to the SUD region in the
SARS-CoV proteome and functions as an ADP-ribose1′′-phosphatase. This finding was a surprise, because the
sequence identity between the 2 domains is only 6%.
Tests for binding of a variety of different ligands, based on
NMR chemical-shift perturbation measurements, revealed
that SUD-M recognizes single-stranded polyadenosine
RNA. A possible function suggested by this observation is
in viral genome replication or transcription, which may
involve the recognition of polyadenylated tails of viral RNA
by one or more viral proteins. SUD-M might thus be a
226 MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
F i g . 1 . Structural coverage of nsp3. The horizontal black line represents the polypeptide segment 1–1318; the initially annotated functional
domains are indicated above the line. Globular domain structures determined so far are shown in ribbon representation, along with color-coded
information on the structure determination method used and the new, structure-based functional annotation. In between the globular domains,
blue lines represent flexibly disordered segments as determined by NMR spectroscopy, and black lines indicate unstructured segments implicated by the absence of x-ray diffraction in protein crystals. Regions of the protein with unknown structures are colored green; these regions
extend to the C terminus of nsp3 at residue 1922.
F i g . 2 . A, Ensemble of 20 conformers representing the solution
structure of the protein domain SUD-M (see also Fig. 1). The conformers were superimposed for minimal root-mean-square deviation
of the backbone N, C α , and C′ atoms of the residues 527–651.
Selected sequence positions relative to the intact nsp3 (Fig. 1) are
indicated by numbers. Helical secondary structures are red, β-strands
are green, and segments with no regular secondary structure are gray.
B, Surface view of SUD-M in the same orientation as in A, with the
regions affected by the binding of single-stranded polyadenosine RNA
in magenta to highlight the probable RNA-binding site.
potential target for the development of antiviral drugs that
disrupt viral replication.
On the basis of phylogenetic and bioinformatics
analyses, nsp3 was initially predicted to consist of 7
functional domains: nsp3a–nsp3g. The NMR structure
determination of SUD-M was just one of many steps
toward elucidating the structure of the much larger
nsp3 polypeptide. The overall structural characterization of the region nsp3a–nsp3e now provides a detailed
picture of the domain organization (Fig. 1), and new
functions have already been identified.
As illustrated in Figure 1, using NMR spectroscopy
and x-ray crystallography with polypeptide constructs
of variable lengths makes it possible not only to determine the structures of the individual segmentally arranged
globular domains but also to characterize the intervening linker regions. With this strategy, which was adapted
from target selection in structural genomics projects, we
can obtain a comprehensive view of the multidomain protein, which shows that the protein has overall a predominantly extended shape. The structural information thus
obtained enabled us to immediately predict different functions of nsp3. We are following up our structural findings
with biomedical and physiologic studies.
PUBLICATIONS
Almeida, M.S., Johnson, M.A., Herrmann, T., Geralt, M., Wüthrich, K. Novel
β-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus. J. Virol.
81:3151, 2007.
Chatterjee, A., Johnson, M.A., Serrano, P., Pedrini, B., Wüthrich, K. NMR assignment of the domain 513-651 from the SARS-CoV nonstructural protein nsp3. Biomol. NMR Assign. 1:191, 2007.
Horst, R., Fenton, W.A., Englander, W.S., Wüthrich, K., Horwich A.L. Folding trajectories of human dihydrofolate reductase inside the GroEL GroES chaperonin cavity and free in solution. Proc. Natl. Acad. Sci. U. S. A. 104:20788, 2007.
Johnson, M.A., Southworth, M.W., Herrmann, T., Brace, L., Perler, F.B.,
Wüthrich, K. NMR structure of a KlbA intein precursor from Methanococcus jannaschii. Protein Sci. 16:1316, 2007.
Johnson, M.A., Southworth, M.W., Perler, F.B., Wüthrich K. NMR assignment of
a KlbA intein precursor from Methanococcus jannaschii. Biomol. NMR Assign.
1:19, 2007.
MOLECULAR BIOLOGY
2008
Pedrini, B., Placzek, W.J., Koculi, E., Alimenti, C., LaTerza, A., Luporini, P.,
Wüthrich, K. Cold-adaptation in sea-water-borne signal proteins: sequence and
NMR structure of the pheromone En-6 from the Antarctic ciliate Euplotes nobilii.
J. Mol. Biol. 372:277, 2007.
Placzek, W.J., Almeida, M.S., Wüthrich, K. NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase. J. Mol. Biol.
367:788, 2007.
Placzek, W.J., Etezady-Esfarjani, T., Herrmann, T., Pedrini, B., Peti, W., Alimenti,
C., Luporini, P., Wüthrich, K. Cold-adapted signal proteins: NMR structures of
pheromones from the Antarctic ciliate Euplotes nobilii. IUBMB Life 59:578, 2007.
Serrano, P., Johnson, M.A., Almeida, M.S., Horst, R., Herrmann, T., Joseph, J.S.,
Neuman, B.W., Subramanian, V., Saikatendu, K.S., Buchmeier, M.J., Stevens,
R.C., Kuhn, P., Wüthrich, K. Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome
coronavirus. J. Virol. 81:12049, 2007.
Nuclear Magnetic Resonance of
3-Dimensional Structure and
Dynamics of Proteins in Solution
P.E. Wright, H.J. Dyson, M. Arai, R. Burge, P. Deka,
J. Ferreon, T.-H. Huang, B.B. Koehntop, M. Kostic, B. Lee,
C.W. Lee, M. Landes, M. Martinez-Yamout, T. Nishikawa,
K. Sugase, J. Wojciak, M. Zeeb, E. Manlapaz, L.L. Tennant,
D.A. Case, J. Gottesfeld
e use multidimensional nuclear magnetic
resonance (NMR) spectroscopy to investigate
the structures, dynamics, and interactions
of proteins in solution. Such studies are essential for
understanding the mechanisms of action of these proteins and for elucidating structure-function relationships.
The focus of our current research is protein-protein and
protein–nucleic acid interactions involved in the regulation of gene expression.
W
TRANSCRIPTION FACTOR–NUCLEIC ACID
COMPLEXES
NMR methods are being used to determine the
3-dimensional structures and intramolecular dynamics
of zinc finger motifs from several eukaryotic transcriptional regulatory proteins, both free and complexed with
target nucleic acid. Zinc fingers are among the most
abundant domains in eukaryotic genomes. They play a
central role in the regulation of gene expression at both
the transcriptional and the posttranscriptional level,
mediated through their interactions with DNA, RNA,
or protein components of the transcriptional machinery.
The C2H 2 zinc finger, first identified in transcription
factor IIIA (TFIIIA), is used by numerous transcription
factors to achieve sequence-specific recognition of DNA.
THE SCRIPPS RESEARCH INSTITUTE
227
Growing evidence, however, indicates that some C 2H 2
zinc finger proteins control gene expression both through
their interactions with DNA regulatory elements and, at
the posttranscriptional level, through binding to RNA.
The best-characterized example of a C2H2 zinc finger protein that binds specifically to both DNA and RNA
is TFIIIA, which contains 9 zinc fingers. We showed
previously that different subsets of zinc fingers are
responsible for high-affinity binding of TFIIIA to DNA
(fingers 1–3) and to 5S RNA (fingers 4–6). To obtain
insights into the mechanism by which the TFIIIA zinc
fingers recognize both DNA and RNA, we have used
NMR methods to determine the structures of the complex formed by zf1-3 (a protein consisting of fingers
1–3) with DNA and by zf4-6 (a protein consisting of
fingers 4–6) with a fragment of 5S RNA.
Three-dimensional structures were determined previously for the complex of zf1-3 with the cognate 15-bp
oligonucleotide duplex. The structures contain several
novel features and reveal that prevailing models of DNA
recognition, which assume that zinc fingers are independent modules that contact bases through a limited
set of amino acids, are outmoded.
In addition to its role in binding to and regulating
the 5S RNA gene, TFIIIA also forms a complex with
the 5S RNA transcript. NMR structures of the complex
formed by zinc fingers 4–6 with a truncated form of 5S
RNA have been completed and give important insights
into the structural basis for 5S RNA recognition. Finger 4 of the protein recognizes both the structure of
the RNA backbone and the specific bases in the loop
E motif of the RNA, in a classic lock-and-key interaction. Fingers 5 and 6, with a single residue between
them, undergo mutual induced-fit folding with the
loop A region of the RNA, which is highly flexible in
the absence of the protein.
NMR studies of 2 alternate splice variants of the
Wilms tumor zinc finger protein (WT1) are in progress.
These proteins differ only through insertion of 3 additional amino acids (the tripeptide lysine-threonine-serine)
in the linker between fingers 3 and 4, yet have marked
differences in their DNA-binding properties and subcellular localization. 15N relaxation measurements indicate that the insertion increases the flexibility of the
linker between fingers 3 and 4 and abrogates binding
of the fourth zinc finger to its cognate site in the DNA
major groove, thereby modulating DNA-binding activity. X-ray and NMR structures of the complexes of the
WT1 zinc fingers with 14- and 17-bp DNA oligonucleo-
228 MOLECULAR BIOLOGY
2008
tides have been determined. Zinc fingers 2–4 are
inserted deeply into the DNA major groove, making
sequence-specific contacts with bases. The structure
provides insights into the mechanism by which diseasecausing mutations in the zinc finger domain interfere
with DNA binding. In contrast to fingers 2–4, zinc finger 1 has mostly nonspecific interactions with the DNA.
High-affinity DNA binding is mediated by fingers 2–4;
incorporation of additional amino acids in the linker
by alternate splicing disrupts the finger 4 interactions
and abrogates DNA binding.
NMR structural studies of a complex of the 4 WT1
zinc fingers with an RNA aptamer are nearing completion. In contrast to DNA binding, the RNA interaction
is dominated by zinc fingers 1–3, which bind in the
widened major groove formed in the vicinity of a bulged
base. The interactions of zinc finger 4 with the RNA
loop make only a secondary contribution to binding
affinity. We have also determined the structure of a
novel double-stranded RNA-binding zinc finger protein
and have commenced experiments to define the mechanism of binding to adenovirus VA1 RNA.
We recently determined the structure of a novel zinc
finger protein named Churchill that is involved in regulation of neural induction during embryogenesis. At
the time of its discovery, it was suggested that the protein contained 2 zinc fingers of the C4 type and functioned as a DNA-binding transcription factor. Our NMR
structure shows that far from containing canonical C4
zinc fingers, Churchill contains 3 bound zinc ions in
novel coordination sites, including an unusual binuclear
zinc cluster, which jointly stabilize a single-layer β-sheet
(Fig. 1). We showed further that Churchill does not bind
DNA and suggest that it may function in embryogenesis by mediating protein-protein interactions.
PROTEIN-PROTEIN INTERACTIONS IN
T R A N S C R I P T I O N A L R E G U L AT I O N
Transcriptional regulation in eukaryotes relies on
protein-protein interactions between DNA-bound factors
and coactivators that, in turn, interact with the basal
transcription machinery. The transcriptional coactivator CREB-binding protein (CBP) and its homolog p300
play an essential role in cell growth, differentiation, and
development. Understanding the molecular mechanisms
by which CBP and p300 recognize their various target
proteins is of fundamental biomedical importance. CBP
and p300 have been implicated in diseases such as leukemia, cancer, and mental retardation and are novel targets for therapeutic intervention.
THE SCRIPPS RESEARCH INSTITUTE
F i g . 1 . Structure of Churchill.
We previously determined the structure of the kinaseinducible activation domain of the transcription factor
CREB bound to its target domain (the KIX domain) in
CBP. Ongoing work is directed toward mapping the
interactions between KIX and the transcriptional activation domains of the proto-oncogene c-Myb and of the
mixed-lineage leukemia protein. The solution structure
of the ternary complex between KIX, c-Myb, and the
mixed-lineage leukemia protein has been completed and
provides insights into the structural basis for the ability
of the KIX domain to interact simultaneously and allosterically with 2 different effectors. Our work has also
provided new understanding of the thermodynamics of
the coupled folding and binding processes involved in
interaction of KIX with transcriptional activation domains.
We used R 2 relaxation dispersion experiments to elucidate the mechanism by which folding of the kinaseinducible activation domain of CREB is coupled to
binding to its KIX target domain. These experiments
revealed formation of an ensemble of transient and
largely unfolded encounter complexes at multiple sites on
the surface of KIX. The encounter complexes are stabilized primarily by nonspecific hydrophobic contacts and
evolve via an intermediate to the fully bound state without dissociation from KIX. The C-terminal helix of the
kinase-inducible domain is only partially folded in the
intermediate and becomes stabilized by intermolecular
interactions formed in the final bound state. Future applications of our method will provide new understanding of
the molecular mechanism by which intrinsically disordered proteins perform their diverse biological functions.
MOLECULAR BIOLOGY
2008
Recently, we determined the structure of the complex between the hypoxia-inducible factor Hif-1α and
the TAZ1 domain of CBP. The interaction between Hif-1α
and CBP/p300 is of major therapeutic interest because
of the central role Hif-1α plays in tumor progression and
metastasis; disruption of this interaction leads to attenuation of tumor growth. A protein named CITED2 functions as a negative feedback regulator of the hypoxic
response by competing with Hif-1α for binding to the
TAZ1 domain of CBP. By determining the structure of the
complex, we showed that the intrinsically unstructured
Hif-1α and CITED2 domains use partly overlapping surfaces of the TAZ1 motif to achieve high-affinity binding
and compete effectively with each other for CBP/p300.
To further elucidate the molecular and structural
basis for CBP-dependent coordinated gene expression,
we have determined the solution structures of the complexes formed by the transactivation domains of the
transcription factors STAT2 and STAT1 with CBP TAZ1
and TAZ2 domains, respectively. Despite the overall
topological similarity of the CBP TAZ domains, the
structures reveal 2 very different modes of complex formation. Our findings suggest that TAZ1 may bind activation domains capable of contacting multiple surface
grooves simultaneously in preference to smaller activation motifs that are restricted to a single, contiguous
binding surface. The latter mode of binding is sufficient
for stable complex formation with TAZ2. Binding of both
STAT activation domains involves coupled folding and
binding processes.
We are continuing to map the multiplicity of interactions between CBP/p300 domains and their numerous biological targets. Our goal is to understand the
complex interplay of interactions that mediate key biological processes in health and disease.
PUBLICATIONS
Ebert, M.-O., Bae, S.-H., Dyson, H.J., Wright, P.E. NMR relaxation study of the
complex formed between CBP and the activation domain of the nuclear hormone
receptor coactivator ACTR. Biochemistry 47:1299, 2008.
Lee, B.M., Buck-Koehntop, B.A., Martinez-Yamout, M.A. Dyson, H.J., Wright,
P.E. Embryonic neural inducing factor Churchill is not a DNA-binding zinc finger
protein: solution structure reveals a solvent-exposed β-sheet and zinc binuclear
cluster J. Mol. Biol. 371:1274, 2007.
Stoll, R., Lee, B.M., Debler, E.W., Laity, J.H., Wilson, I.A., Dyson, H.J., Wright,
P.E. Structure of the Wilms tumor suppressor protein zinc finger domain bound to
DNA. J. Mol. Biol. 372:1227, 2007.
Sugase, K., Landes, M.A., Wright, P.E., Martinez-Yamout, M.A. Overexpression of
post-translationally modified peptides in Escherichia coli by co-expression with
modifying enzymes. Protein Expr. Purif. 57:108, 2008.
Sugase, K., Lansing, J.C., Dyson, H.J., Wright, P.E. Tailoring relaxation dispersion
experiments for fast-associating protein complexes J. Am. Chem. Soc. 129:13406,
2007.
THE SCRIPPS RESEARCH INSTITUTE
229
Folding of Proteins and
Protein Fragments
P.E. Wright, H.J. Dyson, D. Meinhold, C. Nishimura,
D. Felitsky, M. Kostic, S.J. Park, J. Chung, L.L. Tennant,
V. Bychkova,* T. Yamagaki
* Institute of Protein Research, Puschino, Russia
he molecular mechanism by which proteins fold
into their 3-dimensional structures remains one
of the most important unsolved problems in structural biology. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to provide information on the
structure of transient intermediates formed during protein folding. Previously, we used NMR methods to show
that many peptide fragments of proteins have a tendency
to adopt folded conformations in water solution. The
presence of transiently populated folded structures,
including reverse turns, helices, nascent helices, and
hydrophobic clusters, in water solutions of short peptides has important implications for initiation of protein
folding. Formation of elements of secondary structure
probably plays an important role in the initiation of
protein folding by reducing the number of conformations that must be explored by the polypeptide chain
and by directing subsequent folding pathways.
T
A P O M Y O G L O B I N F O L D I N G PAT H WAY
A major program in our laboratory is directed toward
a structural and mechanistic description of the apomyoglobin folding pathway. Previously, we used quenched-flow
pulse-labeling methods in conjunction with 2-dimensional NMR spectroscopy to map the kinetic folding
pathway of the wild-type protein. With these methods,
we showed that an intermediate in which the A, G, and
H helices and part of the B helix adopt hydrogen-bonded
secondary structure is formed within 6 milliseconds of
the initiation of refolding. Folding then proceeds by
stabilization of additional structure in the B helix and
in the C and E helices. We are using carefully selected
myoglobin mutants and both optical stopped-flow spectroscopy and NMR methods to further probe the kinetic
folding pathway. For some of the mutants studied, the
changes in amino acid sequence resulted in changes
in the folding pathway of the protein.
These experiments are providing novel insights into
both the local and long-range interactions that stabilize
the kinetic folding intermediate. Of particular importance,
long-range interactions have been observed that indi-
230 MOLECULAR BIOLOGY
2008
cate nativelike packing of some of the helices in the
kinetic molten globule intermediate. However, folding
is impeded by local nonnative helix packing; the H helix
is translocated relative to the G helix by a single helical turn, and folding cannot proceed until this defect
is repaired.
Apomyoglobin provides a unique opportunity for
detailed characterization of the structure and dynamics
of a protein-folding intermediate. Conditions were previously identified under which the apomyoglobin molten
globule intermediate is sufficiently stable for acquisition
of multidimensional heteronuclear NMR spectra. Analysis of 13C and other chemical shifts and measurements
of polypeptide dynamics provided unprecedented insights
into the structure of this state.
The A, G, and H helices and part of the B helix are
folded and form the core of the molten globule. This
core is stabilized by relatively nonspecific hydrophobic
interactions that restrict the motions of the polypeptide
chain. Fluctuating helical structure is formed in regions
outside the core, although the amount of helix is low
and the chain retains considerable flexibility. The F helix
acts as a gate for heme binding and only adopts stable structure in the fully folded holoprotein.
The acid-denatured (unfolded) state of apomyoglobin
is an excellent model for the fluctuating local interactions
that lead to the transient formation of unstable elements
of secondary structure and local hydrophobic clusters
during the earliest stages of folding. NMR data indicated
substantial formation of helical secondary structure in
the acid-denatured state in regions that form the A and H
helices in the folded protein and also revealed nonnative
structure in the D and E helix regions.
Because the A and H regions adopt stabilized helical
structure in the earliest detectable folding intermediate,
these results lend strong support to folding models in
which spontaneous formation of local elements of secondary structure plays a role in initiating formation of
the A-[B]-G-H molten globule folding intermediate. In
addition to formation of transient helical structure, formation of local hydrophobic clusters has been detected
by using 15N relaxation measurements. Significantly,
these clusters are formed in regions where the average
surface area buried upon folding is large. In contrast to
acid-denatured unfolded apomyoglobin, the urea-denatured state is largely devoid of structure, although
residual hydrophobic interactions have been detected
by using relaxation measurements.
We have measured residual dipolar couplings for
unfolded states of apomyoglobin by using partial align-
THE SCRIPPS RESEARCH INSTITUTE
ment in strained polyacrylamide gels. These data provide novel insights into the structure and dynamics of
the unfolded polypeptide chain. We have shown that the
residual dipolar couplings arise from the well-known statistical properties of flexible polypeptide chains. Residual dipolar couplings provide valuable insights into the
dynamic and conformational propensities of unfolded
and partly folded states of proteins and hold great
promise for charting the upper reaches of protein-folding landscapes.
To probe long-range interactions in unfolded and
partially folded states of apomyoglobin, we introduced
spin-label probes at several sites throughout the polypeptide chain. These experiments led to the surprising
discovery that transient structures with nativelike longrange contacts between hydrophobic clusters exist within
the ensemble of conformations formed by the aciddenatured state of apomyoglobin. They also indicated
that the packing of helices in the molten globule state
is similar to that in the native folded protein. The relative amounts of the transiently collapsed states formed
in the apomyoglobin polypeptide chain are determined
by the entropic cost of loop closure. The specificity of
the long-range contacts in the most structured of these
states suggests that the contacts play a key role in
directing chain collapse and initiating folding.
The view of protein folding that results from our
work on apomyoglobin is one in which collapse of the
polypeptide chain to form increasingly compact states
leads to progressive accumulation of secondary structure and increasing restriction of fluctuations in the
polypeptide backbone. Chain flexibility is greatest at
the earliest stages of folding, when transient elements
of secondary structure and local hydrophobic clusters
are formed. As the folding protein becomes increasingly
compact, backbone motions become more restricted,
the hydrophobic core is formed and extended, and
nascent elements of secondary structure are progressively stabilized. The ordered tertiary structure characteristic of the native protein, with well-packed side
chains and relatively low-amplitude local dynamics,
appears to form rather late in folding.
We recently introduced a variation on the classic
quench-flow technique, which makes use of the capabilities of modern NMR spectrometers and heteronuclear NMR experiments, to study the proteins labeled
along the folding pathway in an unfolded state in an
aprotic organic solvent. This method allows detection
of many more amide proton probes than in the classic
MOLECULAR BIOLOGY
2008
method, which requires formation of the fully folded
protein and the measurement of the protein’s NMR spectrum in water solution. This method is particularly useful
in documenting changes in the folding pathway that
result in the destabilization of parts of the protein in
the molten globule intermediate. We recently showed
that self-compensating mutations designed to change
the amino acid sequence such that the average area
buried upon folding is significantly changed while the
3-dimensional structure of the final folded state remains
the same. These studies showed that the average area
buried upon folding is an accurate predictor of those
parts of the apomyoglobin molecule that will fold first
and participate in the molten globule intermediate.
Quench-flow hydrogen exchange experiments performed
on a series of hydrophobic core mutants indicated that
the overall helix-packing topology of the kinetic folding
intermediate is like that of the native protein, despite
local nonnative interactions in packing of the G and H
helices (Fig. 1). Finally, using a rapid mixing device,
THE SCRIPPS RESEARCH INSTITUTE
231
mechanism known as quorum sensing; bacteria release
extracellular signal molecules, called autoinducers, for
cell-cell communication within and between bacterial
species. A number of bacteria appear to use quorum
sensing for regulation of gene expression in response
to fluctuations in cell population density. Processes
regulated in this way include symbiosis, virulence,
competence, conjugation, production of antibiotics,
motility, sporulation, and formation of biofilms.
We determined the 3-dimensional solution structure
of a complex composed of the N-terminal 171 residues
of the quorum-sensing protein SdiA of Escherichia coli
and an autoinducer molecule, N-octanoyl-L-homoserine lactone (HSL). The SdiA-HSL system shows the
“folding switch” behavior associated with quorum-sensing factors produced by other bacterial species. In the
presence of HSL, SdiA is stable and folded and can be
produced in good yields from an E coli expression system. In the absence of the autoinducer, the SdiA is
expressed into inclusion bodies. Samples of the SdiAHSL complex can be denatured but cannot be refolded
in aqueous buffers. The solution structure of the complex provides a likely explanation for this behavior. The
autoinducer molecule is tightly bound in a deep pocket
in the hydrophobic core and is bounded by specific
hydrogen bonds to the side chains of conserved residues. The autoinducer thus forms an integral part of
the hydrophobic core of the folded SdiA.
CHAPERONE–COCHAPERONE–CLIENT PROTEIN
INTERACTIONS
F i g . 1 . Schematic representation of the amide proton occupancies in the kinetic intermediate state formed in the burst phase of
apomyoglobin folding (solid ribbons), mapped onto the structure of
fully folded myoglobin. Areas of the protein that are not folded until
later stages are shown as dotted lines.
we have reduced the dead time of the kinetic refolding
experiments and have shown that a compact helical
intermediate is formed within 400 microseconds after
initiation of apomyoglobin refolding. The new measurements reveal that folding occurs by a hierarchical process: the A, G, and H helices fold rapidly to form a
compact core, and the other helices fold more slowly
by docking onto the preformed core.
FOLDING-UNFOLDING TRANSITIONS IN CELLULAR
M E TA B O L I S M
Many species of bacteria sense and respond to their
own population density by an intricate autoregulatory
Understanding the role of unfolded states in cellular
processes will require an understanding of the structural basis of their interactions, but unfolded proteins
are impossible to characterize structurally by x-ray crystallography, and spectroscopic methods of all kinds are
limited. Unfolded proteins must be explored under conditions that approximate the proteins’ physiologic milieu:
in solution, at physiologic pHs and salt concentrations,
and in the presence of specific cofactors. Structural
insights will be obtained not only from the delineation
of 3-dimensional structures but also from the description of conformational ensembles and of the motions
of polypeptide chains under various conditions.
To gain new insights into the structural basis for the
ability of unfolded and partly folded proteins to function in living systems, we study the interactions of
“client” proteins and cochaperones with a well-known
eukaryotic chaperone, Hsp90. Some of the protein components are much larger than have traditionally been
232 MOLECULAR BIOLOGY
2008
studied by using solution NMR. However, we have
designed a set of experiments that will allow us to draw
valid conclusions about the extent and role of disorder
in Hsp90 interactions. In particular, we will apply techniques recently developed in our laboratory for analyzing hydrogen-deuterium exchange from unstable partially
folded proteins by trapping the 2H-labeled species in
the aprotic solvent dimethyl sulfoxide. This powerful
new technique will be used to probe the structure, stability, and interactions of client proteins and cochaperones with Hsp90.
PUBLICATIONS
Felitsky, D.J., Lietzow, M.A., Dyson, H.J., Wright, P.E. Modeling transient collapsed states of an unfolded protein to provide insights into early folding events.
Proc. Natl. Acad. Sci. U. S. A. 105:6278, 2008.
Nishimura, C., Dyson, H.J., Wright, P.E. The kinetic and equilibrium molten globule
intermediates of apoleghemoglobin differ in structure. J. Mol. Biol. 378:715, 2008.
Schwarzinger, S., Mohana-Borges, R., Kroon, G.J.A., Dyson, H.J., Wright, P.E.
Structural characterization of partially folded intermediates of apomyoglobin H64F.
Protein Sci. 17:313, 2008.
Nuclear Magnetic Resonance
Studies of the Structure and
Dynamics of Enzymes
H.J. Dyson, P.E. Wright, S.H. Bae, G. Bhabha, D. Boehr,
C. Cervantes,* G. Kroon, M. Martinez-Yamout, S.C. Sue,
L.M. Tuttle, L.L. Tennant, C.L. Brooks, S.J. Benkovic,**
A. Holmgren,*** E.A. Komives*
* University of California, San Diego, California
** Pennsylvania State University, University Park, Pennsylvania
*** Karolinska Institutet, Stockholm, Sweden
e use site-specific information on structure and
dynamics, obtained from nuclear magnetic
resonance (NMR), to further the understanding of protein function. We focus on the mechanism of
enzymes and the relationship between dynamics and
function in a number of medically important systems.
W
DYNAMICS IN ENZYME ACTION
Dynamic processes are implicit in the catalytic function of all enzymes. We use state-of-the-art NMR methods to elucidate the dynamic properties of several
enzymes. New methods have been developed for analysis of NMR relaxation data for proteins that tumble
anisotropically and for analysis of slow timescale motions.
Dihydrofolate reductase (DHFR) plays a central
role in folate metabolism and is the target enzyme for
a number of antibacterial and anticancer agents. 15N
THE SCRIPPS RESEARCH INSTITUTE
relaxation experiments on DHFR from Escherichia coli
have revealed a rich diversity of backbone dynamical
features for a broad range of timescales (picoseconds
to milliseconds).
A major focus is the characterization of all intermediates in the DHFR reaction cycle. We have identified functionally important motions in loops that control
access to the active site of the reductase. These motions
differ in amplitude and timescale depending on the
presence of substrate and/or cofactor in the active site.
In addition, measurements of the population distribution of aliphatic side-chain rotamers provide evidence
for coupled motion of active-site side chains that could
enhance the catalytic process.
Most recently, we used relaxation dispersion measurements to obtain direct information on microsecond to
millisecond timescale motions in DHFR, allowing us to
characterize the structures of excited states involved in
some of these catalysis-relevant processes. Each intermediate in the catalytic cycle samples low-lying excited
states whose conformations resemble the ground-state
structures of the preceding and following intermediates.
Fluctuations between these states occur on a timescale
that is directly relevant to the structural transitions
involved in progression through the catalytic cycle.
Substrate and cofactor exchange occurs through
these excited substates. The maximum hydride transfer and steady-state turnover rates are governed by the
dynamics of transitions between the ground and excited
states of the intermediates. The modulation of the energy
landscape by the bound ligands funnels the enzyme
through its reaction cycle along a preferred kinetic path.
DHFR is also the test system for a series of experiments to address the question, If all of the chemistry
goes on at the active site, what is the purpose of the
rest of the enzyme? We are using chimeric mutants,
synthesized by our collaborator S.J. Benkovic, Pennsylvania State University, by using a library approach.
The purpose of these experiments is to test the hypothesis that local variations in amino acid sequence,
3-dimensional structure, and polypeptide chain dynamics strongly influence the local interactions that mediate enzyme catalysis and may constitute the essential
circumstance that allows enzymes to achieve high
turnover rates as well as exquisite specificity in their
reactions. A combination of NMR structure and dynamics measurements, single-molecule fluorescence measurements, and analysis of the catalytic steps in these
mutant proteins provides new insights into the role of
the protein in enzyme catalysis.
MOLECULAR BIOLOGY
2008
S T R U C T U R E A N D D Y N A M I C S O F P R I O N VA R I A N T S
Onset of prion diseases is caused by conversion of
the cellular prion protein PrPC into an abnormally folded
isoform, PrPSc, that has the same primary structure as
PrPC but a totally different 3-dimensional conformation.
The abnormally folded (“scrapie”) form of the protein
is associated with several diseases, including scrapie
in sheep, bovine spongiform encephalopathy (mad cow
disease), and human Creutzfelt-Jakob disease and other
inherited prion diseases. We are gathering information on
the mechanism of PrPSc formation that can be obtained
from structural and dynamic studies of mutant prion
proteins corresponding to inherited prion diseases.
Individuals carrying familial mutations such as P102L
(P101L in our study) are more susceptible to prion disease. On the other hand, sheep or humans carrying
Q167R and/or Q218K mutations are resistant to scrapie
and Creutzfelt-Jakob disease, respectively. We are using
the protease-resistant cores of wild-type and mutant
mouse prion proteins to study the structural and dynamic
basis of PrPC-to-PrPSc conversion in inherited prion diseases. The core is sufficient to transmit infectivity.
DYNAMICS AND THE FUNCTION OF IκBα
It is becoming increasingly clear that the function
of many systems in living cells depends not only on the
structures of the components but also on the structures’
flexibility. Numerous examples exist in which components
of an important biological interaction are unstructured or
partly structured. In addition, even those interacting molecules that can be classified as “folded” have areas of
mobility. Often, these areas are located precisely in the
active site of an enzyme or in the binding site of an
interacting molecule.
A central molecular interaction in cellular control is
the interaction between the nuclear transcription factor NF-κB and its inhibitor IκBα. IκBα consists of a
series of ankyrin repeats, which appear to have differential mobility. Using hydrogen-deuterium exchange
and mass spectrometry, our collaborator, E.A. Komives,
University of California, San Diego, found that the second, third, and fourth ankyrin repeats of IκBα are well
folded, whereas the fifth and sixth repeats, apparently
with exactly the same structure, are highly dynamic.
These observations prompt a number of questions:
Are the motions inferred from the hydrogen-deuterium
mass spectrometry experiments also reflected in the
backbone and side-chain dynamics of the protein, as
measured by NMR relaxation? Are the motions still present in the IκBα–NF-κB complex? Are they necessary for
complex formation, so that if they are damped out, for
THE SCRIPPS RESEARCH INSTITUTE
233
example by site-directed mutagenesis at appropriate
positions, is the formation of the complex disfavored? To
answer these questions, we are doing a series of NMR
experiments on IκBα and its complexes with NF-κB.
PUBLICATIONS
Boehr, D.D., Dyson, H.J., Wright, P.E. Conformational relaxation following hydride
transfer plays a limiting role in dihydrofolate reductase catalysis. Biochemistry
47:9227, 2008.
Sue, S.C., Cervantes, C., Komives, E.A., Dyson, H.J. Transfer of flexibility between
ankyrin repeats in IκBα upon formation of the NF-κB complex. J. Mol. Biol.
380:917, 2008.
Chaperonin-Mediated
Protein Folding
A.L. Horwich, E. Chapman, S.M. Johnson, E. Koculi
uring the past year, we have continued to investigate the mechanism of action of the large molecular machines known as chaperonins that assist
in protein folding in the cell. These megadalton-sized
double-ring assemblies are found in the cytosol of all
organisms, where they assist in the folding of many
newly translated proteins, effectively carrying out the
final step of information transfer. Chaperonins are also
present in the matrix of mitochondria and the stroma of
chloroplasts, where they assist in the folding of proteins
imported from the cytosol. In all contexts, chaperonins
assist folding by 2 sequential actions involving the central cavity of their rings: (1) binding nonnative proteins
in the cavity of an open ring, forestalling aggregation
that could occur if the proteins were free in solution,
and (2) folding, triggered by association of ATP with the
substrate-bound ring, producing release of the substrate
protein into the now-encapsulated cavity, where the
substrate folds in isolation.
D
F O L D I N G T R A J E C T O R Y O F A P R O T E I N S U B S T R AT E
I N S I D E T H E E N C A P S U L AT E D C AV I T Y O F B A C T E R I A L
GroEL-GroES VS FOLDING FREE IN SOLUTION
In collaboration with K. Wüthrich, Department of
Molecular Biology, we used hydrogen-deuterium exchange
and nuclear magnetic resonance (NMR) analysis to
compare the folding trajectory of the substrate protein
human dihydrofolate reductase (DHFR) inside the
GroES-encapsulated chaperonin cavity of a single-ring
version of GroEL vs folding free in solution (Fig. 1). For
both situations, folding was started in aqueous buffer, and
at various times an excess of deuterium oxide was added
and the folding reaction was allowed to continue to completion (the half-time of the reaction is about 2 min-
234 MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
cavity it was 80%–90%. When DHFR was free in solution, more than half of the protein was lost to aggregation. Thus, folding to the native state in the chaperonin
cavity occurs by the same route as in solution, but
multimolecular aggregation is forestalled in the case of
the chaperonin reaction by the “solitary confinement”
of the folding substrate protein.
The results of additional studies with larger substrate proteins under conditions in which the substrates
can reach native form either free in solution or inside
the chaperonin cavity (so-called permissive conditions)
supported our earlier findings; that is, aggregation
occurred in solution but not in the chaperonin reaction.
Thus, the major action of the encapsulated chaperonin
cavity appears to be provision of a site for confining
the folding protein in a hydrophilic chamber with polar
“nonstick” walls, where the protein can fold in isolation, without the possibility of aggregation.
S T R U C T U R A L S TAT E S O F A G R O U P 2 C H A P E R O N I N
F i g . 1 . Experimental protocol for using amide proton (H)-deuterium (D) exchange monitored by NMR to analyze the refolding trajectory of human DHFR while the reductase is inside the chaperonin cavity
(left) or free in solution (right). T is the refolding time in water before
addition of deuterium oxide (DO2). Methotrexate (Mtx) was added
to both reactions to stabilize the native state of DHFR. Reprinted from
Horst et al. Proc. Natl. Acad. Sci. U. S. A. 104:20788, 2007.
Copyright 2007 National Academy of Sciences U.S.A.
utes). The refolded native DHFR was then recovered and
analyzed by using NMR. A total of 51 amide proton positions in native DHFR are well protected and served as
“probes.” Any amide proton involved in a hydrogen bond
at the time deuterium oxide is added would be expected
to be relatively protected from hydrogen-deuterium
exchange and should be visible in the native protein on
NMR analysis, whereas amide protons in regions lacking
structure at the time the deuterium oxide was added
could be exchanged to deuterons and would be invisible.
We found that the acquisition of protection at the
probe positions followed single exponential kinetics,
and the patterns of protection and rates of acquisition
were virtually identical for DHFR folding in the cavity
or free in solution. That is, the folding trajectories were
identical. The extent of recovery of native DHFR free
in solution, however, was about 40%, whereas in the
Rather than use a detachable cochaperonin (e.g.,
bacterial GroES), the chaperonins in the cytosol of
eukaryotes and in archaea have a built-in “lid” structure composed of α-helical apical domain protrusions
that come together to enclose the cavity. The nature of
subunit movements during the ATP-driven reaction
cycle of these machines is not well understood. In collaboration with B. Carragher and C. Potter, Department
of Cell Biology, we used a homo-oligomeric archaeal
chaperonin from Methanococcus maripaludis, overproduced and purified from Escherichia coli, to address
the nature of such movements.
The apo form of the chaperonin had poorly resolving
wide-open apical domains, essentially pointing straight
upward from the stable equatorial “base” into the bulk
solution, without side-by-side contacts. The different
attitudes of these domains suggested flexible positioning in this apo state. We think that this state is the
substrate-binding proficient state of the machine. The
complex composed of the chaperonin, ADP, and aluminum fluoride populated 3 states: an open state resembling the apo form, a state with one ring partially closed
and the other wide open, and a fully closed state like that
of a crystal structure of the archaeal thermosome (Fig. 2).
The apo form now had regular apical density with the
helical protrusions pointing straight upward. The partially closed state presented clockwise-rotated apical
domains on 1 ring that could partially enclose a substrate of considerable size, up to about 80 kD. This state
may comprise a folding-active state for larger proteins.
MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
235
cules that can be programmed by chemical synthesis
to recognize specific DNA sequences, and (2) histone
deacetylase (HDAC) inhibitors, compounds that alter the
postsynthetic modification states of chromosomal proteins and thereby modulate gene expression. Our goals
are to develop polyamides as therapeutics for human
cancer and HDAC inhibitors as therapeutics for neurodegenerative diseases and cystic fibrosis.
B L O C K I N G C A N C E R C E L L P R O L I F E R AT I O N W I T H A
P O LYA M I D E - C H L O R A M B U C I L C O N J U G AT E
F i g . 2 . Three-dimensional image reconstructions of the 3 ADP·AIFx
states of the M maripaludis chaperonin. Left panels, Open state;
center panels, partially closed state (top ring; bottom ring is open);
right panels, closed state, with fitted domains from the homologous
type 2 thermosome chaperonin. Top panels, End views, viewing
down the 8-fold symmetry axis; bottom panels, side views.
The fully closed state included only approximately half
as much volume, produced by inward rotation of subunits. Whether the fully closed state remains an obligate step in the reaction cycle remains to be addressed.
It may be a folding chamber dedicated solely to smaller
protein substrates.
PUBLICATIONS
Clare, D.K., Stagg, S., Quispe, J., Farr, G.W., Horwich, A.L., Saibil, H.R. Multiple
states of a nucleotide-bound group 2 chaperonin. Structure 16:528, 2008.
Horst, R., Fenton, W.A., Englander, S.W., Wüthrich, K., Horwich, A.L. Folding
trajectories of human dihydrofolate reductase inside the GroEL-GroES chaperonin
cavity and free in solution. Proc. Natl. Acad. Sci. U. S. A. 104:20788, 2007.
Chemical Regulation of Gene
Expression
J.M. Gottesfeld, R. Burnett, J. Chou, D. Herman, S. Ku,
C. Xu, E. Soragni, E.A. Thomas, D. Hutt,* W.E. Balch,*
S. Tsai,** M. Farkas,** P.B. Dervan,** S.L. Perlman,***
G. Coppola,*** D. Geschwind,*** M. Rai,****
M. Pandolfo,**** T. O’Hare,***** B. Druker*****
* Department of Cell Biology, Scripps Research
** California Institute of Technology, Pasadena, California
*** University of California, Los Angeles, California
**** Universite Libre de Bruxelles-Hospital Erasme, Brussels, Belgium
***** Oregon Health and Sciences University, Portland, Oregon
W
e focus on 2 classes of small molecules that
can alter gene expression in human cells: (1)
pyrrole-imidazole polyamides, a class of mole-
DNA alkylators, such as the nitrogen mustard chlorambucil, are among the most common agents used
to treat cancer in humans and act by damaging DNA.
Because chlorambucil alkylates DNA at all available
guanine residues in cellular DNA, coupling chlorambucil
to a sequence-specific polyamide decreases the number
of sites in the genome that are damaged and reduces
unwanted side effects while retaining the ability of the
compound to kill cancer cells.
We recently found that a polyamide-chlorambucil
conjugate called 1R-Chl blocks proliferation of multiple
cancer cell lines in culture by causing the cells to arrest
at the G2/M stage of the cell cycle. The compound blocks
tumor growth in immunocompromised mice, including
cells derived from colon, prostate, chronic myelogenous
leukemia, and lung cancers, and no apparent toxic
effects occur at doses required for a therapeutic effect.
Using microarray analysis, we discovered that the gene
target of 1R-Chl is the gene for histone H4c, a member
of the gene family that encodes a critical component
of cellular chromatin and a gene that is highly expressed
in a wide range of cancer cells. Reduction in histone
H4 protein by polyamide treatment was confirmed in cells
treated with 1R-Chl, which caused chromatin decondensation. Small interfering RNAs to H4c mRNA also
had this effect, providing target validation for the effects
of 1R-Chl. Recent studies indicated that 1R-Chl causes
growth arrest of chronic myelogenous leukemia cells
harboring both wild-type BCR-ABL, the gene translocation that causes the leukemia, and 3 tyrosine kinase
inhibitor–resistant strains, suggesting that 1R-Chl could
be used in combination therapy for chronic myelogenous leukemia or in cases of relapsed disease. We are
using additional animal and cellular models for human
cancer and proliferative diseases to determine the therapeutic potential of 1R-Chl.
G E N E R E G U L AT I O N I N N E U R O L O G I C D I S E A S E S
The neurodegenerative disease Friedreich ataxia is
caused by gene silencing through expansion of GAA•TTC
236 MOLECULAR BIOLOGY
2008
triplet repeats in the first intron of a nuclear gene that
encodes the essential mitochondrial protein frataxin. We
used antibodies to the various modification states of the
core histones and chromatin immunoprecipitation methods to examine the chromatin structure of the gene
frataxin in normal cells and in cells derived from patients
with Friedreich ataxia. We found that gene silencing at
expanded frataxin alleles is accompanied by hypoacetylation of histones H3 and H4 and methylation of histone H3, consistent with a heterochromatin-mediated
repression mechanism. These findings suggest that
HDAC inhibitors, compounds that reverse heterochromatin, might activate frataxin.
On the basis of the structure of a commercial HDAC
inhibitor that partially reverses frataxin silencing, we
synthesized and assayed a series of derivatives of the
inhibitor and identified novel pimelic diphenylamide
HDAC inhibitors that reverse frataxin silencing in primary lymphocytes from patients with Friedreich ataxia.
One molecule, 4b, acts directly on the histones associated with frataxin, increasing acetylation at particular lysine residues on histones H3 and H4. A chemical
proteomics approach revealed that HDAC3 is the likely
cellular target of our inhibitors.
Of note, the HDAC inhibitors cross the blood-brain
barrier and increase levels of frataxin mRNA in the brain
and other organs in a mouse model of the human disease. Genome-wide microarray studies indicated that
our compounds partially correct the transcription pattern of genes affected by Friedreich ataxia in the brains
of these mice and in lymphocytes from patients with
Friedreich ataxia to the transcription pattern of healthy
animals or individuals. The pharmacokinetic and toxic
properties of the compounds are under investigation.
HUNTINGTON’S DISEASE
Transcriptional dysregulation is a core pathologic
feature of Huntington’s disease, one of several CAG
triplet repeat disorders characterized by movement
deficits and cognitive dysfunction. Recent studies have
shown therapeutic effects of commercially available
HDAC inhibitors in several models of Huntington’s disease; however, the therapeutic value of the compounds
is limited by their toxic effects. Chronic oral administration of the HDAC inhibitor 4b, beginning after the onset
of motor deficits in R6/2 300Q transgenic mice, which
have about 300 CAG repeats, significantly improved
motor performance, overall appearance, and body weight.
These effects were associated with significant attenuation of gross decline in brain size and striatal atrophy.
THE SCRIPPS RESEARCH INSTITUTE
Microarray studies revealed that 4b treatment ameliorated, in part, deficits in gene expression caused by the
presence of the mutant huntingtin protein in the striatum, cortex, and cerebellum of the transgenic mice.
For selected genes, we found that 4b treatment reversed
histone H3 hypoacetylation that occurs in the presence of mutant huntingtin, in association with correction of the expression levels of various mRNAs. These
findings suggest that 4b, and possibly related HDAC
inhibitors, may be clinically beneficial for patients with
Huntington’s disease.
CYSTIC FIBROSIS
Cystic fibrosis, a disease that affects the lungs and
organs of the digestive tract, is caused by the loss of
the chloride channel, cystic fibrosis transmembrane
conductance regulator (CFTR), on the cell surface. The
resulting loss of ionic homeostasis leads to a thickening of the airway surface liquid, which clogs the lungs
and the pancreatic duct. Although numerous mutations
have been identified in CFTR, the gene for CFTR, most
patients express a form of the protein that is misfolded
and is therefore retained in the endoplasmic reticulum.
We found that inhibition of HDAC7, either with HDAC
inhibitors or short interfering RNA, resulted in rescue
of this trafficking mutant and restoration of its surface
chloride channel activity. These data suggest that HDAC7
is an attractive target for the development of small-molecule correctors of CFTR-trafficking defects. The clinical
use of general HDAC inhibitors as antitumor agents
suggests that HDAC7-specific inhibitors would have
beneficial effects greater than any adverse side effects
and could be useful in the treatment of cystic fibrosis.
PUBLICATIONS
Chou, C.J., Farkas, M.E., Tsai, S.M., Alvarez, D., Dervan, P.B., Gottesfeld, J.M.
Small molecules targeting histone H4 as potential therapeutics for chronic myelogenous leukemia. Mol. Cancer Ther. 7:769, 2008.
Gottesfeld J.M. Small molecules affecting transcription in Friedreich ataxia. Pharmacol. Ther. 116:236, 2007.
Rai, M., Soragni, E., Jenssen, K., Burnett, R., Herman, D., Coppola, G.,
Geschwind D.H., Gottesfeld, J.M., Pandolfo, M. HDAC inhibitors correct frataxin
deficiency in a Friedreich ataxia mouse model. PLoS ONE 3: e1958, 2008.
Nucleic Acid Dynamics
D.P. Millar, J. Gill, G. Pljevaljcić, R. Robertson, J. Wang,
E.J.C. Van der Schans
T
he focus of our research is the assembly and
conformational dynamics of nucleic acid–based
macromolecular machines and assemblies. We
MOLECULAR BIOLOGY
2008
use single-molecule fluorescence methods to investigate a range of systems, including ribonucleoprotein
complexes and DNA polymerases. Our studies reveal
the dynamic structural rearrangements that occur
during the assembly and function of these complex
macromolecular assemblies.
R I B O N U C L E O P R O T E I N A S S E M B LY
The Rev protein from HIV type 1 is a key regulatory protein that controls the transition from early to
late patterns of viral gene expression. Rev binds to a
highly structured region within the viral mRNA, known
as the Rev response element (RRE), where it forms an
oligomeric ribonucleoprotein complex. The formation
of this complex inhibits splicing and facilitates export
of the viral RNA from the nucleus to the cytoplasm.
Because of its critical role in the viral life cycle, the
Rev-RRE complex provides a novel target for development of therapeutic drugs.
To dissect the mechanism of assembly of ribonucleoproteins, we use single-molecule fluorescence imaging
methods to monitor the formation of oligomeric complexes of Rev on individual RRE molecules immobilized
on a solid surface. We found that a single Rev monomer
binds initially to a high-affinity site in stem loop IIB of
the RRE and that assembly subsequently proceeds by
the stepwise addition of additional Rev monomers. The
elementary rate constants for each step of assembly
were also obtained from the single-molecule data. We
also use single-pair Förster or fluorescence resonance
energy transfer (FRET) to probe changes in the conformation of the RRE during the assembly process. We are
using the results of these mechanistic studies to develop
novel fluorescence-based methods for high-throughput
screening of libraries of chemical compounds. The new
screening tools are being used to identify small molecules that block initial binding of Rev to the RRE or prevent the subsequent Rev-Rev oligomerization.
Another example of ribonucleoprotein assembly
under study is the signal recognition particle. This particle is a fascinating molecular machine responsible for the
cotranslational targeting of secretory or membrane proteins to the endoplasmic reticulum. This large complex,
composed of a 300-nucleotide RNA and 6 proteins,
interacts with both the ribosome, during translational
arrest, and a membrane-bound receptor. We are developing novel spectroscopic techniques (based on multicolor
FRET) to dissect the assembly pathway of the particle,
focusing on the temporal order of protein-binding events
and the associated RNA-folding transitions.
THE SCRIPPS RESEARCH INSTITUTE
237
In parallel, we are developing methods to label large
RNA molecules with donor and acceptor probes for FRET
measurements. These reagents and methods are being
used to monitor assembly reactions of signal recognition
particle proteins on individual immobilized RNA molecules by means of single-molecule FRET microscopy. The
interplay between protein-binding and RNA-folding events
revealed in our studies is providing general insights into
the mechanism of assembly of ribonucleoproteins.
D N A P O LY M E R A S E S
DNA polymerases are remarkable for their ability to
synthesize DNA at rates of several hundred base pairs
per second while maintaining an extremely low frequency
of errors. To elucidate the origin of polymerase fidelity, we
are using single-molecule fluorescence methods to examine the dynamic interactions that occur between a DNA
polymerase and its DNA and nucleotide substrates. The
FRET method is being used to observe conformational
transitions of the enzyme-DNA complex that occur during
selection and incorporation of an incoming nucleotide
substrate. Similar methods are being used to monitor
the proofreading step after synthesis.
Our results reveal that binding of a correct nucleotide substrate induces a slow conformational change
within the polymerase, causing the “fingers” domain to
close over the DNA primer terminus and incoming nucleotide. Our studies are also providing new insights into
the mechanisms used to transfer a DNA substrate from
the synthesis site to the exonuclease active site during
proofreading. The advantage of single-molecule observations is that they eliminate the need to synchronize
a population of molecules, allowing these dynamic
processes to be observed directly.
PUBLICATIONS
Bailey, M.F., Van der Schans, E.J.C., Millar, D.P. Dimerization of the Klenow fragment of Escherichia coli DNA polymerase I is linked to its mode of DNA binding.
Biochemistry 46:8085, 2007.
Debler, E.W., Kaufmann, G.F., Meijler, M.M., Heine, A., Mee, J.M., Pljevaljcić,
G., Di Bilio, A.J., Schultz, P.G., Millar, D.P., Janda, K.D., Wilson, I.A., Gray, H.B.,
Lerner, R.A. Deeply inverted electron-hole recombination in an antibody-stilbene
complex. Science 319:1232, 2008.
Pljevaljcić, G., Millar, D. Single-molecule fluorescence methods for the analysis of
RNA folding and ribonucleoprotein assembly. Methods Enzymol., in press.
Stengel, G., Gill, J.P., Sandin, P., Wilhelmsson, M., Albinsson, B., Nordén, B.,
Millar, D. Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue. Biochemistry 46:12289, 2007.
238 MOLECULAR BIOLOGY
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Single-Molecule Biophysics:
Folding, Assembly, and Function
A.A. Deniz, S.Y. Berezhna, A.C.M. Ferreon, Y. Gambin,
E. Lemke, H.-W. Liu, C. Moran, S. Mukhopadhyay
e develop and use state-of-the-art single-molecule fluorescence methods and high-sensitivity
ensemble methods to address key biological
questions about the structure, dynamics, and function
of biomolecules. These methods offer key advantages
over traditional measurements, allowing us to directly
observe the behavior of individual subpopulations in
mixtures of molecules and to measure the kinetics of
structural transitions during stochastic processes
under equilibrium conditions.
A major goal is to apply single-molecule methods
to studies of protein folding and aggregation. For example, partially folded or misfolded protein structures are
thought to play important cellular roles, and these states
can be studied by using single-molecule methods. In
one instance, we are studying the structural properties
and aggregation of α-synuclein, a protein implicated in
the pathogenesis of Parkinson’s disease and other neurodegenerative diseases. We used single-molecule fluorescence to study the folding induced in this protein by
binding of lipid and membrane mimics. Single-molecule
fluorescence resonance energy transfer (FRET) experiments with sodium dodecyl sulfate, which is used to
denature proteins, revealed an extraordinarily complex
binding-induced folding landscape composed of several folded states and transitions. We found that not
only membranes but also small molecules can trigger
these transitions, significant for the biological function
and disease relevance of this protein.
In collaboration with S.L. Lindquist, Whitehead
Institute, Cambridge, Massachusetts, we are examining
the dynamic interplay between folding and aggregation
of the prion domain (NM) of Sup35, a yeast translational
termination factor whose activity is modulated by conversion to a prion form. Using a combination of single-molecule methods, we previously showed that monomeric
native NM populates a collapsed and rapidly fluctuating ensemble of disordered structures. Recently, using
a combination of fluorescence intensity and polarization
measurements during NM aggregation, we observed rapid
formation of oligomeric species followed by a reaction
phase in which conformation and size change in concert; both occur before the fibril growth phase detected in
W
THE SCRIPPS RESEARCH INSTITUTE
standard aggregation experiments. Our observations shed
new light on how amyloid conformation is sequestered
in the context of oligomers rather than monomers,
behavior that may be key in aggregation and biological
function of Sup35 and other amyloidogenic proteins.
The detailed structural and dynamical mapping of
protein binding-folding and aggregation landscapes provided by single-molecule measurements will be applicable to other natively unfolded and amyloid-forming
proteins. Thus, in collaboration with J.W. Kelly, Department of Chemistry, we are exploring the conformational
and aggregation properties of the amyloid β-peptide
involved in Alzheimer’s disease to better understand
the role of the peptide in biology and disease.
Important events on the folding landscapes of proteins and other biomolecules occur on millisecond or
faster timescales. To facilitate understanding of such
fast processes, in collaboration with A. Groisman, University of California, San Diego, we are developing rapid
microfluidic mixing methods. Experiments with singlemolecule resolution are providing us with new information about how different α-synuclein states are connected
on the energy landscape. Additionally, we are continuing to develop and use multicolor single-molecule FRET
methods to study assembly mechanisms of larger multicomponent molecular machines. For example, 3-color
FRET experiments done in collaboration with J.R.
Williamson, Department of Molecular Biology, are providing novel insights into the coupling between folding
of 2 RNA junctions in the 30S ribosomal subunit.
Finally, we are using multicolor fluorescence imaging to study the cellular pathways of RNA interference. In collaboration with P.G. Schultz, Department
of Chemistry, we probed the time-dependent cellular
locations of short interfering RNA and a key RNA
interference protein, Ago2, and found correlations
with sites of viral replication. We have started ensemble and single-molecule FRET measurements to better
understand the formation of RNA-induced silencing complexes in vitro and in living cells.
PUBLICATIONS
Deniz, A.A., Mukhopadhyay, S., Lemke, E.L. Single-molecule biophysics: at the
interface of biology, physics and chemistry. J. R. Soc. Interface 5:15, 2008.
Mukhopadhyay, S., Deniz, A.A. Fluorescence from diffusing single molecules illuminates biomolecular structure and dynamics. J. Fluoresc. 17:775, 2007.
Wang, H., Duennwald, M.L., Roberts, B.E., Rozeboom, L.M., Zhang, Y.L., Steele,
A.D., Krishnan, R., Su, L.J., Griffin, D., Mukhopadhyay, S., Hennessy, E.J., Weigele,
P., Blanchard, B.J., King, J., Deniz, A.A., Buchwald, S.L., Ingram, V.M., Lindquist,
S., Shorter, J. Direct and selective elimination of specific prions and amyloids by 4,5dianilinophthalimide and analogs. Proc. Natl. Acad. Sci. U. S. A. 105:7159, 2008.
MOLECULAR BIOLOGY
Computer Modeling of Proteins
and Nucleic Acids
D.A. Case, Y. Bomble, J Lätzer, V. Pelmenschikov,
T. Steinbrecher, S. Tang, V. Wong
omputer simulations offer an exciting approach
to the study of many aspects of biochemical interactions. We focus primarily on molecular dynamics simulations (in which Newton’s equations of motions
are solved numerically) to model the solution behavior
of biomacromolecules. Recent applications include
detailed analyses of electrostatic interactions in short
peptides (folded and unfolded), proteins, and oligonucleotides in solution.
In addition, molecular dynamics methods are useful in refining solution structures of proteins by using
constraints derived from nuclear magnetic resonance
(NMR) spectroscopy, and we are continuing to explore
new methods in this area. Our developments are incorporated into the Amber molecular modeling package,
designed for large-scale biomolecular simulations, and
into other software, including Nucleic Acid Builder, for
developing 3-dimensional models of unusual nucleic
acid structures; SHIFTS, for analyzing chemical shifts
in proteins and nucleic acids; RNAmotif, for finding
structural motifs in genomic sequence databases; and
DOCK, for placing inhibitors into enzyme active sites.
C
NMR AND THE STRUCTURE AND DYNAMICS OF
PROTEINS AND NUCLEIC ACIDS
Our overall goal is to extract the maximum amount
of information about biomolecular structure and dynamics from NMR experiments. To this end, we are studying
the use of direct refinement methods for determining
biomolecular structures in solution on the basis of NMR
data, going beyond distance constraints to generate
closer connections between calculated and observed
spectra. We are also using quantum chemistry to study
chemical shifts and spin-spin coupling constants. Other
types of data, such as chemical shift anisotropies, direct
dipolar couplings in partially oriented samples, and
analysis of cross-correlated relaxation, are also being
used to guide structure refinement.
In recent structural studies, we focused on the binding of small ligands to DNA and on models for chemically damaged DNA. In studies on dynamics, we have
concentrated on the effects of overall rotational diffusion on NMR relaxation. We have looked at the extent
to which molecular dynamics simulations show the
2008
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239
expected rotational diffusion behavior, and we have
developed novel models to determine relaxation parameters in the presence of time-varying rotational diffusion coefficients.
NUCLEIC ACID MODELING
Another project centers on the development of novel
computer methods to construct models of “unusual”
nucleic acids that go beyond traditional helical motifs.
We are using these methods to study circular DNA,
small RNA fragments, and nucleosome core particles.
We continue to develop efficient computer implementations of continuum solvent methods to allow simplified
simulations that do not require a detailed description of
the solvent (water) molecules; this approach also provides a useful way to study salt effects.
Recent efforts have made second derivatives of
these energies available, so that normal mode analyses
of nucleic acids with dozens to hundreds of nucleotides
can be analyzed and the predictions compared with
those of simpler, elastic continuum models. These efforts
provide a new avenue for developing and testing lowresolution models that can be used for large molecular
assemblies. Currently, we are applying these methods to
nucleosome core particles and to bending and twisting
flexibility of linear and circular duplex DNA.
D Y N A M I C S A N D E N E R G E T I C S O F N AT I V E A N D
N O N N AT I V E S TAT E S O F P R O T E I N S
Analysis methods similar to those described for
nucleic acids are also being used to estimate electrostatic and thermodynamic properties of proteins. A key
feature is the development of computational methods
that can be used to model pH and salt dependence of
complex conformational transitions such as unfolding
events. A second aspect of this research is a detailed
interpretation of NMR results for proteins through molecular dynamics simulations and the construction of models
for molecular motion and disorder.
All of these modeling activities are based on molecular mechanics force fields, which provide estimates of
energies as a function of conformation. We continue
to work on improvements in force fields; recently, we
focused on adding aspects of electronic polarizability,
going beyond the usual fixed-charge models, and on
methods for handling arbitrary organic molecules that
might be considered potential inhibitors in drug discovery efforts. Overall, the new models should provide a
better picture of the noncovalent interactions between
peptide groups and the groups’ surroundings, leading
ultimately to more faithful simulations.
240 MOLECULAR BIOLOGY
2008
PUBLICATIONS
Bomble, Y., Case, D.A. Multiscale modeling of nucleic acids: insights into DNA
flexibility. Biopolymers 89:722, 2008.
THE SCRIPPS RESEARCH INSTITUTE
Chen, J., Dupradeau, F.-Y., Case, D.A., Turner, C.J., Stubbe, J. DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics. Nucleic Acid
Res. 36:253, 2008.
Quantum Chemical Analysis for
Redox-Active Metalloenzymes
and for Photochemistry
Dupradeau, F.Y., Pissard, S., Coulhon, M.P., Cadet, E., Foulon, K., Fourcade, C.,
Goossens, M., Case, D.A., Rochette, J. An unusual case of hemochromatosis due to a
new compound heterozygosity in HFE (p.[Gly43Asp;His63Asp]+[Cys282Tyr]):
structural implications with respect to binding with transferrin receptor 1. Hum.
Mutat. 29:206, 2008.
L. Noodleman, D.A. Case, W.-G. Han, V. Pelmenschikov,
J.A. Fee, L. Hunsicker-Wang,* T. Liu,** M. Ullmann,***
D. Bashford****
* Trinity University, San Antonio, Texas
Graves, A.P., Shivakumar, D.M., Boyce, S.E., Jacobson, M.P., Case, D.A.,
Shoichet, B. Rescoring docking hit lists for model cavity sites: predictions and
experimental testing. J. Mol. Biol. 377:914, 2008.
Guo, Y., Wang, H., Xiao, Y., Vogt, S., Thauer, R.K., Shima, S., Volkers, P.I.,
Rauchfuss, T.B., Pelmenschikov, V., Case, D.A., Alp, E.E., Sturhahn, W., Yoda,
Y., Cramer, S.P. Characterization of the Fe site in iron-sulfur cluster-free hydrogenase (Hmd) and of a model compound via nuclear resonance vibrational spectroscopy (NRVS). Inorg. Chem. 47:3969, 2008.
Hou, T., Zhang, W., Case, D.A., Wang, W. Characterization of domain-peptide
interaction interface: a case study on the amphiphysin-1 SH3 domain. J. Mol. Biol.
376:1201, 2008.
Lukoyanov, D., Pelmenschikov, V., Maeser, N., Laryukhin, M., Yang, T.C., Noodleman, L., Dean, D.R., Case, D.A., Seefeldt, L.C., Hoffman, B.M. Testing if the
interstitial atom, X, of the nitrogenase molybdenum-iron cofactor is N or C:
ENDOR, ESEEM, and DFT studies of the S = 3/2 resting state in multiple environments. Inorg. Chem. 46:11437, 2007.
Payne, R.J., Ficht, S., Tang, S., Brik, A., Yang, Y.-Y., Case. D.A., Wong, C.H.
Extended sugar-assisted glycopeptide ligations: development, scope, and application. J. Am. Chem. Soc. 129:13527, 2007.
Pelmenschikov, V., Case, D.A., Noodleman, L. Ligand-bound S = 1/2 FeMo-cofactor of nitrogenase: hyperfine interaction analysis and implication for the central
ligand X identity. Inorg. Chem. 47:6162, 2008.
Seabra, G.M., Walker, R.C., Elstner, M., Case, D.A., Roitberg, A.E. Implementation of the SCC-DFTB method for hybrid QM/MM simulations within the Amber
molecular dynamics package. J. Phys. Chem. A 111:5655, 2007.
Steinbrecher, T., Mobley, D.L., Case, D.A. Nonlinear scaling schemes for LennardJones interactions in free energy calculation. J. Chem. Phys. 127:214108, 2007.
Tang, S., Case, D.A. Vibrational averaging of chemical shifts anisotropies in model
peptides. J. Biomol. NMR 38:255, 2007.
Thielges, M.C., Case, D.A., Romesberg, F.E. Carbon-deuterium bonds as probes of
dihydrofolate reductase. J. Am. Chem. Soc. 130:6597, 2008.
Walker, R.C., Crowley, M.F., Case, D.A. The implementation of a fast and accurate
QM/MM potential method in Amber. J. Comput. Chem. 28:1019, 2008.
Wong, V., Case, D.A. Comparing MD simulations and NMR relaxation parameters.
Annu. Rep. Comput. Chem., in press.
Wong, V., Case, D.A. Evaluating rotational diffusion from protein MD simulations.
J. Phys. Chem. B 112:6013, 2008.
** University of Maryland, College Park, Maryland
*** University of Bayreuth, Bayreuth, Germany
**** St. Jude Children’s Research Hospital, Memphis, Tennessee
e are using a combination of modern quantum chemistry (density functional theory,
DFT) and classical electrostatics to describe
the energetics, reaction pathways, and spectroscopic
properties of metalloenzymes. In addition, we are analyzing other systems that have novel catalytic, photochemical, or photophysical properties.
Critical biosynthetic and regulatory processes may
involve catalytic transformations of fairly small molecules or groups by transition-metal centers. The ironmolybdenum cofactor center of nitrogenase catalyzes
the multielectron reduction of molecular nitrogen to 2
molecules of ammonia plus molecular hydrogen. We
are continuing our research on the catalytic cycle of
this enzyme, following up on our earlier work on the
structure and oxidation state of the cofactor complex
in the “resting enzyme” before multielectron reduction
and nitrogen binding.
On the basis of DFT calculated vs experimental
physical properties, including redox potentials, cluster
geometries, and Mössbauer isomer shifts, the core
cluster has a (MoFe7S9X ) prismane active site, where
the central X likely is nitride and the “resting cluster
oxidation state” is Mo(IV)Fe(II)4Fe(III)3. A central carbide is also a possibility, because no clear central ligand
hyperfine signal has been detected for the paramagnetic resting state. We have now examined the 2-electron reduced iron-molybdenum cofactor cluster with
the bound alternative product complex (allylic alcohol
generated from propargyl alcohol plus 2 electrons plus
2 protons). Three different ligand-binding structures
are feasible (Fig. 1), and all are expected to yield a
substantial central ligand hyperfine signal for either
14,15 N or 13 C. This finding would be the first confirmed use of nitride or carbide in any enzyme. Our
current structural and redox evidence strongly argues
against the presence of a central oxide.
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MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
241
F i g . 2 . Proposed active-site structure of methane monooxyge-
nase peroxo intermediate P.
F i g . 1 . DFT-optimized model structures of allyl alcohol bound at
Fe6 of the iron-molybdenum cofactor center of nitrogenase. The
calculated relative energies and hyperfine couplings for central
nitrogen atom are indicated.
Methane monooxygenase catalyzes the oxidation of
methane to methanol. This reaction is extremely important in the biosphere because methane is a greenhouse
gas. Further, the monooxygenase can catalyze reactions
with alternative substrates, a characteristic that has
implications for detoxification of organic pollutants. We
have been examining various steps in the catalytic cycle,
comparing predictions based on DFT with those of
Mössbauer spectroscopy. We have focused particularly
on the critical intermediate Q, which performs the oxygen
insertion reaction. Recently, we evaluated the reaction
pathway from the earlier peroxo intermediate P (Fig. 2)
to the dioxo intermediate Q.
The iron complex at the active site of methane
monooxygenase resembles the complex in ribonucleotide
reductases (RNRs; see following); the resemblance is
particularly close for RNRs in some pathogenic bacteria,
including Chlamydia, in which the tyrosine near the ironoxo dimer complex (commonly found in Escherichia coli
and mammalian RNRs) is replaced by phenylalanine.
Evidence now indicates that in RNRs from Chlamydia
the di-iron center is replaced by a manganese-iron center.
The altered RNR mechanism in these pathogens is of
great interest for exploring feasible drug treatments.
Class I RNRs are aerobic enzymes that catalyze the
reduction of ribonucleotides to deoxyribonucleotides, providing the required building blocks for DNA replication
and repair. These enzymes are targets for anticancer,
antiviral, and antibacterial drugs. These ribonucleotideto-deoxyribonucleotide reactions occur via a long-range
radical (or proton-coupled electron transfer) propagation
mechanism initiated by a fairly stable tyrosine radical,
“the pilot light.” When this pilot light goes out, the tyrosine radical is regenerated by a high-oxidation-state
Fe(III)-Fe(IV)-oxo enzyme intermediate called X. We are
using DFT and electrostatics calculations in combination
with analysis of Mössbauer, electron nuclear double resonance, and magnetic circular dichroism spectroscopies to
search for a proper structural and electronic model for
intermediate X.
In collaboration with K. Hahn, University of North
Carolina at Chapel Hill, we have examined the physical and spectroscopic properties of novel solvent-sensitive fluorescent dyes of interest for imaging live cells.
These dyes can act as biosensors for changes in conformational state in cell-signaling proteins, with potential uses in high-throughput drug screening.
With J.A. Fee, Department of Molecular Biology, we
are exploring the mechanism of 4-electron reduction of
molecular oxygen to 2 molecules of water by cytochrome
oxidase (complex IV of mitochondria and the related
cytochrome ba3 from Thermus thermophilus). The cop-
242 MOLECULAR BIOLOGY
2008
per-iron-heme complex links molecular oxygen reduction
to proton pumping across the mitochondrial membrane.
PUBLICATIONS
Fee, J.A., Case, D.A., Noodleman, L. Toward a chemical mechanism of proton
pumping by the B-type cytochrome c oxidases: application of density functional
theory to cytochrome ba3 of Thermus thermophilus. J. Am. Chem. Soc.
130:15002, 2008.
Han, W.-G., Noodleman, L. Structural model studies for the high-valent intermediate Q of methane monooxygenase from broken-symmetry density functional calculations. Inorganica Chim. Acta 361:973, 2008.
Han, W.-G., Noodleman, L. Structural model studies for the peroxo intermediate P
and the reaction pathway from P → Q of methane monooxygenase using brokensymmetry density functional calculations. Inorg. Chem. 47:2975, 2008.
Lukoyanov, D., Pelmenschikov, V., Maeser, N., Laryukhin, M., Yang, T.C., Noodleman, L., Dean, D.R., Case, D.A., Seefeldt, L.C., Hoffman, B.M. Testing if the
interstitial atom, X, of the nitrogenase molybdenum-iron cofactor is N or C:
ENDOR, ESEEM, and DFT studies of the S = 3/2 resting state in multiple environments. Inorg. Chem. 46:11437, 2007.
Noodleman, L., Case, D.A. Broken symmetry states of iron sulfur clusters. In:
Computational Inorganic and Bioinorganic Chemistry. Solomon, E.I., King, R.B.,
Scott, R.A. (Eds.) Wiley & Sons, New York, in press.
Noodleman, L., Pique, M.E., Roberts, V.A. Properties and functions of iron-sulfur
clusters. In: Wiley Encyclopedia of Chemical Biology. Begley, T.P. (Ed.). Wiley Interscience, New York, in press.
Pelmenschikov, V., Case, D.A., Noodleman, L. Ligand-bound S = 1/2 FeMo-cofactor of nitrogenase: hyperfine interaction analysis and implication for the central
ligand X identity. Inorg. Chem. 47:6162, 2008.
Toutchkine, A., Han, W.-G., Ullmann, M., Liu, T., Bashford, D., Noodleman, L.,
Hahn, K.M. Experimental and DFT studies: novel structural modifications greatly
enhance the solvent sensitivity of live cell imaging dyes. J. Phys. Chem. A
111:10849, 2007.
Computation and Visualization
in Structural Biology
A.J. Olson, D.S. Goodsell, M.F. Sanner, M. Chang,
S. Cosconati,* S. Dallakyan, S. Forli, A. Gillet, R. Harris,
R. Huey, J. Huntoon, G. Johnson, D. Keidel, W. Lindstrom,**
G.M. Morris, A. Omelchenko, A. Perryman, M. Pique,
R. Rosenstein, M. Utsintong,*** G. Vareille, Q. Zhang,
Y. Zhao****
THE SCRIPPS RESEARCH INSTITUTE
and research settings, methods for predicting biomolecular interactions and using these interactions in structurebased inhibitor design, analyzing biomolecular structure
and function, and presenting the biomolecular world
in education and outreach.
C O M P O N E N T - B A S E D V I S U A L I Z AT I O N E N V I R O N M E N T S
To facilitate the integration and interoperation of computational models and techniques from a wide variety of
scientific disciplines, we continue to expand our component-based software environment. The environment is
centered on Python, a high-level, object-oriented, interpretive programming language. This approach allows the
compartmentalization and reuse of software components.
Python provides a powerful “glue” for assembling computational components and, at the same time, a flexible language for the interactive scripting of new applications.
In 2007, we released 4 versions of our software
tools: 1.4.4, 1.4.5, 1.4.6, and 1.5.0. We are able to
release new versions so quickly because of the stateof-the-art overnight software testing environment we
have developed. Starting with version 1.4.6, we modified
our installation mechanism to allow the simultaneous
installation of multiple versions of the software. All software tools have been markedly improved. For example,
we enhanced ADT in supporting the new AutoDock4
(described in more detail later); and in the Python Molecular Viewer, ADT modules no longer require a graphical
user interface, rather they can now also function as
stand-alone scripts or as nodes in Vision, our graphical
network editor. Users can now load their own code as
nodes in Vision. We have improved APBSCommands.py
to use local installations of APBS (a software package
for the numerical solution of the Poisson-Boltzmann
equation) or remote APBS Web services through the
Opal toolkit. We have added support for ambient occlusion for polygonal meshes in the visualization component, making it easier to visualize cavities on molecular
surfaces (Fig. 1).
* University of Naples “Federico II,” Naples, Italy
** Acelot, Inc., Santa Barbara, California
*** Mahidol University, Bangkok, Thailand
**** CambridgeSoft, Cambridge, Massachusetts
n the Molecular Graphics Laboratory, we develop
novel computational methods to analyze, understand,
and communicate the structure and interactions of
complex biomolecular systems. Within our componentbased simulation and visualization environment, we
continue to develop 3-dimensional molecular models
as a tangible human-computer interface in educational
I
F i g . 1 . A molecular surface for HIV protease, with traditional
shading on the left and ambient occlusion on the right. Ambient
occlusion simulates the reduced lighting in sheltered areas and gives
useful cues to the shape of complex molecular surfaces. Image
generated with the Python Molecular Viewer.
MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
243
This software has been widely distributed. We had
more than 132,000 downloads during the past 12
months, and 1761 unique users voluntarily registered
in our user database (a sustained average of 5 users
per day).
Most recently, we created an articulated model of MsbA,
a bacterial ABC transporter that undergoes a large
and complex conformational change in the course of
its transport cycle.
TA N G I B L E I N T E R FA C E S I N S T R U C T U R A L B I O L O G Y
AND INTEGRASE
We have continued to develop autofabricated physical models (“solid printing”) of biological molecules in
the context of an augmented reality environment, with
the goal of using the models in both research and education. In collaboration with E. Keinan, Department of
Molecular Biology, we used tangible models to design
self-assembling chemical structures that mimic the selfassembly of viral capsids. Our hypothesis was first developed and tested by using tangible models and then
further characterized by molecular dynamics simulation.
The models allowed direct physical testing of different
possibilities for chemical complementarity of subunits,
for instance, identifying 2 alternative binding modes
for the self-assembled corannulene currently under
development (Fig. 2).
We have also begun work on larger models that
capture the dynamic characteristics of biomolecules.
We are using several approaches in our continuing
work on the development of inhibitors of HIV protease.
After analyzing the evolution of drug resistance against
previously approved inhibitors, we developed a method
to predict mutations associated with drug resistance
that might be induced by new HIV protease inhibitors.
With this technique, we were able to detect more than
half of the major mutations in drug-resistant HIV proteases. Site-directed mutagenesis validated resistance
of 3 predicted mutations (I47V, F53L, and I84V) for
AB2, a novel inhibitor of HIV protease, and the clinically approved drug amprenavir.
In collaboration with ActiveSite, San Diego, California, and C.D. Stout, Department of Molecular Biology, we are developing methods to interpret electron
density maps from crystallographic fragment screens.
In our method, a combination of peak characterization
and computational docking is used to deconvolute peaks
in the crystallographic electron density maps that correspond to fragment binding in crystals soaked with a
mixture of fragments. The method is very rapid, because
docking is only performed in the immediate vicinity of
the identified peaks, and so can be easily incorporated
into the high-throughput work flow in use at ActiveSite.
To improve the efficiency of finding promising chemical lead compounds, the so-called hit rate in drug
discovery, we developed a method that combines highthroughput screening and virtual screening. We performed a virtual screen of 1476 compounds obtained
from a cell-based anti-HIV inhibitor screen. We used a
novel selection procedure to choose compounds that
bound preferentially either to the active site or to a
putative allosteric exosite, but not to both. A total of 5
compounds showed micromolar binding constants; 3
were predicted to bind in the active site and 2 in the
exosite. Kinetic analysis showed noncompetitive binding for the predicted exosite inhibitors, which are now
being further characterized.
We are also collaborating with researchers at Pfizer,
Inc., Sandwich, England, and with J.A. McCammon
and his group at the University of California, San Diego,
to explore the structure and function of HIV integrase
(Fig. 3). We have modeled a form of integrase with a
F i g . 2 . Physical models were used to study self-assembly of viruses
and a designed corannulene complex. Left, Three frames from a movie
showing self-assembly of a virus model. The subunits were fabricated
on the basis of the molecular structure, and magnets were placed
at the interfaces. When the models are shaken in a small bottle, the
intact virus self-assembles. Right, Using these same structural model
abstractions, a corannulene complex was designed. Two possible
modes of assembly were discovered by model building and subsequent computational simulation.
DEVELOPMENT OF INHIBITORS FOR HIV PROTEASE
244 MOLECULAR BIOLOGY
2008
F i g . 3 . Computational modeling and molecular dynamics were
used to generate models of HIV integrase with a closed active-site
loop and 2 hydrated magnesium ions (green). Two conformations of
the enzyme are shown with ribbons, and water molecules and catalytic residues that coordinate the magnesium ions are shown with
bonds. Image generated with the Python Molecular Viewer.
closed loop and 2 magnesium ions that is expected to
be relevant to structure-based drug design. We are
extending this research to model several drug-resistant
mutants and explore how these mutations modify the
dynamics of the enzyme. When combined with quantum mechanical calculations, these models will be useful for studying the mechanism of catalysis. Long-term
goals are to characterize the interaction of the catalytic
domain with DNA and ultimately use this information
to characterize binding of DNA to the intact enzyme.
PROTEIN-LIGAND DOCKING WITH AUTODOCK
According to recent reports, the computer program
AutoDock is the most widely cited docking method, and
we have continued to make it a central tool for predicting biomolecular interactions. AutoDock4 was released
in 2007 and is available through a convenient opensource license. AutoDock was the first docking code to
be used in a public, Internet-based distributed computing project, and we have continued to use this massive
computing resource to perform experiments that are
beyond the reach of traditional computing. We are currently using the World Community Grid, a large Internet distributed computing project sponsored by IBM,
to support FightAIDS@Home on more than 800,000
client computers. Personal computers are used by the
program when the computers are not in use by their
owners, providing an enormous, and largely untapped,
computational resource.
THE SCRIPPS RESEARCH INSTITUTE
To support our growing user community, we have
hosted tutorials, workshops, and lectures presenting
basic methods in AutoDock and specific application to
virtual screening. We have also continued to expand
the capabilities of AutoDock and the graphical user interface AutoDockTools, for instance, exploring the use of
gradient information in the search, predicting covalent
complexes between ligands and proteins, and streamlining all aspects of virtual screening. Most recently,
we tested a method for using results from reiterated
docking experiments to evaluate an empirical vibrational entropy of binding in ligand-protein complexes
in collaboration with R.K. Bele, University of California,
San Diego, and K.S. Carroll, University of Michigan,
Ann Arbor. In addition, we have added new support
for the relaxed complex method, in which snapshots
are taken from a molecular dynamics simulation and
used to sample the range of conformations available
to a protein target.
We have continued development and application
of AutoLigand, a program used to identify and quantify
optimal binding sites in proteins. We showed that the
method is effective for identifying binding sites in proteins and for optimizing the binding of drugs to protein
targets. We have used the method to design exosite
inhibitors of HIV protease; inhibitors for histidine
deacetylase, in collaboration with J.M. Gottesfeld,
Department of Molecular Biology; and exosite inhibitors
for p38 MAP kinase, in collaboration with J.A. Tainer,
Department of Molecular Biology.
PROTEIN FLEXIBILITY AND PROTEIN-PROTEIN
DOCKING
In collaboration with C. Bajaj, University of Texas,
Austin, we have continued the development of a new protein-protein docking technique that is now implemented in
a software program called F2Dock (which stands for Fast
Fourier-transform–based docking). This program is being
tested on a set of protein-protein complexes to evaluate its performance and identify its shortcomings.
We have continued the development and testing
of a new automated docking program called FLIPDock
that uses a hierarchical and multiresolution representation of the flexibility of biological macromolecules to
model protein motion and induced fit. We have further
developed this software tool and applied it to a variety
of docking problems in which receptor flexibility is known
to cause the failure of rigid receptor-based docking simulations. We have optimized FLIPDock and fixed several
problems in the software; we released version 0.1 beta
to a selected set of users.
MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
245
To test the hypothesis that low-resolution surfaces
can be used to improve the success of protein-protein
docking, we did a systematic study of the effects of
multiresolution blurred surfaces on shape complementarity in a set of 66 protein-protein complexes for which
complexed and unbound structures are available. We
found that medium-resolution smoothing can reproduce
about 88% of the shape complementarity of atomic resolution surfaces, and complexes formed from the free
component structures show many overlaps and gaps
with atomic resolution surfaces, which are improved
by smoothing the surfaces to low resolution.
C O M P U TAT I O N A L M O D E L I N G O F E X T R A C E L L U L A R
INTERACTIONS OF TISSUE FACTOR
Signaling through protease-activated receptor 2
(PAR-2) by the complex composed of tissue factor and
coagulation factor VIIa regulates gene transcription and
protein translation, cell proliferation and survival, and
cell motility and integrin activation. This signaling
involves cleavage of the PAR-2 extracellular N-terminal
tail between arginine at position 36 and serine at position 37 by the protease domain of factor VIIa. However,
it is unclear how the protease domain of factor VIIa
recognizes and binds the PAR-2 tail to facilitate the
cleavage. We used molecular modeling and molecular
dynamics simulations to derive the interactions between
PAR-2 and factor VIIa. We found 3 types of key interactions at noncatalytic sites of the factor VIIa protease
domain. Four of the key factor VIIa residues were then
experimentally shown to be involved in PAR-2 activation.
V I S U A L M E T H O D S F R O M AT O M S T O C E L L S
Understanding structural molecular biology is essential to foster progress and critical decision making among
students, policy makers, and the general public. In the
past year, we continued our long-standing commitment
to science education and outreach with a combination
of presentations, popular and professional illustrations
and animations, 3-dimensional tangible models, and a
presence on the World Wide Web. In these projects,
we use the diverse visualization tools developed in the
Molecular Graphics Laboratory to disseminate results
that range from atomic structure to cellular function.
In the past year, the “Molecule of the Month” at
the Protein Data Bank entered its ninth year of providing an accessible introduction to the central database
of biomolecular structure (Fig. 4). In collaboration with
T. Herman, Milwaukee School of Engineering in Wisconsin, we continued work on “Protein Active Learning
Modules” that provide educational materials for the
F i g . 4 . The adrenergic receptor was presented as the 100th
installment of the “Molecule of the Month” at the Protein Data
Bank (http://www.pdb.org). Each month, a new molecule is presented with a description of its structure, function, and relevance
to health and welfare. Visitors are then given suggestions on to
how to begin their own exploration of the structures in the Protein
Data Bank.
high school and undergraduate level. We are also extending our modeling efforts from the realm of molecules
and complexes into the realm of whole cells. Building
on previous illustrative work, we are developing methods to model significant parts of living cells (Fig. 5).
Currently, we are building these tools in the context of
high-end computer animation software, to allow easy
creation of educational materials.
F i g . 5 . A simulated 3-dimensional model of part of a cell, with a
cell membrane at the top and generic cytoplasm at the bottom.
246 MOLECULAR BIOLOGY
2008
PUBLICATIONS
Amaro, R., Minh, D.L., Cheng, L., Lindstrom, W.M., Jr., Olson, A.J., Lin, J.-H.,
Li, W.W., McCammon, J.A. Remarkable loop flexibility in avian influenza N1 and
its implications for antiviral drug design. J. Am. Chem. Soc. 129:7764, 2007.
Beuscher, A.E., Olson, A.J. Iterative docking strategies for virtual ligand screening.
In: Computational and Structural Approaches to Drug Discovery: Ligand-Protein Interactions. Stroud, R.M., Finer-Moore, J. (Eds.). RSC Publishing, Cambridge, England,
2007, p. 242. A volume in the series RSC Biomolecular Sciences.
THE SCRIPPS RESEARCH INSTITUTE
T a b l e 1 . New drug leads and inhibitors.
Lead or inhibitor
Condition affected
Androgen receptor
antagonist (Fig. 1)
Cancer
Chang, M.W., Lindstrom, W., Olson, A.J., Belew, R.K. Analysis of HIV wild-type
and mutant structures via in silico docking against diverse ligand libraries. J.
Chem. Info. Model. 47:1258, 2007.
Evans, M.J., Morris, G.M., Wu, J., Olson, A.J., Sorensen, E.J., Cravatt, B.F.
Mechanistic and structural requirements for active site labeling of phosphoglycerate
mutase by spiroepoxides. Mol. Biosyst. 3:495, 2007.
J. Dalton, Ohio State
University, Columbus, Ohio
α1-Antitrypsin
polymerization
blocker
α1-Antitrypsin
deficiency,
emphysema
D.A. Lomas,
University of Cambridge,
Cambridge, England
Serotonin
N-acetyltransferase
inhibitor
Sleep and mood
disorders
P.A. Cole,
Johns Hopkins School of
Medicine,
Baltimore, Maryland
Enoyl reductase
inhibitor
Malaria
D.A. Fidock,
Albert Einstein College of
Medicine,
Bronx, New York
Goodsell, D.S. Making the step from chemistry to biology and back. Nat. Chem.
Biol. 3:681, 2007.
J.C. Sacchettini,
Texas A&M University,
College Station, Texas
Goodsell, D.S., Johnson, G.T. Filling in the gaps: artistic license in education and
outreach. PloS Biol. 5:e308, 2007.
Harris, R., Olson, A.J., Goodsell, D.S. Automated prediction of ligand-binding sites
in proteins. Proteins 70:1506, 2008.
Illingworth, C.J.R., Morris, G.M., Parkes, K.E.B., Snell, C.R., Reynolds, C.A.
Assessing the role of polarization in docking. J. Phys. Chem. A, in press.
Morris, G.M., Huey, R., Olson, A.J. Using AutoDock for ligand-receptor docking.
Curr. Protoc. Bioinformatics, in press.
Telomeric
G-quadruplex
Cancer
K.Y. Wong,
Hong Kong Polytechnic
University,
Hong Kong, China
Melaninconcentrating
hormone receptor 1
Obesity
F. Monsma,
Schering Plough Research
Institute,
Kenilworth, New Jersey
A. Orry, C. Cavasotto,
MolSoft L.L.C.,
La Jolla, California
Olson, A.J., Hu, Y.H.U., Keinan, E. Chemical mimicry of viral capsid self-assembly. Proc. Natl. Acad. Sci. U. S. A. 104: 20731, 2007.
Zhao, Y., Sanner, M.F. FLIPDock: docking flexible ligands into flexible receptors.
Proteins 68:726, 2007.
Zhao, Y., Sanner, M.F. Protein-ligand docking with multiple flexible side chains. J.
Comput. Aided Mol. Des., in press.
Computational Structural
Proteomics for Drug Discovery
X.K. Zhang,
Burnham Institute,
La Jolla, California
P. Sexton, A. Christopoulos,
Monash University,
Victoria, Australia
Bongini, L., Fanelli, D., Piazza, F., De Los Rios, P., Sanner, M., Skoglund, U. A
dynamical study of antibody-antigen encounter reactions. Phys. Biol. 4:172, 2007.
Chang, M.W., Belew, R.K., Carroll, K.S., Olson, A.J., Goodsell, D.S. Empirical
entropic contributions in computational docking: evaluation in APS reductase complexes. J. Comput. Chem. 29:1753, 2008.
Collaborators
Ubiquitin-like
poxvirus
proteinase I7L
Smallpox
V. Katritch, D. Hruby,
SIGA Technologies, Inc.,
Corvallis, Oregon
tural models of inhibition of sirtuin 2. This foundation
may be helpful in the further development of sirtuin 2
inhibitors for the treatment of Parkinson’s disease.
R. Abagyan, G. Bottegoni, A. Grigoryan, J. Kovacs,
I. Kufareva, G. Nicola, S.-J. Park, K. Reynolds, M. Rueda
e focus on developing and implementing new
mathematical and computational methods
to improve structure prediction and docking
methods for structure-based drug design.
W
DE NOVO DISCOVERY OF DRUG LEADS
Our improved structure-based methods led to the
discovery of new drug leads and inhibitors in collaborative studies with many scientists (Table 1).
A D VA N C E D A P P L I C AT I O N S O F M O L E C U L A R
MODELING AND DOCKING
In collaborations with A.G. Kazantsev, Harvard Medical School, Charlestown, Massachusetts, we built struc-
F i g . 1 . A novel androgen receptor antagonist repurposed from an
antipsychotic drug.
MOLECULAR BIOLOGY
2008
We used the multiple receptor conformation approach
to elucidate the structural mechanism of inhibition of
the 3-phosphoinositide-dependent protein kinase-1 by
3-hydroxyanthranilic acid, a tryptophan metabolite.
We found that 3-hydroxyanthranilic acid inhibited activation of the transcription factor NF-κB upon engagement of T-cell receptors by specifically targeting the
kinase. Our docking models helped rationalize this
interaction, which appears to play a key role in natural regulation of NF-κB activity.
Collaboration with M. Yeager, Department of Cell
Biology, led to the development of techniques and models for complex multisubunit transmembrane proteins.
The detailed atomic models of the gap junction channel
and several other membrane proteins have been assigned
to low-resolution electron density and refined by using
the internal coordinate mechanics protocols. We also
used the internal coordinate mechanics protocols in collaboration with J. Gertsch, ETH Zurich, Zürich, Switzerland, to develop models of supramolecular aggregates of
cannabinomimetics. J. Fernandez-Recio and colleagues,
University of Zaragoza, Zaragoza, Spain, collaborated
with us to improve the prediction for transient proteinprotein complexes. Finally, in collaboration with L.J.
Miller, Mayo Clinic Scottsdale, Scottsdale, Arizona; P.C.
Lam, MolSoft; and P.M. Sexton and A. Christopoulos,
University of Monash; we designed a series of protocols
designed for predicting realistic atomic models of a
ternary complex of the secretin peptide and 2 domains
of its receptor, a family B G protein–coupled receptor.
CHALLENGES IN STRUCTURE-BASED DOCKING AND
SCREENING
Receptor flexibility is a critical issue in structurebased virtual screening methods. Most of the docking
protocols rely on a fixed conformation of the receptor
or on prior knowledge of multiple conformations representing the variation of the pocket. We have developed
an induced-fit docking protocol called SCARE (SCan
Alanines and Refines) that requires only a single initial
pocket conformation for ligand docking and no prior
knowledge about the location of the binding site.
To overcome the error of binding pose and binding
score of ligands with single fixed receptors, we have
introduced multiple receptor conformation, which will
improve the docking calculation overall and account
for receptor flexibility. We used this approach to screen
large ligand databases against p38α kinase and the
nuclear receptor peroxisome proliferator–activated
receptor γ and found dramatic improvement in database enrichment factors against both receptors.
THE SCRIPPS RESEARCH INSTITUTE
247
We also devised a method for evaluating the druggability (i.e., the potential to be targeted by a small-molecule drug) of protein targets at the level of structural
proteomes. The method includes evaluating the structural feasibility of targeting the given protein with a
small molecule (the presence of a sufficiently large
and sufficiently buried binding pocket), predicting the
target genomic instability and escape mutations (evolutionary conservation of the binding site across the
genome of interest and related genomes), and predicting possible drug off-target activities and toxicities (the
lack of homology with human proteins). This method
has been applied to studies of the malarial proteome.
PUBLICATIONS
Bisson, W.H., Cheltsov, A.V., Bruey-Sedano, N., Lin, B., Chen, J., Goldberger, N.,
May, L.T., Christopoulos, A., Dalton, J.T., Sexton, P.M., Zhang, X.K., Abagyan, R.
Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs.
Proc. Natl. Acad. Sci. U. S. A. 104:11927, 2007.
Bottegoni, G., Kufareva, I., Totrov, M., Abagyan, R. A new method for ligand
docking to flexible receptors by dual alanine scanning and refinement (SCARE). J.
Comput. Aided Mol. Des. 22:311, 2008.
Cavasotto, C.N., Orry, A.J., Murgolo, N.J., Czarniecki, M.F., Kocsi, S.A., Hawes,
B.E., O’Neill, K.A., Hine, H., Burton, M.S., Voigt, J.H., Abagyan, R.A., Bayne,
M.L., Monsma, F.J., Jr. Discovery of novel chemotypes to a G-protein-coupled
receptor through ligand-steered homology modeling and structure-based virtual
screening. J. Med. Chem. 51:581, 2008.
Chrencik, J.E., Brooun, A., Recht, M.I., Nicola, G., Davis, L.K., Abagyan, R.,
Widmer, H., Pasquale, E.B., Kuhn, P. Three-dimensional structure of the EphB2
receptor in complex with an antagonistic peptide reveals a novel mode of inhibition. J. Biol. Chem. 282:36505, 2007.
Dong, M., Lam, P.C., Gao, F., Hosohata, K., Pinon, D.I., Sexton, P.M., Abagyan,
R., Miller, L.J. Molecular approximations between residues 21 and 23 of secretin
and its receptor: development of a model for peptide docking with the amino terminus of the secretin receptor. Mol. Pharmacol. 72:280, 2007.
Harikumar, K.G., Lam, P.C., Dong, M., Sexton, P.M., Abagyan, R.,Miller, L.J. Fluorescence resonance energy transfer analysis of secretin docking to its receptor:
mapping distances between residues distributed throughout the ligand pharmacophore and distinct receptor residues. J. Biol. Chem. 282:32834, 2007.
Hayashi, T., Mo, J.H., Gong, X., Rossetto, C., Jang, A., Beck, L., Elliott, G.I.,
Kufareva, I., Abagyan, R., Broide, D.H., Lee, J., Raz, E. 3-Hydroxyanthranilic acid
inhibits PDK1 activation and suppresses experimental asthma by inducing T cell
apoptosis. Proc. Natl. Acad. Sci. U. S. A. 104:18619, 2007.
Katritch, V., Byrd, C.M., Tseitin, V., Dai, D., Raush, E., Totrov, M., Abagyan, R.,
Jordan, R., Hruby, D.E. Discovery of small molecule inhibitors of ubiquitin-like
poxvirus proteinase I7L using homology modeling and covalent docking
approaches. J. Comput. Aided Mol. Des. 21:549, 2007.
Kovacs, J.A., Baker, K.A., Altenberg, G.A., Abagyan, R., Yeager, M. Molecular
modeling and mutagenesis of gap junction channels. Prog. Biophys. Mol. Biol.
94:15, 2007.
Kovacs, J.A., Yeager, M., Abagyan, R. Computational prediction of atomic structures
of helical membrane proteins aided by EM maps. Biophys. J. 93:1950, 2007.
Lee, H.S., Choi, J., Kufareva, I., Abagyan, R., Filikov, A., Yang, Y., Yoon, S. Optimization of high throughput virtual screening by combining shape-matching and
docking methods. J. Chem. Inf. Model. 48:489, 2008.
Ma, D.L., Lai, T.S., Chan, F.Y., Chung, W.H., Abagyan, R., Leung, Y.C., Wong,
K.Y. Discovery of a drug-like G-quadruplex binding ligand by high-throughput docking. ChemMedChem 3:881, 2008.
248 MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
Mallya, M., Phillips, R.L., Saldanha, S.A., Gooptu, B., Brown, S.C., Termine,
D.J., Shirvani, A.M., Wu, Y., Sifers, R.N., Abagyan, R., Lomas, D.A. Small molecules block the polymerization of Z α1-antitrypsin and increase the clearance of
intracellular aggregates. J. Med. Chem. 50:5357, 2007.
Medina, M., Abagyan, R., Gómez-Moreno, C., Fernandez-Recio, J. Docking analysis of transient complexes: interaction of ferredoxin-NADP+ reductase with ferredoxin and flavodoxin. Proteins 72:848, 2008.
Nicola, G., Smith, C.A., Abagyan, R. New method for the assessment of all druglike pockets across a structural genome. J. Comput. Biol. 15:231, 2008.
Nicola, G., Smith, C.A., Lucumi, E., Kuo, M.R., Karagyozov, L., Fidock, D.A.,
Sacchettini, J.C., Abagyan, R. Discovery of novel inhibitors targeting enoyl-acyl
carrier protein reductase in Plasmodium falciparum by structure-based virtual
screening. Biochem. Biophys. Res. Commun. 358:686, 2007.
Outeiro, T.F., Kontopoulos, E., Altmann, S.M., Kufareva, I., Strathearn, K.E.,
Amore, A.M., Volk, C.B., Maxwell, M.M., Rochet, J.C., McLean, P.J., Young,
A.B., Abagyan, R., Feany, M.B., Hyman, B.T., Kazantsev, A.G. Sirtuin 2 inhibitors
rescue α-synuclein-mediated toxicity in models of Parkinson’s disease. Science
317:516, 2007.
Raduner, S., Bisson, W., Abagyan, R., Altmann, K.H., Gertsch, J. Self-assembling
cannabinomimetics: supramolecular structures of N-alkyl amides. J. Nat. Prod.
70:1010, 2007.
F i g . 1 . A novel nonlinear approach that incorporates both ana-
lytical and bioinformatics technologies for quantitative comparisons
of metabolites.
data analysis approaches to identify and structurally
characterize metabolites of physiologic importance.
N A N O S T R U C T U R E - I N I T I AT O R M A S S S P E C T R O M E T R Y
A N A LY S I S , A C T I V I T Y, A N D I M A G I N G
To develop ultra-high-sensitivity approaches in
mass spectrometry (Fig. 2), we are using nanostruc-
Szewczuk, L.M., Saldanha, S.A., Ganguly, S., Bowers, E.M., Javoroncov, M.,
Karanam, B., Culhane, J.C., Holbert, M.A., Klein, D.C., Abagyan, R., Cole, P.A.
De novo discovery of serotonin N-acetyltransferase inhibitors. J. Med. Chem.
50:5330, 2007.
Totrov, M., Abagyan, R. Flexible ligand docking to multiple receptor conformations:
a practical alternative. Curr. Opin. Struct. Biol. 18:178, 2008.
Mass Spectrometry:
Metabolomics Profiling,
Activity, and Imaging
G. Siuzdak, J. Apon, H.P. Benton, B.O. Crews, K. Harris,
L. Hoang, E. Kalisiak, A. Nordström, T. Northen,
M. Sonderegger, S. Trauger, W. Uritboonthai, W. Webb,
W. Wikoff, D. Wong, H.K. Woo, O. Yanes
M E TA B O L O M I C S
ndogenous metabolites, ubiquitous in biofluids,
tissues, and organisms of every kind, are crucial
elements in understanding fundamental biochemistry, disease diagnosis, and drug toxicity. The inherent
advantage of monitoring small molecules rather than
proteins is the relative ease of quantitative analysis with
mass spectrometry. We are implementing novel mass
spectrometry and bioinformatics techniques (Fig. 1) to
investigate the profile of small-molecule metabolites. The
purpose of this effort is to develop mass-based metabolomics technology for a better understanding of fundamental biochemistry, such as in stem cell differentiation,
as well as a diagnostic research tool. Our ultimate goal
is to create analytical and chemical technologies and
E
F i g . 2 . Illustration of the Nimzyme assay: immobilization of metab-
olites in the fluorous ‘‘clathrate’’ phase of the nanostructure-initiator mass spectrometry surface (A), incubation of the surface with
the sample to screen for enzymatic activity (B), and laser irradiation, resulting in vaporization of the fluorous phase, efficiently transferring the immobilized substrate and products into the gas phase.
Based in part on Northen, T.R., Lee, J.-C., Hoang, L., Raymond, J.,
Hwang, D.-R, Yannone, S.M., Wong, C.-H, Siuzdak, G. A nanostructure-initiator mass spectrometry-based enzyme activity assay.
Proc. Natl. Acad. Sci. U. S. A. 105:3678, 2008. Copyright 2008
National Academy of Sciences U.S.A.
tured clatherates to facilitate vaporization and ionization of biomolecules. Using this technology, termed
nanostructure-initiator mass spectrometry, we can analyze a wide range of molecules with unprecedented
sensitivity, in the yoctomole (10-24 moles) range. The
method is also being developed as a platform for biomolecular tissue imaging.
PUBLICATIONS
Benton, H.P., Wong, D.M., Trauger, S.A., Siuzdak, G. XCMS2: processing tandem
mass spectrometry data for metabolite identification and structural characterization. Anal. Chem., in press.
Nordström, A., Want, E., Northen, T., Lehtiö, J., Siuzdak, G. Multiple ionization
mass spectrometry strategy used to reveal the complexity of metabolomics. Anal.
Chem. 80:421, 2008.
Northen, T.R., Lee, J.-C., Hoang, L., Raymond, J., Hwang, D.-R., Yannone, S.M.,
Wong, C.-H, Siuzdak, G. A nanostructure-initiator mass spectrometry-based
enzyme activity assay. Proc. Natl. Acad. Sci. U. S. A. 105:3678, 2008.
MOLECULAR BIOLOGY
2008
Northen, T.R., Woo, H.K., Northen, M.T., Nordström, A., Uritboonthai, W.,
Turner, K., Siuzdak, G. High surface area of porous silicon drives desorption of
intact molecules. J. Am. Soc. Mass Spectrom. 18:1945, 2007.
THE SCRIPPS RESEARCH INSTITUTE
249
at an RNA structure called the Rev responsive element
(RRE; Fig. 1). Binding of Rev to the RRE directs mRNAs
Northen, T.R., Yanes, O., Northen, M.T., Marrinucci, D., Uritboonthai, W., Apon,
J., Golledge, S.L., Nordström, A., Siuzdak, G. Clathrate nanostructures for mass
spectrometry [letter]. Nature 449:1033, 2007.
Trauger, S.A., Kalisiak, E., Kalisiak, J., Morita, H., Weinberg, M.V., Menon, A.L.,
Poole, F.L. II, Adams, M.W.W., Siuzdak, G. Correlating the transcriptome, proteome, and metabolome in the environmental adaptation of a hyperthermophile. J.
Proteome Res. 7:1027, 2008.
Vallon, V., Eraly, S.A., Wikoff, W.R., Rieg, T., Kaler, G., Truong, D.M., Ahn, S.Y.,
Mahapatra, N.R., Mahata, S.K., Gangoiti, J.A., Wu, W., Barshop, B.A., Siuzdak,
G., Nigam, S.K. Organic anion transporter 3 contributes to the regulation of blood
pressure. J. Am. Soc. Nephrol., in press.
Wikoff, W.R., Gangoitti, J.A., Barshop, B.A., Siuzdak, G. Metabolomics identifies
novel perturbations in human disorders of propionate metabolism. Clin. Chem.
53:2169, 2007.
Wikoff, W.R., Pendyala, G., Siuzdak, G., Fox H.S. Metabolomic analysis of the
cerebrospinal fluid reveals changes in phospholipase expression in the CNS of SIVinfected macaques. J. Clin. Invest. 118:2661, 2008.
Woo, H.-K., Northen, T.R., Yanes, O., Siuzdak, G. Nanostructure-initiator mass
spectrometry (NIMS): a protocol for preparing and applying NIMS surfaces for high
sensitivity mass analysis. Nat. Protoc., in press.
RNA-Protein Complexes
Mediating Nuclear Transport
of HIV mRNAs
J.R. Williamson, F. Agnelli, W. Anderson, A. Beck, C. Beuck,
F i g . 1 . Nuclear transport of HIV Rev. The Rev protein shuttles in
and out of the nucleus by sequentially interacting with a series of
human factors in 3 key complexes. First, an import complex is formed
by association of Rev (yellow) with the nuclear transport factor
importin-β (blue). Once in the nucleus, Rev forms an oligomeric
RNA complex by binding to the RRE RNA. Multiple copies of Rev
bind to the RRE to promote efficient export, but the details of the
oligomeric structure are not known. Rev recruits the nuclear transport factor CRM-1 (orange), which facilitates transport of the RevRRE complex back to the cytoplasm. Other factors associate with
this complex, including the helicases DDX1 and DDX3, to form the
export complex. DDX1 (red) interacts directly with Rev, whereas
A. Bunner, A. Carmel, S. Chen, S., Edgcomb, D. Kerkow,
DDX3 (blue) interacts with Rev by indirect binding to CRM-1. The
S. Kwan, E. Menichelli, W. Ridgeway, G. Ring, H. Schultheisz,
Z. Shajani, E. Sperling, M.T. Sykes, B. Szymczyna, J. Wu
helicases may dissociate proteins from the viral RNA after export to
the cytoplasm (lower right) to facilitate translation of the viral mRNA
or packaging of the viral genome. Each of these complexes may be
a new target for intervention against HIV.
evelopment of novel therapeutic strategies against
HIV infection is a pressing need and requires
elucidation of the fundamental mechanisms of
HIV replication. Currently prescribed anti-HIV drugs
inhibit the viral protease, viral reverse transcriptase,
or viral integrase, but these enzymes are only a small
fraction of the viral proteins responsible for some key
steps in viral replication. To develop new therapeutic
strategies, we need to understand additional steps in
viral replication and to identify new potential targets.
Rev is an essential HIV protein that is required to
mediate transport of HIV viral mRNAs from the nucleus,
where they are transcribed, into the cytoplasm, where
they are either translated or packaged into new virions.
Early in infection, viral mRNAs are fully processed in the
nucleus and exported to the cytoplasm, where several
small regulatory proteins, including Rev, are synthesized.
The Rev protein itself is imported back into the nucleus,
where it interacts with newly transcribed viral mRNAs
D
from the nucleus to the cytoplasm before full RNA processing takes place. These longer mRNAs code for the
structural proteins necessary to assemble a new virus.
Thus, binding of Rev to the RRE changes the pattern
of gene expression from production of the early regulatory genes to production of the late structural genes,
and this binding is therefore a potential point of therapeutic intervention.
Several human cellular proteins act in concert with
Rev to mediate the nuclear transport of viral mRNAs.
In collaboration with L.R. Gerace and J.R. Yates, Department of Cell Biology, using a proteomic method to investigate Rev-RRE–associated factors, we identified dozens
of such potential cofactor proteins. In particular, we
discovered several so-called DEAD-box helicases, named
after the conserved sequence signature, that associate
with Rev during viral mRNA transport. The helicase DDX1
250 MOLECULAR BIOLOGY
2008
is thought to associate with Rev while Rev is bound to
the RRE RNA in the nucleus; the helicase DDX3 interacts
indirectly with Rev through the transport protein CRM-1.
The presence of helicases in the ribonucleoprotein
complex for viral mRNA export raises some interesting
questions about how Rev functions. Helicases are ATPdependent motors that can unwind duplex RNAs or
displace proteins from RNA-protein complexes. Possibly these helicases play critical roles in either assembling the proper RNA complex for nuclear transport or
in disassembling the complex in the cytoplasm to allow
translation or packaging of the virus.
We are using biochemical and structural biology
approaches to investigate the interactions of Rev with
the helicases DDX1 and DDX3. The normal human substrates for these helicases are not known, and we must
develop binding and functional assays based on Rev and
Rev-RRE complexes. The protein-protein interactions are
monitored by using fluorescence assays or isothermal
titration calorimetry; formation of RNA-protein complexes
is monitored by using polyacrylamide electrophoretic
mobility shift assays. In addition, we are developing
helicase ATPase assays with a variety of substrates to
determine how the helicase activity is modulated in
the presence of Rev and RRE. Finally, we are working
toward structure determination of protein-protein and
protein-RNA complexes to understand the molecular
basis for this interaction.
Our results will provide the basis for understanding
potentially new targets for antiviral therapy. In addition, although helicases are widespread in human cells,
the authentic substrates for these enzymes are known
in only a few instances. Studying of Rev as a cargo for
nuclear transport and as an authentic substrate for
helicases will provide important insights into helicase
function. The biochemical and structural work may lead
to assays for the discovery of inhibitors of Rev function by a novel mechanism with therapeutic potential.
PUBLICATIONS
Edgcomb, S.P., Aschrafi, A., Kompfner, E., Williamson, J.R., Gerace, L., Hennig,
M. Protein structure and oligomerization are important for the formation of exportcompetent HIV-1 Rev-RRE complexes. Protein Sci. 17:420, 2008.
Hennig, M., Scott, L.G., Sperling, E., Bermel, W., Williamson, J.R. Synthesis of
5-fluoropyrimidine nucleotides as sensitive NMR probes of RNA structure. J. Am.
Chem. Soc. 129:14911, 2007.
Naidoo, N., Harrop, S.J, Sobti, M., Haynes, P.A., Szymczyna, B.R., Williamson,
J.R., Curmi, P.M., Mabbutt, B.C. Crystal structure of Lsm3 octamer from Saccharomyces cerevisae: implications for Lsm ring organisation and recruitment. J. Mol.
Biol. 377:1357, 2008.
Sperling, E., Bunner, A.E., Sykes, M.T., Williamson, J.R. Quantitative analysis of
isotope distributions in proteomic mass spectrometry using least-squares Fourier
transform convolution. Anal. Chem. 80:4906, 2008.
THE SCRIPPS RESEARCH INSTITUTE
Vallurupalli, P., Scott, L., Williamson, J.R., Kay, L.E. Strong coupling effects during X-pulse CPMG experiments recorded on heteronuclear ABX spin systems: artifacts and a simple solution. J. Biomol. NMR 38:41, 2007.
Structure and Biology of RNA
Interference Machinery
I.J. MacRae, P.-W. Lau, A.J. Pratt
NA interference is a widespread eukaryotic
mechanism of gene silencing that plays a fundamental role in many aspects of animal biology, including developmental timing, stem cell division,
memory, and learning. On the cellular level, RNA interference is mediated by a family of ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs),
which silence genes by mediating translational repression and degradation of targeted mRNAs. The versatility and power of RNA interference arises from the fact
that RISCs can be programmed to target any nucleic
acid sequence for silencing. RISC programming is therefore a critical cellular function, requiring the action of
a specialized macromolecular assembly called the RISCloading complex (RLC). We are studying the structure
and catalytic mechanism of the human RLC. We hope
that insight into the function of this complex will facilitate the development of therapeutic agents based on
RNA interference.
On the molecular level, RLCs program RISCs with
target sequence information by mediating the noncovalent binding, or “loading,” of an RNA of approximately
22 nucleotides into the protein Argonaute. Argonaute
forms the core subunit of RISCs, in which the small
RNA functions as a guide for gene silencing through
base-pairing recognition of target mRNAs. One of our
major long-term goals is to visualize RISC programming
by obtaining high-resolution crystal structures of the
RLC at multiple steps in loading. Thus, we have established an efficient method for reconstituting human RLCs
from individually purified recombinant preparations of
the 3 RLC subunits: Dicer, Argonaute 2, and the TARRNA binding protein. The recombinant nature of the
reconstitution will also allow us to produce mutant RLCs
that stall at key steps in loading. We will then use the
mutants to probe the mechanism of RISC loading.
We anticipate that crystallization of the RLC will be
technically challenging and may take years to accomplish. To obtain structural information in the earlier
stages of this study, we have established a collabora-
R
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2008
tion with B. Carragher and C. Potter at Scripps Research
in the National Resource for Automated Molecular
Microscopy. Our goal is to generate structures of the
RLC by using electron cryomicroscopy and single-particle analysis. These structures will reveal the overall
architecture of the RLC and enable us to visualize largescale conformational changes associated with different
steps of RISC loading. Modeling the available crystal
structures of Giardia Dicer, which represents the conserved catalytic core structure of human Dicer, and
Pyrococcus Argonaute, an archeal ortholog of human
Argonaute 2, into electron microscopy maps will answer
some of the basic questions that shape our thinking
about the RLC mechanism. Ideas and hypotheses derived
from structural studies will then be tested biochemically
by using reconstituted mutant and chemically modified
RLCs. The combined structural and functional studies
will provide new insights into RNA interference in
humans and may suggest new approaches for using
RNA interference for the treatment of human disease.
Development of the Genetic
Code and Its Connection to
Human Disease
P. Schimmel, X.-L. Yang, R. Belani, E. Chong, M. Guo,
R.T. Guo, M. Hanan, W.-W. He, I.L. Jung, M. Kapoor, J. Liu,
E. Merriman, M.H. Nawaz, R. Shapiro, M. Vo, W. Zhang,
Q. Zhou
e focus on aminoacyl tRNA synthetases and
how their role as catalysts of aminoacylation
is connected to disease and to broader biological systems through expanded functions that were
developed over the long evolutionary history of the
enzymes. The enzymes are ancient and are thought to
have appeared in the transition from the putative RNA
world to the theater of proteins. By linking amino acids
to tRNAs that bear anticodon triplets encoding the
attached amino acids, the synthetases establish the
algorithm of the genetic code. Thus, alanyl-tRNA synthetases catalyze formation of Ala-tRNAAla, glycyl-tRNA
synthetase catalyzes production of Gly-tRNAGly, and
isoleucyl-tRNA synthetase produces Ile-tRNAIle. Altogether there are 20 tRNA synthetases, 1 for each amino
acid, and the tRNA to which the amino acid is attached
bears the triplet of the code for that amino acid. It is,
therefore, this reaction that establishes the code.
W
THE SCRIPPS RESEARCH INSTITUTE
251
Recent research established that heritable mutations in these 20 enzymes are causally linked to specific human diseases. This linkage occurs in one of two
ways. First is the connection to mistranslation (Fig. 1).
F i g . 1 . Effects of mistranslation.
This connection occurs because many of the enzymes
have an editing activity (encoded by a distinct active
site) that clears amino acids when the amino acids
are attached to the wrong tRNA. For example, alanyltRNA synthetase occasionally confuses glycine or serine
for alanine, so Gly-tRNAAla or Ser-tRNAAla is produced.
However, the occasional mischarged tRNA is cleared
by a specific editing activity that can distinguish serine and glycine from alanine in the context of tRNAAla.
Were these mischarged amino acids not cleared away,
mistranslation would occur.
Recently, we found that disruption of the editing
activity not only is toxic to bacteria but also leads to
serious pathologic changes in mammalian cells, in a
trans-dominant way. Even a mild mutation, which produces only a 2-fold decrease in the activity for editing,
leads to a heritable condition in mice, characterized
by ataxia and neurodegeneration. We also discovered
that mistranslation is mutagenic in aging bacteria. We
are now exploring the possibility that mistranslation is
mutagenic in mammalian cells in a way that could lead
to oncogenic transformation.
How does alanyl-tRNA synthetase distinguish mischarged Ser-tRNA Ala from the correctly charged SertRNASer; that is, how does it use the context of the
tRNA to strip serine from tRNAAla but not from tRNASer?
The enzyme has distinct domains for aminoacylation
(formation of charged tRNAAla) and editing (clearing of
252 MOLECULAR BIOLOGY
2008
mischarged tRNAAla). These domains are encoded by
polypeptides arranged in tandem along the sequence
of alanyl-tRNA synthetase. For aminoacylation, tRNAAla
synthetases throughout evolution are marked for aminoacylation with alanine by a single guanine-uracil base
pair (G3:U70; located in the acceptor stem; Fig. 2).
F i g . 2 . tRNA Ala acceptor stems.
Transfer of this base pair into non-tRNAAla synthetases
converts the enzymes into alanine-accepting tRNAs.
The recognition of a single G3:U70 base pair is through
determinants in the N-terminal aminoacylation domain
of the protein. Remarkably, and to our surprise, this
same base pair is used by the editing domain to pick
out mischarged tRNAAla. Thus, the same base pair is
recognized by distinct domains within the same protein.
Last, in the human and mouse genomes, in addition to being encoded as part of alanyl-tRNA synthetase,
the editing domain is separately encoded as a standalone fragment known as AlaXp. This fragment can also
clear mischarged tRNAAla (Fig. 3). Thus, mammalian
F i g . 3 . Alanyl-tRNA synthetase (AlaRS) and artificial and natural
editing-proficient fragments.
cells have developed 3 ways to prevent confusion of
serine and glycine for alanine: (1) reasonably accurate
(about 99%) selection of alanine in the aminoacylation
step, (2) clearance of occasional errors, of confusing
serine or glycine for alanine, by the editing domain of
THE SCRIPPS RESEARCH INSTITUTE
the enzyme, and (3) clearance of any residual mischarged tRNAAla by the free-standing AlaXp.
In summary, we started with the observation of how
a single mutation in the site for editing led to disease
in mice. From there, we uncovered the first example of
2 distinct domains in the same protein being able to
recognize the same base pair.
The second connection of tRNA synthetases to heritable human diseases is through the expanded functions of the enzymes. Many human tRNA synthetases
have functions beyond aminoacylation. For example,
when activated, tyrosyl-tRNA synthetase and tryptophanyl-tRNA synthetase have opposing activities in
angiogenesis. Lysyl-tRNA synthetase is incorporated
into HIV virions for bringing in tRNALys, which is used
as a primer for reverse transcription of the viral RNA
genome during infection of a host cell. These extratranslation activities link aminoacyl-tRNA synthetases
with boarder biological systems.
Two specific tRNA synthetases, tyrosyl- and glycyltRNA synthetase, are linked to Charcot-Marie-Tooth
(CMT) disease, the most common heritable peripheral
neuropathy; 1 of 2000 persons in the United States
and Europe has the disease. Eleven different mutant
alleles of GARS, the gene that encodes glycyl-tRNA
synthetase, are reported to cause an axonal form of
CMT in a dominant way. About half of the 11 CMTcausing mutants of glycyl-tRNA synthetase are unaffected in their aminoacylation activities. Similarly,
dominant mutations in YARS, the gene that encodes
tyrosyl-tRNA synthetase, have also been detected as
the genetic cause of the disease in patients with CMT.
Again, the mutations are not correlated with an aminoacylation defect. The lack of correlation of CMT mutations with aminoacylation activity suggests additional,
expanded functions for glycyl- and tyrosyl-tRNA synthetases in neuronal cells.
The 11 CMT-linked mutations are spread out on the
primary sequence of glycyl-tRNA synthetase. However,
as revealed in our crystal structure of the synthetase,
all of the CMT-associated residues are located on or near
the dimerization interface of the α2 homodimer (Fig. 4).
Strikingly, 2 CMT-causing mutations, each from a different subunit, are associated with residues in the wildtype enzyme that make contact with each other across
the dimer interface. This finding suggests that the dimer
interface is critical for the potential expanded function
of glycyl-tRNA synthetase. Because both monomer and
dimer forms of the synthetase coexist in equilibrium, we
hypothesized that the monomer form, which is inactive
MOLECULAR BIOLOGY
2008
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253
Kapoor, M., Zhou, Q., Otero, F., Myers, C.A., Bates, A., Belani, R., Liu, J., Luo,
J.-K., Tzima, E., Zhang, D.-E., Yang, X.-L., Schimmel, P. Evidence for annexin IIS100A10 complex and plasmin in mobilization of cytokine activity of human
TrpRS. J. Biol. Chem. 283:2070, 2008.
Waas, W.F., Druzina, Z., Hanan, M., Schimmel, P. Role of a tRNA base modification
and its precursors in frameshifting in eukaryotes. J. Biol. Chem. 282:26026, 2007.
Waas, W.F., Schimmel, P. Evidence that tRNA synthetase-directed proton transfer
stops mistranslation. Biochemistry 46:12062, 2007.
Yang, X.-L., Kapoor, M., Otero, F.J., Slike, B.M., Tsuruta, H., Frausto, R., Bates,
A., Ewalt, K.L., Cheresh, D.A., Schimmel, P. Gain-of-function mutational activation of a human tRNA synthetase procytokine. Chem. Biol. 14:1323, 2007.
Zhou, Q., Kiosses, W.B., Liu, J., Schimmel, P. Tumor endothelial cell tube formation model for determining anti-angiogenic activity of a tRNA synthetase cytokine.
Methods 44:190, 2008.
Directed Evolution of Nucleic
Acid Enzymes
F i g . 4 . Crystal structure of human glycyl-tRNA synthetase homodimer.
for aminoacylation, has a distinct biological role specific
for neuronal cells. Disruption of this expanded role links
glycyl-tRNA synthetase to the etiology of CMT.
Interestingly, when transfected into a mouse neuroblastoma N2a cell line, genes encoding each of the
CMT-causing glycyl-tRNA synthetase mutants were defective in distribution into neurite terminals. This distribution defect is reminiscent of the muscle weakness
around the terminal nerves in patients with CMT. We
have speculated that the CMT-associated mutations
of glycyl-tRNA synthetase affect transportation of the
synthetase into the neurites via a mechanism linked to
the dimerization interface.
Our awareness of the connections of tRNA synthetases, which are intimately associated with the
development of the genetic code, to diseases is only
beginning. We anticipate that many more connections
will be found.
PUBLICATIONS
Bacher, J., Waas, W.F., Metzgar, D., de Crécy-Lagard, V., Schimmel, P. Genetic
code ambiguity confers a selective advantage on Acinetobacter baylyi. J. Bacteriol.
189:6494, 2007.
Beebe, K., Mock, M., Merriman, E., Schimmel, P. Distinct domains of tRNA synthetase recognize the same base pair. Nature 451:90, 2008.
Beebe, K., Waas, W., Druzina, Z., Guo, M., Schimmel, P. A universal plate format
for increased throughput of assays that monitor multiple aminoacyl transfer RNA
synthetase activities. Anal. Biochem. 368:111, 2007.
Greenberg, Y., King, M., Kiosses, W.B., Ewalt, K., Yang, X.-L., Schimmel, P.,
Reader, J.S., Tzima, E. The novel fragment of tyrosyl-tRNA synthetase, mini-TyrRS,
is secreted to induce an angiogenic response in endothelial cells. FASEB J.
22:1597, 2008.
Guo, M., Ignatov, M., Musier-Forsyth, K., Schimmel, P., Yang, X.-L. Crystal structure of tetrameric form of human lysyl-tRNA synthetase: implications for multisynthetase complex formation. Proc. Natl. Acad. Sci. U. S. A. 105:2331, 2008.
G.F. Joyce, S.E. Hamilton, D.P. Horning, T.A. Lincoln,
B.J. Lam, B.M. Paegel, K.L. Petrie, S.B. Voytek
he scientific community will soon celebrate the
200th anniversary of the birth of Charles Darwin
and the 150th anniversary of the publication of
his seminal work On the Origin of Species by Means
of Natural Selection. The principles of darwinian evolution are fundamental to understanding biological organization at the level of populations of organisms and
for explaining the development of biological genomes
and macromolecular function. In our laboratory, darwinian evolution has become a chemical tool for discovering and optimizing functional macromolecules in
the test tube. We have developed powerful methods for
the in vitro evolution of nucleic acids and are applying
those methods to the discovery of molecules of biochemical and biomedical importance. In addition, we
are studying the processes of darwinian evolution itself,
carried out at the level of molecules rather than at the
level of cells or organisms.
T
CONTINUOUS IN VITRO EVOLUTION
We have devised a system for the continuous in
vitro evolution of RNA enzymes that have RNA-joining
activity. The system operates at a constant temperature
within a common reaction vessel. RNA enzymes in a
population of trillions are challenged to attach themselves to an RNA substrate, and as a consequence, the
reacted enzymes become amplified by polymerase proteins (also present in the reaction mixture) to generate
progeny. The progeny enzymes in turn have the opportunity to perform the reaction, causing the population
to expand exponentially. Whenever the supply of sub-
254 MOLECULAR BIOLOGY
2008
strates becomes exhausted, a fresh supply of reactants
can be provided, allowing exponential amplification to
continue indefinitely.
Until recently, all continuous in vitro evolution experiments were done with a single species of RNA enzyme
derived from the CL1 ligase. We recently established a
second continuously evolving enzyme based on descendants of the DSL ligase. This new enzyme was propagated for hundreds of successive generations to optimize
its catalytic activity.
Recently, we challenged the 2 distinct species of
continuously evolving enzymes to operate within the
same environment (Fig. 1). Initially, variants of the CL1
ligase dominated the mixture, prompting us to add an
F i g . 1 . Continuous coevolution of 2 distinct species of RNA enzymes
with RNA-joining activity. Zigzag lines indicate the concentration of
the CL1 (blue) and DSL (red) enzymes before and after each cycle
of exponential growth and dilution (based on the concentration of
the corresponding cDNA).
inhibitory molecule to modulate their growth. Subsequently, variants of the DSL ligase became dominant,
and these too were kept in check by adding a speciesspecific inhibitor. Eventually, we succeeded in maintaining the 2 species without the use of inhibitors by
supplying 5 different RNA substrates. Each of the 2
enzymes evolved to use different substrates as its preferred resource, exploiting distinct niches within the
common environment. This situation is a demonstration
of niche formation at the molecular level, analogous to
processes of biological evolution essential for maintaining species diversity within natural ecosystems.
S E L F - S U S TA I N E D R E P L I C AT I O N O F R N A
The continuous in vitro evolution system depends
on 2 protein enzymes, a retroviral reverse transcriptase
and a bacteriophage RNA polymerase, to bring about
the amplification of reacted RNA enzymes. Recently,
we developed a system in which the RNA enzymes
THE SCRIPPS RESEARCH INSTITUTE
catalyze their own replication. We began with the R3C
ligase developed previously in our laboratory. This molecule has a simple architecture amenable to various
rearrangements. Previously, the R3C ligase was configured so that it would join 2 pieces of RNA to produce
additional copies of itself, thus achieving RNA-catalyzed
self-replication. Next, the enzyme was converted to a
cross-catalytic format whereby 2 RNA enzymes brought
about each other’s synthesis from a total of 4 component RNA substrates. During the past year, we used in
vitro evolution to enhance the activity of the cross-replicating RNA enzymes, improving their catalytic rate by
more than 20-fold and increasing their extent of reaction from 15% to 90%. The resulting enzymes are able
to undergo self-sustained exponential amplification at
a constant temperature, achieving nearly billion-fold
amplification in 30 hours.
The cross-replicating RNA enzymes can amplify
themselves indefinitely in the absence of proteins or
any other biological materials. As in the continuous
evolution system, we allow the molecules to expand
exponentially until the supply of substrates is exhausted
and then provide fresh reactants to allow exponential
amplification to continue indefinitely. We have prepared
several versions of the cross-replicating enzymes that
differ with respect to their genotype and corresponding
phenotype. The genotype specifies the identity of the
cross-replicating partners, and the phenotype is reflected
in the catalytic properties of the molecules. In this way,
we are striving to construct an artificial genetic system
that can undergo self-sustained darwinian evolution.
Thus far, we have shown selection among various crossreplicators that undergo exponential amplification within
a common reaction mixture. Occasionally, a novel recombinant arises that amplifies more efficiently than either
of its parents. With this system, it may be possible to
explore alternative solutions to different environmental
constraints, as occur in the natural evolution of biological organisms.
PUBLICATIONS
Joyce, G.F. Forty years of in vitro evolution. Angew. Chem. Int. Ed. 46:6420, 2007.
Paegel, B.M., Joyce, G.F. Darwinian evolution on a chip. PLoS Biol. 6:e85, 2008.
Voytek, S.B., Joyce, G.F. Emergence of a fast-reacting ribozyme that is capable of
undergoing continuous evolution. Proc. Natl. Acad. Sci. U. S. A. 104:15288 2007.
MOLECULAR BIOLOGY
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Studies at the Interface of
Molecular Biology, Chemistry,
and Medicine
C.F. Barbas III, K. Albertshofer, T. Bui, R.P. Fuller,
C. Gersbach, B. Gonzalez, R. Gordley, J. Guo, D.H. Kim,
R.A. Lerner, W. Nomura, A. Onoda, S.S.V. Ramasastry,
M. Santa Marta, L.J. Schwimmer, D. Shabat,* F. Tanaka,
U. Tschulena, N. Utsumi, K.S. Yi, H. Zhang
* Tel Aviv University, Tel Aviv, Israel
e are concerned with problems in molecular
biology, chemistry, and medicine. Many of
our studies involve learning or improving
Nature’s strategies to prepare novel molecules that perform specific functional tasks, such as regulating a gene,
destroying cancer, or catalyzing a reaction with enzymelike efficiency. We hope to apply these novel insights,
technologies, methods, and their products to provide
solutions to human diseases, including cancer, HIV
disease, and genetic diseases.
W
D I R E C T I N G T H E E V O L U T I O N O F C ATA LY T I C F U N C T I O N
Using reactive immunization, we have developed
antibodies that catalyze aldol as well as retro-aldol reactions of a wide variety of molecules. The catalytic proficiency of the best of these antibodies is almost 1014,
a value 1000 times that of the best catalytic antibodies reported to date and overall the best of any synthetic
protein catalyst. We have shown the efficient asymmetric synthesis and resolution of a variety of molecules,
including tertiary and fluorinated aldols, and have used
these chiral synthons to synthesize natural products
(Fig. 1). These results highlight the potential synthetic
usefulness of catalytic antibodies as artificial enzymes
in addressing problems in organic chemistry that are
not solved by using natural enzymes or more traditional
synthetic methods.
Other advances in this area include the development of the first peptide aldolase enzymes. By using
both design and selection, we have created small peptide catalysts that recapitulate many of the kinetic features of large enzyme catalysts. These smaller enzymes
allow us to address the relationship between the size
of natural proteins and the proteins’ catalytic efficiency.
O R G A N O C ATA LY S I S : A B I O O R G A N I C A P P R O A C H T O
C ATA LY T I C A S Y M M E T R I C S Y N T H E S I S
To further explore the principles of catalysis, we
are studying amine catalysis as a function of catalytic
F i g . 1 . A variety of compounds synthesized with the world’s
first commercially available catalytic antibody, 38C2, produced at
Scripps Research.
scaffold. Using insights garnered from our studies of
aldolase antibodies, we determined the efficacy of simple
chiral amines and amino acids for catalysis of aldol
and related imine and enamine chemistries such as
Michael, Mannich, Knoevenagel, and Diels-Alder reactions. Although aldolase antibodies are superior catalysts in terms of the kinetic parameters, these more
simple catalysts are enabling us to quantify the significance of pocket sequestration in catalysis.
Furthermore, many of these catalysts are cheap,
environmentally friendly, and practical for large-scale
synthesis. With this approach, we showed the scope
and usefulness of the first efficient amine catalysts of
direct asymmetric aldol, Mannich, Diels-Alder, and
Michael reactions. The organocatalyst approach is a
direct outcome of our studies of catalytic antibodies
and provides an effective alternative to organometallic
reactions that use severe reaction conditions and oftentoxic catalysts.
In extensions of these concepts, we designed novel
amino acid derivatives that direct the stereochemical
outcome of reactions in ways not possible with proline.
In other studies, we created the first asymmetric smallmolecule aldol catalysts that are highly effective with
water and seawater as solvent. We think that our results
are also relevant to the prebiotic synthesis of the molecules of life. For example, we have shown that our amino
acid strategy can be used to synthesize carbohydrates
directly, thereby providing a provocative prebiotic route
to the sugars essential for life.
256 MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
I S P R I M O R D I A L A S Y M M E T R I C O R G A N O C ATA LY S I S
A N E X TA N T B I O S Y N T H E T I C M E C H A N I S M ?
We think that in time, research will show that
organocatalysts or “aminozymes” (chiral amines or amino
acids with biosynthetic roles) constitute components of
an unseen biosynthetic apparatus at work in cells today.
As we begin to appreciate the fascinating chemical transformations that are now possible through organocatalysis,
and amino acid catalysis in particular, we need to look at
cellular metabolism and biosynthesis in a new light. Classically, we are trained to search for a “protein” enzyme
for each and every step in the synthesis of a natural
product in vivo. We suggest that many of the more elusive metabolic enzymes are likely to be organocatalysts
and, in many instances, simple amino acids. Because
intracellular concentrations of amino acids can exceed
1 M, many wonderful and diverse exotic natural products
may actually be synthesized in vivo with the aid of
aminozymes and other forms of organocatalysts more
complicated than amino acids (Fig. 2).
ANTIBODY ENGINEERING: THERAPEUTIC
ANTIBODIES, IN AND OUT OF CELLS
We developed the first human antibody phage display libraries and the first synthetic antibodies and
methods for the in vitro evolution of antibody affinity.
The ability to manipulate large libraries of human antibodies and to evolve such antibodies in the laboratory
provides tremendous opportunities to develop new
medicines. Laboratories and pharmaceutical companies
around the world now apply the phage display technology that we developed for antibody Fab fragments. In
our laboratory, we are targeting cancer and HIV disease. One of our antibodies, IgG1-b12, protects animals against primary challenge with HIV type 1 (HIV-1)
and has been further studied by many researchers. We
improved this antibody by developing in vitro evolution
strategies that enhanced its neutralization activity. By
coupling laboratory-evolved antibodies with potent toxins, we showed that immunotoxins can effectively kill
infected cells.
We are also developing genetic methods to halt HIV
by gene therapy. We created unique human antibodies
that can be expressed inside cells to make the cells
resistant to HIV infection. In the future, these antibodies
might be delivered to the stem cells of patients infected
with HIV-1, allowing the development a disease-free
immune system that would obviate the intense regimen
of antiviral drugs now required to treat HIV disease.
Using our increased understanding of antibody-antigen interactions, we extended our efforts in cancer
F i g . 2 . Potential roles of aminozymes in the biosynthesis of the
Daphniphyllum alkaloids (A) and the potential anticancer compound FR182877 (B).
therapy and developed rapid methods for creating
human antibodies from antibodies derived from other
species. We produced human antibodies that should
enable us to selectively starve a variety of cancers by
inhibiting angiogenesis and antibodies that will be used
to deliver radioisotopes to colon cancers to destroy the
tumors. We hope that these antibodies will be used in
clinical trials done by our collaborators at the SloanKettering Cancer Center, New York, New York.
On the basis of our studies on HIV-1, we used intracellular expression of antibodies directed against angiogenic receptors to create a new gene-based approach to
cancer. Our results indicate that this type of gene therapy
can be successfully applied to the treatment of cancer.
T H E R A P E U T I C A P P L I C AT I O N S O F C ATA LY T I C
ANTIBODIES
The development of highly efficient catalytic antibodies opens the door to many practical applications.
One of the most fascinating is the use of such antibodies in human therapy. We think that use of this strategy
can improve chemotherapeutic approaches to diseases
such as cancer and AIDS. Chemotherapeutic regimens
are typically limited by nonspecific toxic effects. To
address this problem, we developed a novel and broadly
applicable drug-masking chemistry that operates in
conjunction with our unique broad-scope catalytic antibodies. This masking chemistry is applicable to a wide
range of drugs because it is compatible with virtually
any heteroatom. We showed that generic drug-masking groups can be selectively removed by sequential
retro-aldol-retro-Michael reactions catalyzed by antibody 38C2 (Fig. 3). This reaction cascade is not catalyzed by any known natural enzyme.
Application of this masking chemistry to the anticancer drugs doxorubicin, camptothecin, and etoposide
produced prodrugs with substantially reduced toxicity.
MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
257
F i g . 3 . Targeting cancer and HIV with prodrugs activated by catalytic
antibodies. A bifunctional antibody is shown targeting a cancer cell for
destruction. A nontoxic analog of doxorubicin, prodoxorubicin, is being
activated by an aldolase antibody to the toxic form of the drug.
These prodrugs are selectively unmasked by the catalytic
antibody when the antibody is applied at therapeutically
relevant concentrations. The efficacy of this approach
has been shown in in vivo models of cancer. Currently,
we are developing more potent drugs and novel antibodies that will allow us to target breast, colon, and prostate
cancers as well as cells infected with HIV-1. On the basis
of our preliminary findings, we think that our approach
can become a key tool in selective chemotherapeutic
strategies. To see a movie illustrating this approach, visit
http://www.scripps.edu/mb/barbas/antibody/antibody.mov.
C H E M I C A L LY P R O G R A M M E D A N T I B O D I E S : T H E
ADVENT OF CHEMOBODIES
We think that combining the chemical diversity of
small synthetic molecules with the immunologic characteristics of antibody molecules will lead to therapeutic
agents with superior properties. Therefore, we developed a conceptually new device that equips small synthetic molecules with both the immunological effector
functions and the long serum half-life of a generic antibody molecule. For a prototype, we developed a targeting device based on the formation of a covalent bond of
defined stoichiometry between (1) a 1,3-diketone derivative of an arginine–glycine–aspartic acid peptidomimetic
that targets the integrins αvβ3 and αvβ5 and (2) the reactive lysine of aldolase antibody 38C2 (Fig. 4). The resulting complex spontaneously assembled in vitro and in vivo,
selectively retargeted antibody 38C2 to the surface of
cells expressing the integrins αvβ3 and αvβ5, dramatically increased the circulatory half-life of the peptidomimetic, and effectively reduced tumor growth in animal
models of human Kaposi sarcoma, colon cancer, and
melanoma. Three novel chemically programmed antibodies, an entirely new class of drugs, are now in phase 1
clinical studies.
F i g . 4 . Designed small-molecule targeting agents (SCS-873 is
shown) program the specificity of the antibody 38C2 (A). The resulting chemobodies (cp38C2, B) have characteristics that are often
superior to those of either the small molecule or the antibody alone.
ZINC FINGER GENE SWITCHES AND ENZYMES
The solutions to many diseases might be simply
turning genes on or off in a selective way or adding or
deleting genes. To accomplish all of these aims, we
are studying molecular recognition of DNA by zinc finger
proteins and methods of creating novel zinc finger DNAbinding proteins. We showed that proteins that contain
zinc fingers can be selected or designed to recognize
novel DNA sequences.
These studies are aiding the elucidation of rules for
sequence-specific recognition within this family of proteins. We selected and designed specific zinc finger
domains that will constitute an alphabet of 64 domains
that will allow any DNA sequence to be bound selectively. The prospects for this “second genetic code”
are fascinating and may have a major impact on basic
and applied biology.
We showed the potential of this approach in multiple mammalian and plant cell lines and in whole organisms. With the use of characterized modular zinc finger
domains, polydactyl proteins capable of recognizing an
18-nucleotide site can be rapidly constructed (see
www.zincfingertools.org). Our results suggest that zinc
finger proteins might be useful as genetic regulators for
a variety of human ailments and provide the basis for a
new strategy of gene therapy. Our goal is to develop this
class of therapeutic proteins to inhibit or enhance
the synthesis of proteins, providing a direct strategy for
fighting diseases of either somatic or viral origin.
We are also developing proteins that will inhibit the
growth of tumors and others that will inhibit the expres-
258 MOLECULAR BIOLOGY
2008
sion of a protein known as CCR5, which is a key to
infection of human cells by HIV-1. We developed an
HIV-1–targeting transcription factor that strongly suppresses HIV-1 replication and another transcription factor that upregulates fetal hemoglobin as a treatment for
sickle cell anemia. More recently, we have focused on
evolving zinc finger enzymes that modify the genome.
These studies have led to the development of programmable zinc finger recombinases (Fig. 5) that promise to
THE SCRIPPS RESEARCH INSTITUTE
Zhang, H., Ramasastry, S.S.V., Tanaka, F., Barbas, C.F. III. Organocatalytic antiMannich reactions with dihydroxyacetone and acyclic dihydroxyacetone derivatives:
a facile route to amino sugars. Adv. Synth. Catal. 350:791, 2008.
Catalytic Antibodies,
Synthetic Enzymes,
Biomolecular Computing,
and Synthetic Capsids
E. Keinan, O. Reany, N. Metanis, E. Kossoy, M. Soreni,
R. Piran, M. Sinha, I. Ben-Shir, T. Shekhter, T. Ratner,
T. Mejuch, E. Solel, S. Shoshani, R. Gershoni, A. Karmakar,
D. Pappo, G. Parvari
C ATA LY T I C A N T I B O D I E S
F i g . 5 . Model of a zinc finger recombinase with programmable
specificity created by using rational design and directed molecular
evolution.
reshape the way scientists manipulate the genome for
study and therapy of disease.
PUBLICATIONS
Barbas, C.F. III. Organocatalysis lost: modern chemistry, ancient chemistry, and an
unseen biosynthetic apparatus. Angew. Chem. Int. Ed. 47:42, 2008.
Blancafort, P., Tschan, M.P., Bergquist, S., Guthy, D., Brachat, A., Sheeter, D.A.,
Torbett, B.E., Erdmann, D., Barbas, C.F. III. Modulation of drug resistance by artificial transcription factors. Mol. Cancer Ther. 7:688, 2008.
Jiang, L., Althoff, E.A., Clemente, F.R, Doyle, L., Röthlisberger, D., Zanghellini,
A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F. III, Hilvert, D., Houk,
K.N., Stoddard, B.L., Baker D. De novo computational design of retro-aldol
enzymes. Science 319:1387, 2008.
Magnenat, L., Schwimmer, L.J., Barbas, C.F. III. Drug-inducible and simultaneous
regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger
transcription factor gene switches. Gene Ther. 15:1223, 2008.
Nomura, W., Barbas, C.F. III. In vivo site-specific DNA methylation with a designed
sequence-enabled DNA methylase. J. Am. Chem. Soc. 129:8676, 2007.
Ramasastry, S.S.V., Albertshofer, K., Utsumi, N., Barbas, C.F. III. Water-compatible organocatalysts for direct asymmetric syn-aldol reactions of dihydroxyacetone
and aldehydes. Org. Lett.10:1621, 2008.
Ramasastry, S.S.V., Albertshofer, K., Utsumi, N., Tanaka, F., Barbas, C.F. III.
Mimicking fructose and rhamnulose aldolases: organocatalytic syn-aldol reactions
with unprotected dihydroxyacetone. Angew. Chem. Int. Ed. 46:5572, 2007.
Tanaka, F., Fuller, R.P., Asawapornmongkol, L., Warsinke, A., Gobuty, S., Barbas,
C.F. III. Development of a small peptide tag for covalent labeling of proteins. Bioconjug. Chem. 18:1318, 2007.
relatively unexplored opportunity in the science
of catalytic antibodies is modifying the phenotype of an organism by incorporating the gene
for a catalytic antibody into the genome of that organism. An attractive application of this concept would be
the expression of such a catalyst in transgenic plants
to provide a beneficial trait. For example, introduction
of a herbicide-resistance trait in commercial plants is
highly desirable because plants with the trait could be
grown in the presence of a nonselective herbicide that
affects only weeds and other undesired plant species.
We have shown that herbicide-resistant plants can
be engineered by designing both a herbicide and a catalytic antibody that destroys the herbicide within the
plants. Such a transgenic plant was achieved via a
3-step maneuver: (1) development of a new carbamate
herbicide, one that can be catalytically destroyed by
the aldolase antibody 38C2; (2) separate expression
of the light chain and half of the heavy chain (Fab) of
the catalytic antibody in the endoplasmic reticulum of
2 plant lines of Arabidopsis thaliana; and (3) crosspollination of these 2 transgenic plants to produce a
herbicide-resistant F1 hybrid (Fig. 1). In vivo expression
of catalytic antibodies could become a useful, general
strategy to achieve desired phenotype modifications not
only in plants but also in other organisms.
A
SYNTHETIC ENZYMES
Utsumi, N., Imai, M., Tanaka, F., Ramasastry, S.S.V., Barbas, C.F. III. Mimicking
aldolases through organocatalysis: syn-selective aldol reactions with protected dihydroxyacetone. Org. Lett. 9:3445, 2007.
Zhang, H., Mitsumori, S., Utsumi, N., Imai, M., Garcia-Delgado, M., Mifsud, M.,
Albertshofer, K., Tanaka, F., Barbas, C.F. III. Catalysis of 3-pyrrolidinecarboxylic
acid and related pyrrolidine derivatives in enantioselective anti-Mannich-type reactions: importance of the 3-acid group on pyrrolidine for stereocontrol. J. Am.
Chem. Soc. 130:875, 2008.
Selenoenzymes have a central role in maintaining
cellular redox potential. These enzymes have selenylsulfide bonds in their active sites that catalyze the
reduction of peroxides, sulfoxides, and disulfides. The
selenol-disulfide exchange reaction is common to all of
the enzymes, and the active-site redox potential reflects
MOLECULAR BIOLOGY
2008
F i g . 1 . Influence of herbicide (4) on the rooting and development
of seedlings of F 1 hybrids and control A thaliana plants. The control plants are shown in A and C; the hybrid plant lines (F1) expressing both light and heavy chains of catalytic antibody 38C2, in B
and D. Plantlets grown on medium without the herbicide are shown
in A and B; those grown with the herbicide are shown in C and D.
the ratio between the forward and reverse rates of this
reaction. The preparation of enzymes containing selenocysteine is experimentally challenging. As a result, little
is known about the kinetic role of selenols in enzyme
active sites, and the redox potential of a selenylsulfide
or diselenide bond in a protein has not been experimentally determined.
To fully evaluate the effects of selenocysteine on
oxidoreductase redox potential and kinetics, we chemically synthesized glutaredoxin 3 (Grx3) and all 3 selenocysteine variants of its conserved 11CXX14C active site
and determined their redox potentials. The position of
redox equilibrium between Grx3(C11U-C14U) (–308 mV)
and thioredoxin (–270 mV) suggests a possible role for
diselenide bonds in biological systems. Kinetic analysis
showed that the lower redox potentials of the selenocysteine variants are due primarily to the greater nucleophilicity of the active-site selenium. The 102- to 104-fold
increase in the rate of thioredoxin reduction by the selenoGrx3 analogs indicates that oxidoreductases containing
either selenylsulfide or diselenide bonds can have physiologically compatible redox potentials and enhanced
reduction kinetics in comparison with their sulfide counterparts. This research was done in collaboration with
P.E. Dawson, Department of Cell Biology.
BIOMOLECULAR COMPUTING DEVICES
In fully autonomous molecular computing devices,
all components, including input, output, software, and
THE SCRIPPS RESEARCH INSTITUTE
259
hardware, are specific molecules that interact with each
other through a cascade of programmable chemical
events, progressing from the input molecule to the
molecular output signal. DNA molecules and DNA
enzymes have been used as convenient, readily available components of such computing devices because
the DNA materials have highly predictable recognition
patterns, reactivity, and information-encoding features.
Furthermore, DNA-based computers can become part
of a biological system, generating outputs in the form
of biomolecular structures and functions.
Our previously reported 2-symbol–2-state finite
automata computed autonomously, and all of their
components were soluble biomolecules mixed in solution. The hardware consisted of 2 enzymes, an endonuclease and a ligase, and the software and the input
were double-stranded DNA oligomers. More recently,
we designed and created 3-symbol–3-state automata
that can carry out more complex computations. In
addition, we found that immobilization of the input
molecules on chips allowed parallel computation, a
system that can be used encrypt information.
The main advantage of autonomous biomolecular
computing devices compared with electronic computers is the ability of the devices to interact directly with
biological systems. No interface is required because all
components of molecular computers, including hardware,
software, input, and output, are molecules that interact
in solution along a cascade of programmable chemical
events. We showed for the first time that the output of
a molecular finite automaton can be a visible bacterial
phenotype. Our 2-symbol–2-state finite automaton uses
linear double-stranded DNA inputs prepared by inserting a string of 6-bp symbols into the lacZ gene on plasmid pUC18. The computation resulted in a circular
plasmid that differed from the original pUC18 by either
a 9-bp (accepting state) or an 11-bp (unaccepting state)
insert within the lacZ gene. Upon transformation and
expression of the resultant plasmids in Escherichia coli,
either blue colonies or white colonies, respectively, were
formed (Fig. 2).
SYNTHETIC CAPSIDS
Stable structures of icosahedral symmetry can have
numerous functional roles, including chemical microencapsulation and delivery of drugs and biomolecules, a
way to observe encapsulated reactive intermediates,
presentation of epitopes for efficient immunization, synthesis of nanoparticles of uniform size, and formation
of structural elements for molecular supramolecular con-
260 MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
Macromolecular Interactions:
Evolution, Engineering, and
Detection
V.V. Smider, C.C. Liu, J. Mills, B. Hutchins, B. Leonard
e are involved in several projects related to
molecular recognition. These include understanding the evolution and biochemical characteristics of the human germ-line antibody repertoire,
chemically enhancing antibody properties, analyzing
RNA hairpin interactions that regulate DNA replication,
and developing new chemical technologies to detect
interactions between macromolecules.
W
ENHANCED AND NOVEL INTERACTIONS:
ENGINEERED ANTIBODIES
F i g . 2 . Computation with aaba input in the presence (A) and
absence (B) of transition molecules results in white bacteria when
the transition molecules are present. Computation with abba input
in the presence (C) and absence (D) of transition molecules results
in blue bacteria when the transition molecules are present.
structs and molecular computing. By examining physical models of spherical virus assembly, we developed
a general synthetic strategy for producing chemical capsids at size scales between fullerenes and spherical
viruses. Such capsids can be formed by self-assembly
from a class of novel symmetric molecules developed
from a pentagonal core. By designing chemical complementarity into the 5 interface edges of the molecule, we can produce self-assembling stable structures
of icosahedral symmetry.
We considered 3 different binding mechanisms:
hydrogen bonding, metal binding, and formation of
disulfide bonds. These structures can be designed to
assemble and disassemble under controlled environmental conditions. We have conducted molecular
dynamics simulation on a class of corannulene-based
molecules to demonstrate the characteristics of selfassembly and to aid in the design of the molecular
subunits. This research was done in collaboration
with A.J. Olson, Department of Molecular Biology.
Antibodies bind their antigens noncovalently. However, for certain diagnostic or therapeutic applications,
antibodies that irreversibly bind or modify their antigens
would be useful. We recently succeeded in engineering
a metal-dependent antibody that irreversibly binds its
antigen, TNF-α, perhaps through an exchange inert
cobalt complex (Fig. 1). In collaboration with P.G.
Schultz, Department of Chemistry, we are engineering
other metalloantibodies and are incorporating amino
acids with unique side chains (unnatural amino acids)
into antibodies. Similarly, we are creating unique multivalent protein constructs. This technology should allow
both genetic and chemical expansion of the antibody
repertoire for therapeutic and diagnostic applications.
R N A H A I R P I N S R E G U L AT I N G D N A R E P L I C AT I O N
RNA molecules are now recognized as important
regulators of many cellular properties via RNA-RNA
interactions. One of the simplest and evolutionarily
ancient mechanisms of RNA-mediated regulation occurs
at plasmid origins of replication. We are applying molecular evolution techniques to these regions and using
simple copy number and compatibility assays to ascertain the regulatory and recognition features of these
hairpins in Escherichia coli.
B I O M O L E C U L A R D E T E C T I O N : O X A L AT E E S T E R
CHEMIFLUORESCENCE
PUBLICATIONS
Kossoy, E., Lavid, N., Soreni-Harari, M., Shoham, Y., Keinan, E. A programmable
biomolecular computing machine with bacterial phenotype output. Chembiochem
8:1255, 2007.
Olson, A.J., Hu, Y.H.E., Keinan, E. Chemical mimicry of viral capsid self-assembly.
Proc. Natl. Acad. Sci. U. S. A. 104:20731, 2007.
Sensitive methods such as fluorescence can be
used to detect multiple macromolecular interactions
simultaneously (multiplex detection). An alternative
approach is detection via chemifluorescence, in which
a fluorescent dye is activated through chemical transfer of energy. We recently synthesized aqueous oxalate
MOLECULAR BIOLOGY
2008
THE SCRIPPS RESEARCH INSTITUTE
261
F i g . 2 . A water-soluble oxalate ester can activate fluorescently
labeled protein. Top, The postulated reaction between an oxalate
ester and hydrogen peroxide to produce a dioxetane intermediate,
which can transfer energy to a fluorescent dye to produce carbon
dioxide and light emission at the dye’s characteristic wavelength.
Bottom, Samples of fluorescently labeled bovine serum albumin
(BSA-ROX, BSA-JOE, BSA-Cy3, and BSA-Cy5) adsorbed to a membrane and exposed to activated water-soluble oxalate ester emit
light of characteristic wavelengths.
F i g . 1 . An antibody scFv that binds its antigen irreversibly. Top,
Western blot shows a complex between an engineered scFv 21H9 and
its TNF antigen at 44 kD (lanes 6 and 9) that only forms in the
presence of cobalt. Both scFv and TNF are in the complex, as indicated by staining with antibody to TNF (lane 9a) and antibody to
scFv (lane 9b). The parental scFv RA72, which binds TNF nonco-
valently, does not form the 44-kD complex (lanes 1–4). Bottom,
Close-up model of the 21H9 scFv (green) with its metal binding site
(red) in proximity to mapped linkage regions (pink) of TNF. The TNF
trimer is shown as an outset with linkage residues in cyan and pink.
ester compounds capable of exciting fluorescent dyes
conjugated to biomolecules (Fig. 2). Unlike fluorescence, chemifluorescence allows integration of the emission signal over time, a situation that may result in
highly sensitive detection of molecular interactions.
Applications include microarrays, blots, and solid-phase
immunoassays in research and diagnostic settings.
PUBLICATIONS
Grünewald, J., Tsao, M.L., Perera, R., Dong, L., Niessen, F., Wen, B.G., Kubitz,
D.M., Smider, V.V., Ruf, W., Nasoff, M., Lerner, R.A., Schultz, P.G. Immunochemical
termination of self-tolerance. Proc. Natl. Acad. Sci. U. S. A. 105:11276, 2008.
Trisler, K., Looger, L.L., Sharma, V., Baker, M., Benson, D.E., Trauger, S.,
Schultz, P.G., Smider, V.V. A metalloantibody that irreversibly binds a protein antigen. J. Biol. Chem. 282:26344, 2007.
Functional Characterization
of Proteases via
Combinatorial Libraries
J.L. Harris, J. Alves
he human genome encodes more than 500 proteases. These enzymes play important roles in
all aspects of biology, from the beginning of life
(fertilization, development, differentiation) to the end
of life (cell death, apoptosis, degradation) and most
biological processes in between. Proteases are also
implicated in many pathologic states, such as cancer,
inflammation, infectious disease, and neurodegeneration.
Proteases exert their activity by cleaving peptide
bonds in proteins. These cleavage events can be exquisitely selective, depending on the substrate specificity
of the protease. Identifying the substrate specificity of
a protease aids in understanding the role of the protease in a specific biological process.
T
262 MOLECULAR BIOLOGY
2008
To define the substrate specificity of proteases, we
have developed peptide nucleic acid (PNA)–encoded
substrate and inhibitor libraries. Encoding substrate and
inhibitor libraries with PNA tags allows not only for capture of the synthetic history of the library in the resulting molecule but also for spatial deconvolution of the
molecules on DNA microarrays. The PNA tag is covalently cosynthesized with the small-molecule compound
that can interact with enzymes in biological samples.
With this approach, we can efficiently synthesize thousands of molecules in the same time it would take to
synthesize a single individual molecule. Although the
resulting library of thousands of compounds is screened
as a mixture, the specific molecules within the library
can be easily identified on a DNA microarray by using
the hybridization properties of the encoded PNA tag.
Another technique for defining the substrate specificity of proteases includes the use of positional scanning
combinatorial libraries with fluorogenic latent molecules
based on the coumarin or rhodamine scaffold. Using this
technique, we have developed substrates that can be
used to monitor not only the substrate specificity of
proteases in enzymatic assays but also proteolytic activity in live cells for the development of diagnostic agents
or inhibitors. For example, we developed a series of
rhodamine-based substrates to monitor the specific
action of cathepsin C, a protease implicated in immunity and immunologic diseases, in live cells by using
flow cytometry.
Another example of functional characterization of
protease activity is the profiling of the substrate specificity of kallikreins. In collaboration with E.P. Diamandis,
University of Toronto, Toronto, Ontario, we used a substrate library of approximately 160,000 fluorogenic
substrates to characterize the structure-activity relationship of kallikreins, a protease class that has been
strongly linked to cancer progression.
PUBLICATIONS
Borgoño, C.A., Gavigan, J.A., Alves, J., Bowles, B., Harris, J.L., Sotiropoulou, G.,
Diamandis, E.P. Defining the extended substrate specificity of kallikrein 1-related
peptidases. Biol. Chem. 388:1215, 2007.
Denault, J.B., Drag, M., Salvesen, G.S., Alves, J., Heidt, A.B., Deveraux, Q., Harris, J.L. Small molecules not direct activators of caspases [letter]. Nat. Chem. Biol.
3:519, 2007.
Hampton, E.N., Knuth, M.W., Li, J., Harris, J.L., Lesley, S.A., Spraggon, G. The
self-inhibited structure of full-length PCSK9 at 1.9 Å reveals structural homology
with resistin within the C-terminal domain. Proc. Natl. Acad. Sci. U. S. A.
104:14604, 2007.
Li, J., Petrassi, H.M., Tumanut, C., Masick, B.T., Trussell, C., Harris, J.L. Substrate optimization for monitoring cathepsin C activity in live cells. Bioorg. Med.
Chem., in press.
THE SCRIPPS RESEARCH INSTITUTE
Li, J., Tumanut, C., Gavigan, J.A., Huang, W.J., Hampton, E.N., Tumanut, R.,
Suen, K.F., Trauger, J.W., Spraggon, G., Lesley, S.A., Liau, G., Yowe, D., Harris,
J.L. Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity. Biochem. J. 406:203, 2007.
Anticancer Agents: Synthesis
and Selective Delivery
S.C. Sinha, R.A. Lerner, R. Goswami, V. Erigala, D. Xue,
K.M. Bajjuri, Z. Chen, Z.-Z. Huang
ur main research interests include antibody
catalysis, synthesis of natural and unnatural
molecules, drug development, and selective
drug delivery. This year, we mainly focused on the
construction and evaluation of novel antibody conjugates for the development of prodrug therapy and
chemically programmed antibody approaches to cancer therapy. In addition, we synthesized the naturally
occurring anticancer adjacent bis-tetrahydrofuran acetogenins and their analogs.
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S Y N T H E S I S O F C E L L - TA R G E T I N G A N T I B O D I E S
For the development of the prodrug therapy approach,
we used the catalytic aldolase antibody 38C2 and a
small-molecule inhibitor of integrin α v β 3 to prepare 2
cell-targeting catalytic antibodies (Fig. 1A). In these
conjugates, the small molecules combined with 38C2
through the surface lysine residues or the sulfide groups
obtained by reduction of the disulfide bridge in the
antibody hinge region. Mass spectrometry indicated
that each conjugate had approximately 2 molecules of
the small molecule. In flow cytometry assay, both conjugates bound cells expressing αvβ3 with high affinity.
Because the small molecule used for the conjugates also
had weak affinity for the integrin α v β 5 , the resulting
conjugates also bound to cells expressing that integrin.
We found that the conjugates efficiently catalyzed
the activation of several doxorubicin prodrugs, and the
released drug was cytotoxic for MDA-MB-231 breast
cancer cells in vitro. We have also designed a series of
doxorubicin prodrugs (Fig. 1C) and evaluated them in
vitro. Now we are using the prodrugs and antibody
conjugates to evaluate the antibody-prodrug therapy
approach in vivo.
Using αvβ3 antagonists, we have also prepared and
evaluated noncatalytic 38C2 conjugates or the chemically programmed 38C2 (cp38C2) in which the compound reacted to the antibody-binding sites through a
diketone or the vinylketone linker (Fig. 1A). We are now
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263
the most cytotoxic compounds that target the mitochondrial enzyme complex I. We have prepared 10 stereoisomeric asimicin analogs and a new naturally occurring
acetogenin, 27-hydroxybullatacin (Fig. 1D), and its
15-epimer. To explore the binding pocket of these acetogenins in complex I, we also prepared a fluorescent
analog of 15-epi-rolliniastatin. In preliminary studies,
these compounds appeared to bind in the complex I
binding pocket, additional studies are required to confirm the results. The in vitro evaluations and complex
I binding studies are carried out in collaboration with
F. Valeriote, Henry Ford Health System, Detroit, Michigan,
and M. Verkhovskaya, University of Helsinki, Finland.
B I S - T E T R A H Y D R O F U R A N D E R I VAT I V E S
The demands for novel chemical entities with
unknown biological functions are ever growing. Compounds with tetrahydrofuran fragments in the molecules
have important biological properties, including antitumor and antibacterial activities. However, a library of
these compounds had never been attempted. In collaboration with P. Wipf, University of Pittsburgh, Pennsylvania, we are developing multicomponent reaction products
based on mono- and bis-tetrahydrofurans. Using the
tetrahydrofurans and their multicomponent reaction products, we are also synthesizing a library of the azidealkyne “click” products. These compounds will undergo
systematic biological evaluations.
F i g . 1 . Structures of compounds that target integrins αvβ3/αvβ5
(A) and αvβ3/αvβ6 (B), doxorubicin prodrugs (C), and the adjacent
bis-tetrahydrofuran 27-hydroxybullatacin (D).
focusing on compounds and antibody conjugates that
would target other integrins, including αvβ6, α5β1, and
αIIbβ3, which may be useful in the treatment and diagnosis of various human diseases. For example, we prepared
noncatalytic 38C2 conjugates by using the diketone and
vinylketone derivatives of an analog of peptoids (Fig. 1B)
that are known to bind integrins αvβ3 and αvβ6. We
are evaluating these conjugates in vitro and in vivo. These
studies are carried out in collaboration with C.F. Barbas,
Department of Molecular Biology, B. Felding-Habermann, Department of Molecular and Experimental
Medicine, and C. Liu, Department of Immunology and
Microbial Science.
SYNTHESIS OF ANNONACEOUS ACETOGENIN
ANALOGS
We are developing a library of annonaceous acetogenin analogs, chemical entities based on the novel
mono- and bis-tetrahydrofurans, and of protease inhibitors. Asimicin-type annonaceous acetogenins are among
PROTEASE INHIBITORS
Numerous proteases are overexpressed in several
cancer cell lines and are highly implicated in tumor
growth and metastases. Inhibition of such proteases
has potential application in cancer therapy. In collaboration with Dr. Liu, we are developing inhibitors of
legumain protease, which is highly expressed in several
cancer cell lines, including prostate and breast cancer
cells. Although our main goal is to discover a novel
protease with previously unknown structure, we are
also determining the structure-activity relationship of
the already known nanomolar inhibitors of the legumain protease.
PUBLICATIONS
Chen, Z., Sinha, S.C. Total synthesis of 27-hydroxy-bullatacin and its C-15 epimer,
and studies on their inhibitory effect on bovine heart mitochondrial complex I functions. Tetrahedron 64:1603, 2008.
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Chemical Transformations of
Small Molecules and Proteins
and Reactions Between Them
F. Tanaka, K. Albertshofer, C.F. Barbas III, R.P. Fuller,
H.-M. Guo, M. Minakawa, S.S.V. Ramasastry, N. Utsumi,
H. Zhang
e create and develop molecules and methods
that contribute to basic biomedical research
and to the development of drug candidates
and therapeutic strategies.
One aspect of our research is the development of
new strategies and methods for labeling proteins with
small molecules at certain positions. Covalent labeling
reactions are required for preparing protein–small molecule conjugates that are useful in research and medicine. For example, protein-drug conjugates are often
safer and more effective therapeutics than is drug or
protein alone. Small molecules conjugated to proteins
have been used to direct the localization of conjugates
in living cells according to the binding specificity of
the small molecules.
We are developing synthetic labeling molecules that
selectively and covalently react with tyrosine. Labeling
reactions specific for tyrosine can be used to label a
tyrosine naturally present in a protein and/or a tyrosine
within a tag fused to a protein. Tyrosine residues occur
often less on the surface of folded proteins than do
residues containing lysine or carboxylic acid. Thus,
accessible tyrosine is an attractive covalent conjugation site. For proteins that do not have accessible tyrosine on the surface, the phenol group is orthogonal;
these proteins should not be affected by reagents or
compounds that react with phenols or tyrosine.
When a tag that contains accessible tyrosine is fused
to a protein, the tyrosine within the tag can be used as
a selective labeling site. Whereas incorporation of unusual
amino acids into proteins often requires special conditions and reagents, preparation of protein fusions with
tyrosine-containing tags does not. Therefore, reactions
at tyrosines will be a convenient way to specifically
label proteins.
We have developed cyclic imine derivatives that
react with phenols, including tyrosine-containing peptides, in water over a wide pH range without the need
for additional catalysts at room temperature to 37°C.
The reaction is the formation of a carbon-carbon bond
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THE SCRIPPS RESEARCH INSTITUTE
at the carbon of the phenol benzene ring; bonds formed
via this reaction were stable. The imine derivatives can
be conjugated with fluorescent molecules and drug
molecules by using chemical synthesis. Therefore,
through reactions at tyrosines with these molecules, a
variety of molecules can be covalently and selectively
attached to a protein of interest.
When a labeling molecule contains a moiety that
selectively and covalently reacts with a specific amino
acid residue and a moiety that noncovalently interacts
with a target protein, the labeling molecule can be used
to tag naturally occurring proteins of interest in natural
environments, including cells. When a labeling molecule contains a moiety that binds to the surface of a
protein, the protein can be labeled without affecting the
protein’s function. On the other hand, when the specificity of a labeling molecule is imparted through a ligand
that binds to the active site of an enzyme, the resulting molecule can be an efficient, selective, irreversible
inhibitor of the enzyme. Accordingly, we are developing imine-derived molecules for these types of uses.
We are also developing protein and peptide tags
that selectively form covalent bonds with designed synthetic molecules; these tags are generated by using
reaction-based selections from combinatorial libraries
of proteins and peptides. When the tag is fused to a
protein of interest, the fusion protein can be modified
at the tag sequence through the reaction with the
designed synthetic compound.
We are also developing synthetic methods for concise access to functionalized molecules in regioselective,
diastereoselective, and enantioselective fashions. These
methods are useful for synthesizing bioactive molecules,
ligands of proteins, and candidates of these molecules.
Overall, our research provides insight into the construction of functional molecules and into biological
functions of naturally occurring molecules such as proteins and enzymes.
PUBLICATIONS
Guo, H.-M., Minakawa, M., Tanaka, F. Fluorogenic imines for fluorescent detection of
Mannich-type reactions of phenols in water. J. Org. Chem. 73:3964, 2008.
Hayashi, I., Mizuno, H., Tong, K.I., Furuta, T., Tanaka, F., Yoshimura, M.,
Miyawaki, A., Ikura, M. Crystallographic evidence for water-assisted photo-induced
peptide cleavage in the stony coral fluorescent protein Kaede. J. Mol. Biol.
372:918, 2007.
Jiang, L., Althoff, E.A., Clemente, F.R., Doyle, L., Röthlisberger, D., Zanghellini,
A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F. III, Hilvert, D., Houk,
K.N., Stoddard, B., Baker, D. De novo computational design of retro-aldol
enzymes. Science 319:1387, 2008.
Ramasastry, S.S.V., Albertshofer, K., Utsumi, N., Tanaka, F., Barbas, C.F. III.
Mimicking fructose and rhamnulose aldolases: organocatalytic syn-aldol reactions
with unprotected dihydroxyacetone. Angew. Chem. Int. Ed. 46:5572, 2007.
MOLECULAR BIOLOGY
2008
Tanaka, F., Hu, Y., Sutton, J., Asawapornmongkol, L., Fuller, R., Olson, A.J., Barbas, C.F. III, Lerner, R.A. Selection of phage-displayed peptides that bind to a particular ligand-bound antibody. Bioorg. Med. Chem. 16:5926, 2008.
Utsumi, N., Imai, M., Tanaka, F., Ramasastry, S.S.V., Barbas, C.F. III. Mimicking
aldolases through organocatalysis: syn-selective aldol reactions with protected dihydroxyacetone. Org. Lett. 9:3445, 2007.
Zhang, H., Mitsumori, S., Utsumi, N., Imai, M., Garcia-Delgado, N., Mifsud, M.,
Albertshofer, K., Cheong, P.H.-Y., Houk, K.N., Tanaka, F., Barbas, C.F. III. Catalysis of 3-pyrrolidinecarboxylic acid and related pyrrolidine derivatives in enantioselective anti-Mannich-type reactions: importance of the 3-acid group on pyrrolidine
for stereocontrol. J. Am. Chem. Soc. 130:875, 2008.
THE SCRIPPS RESEARCH INSTITUTE
265
linker length, and chemical composition. The structures
reveal a significant structural plasticity of the distal substrate-binding channel of the enzyme that arises from
combinations of several modes of movements in the F, G,
and B′ helices. Changes in the hydrogen-bonding interactions between wire and protein affected ligand orientation
more than did binding affinity (Fig. 1). The linker length
Zhang, H., Ramasastry, S.S.V., Tanaka, F., Barbas, C.F. III. Organocatalytic antiMannich reactions with dihydroxyacetone and acyclic dihydroxyacetone derivatives:
a facile route to amino sugars. Adv. Synth. Catal. 350:791, 2008.
Metalloenzyme Engineering
D.B. Goodin, C.D. Stout, H.B. Gray,* E.C. Glazer,
R.F. Wilson, A. Annalora, S. Vetter
* California Institute of Technology, Pasadena, California
he primary purposes of our research are to understand the diversity of metalloenzyme catalysts
and to develop methods for directed evolution of
enzymes with novel function. Our goal is to gain sufficient control over substrate-enzyme interactions and
the oxidative chemistry catalyzed at the active site to
allow directed evolution of catalysts capable of regiospecific and stereospecific oxidation of a given target substrate. We use a number of techniques in structural
biology and spectroscopy and strategies of rational
protein redesign and molecular evolution.
In the past year, we focused on developing synthetic
molecular wires to probe the active site and function
of the heme enzymes P450cam and nitric oxide synthase
(NOS). These wires, which consist of substrate analogs
tethered to a reporter or sensitizer, are designed to bind
specifically to the active-site channel of a given enzyme
and are effective tools for inhibitor discovery, phototriggered enzyme turnover, and molecular evolution strategies.
We have made significant progress in obtaining a
detailed structural characterization of a library of tethered substrate wires that bind the substrate access channel of P450 cam . In the past, we showed that alkyl
adamantane wires bind to the active site of P450cam
and induce a large conformational change at the enzyme’s
active site. On the basis of these findings, we have
designed a new generation of P450 wires, and we
recently solved the crystal structures of P450cam in
complex with more than 12 variants that differ from
each other in the position of hydrogen-bonding groups,
T
F i g . 1 . Crystal structures of 3 tethered substrate wires bound to
the active site of P450cam. The linkers differ in the location of an
amide that provides a critical hydrogen-bonding interaction with
the substrate.
and hydrophobicity have a significant effect on the
conformation disorder in both the wire and the protein.
We have also made significant progress in characterizing photoactive probes specific for the pterin site
of murine inducible NOS. Addressing the pterin site
through a molecular wire will help define the role of
the cofactor in catalysis by allowing photochemical
triggering of enzyme turnover. We have synthesized a
series of ruthenium(II)-pterin wires and have characterized their interactions with murine inducible NOS.
Binding was confirmed by heme-induced quenching of
either pterin or ruthenium(II) fluorescence upon interaction with the heme domain of inducible NOS, and
this quenching was reversed by binding of the natural
pterin cofactor. Time-resolved emission experiments
showed bound and free forms of the wire and allowed
estimation of the affinity and the distance between the
ruthenium(II) and heme. Our findings are consistent
with the modeled geometry of the wire within the
pterin-binding site. We recently observed photoinduced reduction of ferric NOS after 450-nm illumination of the ruthenium(II) center in the presence of
reductive quenchers. These wires are being examined
for their potential to generate unstable intermediates
in the NOS reaction cycle.
To explore the potential of generating novel P450like catalysts, we have introduced thiolate coordination
into the heme of the lipocalin nitrophorin 1. UV and visible light, resonance Raman spectroscopy, and magnetic
266 MOLECULAR BIOLOGY
2008
circular dichroism spectra suggest weak thiolate coordination only in the ferric state of the H60C mutant of
nitrophorin 1. Two crystal structures of the mutant in
complex with imidazole and histamine were solved to
1.7- and 1.96-Å resolution, respectively. Both structures
show that the H60C mutation is well tolerated by the
protein scaffold and suggest that heme-thiolate coordination in the mutant nitrophorin 1 requires some movement of the heme within its binding cavity.
PUBLICATIONS
Contakes, S.M., Nguyen, Y.H.L., Gray, H.B., Glazer, E.C., Hays, A.-M., Goodin,
D.B. Conjugates of heme-thiolate enzymes with photoactive metal-diimine wires.
Struct. Bond. 123:177, 2007.
Glazer, E.C, Nguyen, Y.H., Gray, H.B., Goodin, D.B. Probing inducible nitric oxide
synthase with a pterin-ruthenium(II) sensitizer wire. Angew. Chem. Int. Ed.
47:898, 2008.
Structure, Function, and
Applications of Virus Particles
J.E. Johnson, M. Banerjee, C.-Y. Fu, I. Gertsman, R. Huang,
R. Khayat, G. Lander, J. Lanman, K.K. Lee, T. Matsui,
A. Odegard, J. Speir, R. Taurog
e investigate model virus systems that provide insights for understanding viral assembly, maturation, entry, localization, and
replication. We have also developed viruses as reagents
for applications in nanomedicine, nanochemistry, and
nanobiology. We investigate viruses that infect bacteria, insects, plants, and the extreme thermophile Sulfolobus. These viruses have genomes of single-stranded
RNA and double-stranded DNA.
We use a variety of physical methods to investigate
structure-function relationships, including single-crystal x-ray diffraction, static and time-resolved solution
x-ray diffraction, electron cryomicroscopy and image
reconstruction, mass spectrometry, structure-based computational analyses, and methods associated with
thermodynamic characterization of virus particles and
the particles’ transitions. Biological methods we use
include the genetic engineering of viral genes and their
expression in Escherichia coli, mammalian cells, insect
cells, and yeast and the characterization of these gene
products by physical methods. For cytologic studies of
viral entry and infection, we use fluorescence and electron microscopy and particles assembled in heterologous
expression systems. Our studies depend on extensive
consultations and collaborations with others at Scripps
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THE SCRIPPS RESEARCH INSTITUTE
Research, including the groups led by B. Carragher,
M.G. Finn, M. Manchester, R.A. Milligan, C. Potter,
V. Reddy, A. Schneemann, G. Siuzdak, and J.R. Williamson, and a variety of groups outside of Scripps.
DOUBLE-STRANDED DNA VIRUSES
HK97 is a double-stranded DNA virus similar to
bacteriophage λ. It undergoes a remarkable morphogenesis in its assembly and maturation, and this process
can be recapitulated in vitro. We determined the atomic
resolution structure of the 650-Å mature head II particle and discovered the mechanism used to concatenate
the subunits of the particle into a chain-mail structure
similar to that seen in armor of medieval knights.
In the past year, we focused on the structures of
prohead I and prohead II, the first and second intermediates in the assembly pathway. The prohead II structure
is at 3.7-Å resolution, and the subunit fold and the
location of many side chains have been determined.
The tertiary structure of the subunit in prohead II differs from that of the subunit in head II, an unexpected
result. At lower resolution, the transition from prohead
II to head II appeared to be rigid body motions. It is now
clear that contacts near the 3-fold particle axes are
fixed and that a dramatic change occurs in the subunit structure, with a twist about 3 β-strands and the
bending of a long helix, although the domains remain
largely rigid. The change in tertiary structure may be
the energy storage mechanism that propels the maturation of the particle.
We used electron cryomicroscopy to study bacteriophage λ in the prohead and head states. The crystal
structure of the HK97 bacteriophage capsid fits most
of the T = 7 λ particle density with only minor adjustment. A prominent surface feature at the 3-fold axes
corresponds to the cementing protein glycoprotein D,
necessary for stabilization of the capsid shell. The position of the glycoprotein coincides with the location of
the covalent cross-link formed in the docked HK97 crystal structure, suggesting an evolutionary replacement
of this gene product in bacteriophage λ by autocatalytic
chemistry in HK97.
SINGLE-STRANDED RNA VIRUSES
Flock House virus is a T = 3, single-stranded RNA
virus that infects Drosophila. Infectivity of Flock House
virus requires the autocatalytic cleavage of the capsid
protein at residue 363, liberating the C-terminal 44-residue γ peptides that remain associated with the particle.
In vitro studies indicated that the amphipathic-helical
part (residues 364–385) is membrane active, suggesting
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its role in RNA membrane translocation during infection.
We have now shown that a maturation-defective mutant
of Flock House virus can be rescued by viruslike particles that lack the genome but undergo maturation cleavage in a baculovirus expression system. We propose that
colocalization of the 2 defective particle types in an entry
compartment allows the rescue by γ peptides.
We used time-resolved electron cryomicroscopy for
structural studies of the T = 4 tetravirus Nudaurelia
capensis ω virus. We found that a large-scale structural
change induced by lowering the pH from 7 to 5 occurs
in less than 100 milliseconds, but the annealing of the
polypeptide chains to form active autocatalytic sites
varies dramatically with the position of the subunit in the
surface lattice. Subunits adjacent to 5- and 3-fold axes
form active sites in less than 3 minutes, whereas the
other 2 quasi-equivalent subunits are much slower. One
of the latter subunits forms active sites in 30 minutes;
the other requires more than 2 hours. These data explain
well the unusual kinetics of the cleavage reaction.
Speir, J.A., Johnson, J.E. Tetravirus structure. In: Encyclopedia of Virology, 3rd ed.
Mahy, B.W.J., van Reganmortel, M.H.V. (Eds.). Academic Press/Elsevier, New York,
2008, Vol. 5, p. 27.
PUBLICATIONS
Banerjee, M., Johnson, J.E. Activation, exposure and penetration of virally
encoded, membrane-active, polypeptides during non-enveloped virus entry. Curr.
Protein Pept. Sci. 9:16, 2008.
e are interested in identifying and understanding the structural underpinnings and requirements for the self-assembly, stability, and
targeting specificities of viral capsids. We use this information to design novel protein shells that polyvalently
display multiple copies of peptides or proteins of interest. We use structural, computational, bioinformatics,
and genetic methods.
Viruses are highly evolved macromolecular assemblages that perform a variety of functions during their
life cycle, including self-assembly to form uniform capsids, selective packaging of the genome, binding to susceptible host cells, and delivering the genetic material to
the targeted cells Simple viruses, such as nonenveloped
viruses, form capsids with homogeneous composition
and quaternary architecture. Hence, these viruses are
useful for structural and functional analyses.
We recently determined the structure of Seneca
Valley virus (Fig. 1), which belongs to a new genus
(Senecavirus) of the Picornaviridae family. Seneca Valley
virus is the first known naturally occurring nonpathogenic
picornavirus shown be selectively cytotoxic to tumor cells
with features of neuroendocrine cancer. This research
was done in collaboration with scientists at Neotropix,
Inc., Malvern, Pennsylvania.
In collaboration with G.R. Nemerow, Department
of Immunology, we have made progress in determining
the structure of the entire human adenovirus at 5.5-Å
Gan, L., Johnson, J.E. An optimal exposure strategy for cryoprotected virus crystals
with lattice constants greater than 1000 Å. J. Synchrotron Radiat. 15:223, 2008.
Johnson, J.E. Multi-disciplinary studies of viruses: the role of structure in shaping
the questions and answers. J. Struct. Biol., in press.
Johnson, J.E., Chiu, W. DNA packaging and delivery machines in tailed bacteriophages. Curr. Opin. Struct. Biol. 17:237, 2007.
Johnson, J.E., Speir, J.A. Principles of virus structure. In: Encyclopedia of Virology,
3rd ed. Mahy, B.W.J., van Reganmortel, M.H.V. (Eds.). Academic Press/Elsevier,
New York, 2008, Vol. 5, p. 393.
Kang, S., Lander, G., Johnson, J.E., Prevelige, P.E. Development of bacteriophage
P22 as a platform for molecular display: genetic and chemical modifications of the
procapsid exterior surface. Chembiochem 9:514, 2008.
Lanman, J., Crum, J., Deerinck, T.J., Gaietta, J.M., Schneemann, A., Sosinsky,
G., Ellisman, M.H., Johnson, J.E. Visualizing Flock House virus infection in
Drosophila cells with correlated fluorescence and electron microscopy. J. Struct.
Biol. 161:439, 2008.
Lee, J., Doerschuk, P.C., Johnson, J.E. Exact reduced-complexity maximum likelihood
reconstruction of multiple 3-D objects from unlabeled unoriented 2-D projections and
electron microscopy of viruses. IEEE Trans. Image Process. 16:2865, 2007.
Lee, K.K., Gan, L., Tsuruta, H.C.M., Conway, J.F., Duda, R.L., Hendrix, R.W.,
Steven, A.C., Johnson, J.E. Virus capsid expansion driven by the capture of mobile
surface loops. Structure, in press.
Sosinsky, G.E., Crum, J., Jones, Y.Z., Lanman, J., Smarr, B., Terada, M., Martone,
M.E., Deerinck, T.J., Johnson, J.E., Ellisman, M.H. The combination of chemical
fixation procedures with high pressure freezing and freeze substitution preserves
highly labile tissue ultrastructure for electron tomography applications. J. Struct.
Biol. 161:359, 2008.
Speir, J.A., Johnson, J.E. Nonenveloped virus structure. In: Encyclopedia of Virology, 3rd ed. Mahy, B.W.J., van Reganmortel, M.H.V. (Eds.). Academic Press/Elsevier, New York, 2008, Vol. 5, p. 380.
Steinmetz, N.F., Lin, T., Lomonossoff, G. P., Johnson, J.E. Structure-based engineering of an icosahedral virus for nanomedicine and nanotechnology. In: Viruses
as Nanomaterials for Biomedicine and Bioengineering. Manchester, M., Steinmetz,
N.F. (Eds.). Springer, New York, in press.
Szymczyna, B.R., Gan, L., Johnson, J.E., Williamson, J.R. Solution NMR studies
of the maturation intermediates of a 13 MDa viral capsid. J. Am. Chem. Soc.
129:7867, 2007.
Walukiewicz, H.E., Banerjee, M., Schneemann, A., Johnson, J.E. Rescue of maturation-defective Flock House virus infectivity with noninfectious, mature, viruslike
particles. J. Virol. 82:2025, 2008.
Wickner, R.B., Tang, J., Gardner, N., Johnson, J.E. The yeast double-stranded
RNA virus L-A resembles mammalian dsRNA virus cores. In: Segmented DoubleStranded RNA Viruses: Structure and Molecular Biology. Patton, J.T. (Ed.). Caister
Academic Press, Portland, OR, 2007, p. 105.
Structure, Informatics, and
Design in Virology
V.S. Reddy, M. Tripp, S. Venkataraman
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enables the attachment of peptides or proteins of interest to the C terminus in a linear fashion and their
display on the capsid surface. Such reagents act as
multivalent decoys of the pathogenic molecules and so
can be used as potential vaccines.
PUBLICATIONS
Carrillo-Tripp, M., Brooks, C.L. III, Reddy, V.S. A novel method to map and compare protein-protein interactions in spherical viral capsids. Proteins, in press.
F i g . 1 . Protomer structure (left) and capsid surface (right) of
Seneca Valley virus, an oncolytic picornavirus representative of a
new genus.
resolution by x-ray crystallography. Adenoviruses are
common human pathogens and major causative agents
of acute respiratory and ocular infections. Currently,
adenoviruses are being used as vectors in gene transfer
studies. The structure of an adenovirus at high resolution will provide insight into reengineering of advenovirus vectors to improve vaccine or gene delivery to
specific host cell types. Data acquisition to 4-Å resolution is under way. We have also determined the x-ray
structures of 2 fiber knobs of adenovirus serotypes 35
and 16 and have identified structural requirements for
binding of the knobs to the cellular receptor CD46.
We continue to maintain and expand the virus
structure database, VIPERdb (http://viperdb.scripps.edu),
a Web portal for structures and associated structural
properties of viral capsids. The capsid structures in
the database were analyzed in terms of protein-protein interactions, contacting residue pairs, association
energies, individual residue contributions, and surface
characteristics by using computational methods. We
recently developed a novel method to map and compare protein-protein interactions in viral capsids irrespective of the size and the architecture of the capsids.
The resultant maps can be used as road maps to visualize the extent and distribution of interactions required
for the formation of viral capsids. The results of the
analysis are stored in the database. VIPERdb is being
developed and maintained as part of the National Institutes of Health research resource Multiscale Modeling
Tools for Structural Biology, headed by C.L. Brooks,
University of Michigan, Ann Abor.
We are also actively involved in generating novel
vaccines against cytotoxins such as ricin and against
pathogens by expressing antigenic regions of pathogenic
molecules on the surfaces of viral capsids. Tomato
bushy stunt virus–like capsids are our display platform
of choice. A unique subunit fold of the viral subunit
Lad, S.P., Yang, G., Scott, D.A., Wang, G., Nair, P., Mathison, J., Reddy, V.S., Li, E.
Chlamydial CT441 Is a PDZ domain-containing tail-specific protease that interferes
with the NF-κB pathway of immune response. J. Bacteriol. 189:6619, 2007.
Manayani, D.., Thomas, D., Dryden, K.A., Reddy, V., Siladi, M.E., Marlett, J.M.,
Rainey, G.J., Pique, M.E., Scobie, H.M., Yeager, M., Young, J.A., Manchester, M.,
Schneemann, A. A viral nanoparticle with dual function as an anthrax antitoxin
and vaccine. PLoS Pathog. 3:1422, 2007.
Pache, L., Venkataraman, S., Nemerow, G.R., Reddy, V.S. Conservation of fiber
structure and CD46 usage by subgroup B2 adenoviruses. Virology 375:573, 2008.
Venkataraman, S., Reddy, S.P., Loo, J., Idamakanti, N., Hallenbeck, P.L., Reddy,
V.S. Crystallization and preliminary x-ray diffraction studies of Seneca Valley virus001, a new member of the Picornaviridae family. Acta Crystallogr. Sect. F Struct.
Biol. Cryst. Commun. 64:293, 2008.
Biology and Applications of
Icosahedral Viral Capsids
A. Schneemann, N. Brunn, J. Jovel, J.E. Petrillo, P.A. Venter
oat proteins of nonenveloped, icosahedral viruses
perform multiple functions during viral infection,
including capsid assembly, specific encapsidation of the viral genome, binding to a cellular receptor,
and uncoating. In some viruses, a single type of protein
is sufficient to carry out these functions; we are interested in the determinants that endow a polypeptide
chain with such versatility. We seek to harness this
versatility for novel applications of viruses in biotechnology and nanotechnology.
We focus on a structurally and genetically well-characterized virus family, the T = 3 nodaviruses. Nodaviruses
are composed of 180 copies of a single coat protein and
2 strands of positive-sense RNA. Currently, we are elucidating the mechanism by which the 2 genomic RNAs are
packaged into a single virion. Our data indicate that the
2 viral RNAs are recognized separately, but it is not yet
known whether packaging occurs sequentially and
whether one or more coat protein subunits are involved
in this process. Interestingly, we found that RNA genome
packaging is coupled to genome replication and that specific packaging of the viral genome also requires coat protein translated from newly synthesized viral RNA. The
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coupling of these processes may be a safety mechanism
for the virus to ensure efficient and accurate formation
of progeny virions in infected cells.
We are also investigating the mechanism by which
nodaviral protein B2 suppresses RNA silencing. We are
identifying the double-stranded RNAs that serve as substrates for B2 binding during nodavirus infection, and
we are correlating these results with data from confocal
microscopy and immunoprecipitation studies. We have
identified amino acid residues in B2 that are critical for
the protein’s function as an RNA-binding protein; preliminary data suggest that B2 may have a second function during the viral replication cycle that is unrelated
to its function as a suppressor of RNA silencing.
We are also using nodavirus particles as a novel
platform to develop improved vaccines. In collaboration
with researchers at Scripps Research, the Salk Institute,
La Jolla, California, and Harvard University, Boston,
we displayed the VWA domain of capillary morphogenesis protein 2, the cellular receptor for anthrax toxin, in
a multivalent fashion on the surface of the virion. Two
insertion sites yielding different patterns of 180 copies
of the VWA domain were selected on the basis of computational modeling of the high-resolution crystal structure
of Flock House virus, an insect nodavirus. The resulting
chimeric viruslike particles functioned as a potent anthrax
antitoxin in cell culture and protected rats from challenge with lethal toxin.
This research is important because it shows that
protein domains containing more than 150 amino acids
can be displayed on Flock House virus in a biologically functional form, suggesting numerous additional
applications. Moreover, chimeric particles decorated
with anthrax protective antigen elicited a potent neutralizing antibody response against the antigen that
protected rats from challenge with lethal toxin 4 weeks
after a single immunization without adjuvants. This
chimeric particle platform is a dually acting reagent
for the treatment of and protection against anthrax.
PUBLICATIONS
Lanman, J., Crum, J., Deerinck, T.J., Gaietta, G.M., Schneemann, A., Sosinsky,
G.E., Ellisman, M.H., Johnson, J.E. Visualizing Flock House virus infection in
Drosophila cells with correlated fluorescence and electron microscopy. J. Struct.
Biol. 161:439, 2008.
Manayani, D.J., Thomas, D., Dryden, K.A., Reddy, V., Siladi, M.E., Marlett, J.M.,
Rainey, G.J.A., Pique, M.E., Scobie, H.M., Yeager, M., Young, J.A.T., Manchester,
M., Schneemann, A. A viral nanoparticle with dual function as an anthrax antitoxin
and vaccine. PLoS Pathog. 3:1422, 2007.
Walukiewicz, H.E., Banerjee, M., Schneemann, A., Johnson, J.E. Rescue of maturation-defective Flock House virus infectivity with noninfectious, mature, viruslike
particles. J. Virol. 82:2025, 2008.
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Molecular Mechanism of
Autophagosome Formation
T. Otomo, C. Otomo
utophagy is a conserved degradative mechanism
triggered in response to various stresses in
eukaryotic cells. Cells use autophagy to break
down cytoplasmic fractions in bulk and large cytotoxic
components such as protein aggregates, organelles, and
intracellular pathogens. The hallmark of autophagy is
de novo formation of a large double-membrane–enclosed
transport vesicle called an autophagosome, which
sequesters a fraction of cytoplasm and delivers the
fraction to a lysosome for degradation. Formation of
autophagosomes is thought to involve new molecular
mechanisms that differ from those in classical vesicular transport systems.
We are interested in the molecular basis of the
membrane dynamics and the cargo recognition rules
in autophagy. Our goals are to explain the mechanisms
by revealing atomic structures of relevant protein complexes and establishing in vitro biochemical reconstitutions and to predict potential cargos on the basis of
structural data. Targeting autophagy in therapies and
drug development for various diseases such as cancer,
neurodegeneration, and infectious disease, has been
discussed. Our studies on the mechanistic basis would
facilitate development of such strategies.
Among many autophagy-related proteins, we are
currently focusing on 2 ubiquitin-like protein modifiers,
Atg12 and Atg8, which are autophagy specific. These
molecules are important because they play key roles
in membrane dynamics. Atg12 is conjugated to another
autophagy protein, Atg5, and Atg8 is conjugated to
phosphatidylethanolamine through ubiquitin-like enzymatic reactions. The Atg12-Atg5 conjugate has 3 functions. First, it acts as an E3 ligaselike enzyme in the
conjugation of Atg8 to phosphatidylethanolamine. Second, the Atg12-Atg5 conjugate forms large oligomers
when bound to a coiled coil protein, Atg16. The resulting
Atg12-Atg5-Atg16 complex may be the membrane
scaffold for autophagosomes. Third, Atg12-Atg5 interacts
with a bacterial protein, VirG, from Shigella flexneri,
which is the key for autophagosomes to recognize this
shigella species. We have succeeded in generating the
Saccharomyces cerevisiae and human Atg12-Atg5 conjugates by developing bacterial coexpression systems,
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which will allow us to investigate structural mechanisms
of the functions of the conjugates.
The Atg8-phosphatidylethanolamine conjugate functions in cargo recognition and membrane tethering.
Membrane tethering is intriguing because it may explain
how autophagosomal membranes elongate into doublemembrane vesicles. Biophysical and structural characterization of the conjugate would open the door to
understanding this mysterious membrane dynamics.
Currently, we are producing proteins critical for these
studies. Overall, the combination of structural study and
biochemical reconstitution would answer the mechanistic questions in autophagosome formation.
Control of Cell Division
THE SCRIPPS RESEARCH INSTITUTE
late the proteasome. Proteasomes are complex proteases
that target ubiquitylated proteins, including important
cell-cycle regulatory proteins. Surprisingly, we found
that Cks1/Cdk1 regulates a nonproteolytic function of
proteasomes, the transcriptional activation of the gene
CDC20. Specifically, Cks1 and Cdk1 are required to
recruit proteasomes to CDC20 for efficient transcriptional elongation. Our investigations of CDC20 have
led to the conclusion that Cks1 and Cdk1 are required
for recruitment of proteasomes to and transcriptional
elongation of many other genes as well. Currently, we
are elucidating the mechanism whereby the Cks1/Cdk1proteasome complex facilitates transcriptional elongation. Our most recent results suggest that Cks1 and
proteasomes in conjunction with Cdk1 mediate remodeling of chromatin by removing nucleosomes.
CONTROL IN MAMMALIAN CELLS
S.I. Reed, L.-C. Chuang, M. Henze, V. Liberal, K. Luo,
S. Ekholm-Reed, L. Teixeira, H.-S. Martinsson-Ahlzén
iological processes of great complexity can be
approached by beginning with a systematic
genetic analysis in which the relevant components are first identified and then the consequences of
selectively eliminating the components via mutation
are investigated. We have used yeast, which is uniquely
tractable to this type of analysis, to investigate control
of cell division. In recent years, it has become apparent that the most central cellular processes throughout
the eukaryotic phylogeny are highly conserved in terms
of both the regulatory mechanisms used and the proteins involved. Thus, it has been possible in many
instances to generalize from yeast cells to human cells.
B
CONTROL IN YEAST
We have focused on the role and regulation of the
Cdc28 protein kinase (Cdk1). Initially identified by
means of a mutational analysis of the yeast cell cycle,
this protein kinase and its analogs are ubiquitous in
eukaryotic cells and central to a number of aspects of
control of cell-cycle progression.
One current area of interest is regulation of cellular morphogenesis by Cdk1. The activity of Cdk1 driven
by mitotic cyclins modulates polarized growth in yeast
cells. Specifically, these activities depolarize growth by
altering the actin cytoskeleton. We found that several
proteins that modulate actin structure are targeted by
Cdk1, and we are investigating how these phosphorylation events control actin depolarization and cell shape.
While investigating mitosis in yeast, we found that
Cks1, a small Cdk1-associated protein, appears to regu-
We showed previously that the human homologs
of the Cdc28 protein kinase are so highly conserved,
structurally and functionally, relative to the yeast protein kinase, that they can function and be regulated
properly in a yeast cell. Analyzing control of the cell
cycle in mammalian cells, we produced evidence for
the existence of both positive and negative regulatory
schemes, similar to those elucidated in yeast.
A principal research focus is the positive regulator
of Cdk2, cyclin E. Cyclin E is often found overexpressed
and/or deregulated in human cancers. Using a tissue
culture model, we showed that deregulation of cyclin
E confers genomic instability, probably explaining the
link to carcinogenesis. The observation that deregulation of cyclin E confers genomic instability has led us to
hypothesize a mechanism of cyclin E–mediated carcinogenesis based on accelerated loss of heterozygosity at
tumor suppressor loci. We are testing this hypothesis
in transgenic mouse models. We showed that a cyclin
E transgene expressed in mammary epithelium significantly increases loss of heterozygosity at the p53 locus,
leading to enhanced mammary carcinogenesis. We are
extending these investigations by using mouse prostate,
testis, and skin models.
In an attempt to understand cyclin E–mediated genomic instability, we are investigating how deregulation of
cyclin E affects both S phase and mitosis. Recent data
suggest that deregulation of cyclin E impairs DNA replication by interfering with assembly of the prereplication
complex. We found that cells deregulated for cyclin E
expression progress through S phase abnormally slowly
and tend to enter mitosis prematurely. Cyclin E deregula-
MOLECULAR BIOLOGY
2008
tion also impairs the metaphase-anaphase transition by
promoting the accumulation of inhibitors of anaphase.
Our interest in cyclin E deregulation in cancer led us
to investigate the pathway for turnover of cyclin. We
showed that phosphorylation-dependent proteolysis of
cyclin E depends on a protein-ubiquitin ligase known as
SCFhCdc4. The F-box protein hCdc4 is the specificity factor that targets phosphorylated cyclin E. We are investigating how ubiquitylation of cyclin E is coordinated with
other processes required for its degradation, including
prolyl isomerization. We are also investigating SCFhCdc4
ubiquitylation of other important cellular proteins.
Recently, we began determining the role of SCFhCdc4
in neurodegenerative disease. We found that parkin, a
protein often mutated in hereditary Parkinson’s disease,
regulates the stability of hCdc4, possibly leading to neuropathologic changes. We discovered an SCFhCdc4 substrate, peroxisome proliferator–activated receptor γ
coactivator 1, that may be the effector of hCdc4 deregulation in Parkinson’s disease. In addition, we showed that
SCF hCdc4 regulates the turnover of presenilins in the
brain, proteins strongly implicated in Alzheimer’s disease.
Another area of interest is the role of Cks proteins
in mammals, complementing our research in yeast.
Mammals express 2 paralogs of yeast Cks1, known as
Cks1 and Cks2. Experiments in mice lacking the genes
for Cks1 and Cks2 revealed that each paralog has a
specialized function. Cks1 is required as a cofactor for
Skp2-mediated ubiquitylation and turnover of inhibitors
p21, p27, and p130. Cks2 is required for the transition from metaphase to anaphase in both male and
female meiosis I. Nevertheless, mice nullizygous at
the individual loci are viable. However, doubly nullizygous mice have not been observed because embryos
die at the morula stage, a finding consistent with an
essential redundant function. We found that this function is probably is involved in regulation of transcription and linked to chromatin remodeling, as in yeast.
Furthermore, we found that deregulation of Cks1 and
Cks2, which often occurs in cancer, results in override
of the intra–S phase checkpoint, which protects cells
from DNA damage while the cells are undergoing DNA
replication. Failure of the checkpoint would most likely
make cells cancer prone.
PUBLICATIONS
Akhoondi, S., Sun, D., von der Lehr, N., Apostolidou, S., Klotz, K., Maljukova,
A., Cepeda, D., Fiegl, H., Dafou, D., Marth, C., Mueller-Holzner, E., Corcoran,
M., Dagnell, M., Nejad, S.Z., Nayer, B.N., Zali, M.R., Hansson, J., Egyhazi, S.,
Petersson, F., Sangfelt, P., Nordgren, H., Grander, D., Reed, S.I., Widschwendter,
M., Sangfelt, O., Spruck, C. FBXW7/hCDC4 is a general tumor suppressor in
human cancer. Cancer Res. 67:9006, 2007.
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271
Baskerville, C., Segal, M., Reed, S.I. The protease activity of yeast separase
(esp1) is required for anaphase spindle elongation independently of its role in
cleavage of cohesin. Genetics 178:2361, 2008.
Keck, J.M., Summers, M.K., Tedesco, D., Ekholm-Reed, S., Chuang, L.-C., Jackson, P.K., Reed, S.I. Cyclin E overexpression impairs progression through mitosis
by inhibiting APCCdh1. J. Cell Biol. 178:371, 2007.
Martinsson-Ahlzén, H.S., Liberal, V., Grünenfelder, B., Chaves, S.R., Spruck,
C.H., Reed, S.I. Cyclin-dependent kinase-associated proteins Cks1 and Cks2 are
essential during early embryogenesis and for cell cycle progression in somatic cells.
Mol. Cell. Biol. 28:5698, 2008.
Olson, B.L., Hock, M.B., Ekholm-Reed, S., Wohlschlegel, J.A., Dev, K.K., Kralli,
A., Reed, S.I. SCFCdc4 acts antagonistically to the PGC-1α transcriptional coactivator by targeting it for ubiquitin-mediated proteolysis. Genes Dev. 22:252, 2008.
Reed, S.I. Deathproof: new insights on the role of skp2 in tumorigenesis. Cancer
Cell 13:88, 2008.
Sangfelt, O., Cepeda, D., Maljukova, A., van Drogen, F., Reed, S.I. Both SCFCdc4α
and SCFCdcrγ are required for cyclin E turnover in cell lines that do not overexpress
cyclin E. Cell Cycle 7:1, 2008.
Control of Gene Expression
During the Cell Cycle
and in Response to
DNA Replication Stress
C. Wittenberg, R.A.M. de Bruin, M. Guaderrama,
T.I. Kalashnikova
he ability of cells to proliferate and to respond
to internal and environmental stimuli requires
the capacity to rapidly change the abundance
and activity of the proteins that mediate those processes. That regulation depends mainly on the ability
to modulate gene expression. Recently, we have focused
on the mechanisms by which cells exert control over
gene expression to regulate cell proliferation and intracellular and environmental signals.
In most cells, the initiation of a new round of cell
division during the G1 phase of the cell cycle is accompanied by the activation of a large family of genes that
encode activities involved in the duplication and segregation of cellular components. G1-specific (also known
as G1/S) genes also encode regulatory factors that promote subsequent events in the cell cycle. In the budding
yeast Saccharomyces cerevisiae, G1-specific genes are
regulated by the transcription factors SBF and MBF.
SBF acts as a transcriptional activator and promotes
gene expression during the G1 interval. In contrast, MBF
acts primarily as a transcriptional repressor and limits
transcription of target genes to the G1 phase. Together
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272 MOLECULAR BIOLOGY
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SBF and MBF regulate the expression of nearly 200
genes that promote cell proliferation.
Using mass spectrometry–based multidimensional
protein identification technology, in collaboration with
J.R. Yates, Department of Cell Biology, we have identified
novel regulators of SBF and MBF. We established that
promoter-bound SBF associates with the Whi5 repressor during early G1 phase and that Whi5 is inactivated
via phosphorylation by a G1-specific cyclin-dependent
protein kinase, thereby activating transcription. This
regulation is analogous to the regulation of genes dependent on the transcription factor E2F by the retinoblastoma tumor suppressor in humans. In addition, we
identified Nrm1, a novel MBF-associated corepressor.
When expressed as an MBF target during late G1 phase,
Nrm1 associates with MBF at target promoters and
represses expression as cells enter S phase. In addition
to the Whi5 and Nrm1 transcriptional coregulators, we
identified Msa1, Msa2, and Stb1 as modulators of the
transcriptional activity of SBF- and MBF-regulated genes.
We have now extended our studies of cell-cycle
regulated transcription to the distantly related fission
yeast Schizosaccharomyces pombe, in collaboration
with P. Russell, Department of Molecular Biology. G1/S
transcription in fission yeast is mediated by a single
transcription factor called MBF that, like MBF in budding yeast, acts primarily as a transcriptional repressor
of G1/S genes. We are studying fission yeast homologs
of budding yeast Nrm1 and Whi5. The Nrm1 homolog,
SpNrm1, is a target of MBF and, like budding yeast
Nrm1, acts as a corepressor with MBF as cells exit G1
phase. Inactivation of fission yeast Nrm1, unlike inactivation of its budding yeast homolog, has a dramatic
effect on G1/S gene expression that results in significant cellular phenotypes.
In addition to being regulated during the cell cycle,
the G1-specific transcriptional machinery is regulated
by checkpoints that monitor the integrity of cellular
structures and processes. In fission yeast, when DNA
replication forks are stalled during S phase, repression
of MBF-regulated transcription is disrupted, and expression of MBF target genes is induced. We have shown
that SpNrm1 is a direct target for phosphorylation by
the checkpoint protein kinase Cds1, a functional homolog of human Chk1, and that its phosphorylation leads
to dissociation from MBF and activation of G1/S gene
expression (Fig. 1). That response requires the ATM/ATRlike checkpoint kinase Rad3 and other elements of DNA
replication checkpoint signaling. We also found that
THE SCRIPPS RESEARCH INSTITUTE
F i g . 1 . Regulation of G 1-specific transcription by the DNA replication checkpoint in fission yeast, budding yeast, and humans. In
budding and fission yeast, activation of the DNA replication checkpoint by stalling the progression of DNA replication prevents the
repression of G 1 /S transcription as cells enter S phase. Similarly,
in human cells, the Chk1 checkpoint kinase is required for maintenance of G1 /S transcription. This mechanism promotes the repair
and reactivation of DNA replication forks and prevents the loss of
genome integrity.
derepression of MBF target genes occurs in budding
yeast after activation of the DNA replication checkpoint. As in fission yeast, transcriptional activation
occurs via inactivation of Nrm1 (Fig. 1).
G1/S transcription in mammalian cells is regulated
by the E2F family of transcription factors. Although the
components of the G1-specific transcriptional regulatory system in mammalian cells are apparently unrelated to the yeast regulators, as indicated by amino acid
sequence, the architecture of the mammalian and the
yeast systems is strikingly similar. We hypothesized that
a checkpoint regulatory cascade like that we discovered in the yeast systems controls G1-specific genes via
E2F family members in human cells and that the cascade might therefore play a crucial role in the maintenance of genome integrity. We are testing that hypothesis
in collaboration with C.H. McGowan, Department of
Molecular Biology. We found that human G 1 -specific
genes, like those in budding and fission yeasts, remain
active during S phase when DNA replication forks stall
(Fig. 1). That response, which promotes the expression of genes required for DNA replication and repair, is
abrogated when the Cds1 homolog Chk1 is inactivated
by chemical inhibitors or short interfering RNA. We
are investigating the mechanism by which human G1specific genes are repressed upon entry into S phase
cells and how the DNA replication checkpoint machinery interrupts that regulation.
Together, our recent findings establish that the
mechanisms that govern the expression of G1/S gene
expression are conserved among eukaryotes. Furthermore, we have shown that DNA replication stress leads
to the activation of those genes during S phase, rein-
MOLECULAR BIOLOGY
2008
forcing the cells’ ability to cope with that stress and
avoid genomic instability. We expect our continuing
research on both yeasts and human cells to yield information critical for our understanding of the cellular
response to replication stress, a process central to
both the genesis and the treatment of human cancer.
PUBLICATIONS
Ashe, M., de Bruin, R.A.M., Kalashnikova, T.I., McDonald, W.H., Yates, J.R. III,
Wittenberg, C. The SBF- and MBF-associated protein Msa1 is required for proper
timing of G1-specific transcription in Saccharomyces cerevisiae. J. Biol. Chem.
283:6040, 2008.
de Bruin, R.A.M., Kalashnikova, T.I., Aslanian, A., Wohlschlegel, J.A., Chahwan,
C., Yates, J.R. III, Russell, P., Wittenberg, C. DNA replication checkpoint promotes
G1/S transcription by inactivating the MBF repressor Nrm1. Proc. Natl. Acad. Sci.
U. S. A. 105:11230, 2008.
de Bruin, R.A.M., Kalashnikova, T.I., Wittenberg, C. Stb1 collaborates with other regulators to modulate the G1-specific transcriptional circuit. Mol. Cell. Biol., in press.
Limbo, O., Chahwan, C., Yamada, Y., de Bruin, R.A.M., Wittenberg, C., Russell, P.
Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control
double-strand break repair by homologous recombination. Mol. Cell 28:134 2007.
Cell-Cycle Checkpoints, DNA
Damage, and Cytotoxic Stress
Responses
P. Russell, S. Cavero, C. Dovey, G. Dodson, P. Kennedy,
O. Limbo, E. Mejia, S. Rozenzhak, A. Vashisht, J. Williams,
Y. Yamada
NA damage and cytotoxic stress elicit cellular
responses that are remarkably similar in yeast
and humans. Consequently, studies of genetically tractable microorganisms such as the fission yeast
Schizosaccharomyces pombe are highly relevant to
understanding these cellular processes in more complex multicellular organisms. We study DNA damage
and cytotoxic stress responses because defects in
these mechanisms underlie a wide range of human
diseases, including cancer.
D
THE SCRIPPS RESEARCH INSTITUTE
273
but also mental retardation, neurodegeneration, premature aging, immunologic defects, and infertility.
The mechanism used to repair double-strand breaks
depends on the circumstances in which the damage
occurs. Breaks that arise in the postreplicative (G 2 )
phase of the cell cycle are most effectively repaired by
homologous recombination, an error-free mechanism
in which the undamaged sister chromatid is used as a
template for repair of the broken chromosome. In the
prereplicative G1 phase, the only option for repair of
double-strand breaks is the error-prone mechanism of
nonhomologous end joining. In contrast, homologous
recombination is required for repair when double-strand
breaks are formed by collapse of replication forks during S phase.
We recently investigated how cells regulate repair
of double-strand breaks during the cell cycle. Because
S phase marks the point in the cell cycle at which homologous recombination can repair these breaks, we hypothesized that expression of a homologous recombination
factor might coincide with the onset of S phase. Metaanalysis of genome-wide expression profiling studies
indicated candidate genes. We found that one of these
genes, which we named ctp1, is required for repair of
double-strand breaks by homologous recombination.
Genetic and biochemical studies revealed that ctp1
mutants cannot resect double-strand breaks, a critical
first step of homologous recombination. Other studies
indicated that the protein Ctp1 is a cofactor of the
Mre11-Rad50-Nbs1 complex, a tumor suppressor in
humans (Fig. 1). Ctp1 is so named because it is related
to the human protein CtIP. Interestingly, CtIP can interact
CHECKPOINTS AND DNA DAMAGE RESPONSES
DNA double-strand breaks are among the most lethal
and genome-destabilizing types of DNA damage. The
breaks arise from exogenous sources such as ionizing
radiation, from endogenous sources such as processing of DNA damaged by free radicals, and through errors
involving DNA replication. Rapid and accurate repair of
double-strand breaks is essential for preserving genome
integrity. Indeed, defects in DNA repair are responsible
for a multitude of human diseases, most notably cancer,
F i g . 1 . Regulation of Ctp1 expression controls repair of double-
strand breaks during the cell cycle. Ctp1 acts with the Mre11-Rad50Nbs1 (MRN) complex to promote the resection of double-strand
breaks, which is required for repair by homologous recombination
(HR). Expression of Ctp1 commences at the transition from G1 to
S phase, thereby favoring homologous recombination as the preferred
mode of repair during S and G2 phases. Ctp1 is not expressed in
G1 phase, leaving nonhomologous end joining (NHEJ) as the only
option for repair of double-strand breaks.
274 MOLECULAR BIOLOGY
2008
with BRCA1. The function of BRCA1 has remained an
enigma since its discovery as a tumor suppressor that
is mutated in a large fraction of hereditary breast cancers. Our findings suggest that interactions between
CtIP and BRCA1 may preserve genome stability and
prevent cancer by controlling repair of double-strand
breaks by homologous recombination.
In another study, we investigated the role of Mus81
in repair of single-end breaks. When we discovered
Mus81 several years ago, we proposed that it is a
specialized DNA-cutting enzyme known as a Holliday
junction resolvase. Holliday junctions are the cruciformshaped DNA structures that form at sites of DNA crossovers. Holliday junctions are also proposed to form when
broken replication forks are restored. Using a yeast strain
in which replication forks break at a specific site on a
chromosome, we collaborated with B. Arcangioli, Institut
Pasteur, Paris, France, to show that Mus81 DNA cleavage activity is essential for the survival of a broken fork
(Fig. 2). We also found that Mus81 specifically associates
with the chromosomal region where the Holliday junctions form.
These data strengthen the evidence that Mus81 is
a Holliday junction resolvase. Interestingly, in human
THE SCRIPPS RESEARCH INSTITUTE
cells, Mus81 is specifically required for survival in the
presence of cisplatin, a drug used to treat various types
of cancers such as sarcomas (e.g., bone cancer), some
carcinomas (e.g., ovarian cancer), and lymphomas.
Drugs that target Mus81 may be useful when used in
combination with cisplatin.
CYTOTOXIC STRESS RESPONSE AND SYSTEMS
BIOLOGY
Production of reactive oxygen species (ROS) is a
normal byproduct of aerobic metabolism. Intracellular
ROS can also arise through exposure to environmental
toxicants, such as heavy metals or metalloids (e.g., cadmium and arsenic) and some pesticides. Oxidative stress
in the form of ROS can be highly toxic, causing damage
to proteins, lipids, and nucleic acids. Indeed, the cumulative effects of exposure to ROS are thought to be a
causative factor in many of the most widespread and
debilitating human diseases, such as atherosclerosis,
Alzheimer’s disease, Parkinson’s disease, and cancer,
and in the aging process itself.
In the past year, we used systems biology to define
all the nonessential genes required for tolerance to cadmium and arsenic in fission yeast. Using the fission
yeast haploid deletion library, we have defined approximately 250 genes involved in cadmium or arsenic tolerance. Prominent among these are the gene required
for biosynthesis of coenzyme Q10. Coenzyme Q10 is
an antioxidant commonly used as a dietary supplement for treatment of hypertension, Parkinson’s disease, and other maladies. Our studies are helping us
understand the role of coenzyme Q10 in the physiologic response to environmental toxicants.
PUBLICATIONS
de Bruin, R.A.M., Kalashnikova, T.I., Aslanian, A., Wohlschlegel, J., Chahwan,
C., Yates, J.R. III, Russell, P., Wittenberg, C. The DNA replication checkpoint promotes G1-S transcription by inactivating the MBF repressor Nrm1. Proc. Natl.
Acad. Sci. U. S. A. 105:11230, 2008.
Dovey, C.L., Russell, P. Mms22 preserves genomic integrity during DNA replication
in Schizosaccharomyces pombe. Genetics 177:47, 2007.
Kennedy, P.J., Vashisht, A.A., Hoe, K.-L., Kim, D.U., Park, H.O., Hayles, J., Russell, P. A genome-wide screen of genes involved in cadmium tolerance in
Schizosaccharomyces pombe. Toxicol. Sci. 106:124, 2008.
F i g . 2 . Mus81 is required for recovery from breakage of replication
forks. A, Replication fork encounters a nick in the leading strand
template, leading to breakage of the replication fork. B, Breakage
of the replication fork creates a single-end break. C, The single-end
break is resected, leaving a single-strand tail that invades the sister
chromatid. Strand invasion creates a D-loop that in principle can be
cleaved by the Mus81-Eme1 complex. D, Alternatively, strand invasion can lead to reformation of the replication fork before cleavage
of the resulting Holliday junction by Mus81-Eme1. E, Either pathway can lead to restoration of the replication fork.
Limbo, O., Chahwan, C., Yamada, Y., de Bruin, R.A.M., Wittenberg, C., Russell, P.
Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control
double-strand break repair by homologous recombination. Mol. Cell 28:134, 2007.
Martin, V., Du, L.-L., Rozenzhak, S., Russell, P. Protection of telomeres by a conserved Stn1-Ten1 complex. Proc. Natl. Acad. Sci. U. S. A. 104:14038, 2007.
Noguchi, E., Ansbach, A.B., Noguchi, C., Russell, P. Assays used to study the
DNA replication checkpoint in fission yeast. Methods Mol. Biol., in press.
Rodríguez-Gabriel, M.A., Russell, P. Control of mRNA stability by SAPKs. Top.
Curr. Genet. 20:159, 2008.
MOLECULAR BIOLOGY
2008
Roseaulin, L., Yamada, Y., Tsutsui, Y., Russell, P., Iwasaki, H., Arcangioli, B.
Mus81 is essential for sister chromatid recombination at broken replication forks.
EMBO J. 27:1378, 2008.
Williams, R.S., Moncalian, G., Williams, J.S., Yamade, Y., Limbo, O., Shin, D.S.,
Groocock, L.M., Cahill, D., Hitomi, C., Guenther, G., Moiani, D., Carney, J.P.,
Russell, P., Tainer, J.A. Mre11 dimers coordinate DNA end bridging and nuclease
processing in double-strand-break repair. Cell 135:97, 2008.
DNA Damage Responses in
Human Cells
C.H. McGowan, M. Duquette, E. Langley, J. Scorah,
D. Slavin, E. Taylor
n long-lived animals, including humans, the advantages of being able to replace damaged or aged
cells are offset by the inherent susceptibility of
dividing cells to acquire mutations and become cancerous. DNA is inherently vulnerable to many sorts of
chemical and physical modifications; thus, as cells
duplicate and divide, they can acquire mutations. Cells
have evolved with a complex network of DNA repair
processes and cell-cycle checkpoint responses that
ensure that damaged DNA is repaired before it is replicated and becomes fixed in the genome. Our overall
objective is to understand how mammalian cells protect themselves from DNA damage and thus from cancer. We are especially interested in understanding basic
cellular responses to clinically relevant agents. We use
a combination of molecular, cellular, and genetic techniques to determine how these DNA repair pathways
operate in human cells.
Checkpoints control the order and timing of events
in the cell cycle; they ensure that independent processes
are appropriately coupled. In addition, checkpoints
promote the use of the most appropriate repair pathway. We used genetic models to identify 2 checkpoint
kinases in humans that limit progression of the cell
cycle when DNA is damaged. We are studying the function of both of these kinases and of a number of their
substrates. One of the kinases, Chk2, is activated in
response to DNA damage. Chk2 physically interacts
with Mus81-Eme1, a conserved DNA repair protein
that has endonuclease activity against structure-specific
DNA substrates, including Holliday junctions. Enzymatic
analysis, immunofluorescence studies, and the use of
RNA interference have all contributed to the conclusion that Mus81-Eme1 is required for recombination
repair in human cells. We are also using gene targeting to study the function of the Mus81-Eme1 endonu-
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clease in mice. Inactivation of Mus81 in mice increases
genomic instability and sensitivity to DNA damage but
does not promote tumorigenesis. In addition, we have
shown that Mus81-Eme1 is specifically required for
survival after exposure to cisplatin, mitomycin C, and
other commonly used anticancer drugs. Using laseractivated psoralens to create DNA damage in specific
subnuclear regions, we are defining the mechanism by
which Mus81-Eme1 and other enzymes function in
the repair of chemotherapeutic agents.
Anticancer therapy is mainly based on the use of
genotoxic agents that damage DNA and thus kill dividing
cells. Coordination of cell-cycle checkpoints and DNA
repair is especially important when unusually high
amounts of DNA damage occur after radiation or genotoxic chemotherapy. Hence, a detailed understanding
of cellular responses to DNA damage is essential in
understanding both the development and the treatment of disease in humans.
PUBLICATIONS
Prudden, J., Pebernard, S., Raffa, G., Slavin, D.A., Perry, J.J., Tainer, J.A.,
McGowan, C.H., Boddy, M.N. SUMO-targeted ubiquitin ligases in genome stability.
EMBO J. 26:4089, 2007.
Scorah, J., Dong, M.Q., Yates, J.R. III, Scott, M., Gillespie, D., McGowan, C.H. A
conserved proliferating cell nuclear antigen-interacting protein sequence in Chk1 is
required for checkpoint function. J. Biol. Chem. 283:17250, 2008.
Taylor, E.R., McGowan, C.H. Cleavage mechanism of human Mus81-Eme1 acting
on Holliday-junction structures. Proc. Natl. Acad. Sci. U. S. A. 105:3757, 2008.
DNA Repair and the
Maintenance of
Genomic Stability
M.N. Boddy, J. Heideker, S. Pebernard, J. Prudden
NA repair pathways have evolved to protect
the genome from ever-present genotoxic agents.
Highlighting the importance of the pathways,
defects in DNA repair mechanisms strongly predispose
the host to cancer and to neurologic and developmental
disorders. The DNA repair systems we study in fission
yeast are evolutionarily conserved, and therefore our
studies provide a valuable framework for understanding genome maintenance in human cells. Although
many DNA repair mechanisms have been described,
information on how they are coordinated with necessary changes in chromatin structure is limited.
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THE SMC5-SMC6 COMPLEX
In collaboration with J.R. Yates, Department of Cell
Biology, we purified the structural maintenance of chromosomes (SMC) complex Smc5-Smc6 to identify its core
components. The holocomplex consists of the Smc5Smc6 heterodimer and 6 additional non-SMC elements,
Nse1–Nse6 (Fig. 1A). We showed that Smc5-Smc6
F i g . 1 . Smc5-Smc6 holocomplex and STUbLs. A, Nse1, Nse3,
and Nse4 form a stable heterotrimer that associates with Smc5.
Nse2 interacts directly with Smc5 in the absence of the other Nse
proteins. Smc6 interacts directly with Smc5 but with none of the
other components. Nse5 and Nse6 form a stable heterodimer that
also binds directly to Smc5. Double-headed arrows indicate interactions between subcomplexes. Nse5 and Nse6 may recruit the holocomplex to stalled replication forks and certain DNA damage sites.
B, A target protein is sumoylated, an event that leads to recruitment
of STUbLs via interactions with SUMO and the substrate. For Rad60
or NIP45, STUbLs interact via the SUMO-like domains. STUbLs
then ubiquitinate the substrate and target it for degradation.
prevents the deleterious engagement of an ordinarily
beneficial DNA repair pathway called homologous
recombination. Smc5-Smc6 either prevents initiation of
homologous recombination or separates physically linked
chromosomes that arise late in the process. Spontaneous DNA damage in Smc5-Smc6 mutant cells is due
to the attempted separation of chromosomes into
daughter cells while the chromosomes are still physically
linked. Such defective chromosome separation in humans
could result in cancer and other diseases.
A N U N P R E C E D E N T E D S U M O - TA R G E T E D U B I Q U I T I N
LIGASE
The covalent attachment of ubiquitin and the small
ubiquitin-like protein SUMO to target proteins plays key
roles in genome stability; each of the 2 moieties (i.e.,
ubiquitin and SUMO) has physiologically distinct effects
on the function of target proteins. We have identified
the SUMO-targeted ubiquitin ligase (STUbL) family,
which provides a novel and unanticipated regulatory
link between the ubiquitination and sumoylation pathways. Members of the STUbL family include Slx8-Rfp1
THE SCRIPPS RESEARCH INSTITUTE
in fission yeast, RNF4 in humans, MIP1 in slime molds,
and SLX5/8 in budding yeast. STUbLs are recruited
to sumoylated proteins and proteins containing SUMOlike domains to mediate ubiquitination and degradation of these proteins (Fig. 1B).
Cells with mutations in Slx8-Rfp1 accumulate
sumoylated proteins, have genomic instability, and
are hypersensitive to genotoxic stress. These Slx8-Rfp1
mutant phenotypes are suppressed by concomitant deletion of the major SUMO ligase Pli1, demonstrating the
specificity of STUbLs as regulators of sumoylated proteins. Expression of human RNF4 restores homeostasis
of the SUMO pathway in fission yeast that lack Slx8Rfp1, underscoring the evolutionary functional conservation of STUbLs. The DNA repair factor Rad60 (an
accessory factor of the Smc5-Smc6 complex) and its
human homolog NIP45, which contain SUMO-like
domains, are candidate STUbL targets. Consistently,
mutations in Rad60 and Slx8-Rfp1 cause similar DNA
repair defects.
PUBLICATIONS
Pebernard, S., Perry, J.J., Tainer, J.A., Boddy, M.N. Nse1 RING-like domain supports functions of the Smc5-Smc6 holocomplex in genome stability. Mol. Biol. Cell
19:4099, 2008.
Perry, J.J., Tainer, J.A., Boddy, M.N. A SIM-ultaneous role for SUMO and ubiquitin. Trends Biochem. Sci. 33:201, 2008.
Prudden, J., Pebernard, S., Raffa, G., Slavin, D.A., Perry, J.J., Tainer, J.A.,
McGowan, C.H., Boddy, M.N. SUMO-targeted ubiquitin ligases in genome stability.
EMBO J. 26:4089, 2007.
Signal Transduction Pathways
Mediating Cellular Responses
to Oncogenic Mutations
P. Sun, J. Kwong, R. Liao, A. Seit-Nebi, N. Yoshizuka
evelopment of cancer is a result of multiple
oncogenic genetic alterations, including activation of oncogenes and inactivation of tumor
suppressors. Although these oncogenic mutations contribute to tumorigenic phenotypes, normal cells usually
respond to oncogenic changes by initiating tumor-suppressing defense mechanisms such as apoptosis and
premature senescence (a stable form of growth arrest).
Consequently, tumor development requires additional
mutations that compromise these antioncogenic responses.
Our main interests are to delineate the signal transduction pathways that mediate these tumor-suppress-
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ing responses and to determine how these responses
are evaded during cancer development. Currently, we
are focusing on 2 well-known oncogenes: ras and mdm2.
The oncogene ras encodes a family of small GTPbinding proteins that are often activated in human
tumors and contribute to tumor development. In normal cells, however, the initial response to ras activation is premature senescence. Recent studies have
shown that like apoptosis, oncogene-induced senescence is a bona fide tumor-suppressing mechanism in
vivo that must be compromised in order for cancer to
develop. However, the signaling pathways responsible
for this important antitumorigenic response are poorly
understood. We have shown that ras induces senescence through sequential activation of 2 MAP kinase
pathways. Initially, ras activates the MAP kinase kinase
(MEK)–extracellular signal–regulated kinase (ERK)
pathway. Sustained activation of MEK-ERK turns on
the stress-induced p38 pathway, which subsequently
causes senescence. These studies have revealed a novel,
tumor-suppressing function of p38, in addition to its
known roles in inflammation and stress responses.
The functions of p38 are mediated by its downstream substrates, including a family of serine/threonine
protein kinases. We found that one of these substrate
kinases of p38, p38-regulated/activated protein kinase
(PRAK), mediates senescence upon activation by p38
in response to oncogenic ras. In mice, PRAK deficiency
enhances skin carcinogenesis induced by the environmental mutagen 7,12-dimethylbenz(a)anthracene, coinciding with compromised induction of senescence. In
primary cells, inactivation of PRAK prevents senescence and promotes oncogenic transformation. Moreover, PRAK activates p53 by direct phosphorylation
of the serine at position 37 in p53. We propose that
phosphorylation of p53 by PRAK after activation of
p38 MAP kinase by ras plays an important role in rasinduced senescence and tumor suppression.
Experiments are under way to characterize additional downstream substrates and upstream regulators
of PRAK that contribute to the induction of senescence
and tumor suppression. In addition, we are attempting
to systematically analyze the signaling components of
the p38 pathway to determine their roles in different
cellular functions involving p38, including inflammation
and tumor suppression. Results from these studies will
provide the basis for developing anti-inflammatory and
anticancer drugs that target the p38 pathway.
Another focus of our research is mdm2, an oncogene that can mediate transformation primarily through
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inactivation of the tumor suppressor p53. Previously,
we found that MDM2, the protein encoded by mdm2,
confers resistance to cell-cycle arrest induced by transforming growth factor β (TGF-β), a growth-inhibitory
cytokine. In studies on the molecular mechanism that
underlies MDM2-mediated resistance to TGF-β, we
found that MDM2 makes cells refractory to the cytokine by overcoming a TGF-β–induced arrest of the cell
cycle in G1.
Investigation of the structure-function relationship
of MDM2 revealed 3 elements essential for MDM2 to
confer resistance to TGF-β. One element was the C-terminal half of the p53-binding domain, which at least
partially retained p53 binding and inhibitory activity.
Second, the ability of MDM2 to mediate resistance to
TGF-β was disrupted by mutation of the nuclear localization signal. Finally, mutations of the zinc coordination residues of the RING finger domain abrogated
resistance to TGF-β but not the ability of MDM2 to
inhibit p53 activity or to bind MDMX, another p53
regulator. These data suggest that RING finger–mediated p53 inhibition and MDMX interaction are not sufficient to cause TGF-β resistance and imply a crucial
role of the E3 ubiquitin ligase activity of this domain
in MDM2-mediated TGF-β resistance.
PUBLICATIONS
Han, J., Sun, P. The pathways to tumor suppression via route p38. Trends
Biochem. Sci. 32:364, 2007.
Liu, E., Lee, A.Y., Chiba, T., Olson, E., Sun, P., Wu, X. The ATR-mediated S phase
checkpoint prevents DNA rereplication in mammalian cells when licensing control
is disrupted. J. Cell Biol. 179:643, 2007.
Wang, K., Cheng, C., Li, L., Liu, H., Huang, Q., Xia, C.H., Yao, K., Sun, P., Horwitz, J., Gong, X. γD-crystallin-associated protein aggregation and lens fiber cell
denucleation. Invest. Ophthalmol. Vis. Sci. 48:3719, 2007.
Genetic Modifiers of Behavioral
Despair as Targets for New
Antidepressants
J.G. Sutcliffe, P.B. Hedlund, F.E. Bloom,* B.S. Hilbush*
* ModGene, L.L.C., La Jolla, California
he forced-swim test and the tail-suspension test
are mouse models with high value for predicting
the antidepressant activity of a drug. In both tests,
a state of behavioral despair is created in which mice
cease attempts to escape and become immobile because
of the adverse situation. Known human antidepressants
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increase the length of time the mice attempt to escape,
thus decreasing the immobility times. Significant behavioral differences exist among inbred mouse strains in
these tests. For example, during a 4-minute forced-swim
test, unmedicated, inbred C57BL/6J mice spent 240
seconds or 63% of the swim immobile. In contrast,
inbred DBA/2J mice spent only 47 seconds or 20% of
the time immobile. In collaboration with researchers
at ModGene, L.L.C., we used these strain differences
to identify genes whose activities contribute to relative
basal despair status.
We measured baseline immobility times of both
males and females of 27 strains of recombinant inbred
mice that were produced from C57 x DBA matings.
Each strain had a characteristic immobility time, ranging from 2% (5 seconds) to 70% (168 seconds) of the
swim, and differences between males and females were
detected within strains. We correlated these data with
the haplotype data from these mouse strains and detected
despair modifier genes on chromosome 4 in all mice
and additionally on chromosomes 11 and 13 in female
mice and chromosome 18 in male mice, indicating
some sexual dimorphism in determinants of this behavioral despair, as is known for depression in humans.
In analogous studies with the tail-suspension test, we
detected the same chromosome 4 genes in both males
and females and the same chromosome 11 and 13
genes in females, suggesting that these 3 genes are
directly related to the despair behavior rather than to
the ability to perform in one of the behavioral tests.
We then tested the hypothesis that at least some
of the strain differences occur because of the amount
of mRNA that accumulates from a single gene in each
of the mapped chromosomal regions. We examined the
concentrations of each of more than 30,000 mRNAs in
several regions of the brains of the recombinant inbred
mice. We identified a single gene within the chromosome 4 region for which the amount of mRNA was high
in all strains that inherited the C57 genotype and low
in all strains that inherited the DBA genotype. Similarly, we found a single chromosome 11 gene and a
single chromosome 13 gene for which the mRNA output was inherited in the same mendelian fashion as
the corresponding despair modifier gene.
The identities of the genes responsible for modifying the despair phenotype offer a powerful point of
departure, serving as targets for the development of
new pharmaceutical agents to treat depression. The
studies used to detect the genes provide evidence that
THE SCRIPPS RESEARCH INSTITUTE
differences in the activities of the protein products of
the genes directly contribute to differences in phenotype. Thus, a drug that altered the activity of a protein
encoded by a specific gene and did not have other
deleterious side effects would be suitable candidate to
test for antidepressant activity.
The target gene on chromosome 4 encodes a previously known protein that has never been associated
with brain disorders. The activity of the protein is regulated by phosphorylation by a specific protein kinase.
The concentration of the mRNA of the target gene is
higher in C57 mice (the mice that exhibit greater despair)
than in DBA mice. Therefore, we sought a way to reduce
the activity of the gene to make the behavior of the
C57 mice more like that of the DBA mice.
Compounds that inhibit the activity of the specific
kinase in cultured tumor cells have recently been synthesized. These compounds also inhibit target phosphorylation in the brains of treated mice. When these
compounds were administered to C57 or DBA mice,
immobility times in the forced-swim test and the tailsuspension test were reduced in a dose-dependent fashion, indicative of an antidepressant-like effect. The
compounds had no effects in anxiety or locomotor tests.
These observations suggest that these compounds can
be considered as leads for testing as antidepressants.
PUBLICATIONS
Desplats, P.A., Denny, C.A., Kass, K.E., Gilmartin, T., Head, S.R., Sutcliffe, J.G.,
Seyfried, T.N., Thomas, E.A. Glycolipid and ganglioside metabolism imbalances in
Huntington’s disease. Neurobiol. Dis. 27:265, 2007.
Semenova, S., Geyer, M.A., Sutcliffe, J.G., Markou, A., Hedlund, P.B. Inactivation
of the 5-HT7 receptor partially blocks phencyclidine-induced disruption of prepulse
inhibition. Biol. Psychiatry 63:98, 2008.
Molecular Neurobiology
of CNS Disorders
E.A. Thomas, B. Tang, J.G. Sutcliffe
MOLECULAR BASIS FOR DISEASE PROGRESSION
IN SCHIZOPHRENIA
chizophrenia is a devastating mental illness that
occurs in 1% of the general population. The molecular factors that influence the course of illness
in schizophrenia and how treatment modifies these factors are areas of interest in our group. We have generated genome-wide RNA expression profiles from tissue
samples obtained at autopsy from the prefrontal cortex
of patients who had had schizophrenia for various
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lengths of time, including a cohort of patients who died
within 5 years after the initial diagnosis. We found that
the early stages of disease are associated with the greatest derangement in gene expression and that these genes
are associated with diverse systems and pathways. In
particular, we are focusing on genes that encode proteins with transcriptional regulatory activity, because
these proteins may drive further pathologic or compensatory changes detected later in illness. We are also interested in genes correlated with age in humans who have
schizophrenia and in those regulated by antipsychotic
drug treatment in mouse models. The identification of
genes associated with the early vs later stages of schizophrenia will be important for understanding disease
progression and might lead to the development of agents
that modify the course of disease.
H U N T I N G T O N ’ S D I S E A S E : T R E AT M E N T
A P P R O A C H E S T H AT TA R G E T G E N E T R A N S C R I P T I O N
Huntington’s disease is an autosomal-dominant
neurologic disorder caused by a CAG repeat expansion
within the coding region of the gene for the disease,
resulting in a mutant protein with an expanded polyglutamine tract. Mutant huntingtin protein can disrupt
transcription by diverse mechanisms, including loss of
function of transcription factors and chromatin-mediated
repression. We are exploring mechanisms for transcriptional dysregulation in Huntington’s disease and testing
therapeutic strategies aimed at improving transcriptional output via modulation of chromatin structure.
We have identified target genes for 2 transcriptional
regulatory proteins, Bcl11b and Foxp1, which have
highly enriched expression in the striatum, the brain
region most affected in Huntington’s disease. We found
that these transcription factors interact with huntingtin
protein, suggesting a role in the dysregulation of striatal
gene expression in patients with Huntington’s disease.
In collaboration with J.M. Gottesfeld, Department of
Molecular Biology, we are testing the therapeutic effects
of novel histone deacetylase inhibitors in mouse models of Huntington’s disease. One of these inhibitors,
HDACi 4b, prevents the disease phenotypes in R6/2
transgenic mice. Using microarray analysis, we found
that treatment with HDACi 4b ameliorated abnormalities in gene expression in these mice and caused complete normalization of expression of subsets of genes,
which may be considered biomarkers for treatment
effectiveness. Our findings suggest that HDACi 4b
treatment may be useful in slowing the progression of
symptoms in patients with Huntington’s disease.
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279
PUBLICATIONS
Dean, B., Digney, A., Sundram, S., Thomas, E.A., Scarr E. Plasma apolipoprotein
E is decreased in schizophrenia spectrum and bipolar disorder. Psychiatry Res.
158:75, 2008.
Desplats, P.A., Denny, C.A., Kass, K.E., Gilmartin, T., Head, S.R., Sutcliffe, J.G.,
Seyfried, T.N., Thomas, E.A. Glycolipid and ganglioside metabolism imbalances in
Huntington’s disease. Neurobiol. Dis. 27:265, 2007.
Desplats, P.A., Lambert, J.R., Thomas, E.A. Functional roles for the striatalenriched transcription factor, Bcl11b, in the control of striatal gene expression and
transcriptional dysregulation in Huntington’s disease. Neurobiol. Dis., in press.
Narayan, S., Head, S.R., Gilmartin, T.J., Dean, B., Thomas, E.A. Evidence for disruption of sphingolipid metabolism in schizophrenia. J Neurosci Res., in press.
Narayan, S., Tang, B., Head, S.R., Gilmartin, T.J., Sutcliffe, J.G., Dean, B.,
Thomas, E.A. Molecular profiles of schizophrenia in the CNS at different stages of
illness. Brain Res., in press.
Stuart Gibbons, A., Scarr, E., McOmish, C., Hannan, A.J., Thomas, E.A., Dean,
B. Regulator of G-protein signalling 4 expression is not altered in the prefrontal cortex in schizophrenia. Aust. N. Z. J. Psychiatry 42:740, 2008.
Thomas, E.A., Coppola, G., Desplats, P.A., Tang, B., Soragni, E., Burnett, R.,
Gao, F., Fitzgerald, K.M., Borok, J.F., Herman, D., Geschwind, D.H., Gottesfeld,
J.M. The HDAC inhibitor 4b ameliorates the disease phenotype and transcriptional
abnormalities in Huntington's disease transgenic mice. Proc. Natl. Acad. Sci. U. S. A.
105:15564, 2008.
The 5-HT7 Receptor in
Neuropsychiatric Disorders
P.B. Hedlund, G. Sarkisyan, P.E. Danielson, J.G. Sutcliffe
nterest in the serotonin 5-HT7 receptor as a putative target for the treatment of neuropsychiatric
disorders has been growing continually. The interest was prompted by the finding that several classes
of drugs used to treat disorders such as depression
and schizophrenia have high affinity for this receptor.
We have established evidence that supports a role for
the 5-HT7 receptor in depression, obsessive-compulsive disorder, and schizophrenia.
I
DEPRESSION
The forced swim test and the tail suspension test
are animal models of behavioral despair that have high
value for predicting the antidepressant efficacy of drugs.
The tests can also be used to characterize animals in
which genes have been inactivated. In both of these
tests, we showed that mice lacking the 5-HT7 receptor
have a behavioral profile similar to that of mice treated
with antidepressants. We replicated these findings by
using a compound that acts as a selective antagonist
at the receptor. Thus, both blockade and inactivation
of the 5-HT 7 receptor yield the same result. New evidence suggests that the effects of antidepressants that
act as either serotonin reuptake inhibitors or norepi-
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THE SCRIPPS RESEARCH INSTITUTE
nephrine reuptake inhibitors are potentiated by selective antagonism of the 5-HT7 receptor. These synergistic
interactions provide valuable new insights into the
mechanism of action of antidepressants and open up
new possibilities for the treatment of mood disorders.
consequences of aneuploidy in the brain. While continuing to characterize the extent and regional variations of aneuploidy in the brains of healthy humans
and normal mice, we are determining the link between
neural aneuploidy and human brain disorders.
OBSESSIVE-COMPULSIVE DISORDER
LY S O P H O S P H O L I P I D S I G N A L I N G
Obsessive-compulsive disorder is commonly treated
with antidepressants and thus is related to depression.
In an animal model of obsessive-compulsive disorder
(marble burying), we showed that blockade or inactivation of the 5-HT7 receptor results in less stereotypic
behavior. These findings further support the hypothesis that the 5-HT7 receptor is an important alternative
or supplemental target for antidepressants.
Lysophospholipids are classically known as metabolites in the biosynthesis of cell membranes; however,
their newly discovered functions in a diverse array of
biological processes have highlighted their importance
in health and disease. We discovered the first lysophospholipid receptor, LPA1, and we now know of 10 receptors. We have continued to explore the cellular and
physiologic functions of receptor-mediated lysophospholipid signaling, primarily by generating and examining mice that lack the receptors. In ongoing studies,
we are deciphering the downstream signaling cascades
that mediate the different cellular functions of lysophospholipid receptor activation, determining the contribution of receptor-mediated lysophospholipid signaling to
mammalian brain development, analyzing the neuroanatomic and neurobehavioral phenotypes of mice that
lack lysophospholipid receptors, and examining how
lysophospholipids and their receptors are involved in
myelination and myelination-related diseases, including multiple sclerosis.
SCHIZOPHRENIA
Prepulse inhibition (PPI) of the acoustic startle reflex
is a well-characterized model of schizophrenia. The
model is especially relevant because similar responses
can be observed in patients with schizophrenia. We
showed that PPI per se is not altered in mice lacking
the 5-HT7 receptor but that when PPI is disrupted by
phencyclidine, these mice are significantly less affected
than are mice that have the receptor. Phencyclidineinduced disruption involves a glutamatergic component
of PPI that is relevant for the action of atypical antipsychotic agents such as clozapine. Clozapine is a drug with
relatively high affinity for the 5-HT7 receptor.
NEURAL ANEUPLOIDY
PUBLICATIONS
Semenova, S., Geyer, M.A., Sutcliffe, J.G., Markou, A., Hedlund, P.B. Inactivation
of the 5-HT7 receptor partially blocks phencyclidine-induced disruption of prepulse
inhibition. Biol. Psychiatry 63:98, 2008.
Lysophospholipid Signaling and
Neural Aneuploidy
J. Chun, S. Barral, J. Choi, A. Dubin, S. Gardell, D. Herr,
G. Kennedy, M. Kingsbury, C.W. Lee, D. Letourneau, D. Lin,
M. Lu, T. Mutoh, K. Noguchi, C. Paczkowski, S. Peterson,
R. Rivera, S. Teo, S. Tunaru, W. Westra, X. Ye, Y. Yung, L. Zhu
indings in the past year led to new directions in
our long-term projects. In our studies on lysophospholipid signaling, we discovered roles for lysophosphatidic acid (LPA) receptors in lung and kidney
fibrosis and continued our studies on the functions of
the receptors for sphingosine 1-phosphate (S1P) in the
immune, cardiovascular, and nervous systems.
In our research on neural aneuploidy, we are moving toward a deeper understanding of the functional
F
Since our initial discovery that many cells in the
brain are aneuploid, we have been developing new
methods to investigate aneuploidy to address its anatomic and functional significance. During the past year,
we extended analyses of the genomic complement of
neural cells to the neural cells of teleost fishes, showing the evolutionary conservation of aneuploidy. We
are continuing to investigate how neural aneuploidy
affects brain function at both cellular and system-wide
levels, including the possible contribution to dysfunction in neurologic disorders.
PUBLICATIONS
Chan, L.C., Peters, W., Xu, Y., Chun, J., Farese, R.V., Jr., Cases, S. LPA3 receptor
mediates chemotaxis of immature murine dendritic cells to unsaturated lysophosphatidic acid (LPA). J. Leukoc. Biol. 82:1193, 2007.
Chun, J. Extracellular lipid signals. In: Wiley Encyclopedia of Chemical Biology.
Begley, T.P. Wiley-Interscience, New York, in press.
Chun, J. Genomic disorder and gene expression in the developing CNS. In: The
New Encyclopedia of Neuroscience. Squire, L.R. (Ed. in Chief). Elsevier, Philadelphia, in press.
Chun, J. How the lysophospholipid got its receptor. Scientist 21:48, September 2007.
Chun, J. The sources of a lipid conundrum. Science 316:208, 2007.
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Hama, K., Aoki, J., Inoue, A., Endo, T., Amano, T., Motoki, R., Kanai, M., Ye, X.,
Chun, J., Matsuki, N., Suzuki, H., Shibasaki, M., Arai, H. Embryo spacing and
implantation timing are differentially regulated by LPA3-mediated lysophosphatidic
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