Biophysical and Structural Characterization of Components
From The Nuclear Pore Complex and the Ubiquitin Pathway
by
James R. Partridge
B.S. University of California at Davis
Davis, CA 2004
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DOCTOR OF PHILOSOPHY
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Signature of Author....................
James R. Partridge
Department of Biology
May 4, 2010
.
Certified by....................
............................................................
.
Thomas U. Schwartz
Associate Professor of Biology
Thesis Supervisor
Accepted by...............
Lu7-
Stephen P. Bell
Professor of Biology
Chair, Biology Graduate Committee
Biophysical and Structural Characterization of Components
From The Nuclear Pore Complex and the Ubiquitin Pathway
by
James R. Partridge
Abstract
Formation of an endomembrane system in the eukaryotic cell is a hallmark of biological
evolution. One such system is the nuclear envelope (NE), composed of an inner and outer
membrane, used to form a nucleus and enclose the cell's genome. Access to the nucleus from
the cytoplasm is mediated by a massive macromolecular machine called the nuclear pore
complex (NPC). The NPC resides as a circular opening embedded in the NE and is composed
of only -30 proteins that assemble with octagonal symmetry as biochemically defined
subcomplexes to form the NPC. One such subcomplex is the Nspl / Nup62 complex, composed
of three proteins and stabilized by coiled-coil interactions. Here we reconstitute a tetrameric
assembly between the Nspl-complex and a fourth nucleoporin (Nup) Nic96. Nic96 harbors a 20
kDa coiled-coil domain at the N-terminus followed by a 65 kDa stacked helical domain. The
coiled-coil domain of the Nspl -complex and the N-terminus of Nic96 combine to form a
tetrameric assembly, integrated into the NPC lattice scaffold via the stacked helical domain of
Nic96. We characterized the coiled-coil assembly with size exclusion chromatography and
analytical ultracentrifugation. Deletion experiments and point mutations, directed by hydrophobic
cluster analysis, were used to map connecting helices between members of the protein
assembly.
Although the core of the NPC is a rigid scaffold built for structural integrity, the NPC as a
whole is a dynamic macromolecular machine. Protein transport is regulated by the small G
protein Ran. Ran interacts with the NPC of metazoa via two asymmetrically localized
components, Nupl53 at the nuclear face and Nup358 at the cytoplasmic face. Both Nups
contain distinct RANBP2 type zinc finger (ZnF) domains. We present crystallographic data
detailing the interaction between Nup1 53-ZnFs and RanGDP. A crystal-engineering approach
led to well-diffracting crystals so that all ZnF-Ran complex structures are refined to high
resolution. Each of the four zinc finger modules of Nup1 53 binds one Ran molecule in largely
independent fashion. Nupl53-ZnFs bind RanGDP with higher affinity than RanGTP, however
the modest difference suggests that this may not be physiologically meaningful. ZnFs may be
used to concentrate Ran at the NPC to facilitate nucleocytoplasmic transport.
In a separate study we present a structural analysis of the HECT domain from the E3
ubiquitin ligase HUWE1 and with biophysical data we show that an N-terminal helix stabilizes
the HECT domain. This element modulates activity, as measured by self-ubiquitination induced
in the absence of this helix, distinct from its effects on Ub conjugation of substrate Mcl-1. Such
subtle structural elements in this domain potentially regulate the variable substrate specificity
displayed by all HECT domain type, E3 ubiquitin ligases.
Thesis Supervisor: Thomas U. Schwartz
Title: Associate Professor of Biology
Table of Contents
Abstract...................................................................................................................................................
2
Figure and Table Legend ..............................................................................................................
5
Chapter 1: Introduction ..............................................................................................................
Introduction to the Nuclear Pore Com plex........................................................................................
8
9
Overall structure..............................................................................................................................................................
Modularity.......................................................................................................................................................................11
Protein com position....................................................................................................................................................12
Dynam ics..........................................................................................................................................................................12
Dom ain architec eture ...................................................................................................................................................
FG-repeats .......................................................................................................................................................................
Coiled-coils......................................................................................................................................................................14
p-Propellers ....................................................................................................................................................................
a-Heli cal dom ains ........................................................................................................................................................
ACE1 dom ains................................................................................................................................................................
Structural characterization of NPC subcom plexes ......................................................................
NPC subcom plexes.......................................................................................................................................................18
In vitro nucleoporin subcom plex reassem bly ..........................................................................................
Purification of NPC subcomplexes for crystallographic and biophysical studies ......................
The Nspl / Nup62 subcom plex..............................................................................................................................21
The interaction between Nup153 and the small GTPase Ran ...................................................
Nup153 is a versatile nucleoporin........................................................................................................................23
RanBP2-type zinc fingers in the eukaryotic cell .......................................................................................
Structural and biophysical analysis of Nup153 zinc fingers and RanGDP.......................................
Figures - Chapter 1.......................................................................................................................................28
9
13
13
15
16
17
18
20
20
23
24
26
Chapter 2: Crystallographic and Biochemical Analysis of the Ran-Binding Zinc Finger
34
D om ain ........................ ............................ ....... .......... ..................................................................
35
Introduction...................................................................................................................................................
37
Results and Discussion ...............................................................................................................................
Crystallographic analysis of Nup153-ZnFeRanGDP com plexes.........................................................
Overall structure of the ZnF*RanGDP com plex...........................................................................................
Engineering im proved crystal contacts........................................................................................................
Com parison of the four Nup153 zinc fingers bound to RanGDP ......................................................
Isotherm al titration calorim etry............................................................................................................................43
Conservation of RanBP2-type ZnF cassettes in nucleoporins ............................................................
Com parison of binding interactions am ong RanBP2-type zinc fingers...........................................
Discussion.......................................................................................................................................................
M aterials and M ethods ...............................................................................................................................
Protein purification .....................................................................................................................................................
Crystallization................................................................................................................................................................49
Structure determ ination............................................................................................................................................50
Isotherm al titration calorim etry............................................................................................................................50
Sequence analysis.........................................................................................................................................................51
Protein Data Bank accession num bers................................................................................................................51
Acknow ledgem ents .....................................................................................................................................................
Figures - Chapter 2 ......................................................................................................................................
37
38
40
41
45
46
47
47
48
51
52
Chapter 3: Biophysical Analysis of the Tetrameric Assembly Between Nic96 and the
Trimeric Nspl/Nup57/Nup49 Complex ................................................................................
69
Introduction ...................................................................................................................................................
70
Results..............................................................................................................................................................
75
Reassem bly of the trim eric yNspl / rNup62 subcom plex ....................................................................
Mapping interactions w ithin the Nspl com plex........................................................................................
Biophysical characterization of the Nspl com plex...................................................................................
Assembly of a tetrameric assembly between the Nsp1 complex and Nic96 ................................
Discussion .......................................................................................................................................................
M ethods ...........................................................................................................................................................
Protein purification .....................................................................................................................................................
Hydrophobic cluster analysis..................................................................................................................................85
Mapping helical interactions ...................................................................................................................................
Analytical ultracentrifugation.................................................................................................................................86
Figures - Chapter 3 ......................................................................................................................................
75
77
79
81
82
83
84
85
87
Chapter 4: A Structural Element Within The HUWE1 HECT Domain Modulates Selfand Substrate Ubiquitination A ctivities.................................................................................
95
Introduction ...................................................................................................................................................
96
Mechanism of ubiquitination m ediated by E3 HECT dom ains .................................................................
Structural characteristics of the E3 HECT dom ain ..................................................................................
Results............................................................................................................................................................100
Structure of the HUW E1 HECT dom ain ...........................................................................................................
Catalytic activity of the HECT dom ain ..............................................................................................................
Thioester form ation in the HECT dom ain.......................................................................................................
Substrate ubiquitination catalyzed by the HECT dom ain........................................................................
Catalytic activity of the C4341A m utants................................................................................................
Discussion .....................................................................................................................................................
M ethods .........................................................................................................................................................
Plasm ids .......................................................................................................
...... ............. ..... .. ...................
Bacterial protein expression and purification..............................................................................................
Circular dichroism ....................................................................................................................................................
Biochem ical assays ...................................................................................................................................................
Mcl-1 ubiquitination assay....................................................................................................................................
Thioester assay...........................................................................................................................................................
Single-turnover assay..............................................................................................................................................113
Crystallization of HUW E1 HECT dom ain...................................................................................................
Data collection and processing............................................................................................................................
Acknow ledgem ents ..................................................................................................................................................
Figures - Chapter 4 ....................................................................................................................................
97
98
100
103
105
106
. 107
107
110
110
111
111
111
112
112
113
114
114
116
Chapter 5: Conclusion ..............................................................................................................
127
Sum mary .......................................................................................................................................................
128
Future Directions .......................................................................................................................................
129
A role for Nup153 zinc fingers in nucleocytoplasm ic transport...........................................................129
The tetrameric assembly between Nic96 and the Nspl subcomplex.................................................
131
The HUW E1 HECT dom ain....................................................................................................................................
132
A balance between conformational flexibility and enzyme promiscuity..........................................132
References.........................................................................................................................................135
Figure and Table Legend
28
Figure 1. 1 - Overall structure of the Nuclear Pore Complex .........................................................................
29
Figure 1. 2 - Cryo-electron tomography of the NPC at -60 A resolution .................................................
Figure 1. 3 - Schematic representation of the modular NPC assembly.......................................................30
31
Figure 1. 4 - Inventory of the NPC ....................................................................................................................................
nucleoporins.......................................................................................................................32
Figure 1. 5 - Structures of
33
Figure 1. 6 - Ran regulates nucleocytoplasmic transport................................................................................
Figure 2. 1 - Domain architecture of Nup153.....................................................................................52
Figure 2. 2 - Alignment of the four Ran-binding zinc fingers of Nupl53 from R. norvegicus........... 52
Figure 2. 3 - Ribbon diagram representation of the Nupl53-ZnF2.RanGDP complex........................53
Figure 2.4 - Crystallographic lattice formed in the crystallization of a Nupl53-ZnFRanGDP
54
co m p lex .......................................................................................................................................................................................
Figure 2. 5 - Comparison of the four individual zinc fingers with RanGDP ................................................... 55
57
Figure 2. 6 - Conserved water network and non-conserved hydrogen bond .........................................
NuplS3-ZnF...............58
or
without
with
Figure 2. 7 - Surface representation of RanGDP / RanGTP
Figure 2. 8 - Isothermal titration calorimetry (ITC) for each individual ZnF binding RanGDP ............ 60
Figure 2. 9 - ITC illustrating low micro-molar affinity between RanGDP and two tandem ZnF pairs
62
from the Nup153- and Nup3S8-ZnF domain ..............................................................................................................
unique
to
have
known
zinc
fingers
of
class
Figure 2. 10 - Weblogos from members of the RanBP2
63
b in d in g p artn ers ......................................................................................................................................................................
Figure 2. 11 - Sequence alignment of the human Nup358/RanBP2 and Nupl53 ZnFs, including the
respective C-term inal linker regions..............................................................................................................................64
Figure 2. 12 - Nup153ZnF12.Ran crystal contains Ran and intact Nup153ZnF12.............................. 65
Table 2.1 - Crystallographic Data and Refinement Statistics................................................66
Table 2.2 - Thermodynamic parameters for ZnF-Ran binding in ITC experiments............67
Table 2.3 - Experimental constructs, abbreviations, and descriptions............................... 68
Figure 3. 1 - Domain architecture of both the Nspl and Nup62 trimeric complex...............................87
Figure 3. 2 - Hydrophobic cluster analysis of the Nup62 trimeric assembly...........................................88
Figure 3. 3 - Systematic deletion of helices from Nup57 map an interaction between NupS7 and
90
Nsp l ..............................................................................................................................................................................................
91
Figure 3. 4 - Analytical ultracentrifugation of Nsp1 / Nup62 assemblies .................................................
Nspl
trimeric
and
the
Nic96
between
full-length
assembly
tetrameric
of
a
3.
5
Purification
Figure
93
co m p lex .......................................................................................................................................................................................
94
Table 3.1 - AUC data analysis.................................................................................................
116
Figure 4. 1 - The al helix stabilizes the HUWE1 HECT domain .......................................................................
117
Figure 4.2 - Structure of the HUWE1 HECT domain ............................................................................................
HECT
domain................................................................119
of
HUEW1
activity
ligase
E3
Ubiquitin
Figure 4. 3 120
Figure 4. 4 - Detection of Ub-thioesters in the HUWE1 HECT domains .....................................................
Figure 4. 5 - Substrate ubiquitination activity of the HUWE1 HECT domains .......................................... 121
Figure 4. 6 - Ubiquitination activity of the HUWE1 HECT domains at 37*C............................................... 122
Figure 4. 7 - Both HUWE1 Aal and +al HECT domains maintain the _L conformation ........................ 123
Figure 4. 8 - Mutation of the catalytic cysteine (C4341) to alanine abolishes activity of the HECT
12 4
d o m a in ......................................................................................................................................................................................
125
Table 4.1 - Crystallographic data and refinement statistics.............................................
Acknowledgements
The work presented in this dissertation would not possible without the efforts and
dedication of several people and institutions. First and foremost I want to thank my advisor
Thomas Schwartz. Thomas has provided a seemingly limitless amount of guidance and
encouragement during the past five years. With clarity and passion Thomas has trained me as a
crystallographer and I am thankful for his direction. Prior to entering the graduate program at
MIT there were several other scientists that guided my research and instructed me over the
years. As an undergraduate at UC Davis, lacking any research experience, Clark Lagarias was
brave enough to take me into his lab and give me an independent research project. The
experience in his lab intensified my interest in biological research and for this I am grateful. Also
at UC Davis, my work with Jodi Nunnari finally convinced me to pursue a PhD and I am thankful
for her sustained guidance over the years. My short three months of work with Frank Slack at
Yale University convinced me to leave the great state of California and come to the East Coast.
Frank's patience in training me how to work with C. elegans and think about developmental
biology was remarkable. He is a fantastic teacher and research scientist and I am grateful for
his instruction.
Four months after Thomas started his lab at MIT he met three naive graduate students,
Steve Brohawn, James Whittle, and myself. Together the three of us have formed the core of
Thomas' lab and worked day after day as both friends and colleagues. In the presence of some
very gifted undergraduates, fellow grad students, and postdocs we have remained close friends.
I want to thank Steve and James for being great friends, as well as a source of sanity, insanity,
and fresh ideas. Thank you to Brian, Nina, and Silvija for being great work colleagues. Big
thanks to Sandra and T.B. for continuing to be great friends and helping during my formative
years in the Schwartz lab.
The members of my thesis committee, Amy Keating and Mike Yaffe have been a
constant source of guidance of encouragement throughout the years. I appreciate their work,
not only as thesis advisors, but also as teachers inside the classroom. I had the pleasure of
having both Amy and Mike as instructors and I want to thank them for their work and dedication.
None of this work would be possible without my family. I want to say thank you to my
brothers and sister, Tom, Vince, and Roz. Living so far away it is sometimes hard to express
how often I think about you guys. Your visits throughout the years have made me so much
happier than you can imagine. To my dad, I love you more than you will ever know. To my mom,
I want to say thank you for all of your love and support throughout the years. You are always a
source of inspiration for me and I would not have been able to finish this journey without you. To
Greg, I appreciate all that you have done in our lives and especially for showing me how to live
life to the fullest.
To my best friend and wife, Teresa. There is really no way I can adequately thank you.
You have remained my biggest supporter during our time here at MIT. You have always
believed in me, but most importantly you believed in me when I did not. Your unconditional love
has been a constant source of happiness for me and I am forever grateful.
for Cesar and Jack
Chapter 1: Introduction
Chapter 1: Introduction
A portion of the material presented in this chapter was adapted, with permission, from the
following publication:
Brohawn, S.G., Partridge, J.R., Whittle, J.R., and Schwartz, T.U. (2009). The nuclear pore complex has
entered the atomic age. Structure 17, 1156-1168.
Chapter 1: Introduction
Introduction to the Nuclear Pore Complex
The hallmark of eukaryotic cells is an elaborate endomembrane system that creates
membrane-enclosed organelles. The nucleus is a prominent organelle, as it harbors the genetic
material of the cell. The Nuclear Pore Complex (NPC) is the sole gateway into the nucleus and
perforates the nuclear envelope where the inner nuclear membrane (INM) and outer nuclear
membrane (ONM) of the nuclear envelope (NE) are fused. NPCs are among the largest
multiprotein assemblies in the quiescent cell and were first described 50 years ago by electron
microscopy (Watson, 1959). For general reviews the reader is also referred to (D'Angelo and
Hetzer, 2008; Lim et al., 2008b; Tran and Wente, 2006) and for the mechanism of NPC
assembly to (Antonin et al., 2008) and for nucleocytoplasmic transport of proteins and RNAbased molecules to (Carmody and Wente, 2009; Cook et al., 2007; Pemberton and Paschal,
2005; Stewart, 2007; Weis, 2003). The emerging role of the NPC in gene regulation and nuclear
organization is addressed in (Akhtar and Gasser, 2007; Heessen and Fornerod, 2007).
Overall structure
The first electron micrographs of the NPC showed that it forms an octagonal ring whose
central channel is less electron dense then the eight lobes that surround it. Considering overall
shape, scanning electron microscopy experiments (SEM) have recorded some of the most
stunning NPC images (Fig. 1.1). While the architectural core is grossly symmetric about the
plane of the membrane, the peripheral components on the nuclear and cytoplasmic faces are
distinct. These peripheral components recapitulate the eightfold symmetry about the transport
axis exhibited by the architectural core. On the cytoplasmic side, eight knobs, thought to be
attachment sides for fibrous extensions, are visible in NPCs from multicellular species (Kiseleva
et al., 2000). In yeast, these features are less pronounced, but are likely still present (Kiseleva
et al., 2004). On the nucleoplasmic side, a ring termed the nuclear basket is suspended from
eight filaments that join the NPC. Concerning size, the diameter of the NPC appears to be
Chapter 1: Introduction
similar in all eukaryotes, about 90-120nm (Akey and Radermacher, 1993; Beck et al., 2007;
Fahrenkrog, 2000; Hinshaw et al., 1992; Stoffler et al., 2003). However, there is still
considerable uncertainty about the height, determined to be -30-50nm (Alber et al., 2007b; Elad
et al., 2009).
Further work has led to progressively more detailed reconstructions of the NPC. Cryoelectron microscopy that relies on averaging images from many NPCs has been employed to
study the core NPC structure, the part that spans the distance between the faces of INM and
ONM (Akey and Radermacher, 1993; Hinshaw et al., 1992). These studies have shown that the
scaffold ring structure, the electron dense material near the nuclear membrane, has alternating
thicker and thinner regions, hence it is often called the spoke ring (Akey and Radermacher,
1993; Hinshaw et al., 1992). The scaffold structure appears to penetrate the pore membrane to
form a perinuclear ring structure. Using cryo-electron tomography, the best pictures of complete
NPCs have been achieved, extending even to a resolution of ~ 6 nm (Beck et al., 2007; Elad et
al., 2009). With this technique, details of the ring structures become apparent. The scaffold can
be divided into three main ring elements: a central spoke ring is sandwiched between a
cytoplasmic ring and a nucleoplasmic ring. The rings appear to float on top of one another,
indicating that material connecting them is less electron-dense than the rings themselves.
Alternatively, this may be due to technical difficulties, such as the 'missing cone' problem or
poor resolution in the Z-direction.
The central transport cavity of the nuclear pore complex shows no distinct structural
features, consistent with the perception that it is filled by an aqueous meshwork formed by
natively unfolded FG-domains, which are long polypeptide sequences found in several
nucleoporins that contain phenylalanine-glycine (FG) repeats but are otherwise hydrophilic.
These extensions are thought to form a distinct, semi-permeable environment that prevents the
Chapter 1: Introduction
diffusion of large molecules, unless they are bound to nuclear transport factors that facilitate
entry into this central cavity.
In addition to the central channel, the scaffold itself likely harbors additional, peripheral
channels. The spoke ring appears porous in cryo-EM/-ET structures, with gaps of ~ 9 nm
diameter close to the NE membrane (Hinshaw et al., 1992; Stoffler et al., 2003). Peripheral
channels have been discussed in several studies, and postulated to transport small proteins and
ions (Kramer et al., 2007). It also has been suggested that the peripheral channels transport
membrane proteins destined for the INM. These are inserted into the ER membrane following
translation and stay membrane-anchored until they reach their final destination (the ER, ONM,
and INM are all contiguous). Perhaps these membrane proteins pass the NPC into the nucleus
via these peripheral channels (Powell and Burke, 1990; Zuleger et al., 2008). The
nucleoplasmic domains of INM proteins are limited in size to ~ 40 kDa, about as large as
cavities of the observed channels.
While the general NPC architecture is well established, the cryo-EM/-ET structures do
not permit the assignment of individual proteins, since their boundaries are not visible at this
resolution. For this, higher resolution methods are required.
Modularity
A characteristic of the NPC is its high degree of modularity, which manifests itself at
several levels. First, the NPC is organized around a central eightfold rotational symmetry.
Second, only -30 nucleoporins, composed of a limited set of domain topologies, come together
to construct the NPC. Third, nucleoporins have various dwell times at the NPC, with only a
fraction being stably attached at all times. Finally, the stably attached nucleoporins are
arranged into subcomplexes, each of which assembles in multiple copies to build the entire
NPC (Fig. 1.2 and Fig. 1.3). This modularity is the basis for approaching structural determination
of the assembly at atomic resolution (Schwartz, 2005).
Chapter 1: Introduction
Protein composition
Two studies, using S. cerevisiae (Rout et al., 2000) and rat hepatocytes (Cronshaw et
al., 2002) as starting material, determined an inventory of nucleoporins. In both studies, cell
extracts were enriched for NPCs by fractionation and analyzed by mass-spectrometry to identify
the proteins purified thereby. The set of proteins found in both organisms is largely identical.
The nucleoporins can be broadly classified into three categories (Fig. 1.4). -10 contain
disordered N- and/or C-terminal regions that are rich in phenylalanine-glycine (FG) repeats.
These FG-repeat regions emanate into and form the transport barrier in the channel of the NPC.
-15 nucleoporins have distinct architectural functions and form the NPC scaffold structure.
Three nucleoporins have transmembrane domains and anchor the NPC in the circular openings
in the NE. Immunogold-labeling of all nucleoporins shows that the majority of the Nups, notably
scaffold nucleoporins, are symmetrically localized around a two-fold symmetry axis in the plane
of the NE, perpendicular to the eightfold rotational symmetry about the main transport channel
(Rout et al., 2000). Based on simple hydrodynamic and volumetric calculations the size of the
NPC was estimated to range from 66 MDa in S. cerevisiae (Rout and Blobel, 1993) to 125 MDa
in vertebrates (Reichelt et al., 1990). Calculations based on the stoichiometry of nucleoporins
obtained in the proteomic studies, however, indicate that the NPC size is only 44 MDa in S.
cerevisiae and ~ 60 MDa in rat. The discrepancy supports the conclusion that the NPC is a
porous, lattice-like assembly, rather than a solid entity (Brohawn et al., 2008; Hinshaw et al.,
1992), which accounts for the overestimate of mass based on volumetric analysis.
Dynamics
An important aspect of the NPC is that it is not a rigidly defined machine, but a rather
dynamic entity. Inverse fluorescence recovery after photobleaching experiments using GFPtagged nucleoporins showed that different parts of the NPC have drastically different residence
times (Rabut et al., 2004). Some mobile components detach from the NPC within seconds,
Chapter 1: Introduction
while other components are stable throughout the entire cell cycle. Notably, the components of
the structural scaffold - the Y-complex and the Nic96 complex - are stably attached, while FGnucleoporins are more dynamic. These studies on nucleoporin dynamics are consistent with the
very slow protein turnover of scaffold nucleoporins (D'Angelo et al., 2009; Daigle et al., 2001).
The scaffold structure of the NPC can be viewed as a docking site for more mobile
nucleoporins, which often have functional roles at sites away from the NPC (Kalverda and
Fornerod, 2007).
Domain architecture
Until about five years ago, very little high-resolution structural information on
nucleoporins was available. This was largely due to the technical difficulties of obtaining
nucleoporins of sufficient quantity and quality for structural studies, a challenge particularly
severe in the case of scaffold nucleoporins. Despite the scarcity of experimental evidence,
structural predictions grouped nucleoporins into a small set of fold classes (Berke et al., 2004;
Devos et al., 2004; Devos et al., 2006; Schwartz, 2005). First, FG-domains, the primary
transport factor interaction sites, are present in about one third of all nucleoporins. Second,
coiled-coil domains are present in a number of nucleoporins. Third, scaffold nucleoporins are
largely composed of p-propellers, a-helical domains, or a tandem combination of both. Using
this simple classification, about 76 % of the mass of the yeast NPC was accounted for.
FG-repeats
A total of 13 % of the NPC mass is made up of FG-repeat domains. The repeats are
found at the terminal ends of -10 nucleoporins and make up the NPC physical transport barrier.
NTRs specifically interact with the FG-regions, which allow them to enter the central transport
channel. How FG-repeat regions exactly form the transport barrier is vigorously investigated,
and hotly debated (Frey and Gorlich, 2007; Lim et al., 2007; Peters, 2009; Rout et al., 2003).
Chapter 1: Introduction
Systematic deletion of FG-regions from different Nups has shown that the total mass of these
filaments is more important than any one individual FG-filament, arguing for substantial
redundancy in the meshwork (Terry and Wente, 2007). The intrinsic disorder of the FGfilaments stretches is well documented in a series of crystal structures (Bayliss et al., 2000;
Fribourg et al., 2001; Grant et al., 2003; Liu and Stewart, 2005). Only short peptide stretches
are orderly bound to the convex outer surface of the HEAT-repeats that build NTRs, with the
phenylalanine side chains inserting between neighboring helices. Otherwise, the filaments
remain without structure. Little is known about the intervening, non-FG sequences. They are
poorly conserved, but are rich in polar and charged residues, important for the biophysical
properties of the transport barrier.
Coiled-coils
Coiled-coils in the NPC fulfill significant structural roles. The nuclear basket of the NPC
is mainly constructed from the large coiled-coil proteins Mlp-1/2 in yeast and Tpr in vertebrates.
Coiled-coils are often used for protein-protein interactions, thus the nuclear basket may serve as
a general recruitment platform to bring accessory factors close to the NPC. The desumoylating
enzyme Ulpl, for example, is stably associated with the nuclear basket (Li and Hochstrasser,
2000). Lining the central NPC channel are six nucleoporins containing coiled-coil regions. The
FG-Nup Nspl is part of two distinct entities, the Nspl-Nup57-Nup49 complex (Grandi et al.,
1993) and the Nspl-Nup82-Nupl59 complex (Bailer et al., 2001). In both, the proteins are held
together by coiled-coil interactions (Bailer et al., 2001) and the Nspl-Nup57-Nup49 complex is,
in addition, tethered to the NPC scaffold via the N-terminal coiled-coil region of Nic96 (Grandi et
al., 1995). So far, only a homodimerized 10 kDa fragment of Nup58 (the vertebrate orthologue
to Nup57) has been structurally characterized (Melc k et al.). Biochemical analysis suggests
that the network involves specific rather than promiscuous interactions, arguing for a specific
tethering function for the coiled-coil segments. It will be interesting to see these coiled-coil
Chapter 1: Introduction
interactions in atomic detail in order to manipulate them and potentially swap the attached FGdomains within the NPC. Such experiments could provide important insight into the organization
of the FG-network.
fl-Propellers
A large portion of the NPC scaffold is built from p-propellers, one of the most abundant
classes of proteins, especially in eukaryotes, and with diverse functions (Chaudhuri et al., 2008;
Paoli, 2001). Sets of Nups were initially identified as p-propellers based on sequence analysis.
In yeast, only Sec13 and Seh1 contain the signature WD-40 repeat motif and were among the
very first p-propellers to be recognized (Pryer et al., 1993). More Nups have since been
recognized as p-propellers despite the lack of signature sequence motifs. The N-terminal
domain of Nup1 33 was the first experimentally determined p-propeller of the NPC and after this
structure was solved, the additional non-canonical p-propeller domains in the NPC were
identified (Berke et al., 2004). To date, five of the eight universally conserved p-propellers in the
NPC are structurally characterized (Fig. 1.5). In Nup133, Nup120, and Nup159 (hNup214) the
p-propellers are N-terminal and seven-bladed. While forming a distinct entity in Nup133 and
Nupl59 (Weirich et al., 2004), physically tethered but otherwise not interacting strongly with the
C-terminal part of the protein, the p-propeller in Nupl20 is fully integrated with an adjacent
helical domain to build one continuous oblong domain (Leaks et al., 2009). Seh1 and Secl3 are
so far unique variations of p-propellers in that they are open and 6-bladed (Brohawn et al.,
2008; Debler et al., 2008; Fath et al., 2007; Hsia et al., 2007). Their partner proteins insert a
seventh blade into the p-propeller to complete the domain in trans. The function of the
p-
propellers is architectural and it is widely assumed that they serve as protein-protein interaction
sites. Peripheral p-propellers can recruit accessory proteins, like the mRNA export factor Dbp5
Chapter 1: Introduction
(von Moeller et al., 2009), whereas those more centrally located likely are used to connect
subcomplexes.
a-Helical domains
a-Helical domains make up more than half of the mass of the NPC scaffold. Structural
predictions have classified the non-coiled-coil a-helical domains into a strongly related group of
a-helical solenoids (Devos et al., 2006). a-solenoids are characterized by a two or three helix
unit that is repeatedly stacked to form an elongated, often superhelical domain with N and C
terminus at opposite ends of the molecule (Kobe and Kajava, 2000). Such regular, a-helical
repeat structures are, often in combination with p-propellers, common scaffolds in large protein
assemblies such as the clathrin vesicle coat (Edeling et al., 2006), the protein phosphatase 2A
holoenzyme (Xu et al., 2006) and the anaphase promoting complex (Herzog et al., 2009), to
name a few. Surprisingly, structural characterization of a-helical domain containing Nups has
revealed three different a-helical folds, each distinct from a regular a-solenoid arrangement
(Boehmer et al., 2008; Jeudy and Schwartz, 2007; Leksa et al., 2009; Schrader et al., 2008b).
Nic96 was the first experimentally determined a-helical structure of a scaffold nucleoporin and it
showed an unexpected, atypical a-helical topology (Jeudy and Schwartz, 2007; Schrader et al.,
2008). The 30 helices of the -65 kDa domain, excluding the -200 N-terminal coiled-coil domain,
are arranged in a J-like topology, forming an oblong domain. The chain starts in the middle of
the elongated domain, traverses up on one side of the molecule, folds back over a stretch of 7
helices and then continues past the N terminus to the other end of the molecule (Fig. 1.5).
Three other a-helical scaffold nucleoporins (Nup84, Nup85 and Nup145C) have since been
structurally characterized and shown to adopt the same fold as Nic96, pointing to a common
ancestor (Brohawn et al., 2008; see below). A second, distinct a-helical fold has been identified
in structures of Nupl33 and Nup1 70, which are more distantly related, but share an extended
Chapter 1: Introduction
and stretched a-helical stack (Boehmer et al., 2008; Whittle and Schwartz, 2009), substantially
different from the first group. The third was revealed in the structure of Nup120, which forms a
domain that fully integrates a p-propeller with an a-helical domain (Leksa et al., 2009). The ahelical segment is built around a central stalk of two long helices wrapped with 9 additional
helices in an unprecedented fashion. In summary, the a-helical domains that occur in the NPC
fall in unique classes that provide a significant challenge for structure prediction methods. One
obvious challenge is the exceedingly low sequence conservation, even between orthologs,
apparent in the inconsistent nucleoporin nomenclature. Poor sequence conservation is likely
due to some degree of malleability of the scaffold structure and the construction from common
sequence elements (Aravind et al., 2006). Whether poor sequence conservation is further the
result of adaptive evolution, linking several architectural nucleoporins to speciation, is an
intriguing possibility that should be explored in more detail (Presgraves et al., 2003; Tang and
Presgraves, 2009).
ACE1 domains
As mentioned above, the four a-helical scaffold nucleoporins Nic96, Nup145C, Nup85,
and Nup84 are constructed around a common -65 kDa domain composed of 28 helices (there
are some non-canonical additions in each of these nucleoporins). Notably, this domain has to
date only been identified outside the NPC in Sec3l, one of the main building blocks of the
COPIl vesicle coat. The commonality was surprising. Sequence conservation between the five
members is so low that no specific structural relationship was inferred previously (Alber et al.,
2007, Hsia et al., 2007). This domain, which we termed Ancestral Coatomer Element 1 (ACE1),
is a structural manifestation of the likely common origin of the NPC and the COPIl vesicle coat
(Devos et al., 2004). ACE1 is constructed from three modules, crown, trunk and tail, that
together form an elongated molecule of ~ 140 A x 45 A x 45 A. Structural superposition of ACE1
proteins shows that individual modules are closely aligned, while differences in linkers between
17
Chapter 1: Introduction
modules results in significant differences in their relative orientations. These differences, as well
as proteolytic susceptibility data, suggest at least modestly flexible hinges connect the modules,
especially the trunk and the tail. This likely explains why all crystal constructs except for Nic96
contain either the trunk and crown (Brohawn and Schwartz, 2009; Brohawn et al., 2008; Debler
et al., 2008; Hsia et al., 2007) or the tail (Boehmer et al., 2008). Even with the structural
information in hand, it is difficult to find additional ACE1 proteins. Beyond a few residues
conserved between orthologs, ACE1 is not characterized by a distinct sequence motif. The
reason for this amazing degeneracy of sequence is that for folding the ACE1 domain only some
general sequence profiles need to be satisfied. For example, helices a5, a7, al 5 and al 7 are
typically hydrophobic, because they are incased by surrounding helices and are largely buried
and solvent inaccessible. Thus, a combination of sequence profile evaluation, a-helical
prediction, and overall length are currently the only indicators for the ACE1 domain. The two
remaining a-helical scaffold nucleoporins without any crystallographic structural information are
Nupl88 and Nup1 92. Whether they also belong to the ACE1 class, remains to be determined,
but it appears unlikely.
Structural characterization of NPC subcomplexes
NPC subcomplexes
Most nucleoporins are organized into discrete subcomplexes each present in multiple
copies that arrange according to the symmetry elements of the NPC to form the complete
structure. The subcomplexes are biochemically defined and reflect the stable interaction of
subsets of nucleoporins. Interestingly, these subcomplexes are also found as entities in mitotic
extracts of higher eukaryotes, when the nuclear envelope breaks down during open mitosis
(Matsuoka et al., 1999). At the end of mitosis, NPCs reassemble from these subcomplexes in a
defined order (Dultz et al., 2008). The eight spokes are arranged around the central rotational
axis and composed of 5 subcomplexes (Fig. 1.3). Nup82/Nupl59/Nspl form a subcomplex
Chapter 1: Introduction
localized at the cytoplasmic side of the NPC (Fig. 1.3) (Belgareh et al., 1998). A second pool of
Nsp1 complexes with Nup57 and Nup49 and resides in the center of the NPC, forming the bulk
of the central transport barrier (Grandi et al., 1993). The scaffold ring is constructed from two
major subcomplexes: the heptameric Y- or Nup84-complex and the heteromeric Nic96 complex.
The Y-complex is the best-characterized subcomplex of the NPC and is essential for its
assembly, as shown in several organisms (Boehmer et al., 2003; Fabre and Hurt, 1997; Galy et
al., 2003; Harel et al., 2003; Walther et al., 2003a). It has 7 universally conserved components Nup84, Nup85, Nup1 20, Nup1 33, Nupl45C, Secl3 and Seh1 - that assemble stoichiometrically
and exhibit the eponymous Y-shape in electron micrographs (Kampmann and Blobel, 2009;
Lutzmann et al., 2002; Siniossoglou et al., 2000). In many eukaryotes, notably excluding S.
cerevisiae, three additional proteins, Nup37, Nup43, and ELYS/MEL-28, are considered
members of the Y-complex, but their architectural role is unclear (Cronshaw et al., 2002; Franz
et al., 2007; Rasala et al., 2006). In most models, the Y-complex is thought to symmetrically
localize to the cytoplasmic and the nucleoplasmic face of the NPC sandwiching the Nic96
complex. The Nic96 complex is not as well defined as the Y-complex, likely reflecting the fact
that it associates less stably. However, Nic96 interacts directly with Nup53/59 (Hawryluk-Gara
et al., 2005), and co-immunoprecipitation with Nupl88 (Nehrbass et al., 1996) and with Nupl92
have been reported (Kosova et al., 1999). Further, the Nic96 complex is the tether to the Nspl
complex in the center of the NPC. The newest defined subcomplex contains the transmembrane
Nup Ndcl, considered an anchor for the NPC in the pore membrane. This complex contains
Nup1 57/170 and Nup53/59, which connect the Ndcl complex to the Nic96 complex (Makio et
al., 2009; Onischenko et al., 2009). The other two transmembrane Nups, Pom34 and Pom1 52,
are reported to interact with Ndcl as well, albeit less strongly. Mlp1/2 are attached to the NPC
ring via Nup60 (Feuerbach et al., 2002) and likely form the nuclear basket structure (Strambiode-Castillia et al., 1999).
Chapter 1: Introduction
In vitro nucleoporinsubcomplexreassembly
The NPC exhibits an extraordinarily high level of symmetry, such that the study of
modular and biochemically distinct NPC subcomplexes offers a feasible inroad for studying the
entire NPC (Schwartz, 2005). The research described in this dissertation offers a glimpse at our
progress towards reassembly of large protein subcomplexes in vitro, followed by biophysical
and crystallographic analysis that allows for the description of binary nucleoporin interactions at
atomic resolution. Assembly of large NPC subcomplexes in vitro requires significant knowledge
of the domain architecture and secondary structural elements for every nucleoporin in question.
This information allows for the identification of minimal structural elements responsible for binary
protein interactions between nucleoporins. Our research has benefited greatly from increasingly
powerful secondary structure prediction methods that allow us to identify and methodically
probe minimal binding domains prior to recombinant protein expression and purification studies.
Purificationof NPC subcomplexes for crystallographicand biophysicalstudies
Nucleoporins (Nups) are characteristically large proteins that are typically insoluble when
recombinantly expressed in vitro. Although large and difficult to work with on an individual basis,
Nups interact via minimal binding domains that can be isolated and expressed recombinantly in
E. coli to yield milligram amounts of protein from 1 liter of liquid bacteria culture. The ability to
produce milligram amounts of soluble protein is critical for many of the biophysical and
crystallographic studies presented in this dissertation and solubility is typically improved when
working with smaller minimal binding domains rather then the entire nucleoporin. In addition,
many of these binding domains are largely insoluble unless coexpressed from a polycistronic
vector, or coexpressed from two individual plasmids. Polycistronic vectors have become a
critical tool for expression studies of large complexes and are especially useful when
components are insoluble if expressed alone (Selleck and Tan, 2008; Tan, 2001). We reason
Chapter 1: Introduction
that many of these large nucleoporin complexes are unstable or improperly folded in the
absence of appropriate subcomplex binding partners. For instance, the Nup62 / Nspl complex
described herein, solubility of the trimeric complex Nup62 / Nspl complex is significantly
improved with coexpression of all three components. Following the expression and subsequent
purification of a stable trimeric complex, biophysical and crystallographic studies have provided
a powerful means for further characterization of NPC subcomplex assembly.
The Nsp1 / Nup62 subcomplex
As with several other Nups, the Nspl complex was originally identified in extracts of
fractionated rat liver nuclei as a biochemically stable subcomplex composed primarily of three
proteins (Fig. 1.3) (Davis and Blobel, 1986; Rout and Blobel, 1993; Snow et al., 1987). The
nomenclature of Nups varies between species and the Nspl subcomplex is not spared from this
confusion. The yeast nomenclature for the three members of the Nspl complex is as follows:
Nspl, Nup57, and Nup49. In mammals these same proteins are named: Nup62, Nup54, and
Nup58 respectively. In several instances we will compare the two trimeric complexes and we
will invoke the yeast nomenclature for generalizations made between the two complexes. The
size and sequence of Nspl, Nup57, and Nup49, differ among eukaryotes, however the overall
structural characteristics remain concordant. Nspl, Nup57, and Nup49 are symmetrically
localized within the NPC as judged by immunogold electron microscopy (Rout et al., 2000).
Each member of the Nspl complex serves as an essential component for both function and
structure of the NPC, as determined with deletions and point-mutants (Grandi et al., 1995; Hurt,
1988; Wente et al., 1992), as well as being essential for the formation of a functional minimal
NPC structure (Strawn et al., 2004). Additional in vitro experiments, such as the
immunodepletion of the Nspl complex during reconstitution of the NPC with rat nuclei and
Xenopus egg extracts, disrupts transport of NLS tagged cargo and even the formation of NPCs
Chapter 1: Introduction
as revealed by scanning EM (Finlay et al., 1991; Mutvei et al., 1992). In addition to the three
major components of the Nsp1 complex a fourth protein, Nic96 (yeast) / Nup93 (metazoan), is
present as a partially stable component of the Nspl complex (Grandi et al., 1993; Grandi et al.,
1995; Guan et al., 1995). It is believed that Nic96 serves as a bridging molecule, linking
together the heterotrimeric Nspl complex with the more structurally significant five-membered
Nic96 subcomplex.
The Nspl complex occupies the central channel of the NPC and plays a key functional
role in the transport of cargo across the nuclear membrane (Fahrenkrog et al., 1998). Nsp1 is
also known to interact with Nup82 and Nup1 59 on the cytoplasmic face of the NPC as a
separate subcomplex (Belgareh et al., 1998), an interaction that was not part of this thesis.
Domain architecture of each component from the Nspl complex maintains a conserved
arrangement of secondary structure consisting of a coiled-coiled domain, approximately 200
residues in length, flanked by fiber-like extensions of unstructured amino acid sequence
containing Phe-Gly-rich (FG) domains (Hu et al., 1996; Schwarz-Herion et al., 2007). FG-repeat
domains are found in a total of -11 different Nups and remarkably account for almost 13% of
NPC's total mass to fill the central channel of the NPC (Brohawn et al., 2009). It is well
established that FG-repeats are the primary interaction sites between the NPC and nuclear
transport receptors (NTRs) that shuttle cargo during nucleocytoplasmic transport (Denning et
al., 2003; Isgro and Schulten, 2007a, b; Macara, 2001; Peters, 2005). The coiled-coil domains
from each member of the Nspl complex interact to form a trimeric assembly that lines the innerchannel of the NPC, while the FG-repeat regions are largely unstructured and as such are
nonessential for formation of the Nspl complex (Fig. 1.4) (Bailer et al., 2001; Finlay and Forbes,
1990; Hu et al., 1996; Strawn et al., 2004). This large trimeric coiled-coil assembly serves as a
scaffold to support the FG-repeat domains and fuse these functional elements to the core NPC
scaffold.
Chapter 1: Introduction
Although the core of the NPC is a rigid scaffold built for structural integrity, the NPC is at
least in part also a dynamic macromolecular machine. Evidence is emerging to suggest that the
cytoplasmic ring, the nuclear basket, and the luminal spoke ring of NPC undergo significant
conformational rearrangements (Beck et al., 2007). Aside from itself being a dynamic and
flexible machine, some nucleoporins are dynamic and shuttle on and off the NPC (Rabut et al.,
2004). Moreover, following breakdown of the nuclear envelope during cell division in metazoa,
the NPC disassembles into the discrete subcomplexes mentioned above, and is believed to
reassemble in a step-wise manner at the completion of mitosis (Dultz et al., 2008; Matsuoka et
al., 1999). Little is known of the binary interactions between nucleoporins that facilitate
assembly between subcomplexes. Nic96 is a large helical nucleoporin that is believed to be the
"linker" nucleoporin that connects the Nic96 complex with the Nspl complex (Fig. 1.3) (Grandi
et al., 1993). Nic96 contains a coiled-coil domain at the N-terminus that facilitates a binary
interaction with Nspl to bridge together the Nic96 and Nspl complexes to form a tetrameric
coiled-coil assembly. The study of such a large coiled-coil assembly not only offers promise
towards a better understanding of NPC subcomplex assembly, but offers a great deal of insight
towards understanding how large coiled-coil interactions maintain specificity.
The interaction between Nup153 and the small GTPase Ran
Nup153 is a versatile nucleoporin
Although many nucleoporins share common structural domains, Nup153 is one protein
that defies many of these structural classifications. Nup1 53 is an essential protein (Galy et al.,
2003; Harborth et al., 2001) that is found in higher order eukaryotes. Its domain topology is
roughly conserved between species, but distinct differences are observed (Dimaano et al.,
2001; Shah and Forbes, 1998; Sukegawa and Blobel, 1993). Curiously, Nup1 53 is absent in
single cell eukaryotes, including Saccharomyces cerevisiae and Saccharomyces pombe,,
arguing for a specific role in metazoa. Aside from a direct role in regulating transport Nupl53 is
Chapter 1: Introduction
important in coordinating disassembly and reformation of the NPC (Favreau et al., 1996;
Walther et al., 2003b). Current data suggests that Nup1 53 is primarily localized to the
nucleoplasmic side of the NPC and that it does interact directly with the NPC scaffold (Boehmer
et al., 2003; Fahrenkrog et al., 2002; Krull et al., 2004; Pante et al., 2000; Sukegawa and Blobel,
1993).
The unstructured N-terminal domain of Nup1 53 can be divided into 3 smaller subdomains based on function and known interaction partners (Fig. 2.1). The extreme N-terminus
of human Nup1 53, residues 1-144, is known as the nuclear envelope targeting domain (NETD)
and contains a predicted amphipathic helix that directs Nup1 53 to the nuclear envelope
(Enarson et al., 1998). The nuclear pore association region, located between residues 39-339,
is responsible for targeting Nupl53 to the NPC structural scaffold via the Nupl07 complex
(Vasu et al., 2001; Walther et al., 2003a; Walther et al., 2001). Overlapping partially with the
nuclear pore association region is the RNA binding domain between residues 250-400 (Ball et
al., 2007; Bastos et al., 1996; Dimaano et al., 2001). Similar to roughly a third of all nups,,
Nupl53 contains an FG-repeat domain at the C-terminus (residues 881-1475) and thus plays a
direct role in the transport of cargo through the NPC.
RanBP2-type zinc fingers in the eukaryoticcell
Our primary focus for this study was the protein binding zinc finger domain located in the
middle of Nup153 between residues 650-880,. The domain of the human protein consists of four
C2-C2 zinc fingers that bind the small GTPase Ran (Sukegawa and Blobel, 1993). This protein
binding zinc finger domain is found in only two Nups, Nup153 and Nup358, although Nup358
harbors eight zinc fingers instead of four (Wu et al., 1995; Yokoyama et al., 1995). The Ran
binding zinc fingers found in Nupl53 and Nup358 (Nup358 is also known as RanBP2) have
previously been referred to as the RanBP2-type of protein binding zinc fingers, and more
generally fall within the family of Npl4 type zinc fingers (NZF) (Meyer et al., 2002; Plambeck et
Chapter 1: Introduction
al., 2003; Wang et al., 2003). RanBP2-type/NZF zinc fingers conform to a consensus sequence
pattern: W-X-C-X(2,4)-C-X(3)-N-X(6)-C-X(2)-C (Fig. 2.2). Structural studies have described the
overall fold of the RanBP2/NZF class of zinc fingers as two orthogonal p-hairpin loops with two
cysteines each to coordinate a single zinc ion in the central zinc finger core. In addition to four
highly conserved cysteine residues, an absolutely conserved tryptophan residue stabilizes a
hydrophobic core while an absolutely conserved asparagine residue bridges the two hairpins
together (Higa et al., 2007; Plambeck et al., 2003; Yu et al., 2006). Additional studies provide
structural evidence for a diverse set of binding partners associated with the RanBP2 class of
zinc fingers, detailing interactions made with ubiquitin, RanGDP, and RNA (Alam et al., 2004;
Partridge and Schwartz, 2009; Schrader et al., 2008a). Specifically, the mammalian nuclear
protein localization 4 (Np14) protein contains a single RanBP2-type zinc finger that binds
ubiquitin (Meyer et al., 2002), and with ubiquitin fusion degradation 1 (Ufdl), Npl4 forms an
adaptor complex for the AAA ATPase p97 (known as Cdc48 in yeast). Together the complex
between Npl4 and Ufd1 performs several processes, including nuclear envelope closure,
regulated ubiquitin dependent processing, endoplasmic reticulum-associated degradation, and
mitotic spindle disassembly (Cao et al., 2003; Hetzer et al., 2001). Similar to Nup1 53, the yeast
homologue of Npl4 does not contain a ubiquitin binding zinc finger (Meyer et al., 2002; Ye et al.,
2003). Aside from Npl4 other NZF containing proteins bind ubiquitin, including: Vps36 (Alam et
al., 2004), and Tab2/Tab3 (Kanayama et al., 2004). Crystallographic data demonstrates that
ubiquitin and RanGDP share the same binding interface on NZF/RanBP2-type zinc finger
molecules (PDB codes: 1Q5W, 3GJ3, 3CH5) from Npl4 and Nupl53 respectively (Alam et al.,
2004; Schrader et al., 2008a), and it is clear that ubiquitin does not bind with Nup1 53/Nup358
zinc fingers (Higa et al., 2007). These studies clearly demonstrate that only two or three
residues from each zinc finger module, a domain that is only twenty residues long, mediate
ligand specificity. Another protein with a RanBP2-type zinc finger is ZRANB2 and was recently
Chapter 1: Introduction
shown to bind single-stranded RNA in a manner unique from zinc finger interactions described
in structures with Npl4 and Nup153 (Loughlin et al., 2009). Binding between ZRANB2 and
single-stranded RNA (ssRNA) occurs on the side of the zinc finger module, away from the
"knuckle" where both ubiquitin and RanGDP bind. A tryptophan stacking interaction exists
whereby a highly conserved tryptophan side chain from ZRANB2 is stacked between two bases
of target ssRNA sequence. The authors speculate that this particular zinc finger module may
play some role in splicing, as the recognition motif strongly resembles a 5' splice site (Loughlin
et al., 2009), although the details of ZRANB2's role in the splicing mechanism are yet to be
determined. Together this data highlights the significant diversity of the RanBP2-type zinc
finger motif and confirms the relevance of small, modular protein binding zinc fingers in multiple
processes inside the eukaryotic cell.
Structuraland biophysicalanalysis of Nup153 zinc fingers and RanGDP
The master regulator of nucleocytoplasmic transport through the NPC is the small
GTPase Ran. Ran, a member of the Ras superfamily, is a small protein, only 216 amino acids
long and 24.4 kDa in weight, but is one-hundred percent conserved throughout eukaryotes and
essential for regulation of mitosis and nucleocytoplasmic transport (Bischoff and Ponstingl,
1991 a, b; Drivas et al., 1990; Melchior et al., 1993; Moore and Blobel, 1993). Transport
between the nucleus and the cytoplasm occurs through an energy dependent process and the
NPC acts as a physical barrier to block cytoplasmic proteins from entering the nucleus.
However, following translation in the cytoplasm nuclear proteins are actively imported through
the NPC and into the nucleus. This dynamic process is regulated by a concentration gradient of
Ran between the nucleus and the cytoplasm, with a high concentration of RanGTP inside the
nucleus and a high concentration of RanGDP in the cytoplasm. This gradient is regulated by the
asymmetric localization of two regulator proteins, RanGAP and RCC1 (Fig. 1.6).
Chapter 1: Introduction
RanBP2-type zinc fingers from both Nup1 53 and Nup358 interact directly with Ran,
however the details are uncertain. As a canonical small GTPase, Ran has two switch regions
that adopt a significant conformational change when bound to either GDP or GTP (Stewart et
al., 1998). RanGTP hydrolysis at the cytoplasmic face of the NPC releases export cargo from
the ternary cargo-exportin-RanGTP complex, while RanGTP at the nuclear face of the NPC
releases import cargo from the binary cargo-importin complex (Gorlich and Kutay, 1999). This
well established gradient of RanGTP versus RanGDP across the nuclear envelope provides the
energy for nucleocytplasmic protein transport. Since both Nup153 and Nup358 are each
preferentially positioned at either side of the NPC, we asked whether they might influence the
Ran gradient by selectively binding to Ran in one or the other nucleotide-bound state. The
existing literature on the selectivity of RanBP2-type zinc fingers is controversial (Higa et al.,
2007; Nakielny et al., 1999; Yaseen and Blobel, 1999). In this dissertation we present a series
of crystallographic and biophysical experiments detailing the interaction between Nup1 53,
Nup358, and Ran.
Chapter 1: Introduction
Figures - Chapter 1
a
Xenopus
laevis
Drosophila Sachwomyces
melanoaaster
cerevisiae
cytoplasmic side
nucleoplasmic side
b
side view
cytoplasmic view
Figure 1. 1 - Overall structure of the Nuclear Pore Complex
(a) Representative micrographs of NPCs from diverse eukaryotes and obtained by scanning electron
microscopy. The distinct surface features that define cytoplasmic and nucleoplasmic face of the NPC are
conserved, so are the overall dimensions in the plane of the nuclear envelope. Scale bar indicates 100nm.
(b) Cryo-electron tomographic (cryo-ET) reconstruction of the human NPC. The nuclear basket structure
and the cytoplasmic extensions are omitted for clarity. The central eightfold rotational symmetry is
clearly visible. A comparison between cryo-ET reconstructions of NPCs from diverse species (not shown)
reveals substantial differences in the overall height (Elad et al., 2009).
Chapter 1: Introduction
cytoplasmic
filaments
100 nm
50 nm
structural
scaffold
30 nm
nuclear
basket
90 nm
Figure 1.2 - Cryo-electron tomography of the NPC at ~60 A resolution
Cut away view of the NPC (colored in brown/beige) embedded in the nuclear envelope (gray). The three
major domains of the NPC, the cytoplasmic filaments, the structural scaffold, and the nuclear basket, are
each clearly distinguishable. This view highlights the eight-fold rational symmetry exhibited by the NPC
and four distinct sections are shown in this orientation. To better highlight this, one of the repeating
sections has been marked with a black dotted line (Beck et al., 2007).
Chapter1: Introduction
Cytoplasm
Nup42)~
Nsp1 Nup159
Nup82
Sec13
m34
Nup2l4 Nup62
Nup120
Nup133Nu33
Nup84
Nup145C
Nup5
Nup12(
Nup188
Nup157/170
NIc96
om152 Nup59/53
Ndcl
)Ndc
Seh1
Nup120
Nup133
Nup84
Sec13
ELYS
Nuplee
NUplO7 NUP37
Nup98 Sect3
Nup75 5
Nsp1
Nup62 Nup188
Nu85q~pl60Nup43Nu6
Nup49
Nup58 Nup155
Nup57
Nup54
Nup93C
C
poml2l
210
Nup145C
Nupu5
NNucleoplasm
Figure 1. 3 - Schematic representation of the modular NPC assembly
The NPC is built from -30 nucleoporins, organized in a small set of defined subeomplexes. This cartoon
shows the major subcomplexes that make up the lattice-like scaffold (blue), the membrane-attachment
(green), and the FG-network (gray) of the NPC. S. cerevisiae components on the left, metazoan with
specific additional components on the right. A few peripheral Nups are left out for clarity. Simplified
representation and connections are not to be taken literally.
-
. .....................
- - -----------
Chapter 1: Introduction
Nucleoponn
cytosasmc
homolog
Nup192
Nup188
Nup205
Nup188
Nic96
Nupl57/170
Nup53/5
Nup93
Nupl55
Nup35
l6
Nup133
Nup160
Nup107
e
0
Nup85
Nup85
0
Nup145C
Nup96
e
Seh1
Seh1 '
-:
-35%
a
e0
0
20%
m
e
e
u
Sec13 40
Nup37
Nup43
Mip1
MIp2
Nup2
Nup1
Nup6O
total NPC
1 MDa uctured
Mo unstucturaO
0 0
0 0
eW
0000
000
e0 0
Nup120
Nup84
Sec13
=
m
ernosasmec
Nup133
Fraction of
Mass per NPC
Abundance
Domain Architecture
Metazoan
Tpr
Tpr
1 1
Nup50 (ZIDIEII
mm
-
e
0
12%
-
=0
e
Nup153
0
0
Pom152
Ndcl
Pom34
Nsp1
Nup49
Ndc1 (
-C~)-
gpero
Pom121
Nup62
Nup58
Nup54
Nup57
Nup159 Nup214/CAN
O-*=
9%
.
e
0000
e* e
11
m
IIIDUEDIII
*
-
Rae1/Gle2
Nup98 Ce11 HM
I1
Nup98 M
Nup98 dillM
10%
e
7%
a
e e0
0
(hll(ACE1)
Calculated Composition of the NPC from Scereisiae
ue
Wcotd coO
am0 doman
'
0
e
1llilli
C) p-rope
a
mamm
0
0
-11
Gle1
Glel
Nup42 NLP1IhCG1 (0-111111 D
Nup88
Nup82
Nup358/RanBP2
ALADIN
Rae1/Gle2
Nup145N
NuplOO
Nup116
0
0
EM
11.3
6.5
J
6,3
5.3
l
]
2O 1.311
59
66
total
46 MDa
doman
trans
memrn
Figure 1. 4 - Inventory of the NPC
Summary of the nucleoporins that make up the NPC. Domain architecture of nucleoporins from S.
cerevisiae as determined by x-ray crystallography or prediction (where structural information is still
lacking). Abundance and derived mass calculations are based on published Nup/NPC stoichiometries
(Cronshaw et al., 2002; Rout et al., 2000). Nucleoporins specific to metazoa are italicized.
Chapter 1: Introduction
ilk
NIc96
PDB Code: 2QX5, 2RFO
1 18
839
Nup85-Sehl
3EWE, 3F3F
Nup85 1
Seht
1I
hNup1O7-hNup133
314R, 3C0
hNup1O7 1
744
658925
658
101156
M
517
hNup133 11
349
Nup170
315P, 3150
502
79
1
L~rr
~y~I
yNup145C-hSec13
3BG1
yNup145 11
hSec13
7
1317
741 1158
Nup120
3HXR
1
757
hNup133
1XKS
1037
161156
67
Nupl59 (hNup214)
1XIP, 201T
1
514
37
387
11460
1K1322
316
rNup58
20SZ
1
mNup35
1WWH
1[X1
325
585~ss
327-411
NsPl 11
IrmB
1076
974-1012
3 861
1E
156-261
677
920
1[1325
327-411
Nspl-FGS-Importin
2BPT
hNup98
1K06, 2Q5X/Y
156-261
P
rNup153-ZnF-RanGDP
3CH5, 3GJ3-8
rNup153 1
rRan
11502
691-909
1 E216
hNup358-RanBD.RanGDP
1RRP
hNup358
han
11
/103224
216
1171-1304
Figure 1. 5 - Structures of nucleoporins
Comprehensive list of all representative nucleoporin structures published up until July 2009. PDB
accession codes are indicated. Structures are gradient-colored red- or blue-to-white from N to C terminus.
Residue information for each crystallized fragment is given below the structure. Structures are shown in
the assembly state that is supported by crystallographic and biochemical evidence. Structures are from S.
cerevisiae unless noted otherwise (h, human; m, mouse; r, rat). 2QX5(Jeudy and Schwartz, 2007);
........................
Chapter 1: Introduction
2RFO(Schrader et al., 2008b); 3EWE(Brohawn et al., 2008); 3F3F(Debler et al., 2008); 3CQC(Boehmer
et al., 2008); 314R, 315P, 315Q (Whittle and Schwartz, 2009), 3BG1(Hsia et al., 2007); 3HXR(Leksa et
al., 2009); 1XKS(Berke et al., 2004); 1XIP(Weirich et al., 2004); 20IT(Napetschnig et al., 2007);
20SZ(Melcdk et al.); 1WWH(Handa et al., 2006); 1K06(Hodel et al., 2002); 2Q5X/Y(Sun and Guo,
2008); 2BPT(Liu and Stewart, 2005); 3CH5(Schrader et al., 2008a); 3GJ3-8(Partridge and Schwartz,
2009); 1RRP(Vetter et al., 1999).
CRM1
mRnportin-a
Cytopytoplasm
NTF
k"P~rtRanGAP1
Importin-a
GTPr
Nucleus
Export
R
D
r importini
CRMi
Importin-a
Figure 1. 6 - Ran regulates nucleocytoplasmic transport
Ran is the master regulator of nucleocytoplasmic transport. RanGDP is recycled from the cytoplasm and
into the nucleus by NTF2. In the nucleus RCC1I acts as a nucleotide exchange factor to reload Ran with
GTP. In the nucleus RanGTP causes disruption of imported complexes between importins and cargo
containing an NILS. RanGTP interacts directly with importin-P to stimulate this process. Binding of
RanGTP with the exportin CRM1 promotes the assembly of an export complex formed between CRM1
and cargo containing a NES. In the cytoplasm, RanGTP interacts with the Ran GTPase activating protein
(RanGAP) and RanBP2 or RanBP1 to stimulate hydrolysis of GTP and dissociate the exportin complex.
RanGDP, importins, and exportins are all recycled through the NPC and the cycle begins again. Image
taken from (Clarke and Zhang, 2008).
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
Chapter 2: Crystallographic and Biochemical Analysis of the
Ran-Binding Zinc Finger Domain
The material presented in this chapter was adapted, with permission, from the following
publication:
Partridge JR, Schwartz TU. Crystallographic and biochemical analysis of the Ran-binding zinc
finger domain. J. Mol. Biol. 2009, v391, 375-89.
Experimental contributions: James R. Partridge conducted all experiments.
Chapter 2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
Introduction
Nucleocytoplasmic transport is controlled and facilitated by protein assemblies termed
nuclear pore complexes (NPCs). NPCs reside in circular openings of the nuclear envelope
where inner and outer nuclear membranes are fused. The NPC is a large macromolecular
assembly with a calculated mass of -50 MDa (Alber et al., 2007b). Based on electronmicroscopy (EM) studies from X. laevis oocytes and S. cerevisiae, the general shape and
structure of the pore is conserved across eukaryotes (Kiseleva et al., 2004; Kiseleva et al.,
2001). These EM studies define the pore as a ring embedded in the nuclear envelope,
exhibiting 8-fold rotational symmetry around a central axis and imperfect 2-fold symmetry
between the cytoplasmic and nucleoplasmic faces. Despite its size, the NPC is only made up of
-30 proteins, or nucleoporins (Nups), arranged in a few biochemically defined subcomplexes
that assemble the entire structure in a modular fashion (Schwartz, 2005). As the single
constitutive barrier to regulate permeability, the NPC transports a wide range of substrates
across the double membrane of the nuclear envelope (D'Angelo and Hetzer, 2008; Tran and
Wente, 2006; Weis, 2003). Active transport through the NPC is mediated by nuclear transport
receptors (NTRs), also called karyopherins or importins/exportins (Chook and Blobel, 2001;
Cook et al., 2007).
The small G protein Ran is the master regulator of NTR-mediated, nucleocytoplasmic
protein transport (Gorlich and Kutay, 1999). Ran selectively promotes binding or release of
import or export cargos to NTRs by means of a chemical gradient. Ran binds mostly GDP in the
cytoplasm, and mostly GTP in the nucleus. The GTPase-activating protein RanGAP, localized
to the cytoplasmic face of the NPC, and the chromatin-bound GTP exchange factor, RCC1,
together promote this asymmetry by modulating nucleotide hydrolysis and exchange
respectively. The established gradient provides directionality to protein transport. In the nucleus,
RanGTP releases import-cargo from NTRs by competitive binding. RanGTP is recycled back to
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
the cytoplasm via a trimeric complex formed with NTRs and export-cargo. At the cytoplasmic
face of the NPC, RanGTP interacts with RanGAP to hydrolyze GTP and disrupt the trimeric
NTR-mediated export complex. NTF2 (nuclear transport factor 2) recycles RanGDP back into
the nucleus (Stewart, 1998).
Nup153 and Nup358 (RanBP2) are large, metazoan-specific nucleoporins with multiple
roles (Ball and Ullman, 2005; Shah and Forbes, 1998; Sukegawa and Blobel, 1993; Wu et al.,
1995; Yokoyama et al., 1995). Both interact with Ran through a zinc finger cassette composed
of several individual zinc finger (ZnF) motifs (Fig. 2.1) (Nakielny et al., 1999; Yaseen and Blobel,
1999). Nup1 53 is predominantly localized to the nuclear face of the NPC, although recent
studies suggest the three major domains of Nup1 53 are localized to different regions of the NPC
(Fig. 2.1) (Fahrenkrog et al., 2002; Krull et al., 2004; Pante et al., 1994; Walther et al., 2001).
The N-terminal portion contains a pore-targeting region and an RNA binding domain (Bastos et
al., 1996; Boehmer et al., 2003; Dimaano et al., 2001; Ullman et al., 1999; Walther et al., 2001).
The ZnF cassette harbors multiple zinc fingers and defines the center of Nup1 53. The Cterminal region of Nup1 53 harbors -30 phenylalanine-glycine (FG-) repeats, unstructured motifs
found in several Nups lining the inner channel of the NPC that are responsible for NTR
interaction (Denning et al., 2003; Ribbeck and Gorlich, 2001, 2002; Shah et al., 1998). Nup358
has several characterized domains including a cyclophilin homology domain, a SUMO ligase
domain, a structural leucine-rich region, previously characterized Ran binding domains
(RanBDs), and a cassette containing multiple zinc fingers (Joseph et al., 2004; Pichler et al.,
2002; Salina et al., 2003; Wu et al., 1995; Yokoyama et al., 1995). The ZnFs of Nup153 and
Nup358 are representative of the "RanBP2-type" ZnF family, recognized by the conserved
sequence pattern W-X-C-X(2,4)-C-X(3)-N-X(6)-C-X(2)-C (Fig. 2.2)(Hulo et al., 2006). RanBP2type zinc fingers fold into a structure composed of two p-hairpin strands that sandwich a Zn2
ion coordinated with four cysteine residues (Gamsjaeger et al., 2007). The RanBP2-type zinc
Chapter 2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
finger structure is distinct from other zinc fingers, however it only defines a common scaffold,
not a common function. Ran binding has been reported for Nupl53 and Nup358 zinc fingers
and not for the other structural homologs (Alam et al., 2004; Higa et al., 2007). It is unclear what
role these Ran-binding zinc fingers play at the NPC, nor has it been conclusively analyzed
whether they bind Ran in a nucleotide dependent or independent manner (Higa et al., 2007;
Schrader et al., 2008a).
We have determined the crystal structures of all four ZnFs of Nup1 53 in complex with
RanGDP. Our structural data suggests that all ZnF modules preferentially bind to RanGDP
rather than RanGTP, supported by mutational and microcalorimetric data. While the primary
sequence of the ZnF in the cassette is conserved, the number of ZnFs varies among species.
Our data supports a largely uncooperative model for binding of Ran to the individual ZnF
modules within Nup1 53 or Nup358, explaining why the exact number of consecutive ZnFs is not
conserved. Although we detect differences between ZnF binding with RanGDP versus with
RanGTP, they are moderate and may not be of functional consequence. We propose that the
ZnFs within Nupl53 and Nup358 are primarily used to create a 'Ran sink' and thereby increase
the local concentration of Ran at both the nucleoplasmic and cytoplasmic face of the NPC.
Results and Discussion
Crystallographic analysis of Nup153-ZnFeRanGDP complexes
All protein constructs used in this study were from Rattus norvegicus, except Nup358
from Homo sapiens. The central region of Nup1 53 (residues 658 - 885) contains four zinc
fingers; ZnF1 (residues 658 - 686), ZnF2 (residues 723 - 750), ZnF3 (residues 790 - 817), and
ZnF4 (residues 848 - 885). The individual ZnF domains were cloned and recombinantly
expressed as glutathione-S-transferase fusion proteins in Escherichia coi. In addition to
individual ZnF domains, the tandem pairs of ZnF1 and ZnF2 (ZnF12, residues 658 - 750) as
Chapter 2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
well as ZnF3 and ZnF4 (ZnF34, residues 790 - 885) were examined (see Table 2.3 for all
protein constructs used in this study). Full-length Ran was cloned and expressed as a His-tag
fusion protein in E. coli. RanGDP was separated from RanGTP using ion-exchange
chromatography and the nucleotide-loaded state was validated with HPLC. Our RanGDP
structure is solved at 1.48 A resolution with two molecules in the asymmetric unit. Molecule A is
well ordered with all residues in both switch regions defined. However, in molecule B a portion
of the switch 11region (residues 69 - 74) is disordered. In molecule A, a van der Waals
interaction between Phe77 and the neighboring molecule B orders switch II. Without this
packing interaction and in the absence of the gamma-phosphate from GTP switch II is flexible.
Diffraction quality crystals of a Nup1 53-ZnF in complex with RanGDP were initially
obtained for ZnF2, ZnF4, ZnF12, and ZnF34, but only the ZnF2 complex crystals diffracted
satisfactorily. Well-diffracting crystals of all other complexes were obtained after introducing a
structure-based, surface point mutation in Ran, F35S, to stabilize a crystal contact as described
below. Despite significant effort, no crystals were obtained for any ZnF construct in complex
with RanGTP (in the form of the GTPase deficient mutant, RanQ69L).
Overall structure of the ZnF*RanGDP complex
Each Nupl53-ZnF and RanGDP bind with a 1:1 stoichiometry. ZnF-bound RanGDP is
nearly identical to unbound RanGDP with an rmsd of 0.90 A and 0.40 A when compared to two
RanGDP structures (Protein Data Bank (PDB) codes 1BYU (Stewart et al., 1998) and 3GJO
(this study) respectively). The bound ZnF also maintains a structure similar to the unbound ZnF
(rmsd 0.77 A compared to PDB code 2GQE (Higa et al., 2007)). Four short p-strands form two
orthogonal hairpins flanking the hydrophobic core containing the strictly conserved Trp7ZnF
residue (for ZnF residue numbering scheme, see Fig. 2.2). A single Zn2 ion is sandwiched
between the two p-hairpins, and coordinated by four cysteine residues (Cys9, Cysl2, Cys23,
and Cys26). The side chain amide of Asn16 is highly conserved in all RanBP2-type zinc
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
fingers, suggesting the hydrogen bond formed between Asn16 and the backbone amide group
of residue 24 helps maintain the RanBP2-type ZnF fold. Nupl53-ZnF binds to RanGDP at a
region neighboring and partially encompassing switch I, residues 28 to 48 (residues 65 to 84
define the switch II region, Fig. 2.3) (Vetter et al., 1999). The binding interface measures 469 A2
with major hydrophobic interactions made with Ran structural elements P1, P3, and a4, as well
as electrostatic interactions with the N-terminus and switch I region of RanGDP. Hydrophobic
interactions mediated by the "LVA" motif of Nup1 53 ZnFs, have been highlighted in previous
biochemical and structural studies (Higa et al., 2007; Schrader et al., 2008a). Leu13Nup15 3
Val14Nup15 3 , and Ala25Nup1 53 from Nupl53-ZnF2 interact with RanGDP between switch I and
strand P1 of RanGDP, with some interaction at the switch 11region. Leul3Nup15 3 is situated in a
hydrophobic pocket formed by the aliphatic carbon chain of Lys38Ran, Val47Ran, and Pro49Ran.
Val14Nup153 interacts primarily with Trp64Ran, Lysl2Ran, and with the carbon chain of Gln82Ran.
Ala25Nup 15 3 forms hydrophobic interactions with lle81 Ran, and Trp64Ran, and Leu43Ran. Critical
hydrogen bonds include the backbone carbonyl group of residue
8 Nup153
binding with the side
chain Lys38Ran, and the backbone carbonyl of Cys26Nup15 3 , binding with the side chain of
Thr42Ran. Both Ran residues are found in the switch I region, and are known to undergo
significant rearrangement in the RanGTP conformation. To summarize, the Nup1 53ZnF2-RanGDP structure is principally stabilized by hydrophobic interactions with a constant
region of Ran, and two hydrogen bonds with the switch I region of Ran.
Our 1.8 A Nupl53-ZnF2eRanGDP structure superimposes well with the 2.1 A crystal
structure from Schrader et al., solved in an unrelated space group (rmsd 0.55 A compared to
PDB code 3CH5 (Schrader et al., 2008a)). However, the alternative crystal-packing observed in
this study results in a significantly different interpretation of parts of the ZnF*RanGDP interface.
The contact between Phe 7 14Nupl 5 3 -ZnF2 and a hydrophobic pocket of Ran near P1 and 14, has
been suggested to be a significant contact between Nup1 53-ZnF2 and RanGDP (Schrader et
Chapter 2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
al., 2008a). In our Nupl53-ZnF2-RanGDP structure, Phe72Ran from a symmetry-related Ran
molecule occupies the hydrophobic pocket in question. The ZnF2 construct used in our study
does not include residues 703-722, N-terminal to ZnF2 as used in Schrader et al., but is
representative of the ZnF2 construct used to determine the unbound Nup1 53-ZnF2 NMR
structure (Higa et al., 2007). In addition, the constructs and solved structures of ZnF12 and
ZnF34 do include this region, yet do not bind to the hydrophobic pocket as observed in the
study by Schrader et al.. Our structural data, in accordance with our binding data, suggests the
linker region between the ZnF domains has a rather small contribution in ZnF binding with
RanGDP.
Engineering improved crystal contacts
The structures of RanGDP in complex with ZnF1, ZnF3, ZnF4, ZnF12 and ZnF34,
respectively, were solved at high resolution only after re-engineering a crystal contact involving
Ran, to improve crystal packing. In the initial orthorhombic Nupl53-ZnF2-RanGDP crystals, the
packing interaction was very weak along one crystallographic axis, where a single contact point
was observed. We reasoned that this sub-optimal packing might be the cause for weak
diffraction, high temperature factors, and high mosaicity initially observed (data not shown). The
weak contact is formed by a Ran-Ran interaction, involving a single hydrogen-bond and a small,
strained vdW interface between Phe35Ran and Pro 5 8 Ran' (Ran' denotes the symmetry mate) (Fig.
2.4). Several studies have shown that site-directed surface mutagenesis can be used to
improve crystal quality (Banatao et al., 2006; Cooper et al., 2007; Mizutani et al., 2008). In our
case, we reasoned that changing Phe35Ran to serine would reduce the energetic penalty of a
clash between Phe35Ran and Pro58Ran', and be compatible with the hydrophilic character of the
neighboring His53Ran'. In similar fashion Phe35Ran was mutated to aspartic acid in hopes of
forming an additional H-bond with His53Ran'. Although the RanF35D mutation proved unable to
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc Finger Domain
crystallize, the RanF35S point mutation crystallized and improved the diffraction limit of ZnF4,
ZnF12, and ZnF34 crystals by 1 A, and enabled crystallization of ZnF1 and ZnF3.
The modified interface between neighboring RanF35S molecules generates a stronger
network of hydrogen bonds to stabilize the Nupl53-ZnF-RanGDP crystal lattice, and although
Ser35Ran is just out of range for making an H-bond with His 5 3Ran, the predominant contact
remains between Thr32Ran and His53Ran'. By removing Phe35Ran we have alleviated the clash
with neighboring Pro58Ran' and stabilized the critical hydrogen bond between Thr32Ran and
His53Ran'. The overall structure of Nup1 53-ZnF-RanGDP is unchanged by the F35S mutation,
but the packing of complexes with RanF35S results in slightly different cell dimensions and
orientations of symmetry axes (reducing in some crystal forms the orthorhombic to a monoclinic
space group with a non-crystallographic symmetry axis).
Comparison of the four Nup153 zinc fingers bound to RanGDP
The RanF35S mutant enabled us to individually crystallize all four ZnF modules of
Nupl53 with Ran, allowing for a comprehensive analysis of the interaction. Data collection and
refinement statistics for all constructs are listed in Table 1. Crystals of RanGDP alone were
obtained and the structure refined to 1.48 A (Rwork/Rfree = 18.0%/20.3%). Each of the ZnF
structures in complex with RanGDP contains one Nupl53-ZnF molecule bound to one molecule
of RanGDP (Fig. 2.5a). The orientations of each Nup1 53-ZnF with respect to RanGDP are
similar in the various structures (Table 2.4).
We originally reported that ZnF1 has the highest deviation (mean rmsd value of 1.07 A)
from the other ZnFs, concomitant with a very high B factor of -200 A2 (Partridge and Schwartz,
2009). In the meantime we found a better crystal, judged by higher resolution limit (2.4 vs. 2.7
A) and better Rsym (6.5 v. 12.3). Importantly, the new crystal is also better ordered (judged by
lower mosaicity (0.32. vs. 0.85)and lower Wilson B-factor (53). The ZnF domain is better
resolved, but no major novelties are observed.
Chapter 2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc Finger Domain
In addition to conserved hydrophobic interactions, the backbone carbonyl of residue
1
ZnF
and the side chain of Lys 3 8 Ran, as well as the backbone carbonyl of Cys26ZnF and the
hydroxyl group of Thr42Ran form two conserved H-bonds (Fig. 2.5a-c). A water network,
identically observed in each of the four individual ZnF*RanGDP structures, further stabilizes the
ZnF.Ran interaction by mediating hydrogen bonds (Fig. 2.6).
Nucleotide-dependent changes in the conformation of Ran occur at the switch I and
switch 11regions (Vetter et al., 1999). When bound to GDP, Ran is in the open conformation
with switch I swung out, away from the nucleotide and closer to P1 (Fig. 2.3). Upon binding of
GTP, the two switch regions close to accommodate the y-phosphate. As highlighted in Fig. 2.7,
residues of Ran critical for hydrogen bonding with Nup1 53-ZnF shift away from the ZnF binding
site when Ran is in the GTP-bound conformation. Superimposing RanGTP (PDB code 1WA5)
and our RanGDP structure bound to ZnFs, shows that Lys38Ran is shifted 26 A away from the
ZnF binding site when Ran is bound to GTP. Thr42Ran is buried in the RanGTP structure to
interact with the magnesium ion and thus the H-bond with ZnF cannot be maintained. To
highlight the influence of the conformational shift between RanGDP/RanGTP and binding of
ZnFs, we have modeled a putative Nupl53-ZnF2-RanGTP complex, replacing RanGDP in our
structure with RanGTP (PDB code 1WA5) (Fig. 2.7) (Matsuura and Stewart, 2004). In this
modeled complex, the calculated binding interface area is reduced by 27% to 345 A2 . H-bonds
to switch I residues are not only lost, but in the case of Thr42Ran, are mutually exclusive between
the RanGDP and RanGTP conformations, suggesting a mechanism for preferential binding of
ZnF to RanGDP over RanGTP.
Apart from these interactions in the switch I region, some interactions remain unique to
the individual ZnFs. The principle distinction between the binding modes of the Nup1 53-ZnFs is
the ability or inability to form a H-bond with Gin1ORan. Both Gln8ZnF1 and GIu8ZnF3 H-bond via the
side chain carbonyl, while Asp 8ZnF 2 and Asp 8 ZnF 4 do not, because the shorter aspartic acid side
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
chain does not reach far enough (Fig. 2.6). This single hydrogen bond accounts in large part for
the differences in binding affinity measured by ITC as described below and in Table 2.2.
Protein constructs containing tandem ZnF pairs (ZnF12, ZnF34) have been crystallized
in the same condition as the individual zinc finger domains. The electron density map for each
crystal shows only one ZnF bound to RanGDP in agreement with the structural interactions
described above for single ZnF constructs. In these structures, predominantly the second ZnF
(ZnF2 and ZnF4) is seen to bind with RanF35SGDP, even though, considering the crystal
packing, ZnF1 and ZnF3 could be accommodated as well. Since ZnF2 and ZnF4 tentatively
bind weaker to RanGDP than ZnF1 and ZnF3 (see below), we would expect that binding of the
N-terminal ZnF should be preferred. We conclude that the crystal packing favors binding of the
C-terminal ZnF in the tandem constructs. Since a second zinc finger is not visible in the electron
density we confirmed that the tandem Nup1 53-ZnF1 2 polypeptide is intact based on SDS-PAGE
analysis of the Nupl53-ZnF12-RanGDP crystals (Fig. 2.12). We also analyzed the anomalous
signal from Zn at the measured (high-energy remote) wavelength of 0.98 A (Zn K-edge 1.2837
A), but could only make out the signal of the Zn atom in the bound ZnF (data not shown). To
distinguish which ZnF was bound, both possibilities were modeled and refined. R factors were
consistently lower for the C-terminal ZnF and the difference density showed reduced noise (not
shown). Interestingly, and in contrast to the study by Schrader et al., we do not observe any
interaction of the linker residues between ZnF1 and ZnF2 with Ran.
Isothermal titrationcalorimetry
To expand upon our crystallographic analysis of the Nup1 53-ZnF interaction with
RanGDP, we performed binding assays using isothermal titration calorimetry (ITC). These
experiments demonstrate that individual Nupl53-ZnF domains bind to RanGDP with modestly
varying affinity (Table 2.2). ZnF1 / ZnF3 have comparable binding characteristics that differ from
those measured for ZnF2 / ZnF4. ZnF1 is shown to bind RanGDP with a measured KD of
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
6.5 pM and ZnF3 binds with a KD of 6.6 pM, in an exothermic reaction. ZnF2 / ZnF4 have a
lower binding affinity for RanGDP, with 49 pM and 47 pM respectively, in an endothermic
reaction. The enthalpic differences in RanGDP binding of these ZnFs is likely due to
conformational contributions, thus not readily explainable based on our structures. The variation
in enthalpy between sites is also measured with the ZnF pairs and the full Nupl53 ZnF domain
as described below and in Table 2.2.
Structural data suggests that the missing hydrogen bond between GIn1 0 RanGDP and
residue
8ZnF
may be responsible for this differences. In ZnF2 / ZnF4 Asp8 is too short to H-bond
with Gln1ORanGDP, in contrast to Glu/Gln8 in ZnF1 / ZnF3 which forms this bond (Fig. 2.6). When
residue
8 ZnF
was mutated to a glutamine in ZnF2 and ZnF4, the binding affinity for RanGDP
increased to values very close to those measured for ZnF1 and ZnF3 (Fig. 2.8a, 6b, 6c, 6d).
Nup1 53 was also tested to compare the differences in affinity for RanGDP vs. RanGTP (in the
form of the GTPase deficient mutant, RanQ69L) (Fig. 2.8e). We next asked whether individual
ZnF domains exhibit an allosteric effect on their neighbors. Pairs of ZnFs from Nup1 53 and
Nup358 (Nupl53-ZnF12, Nupl53-ZnF34, and Nup358-ZnF12) were assayed for RanGDP
binding (Fig. 2.9). A two-site model best fit the data in each experiment with Nup1 53. The two
binding sites in the tandem constructs exhibit affinities that are similar to the values measured
for the individual ZnFs, indicating no significant allosteric effect. The data also suggests that the
flexible linker between ZnF domains does not significantly contribute to binding affinity for
Nup1 53-ZnF2, as our data measured with and without the linker agree with previously
measured affinity (Schrader et al., 2008a). Additionally, the binding of Nup358-ZnF12 to
RanGDP was measured to characterize ZnFs of Nup358 in comparison with those of Nup1 53.
Based on the high level of sequence conservation between Nupl53 and Nup358 (Fig. 2.10), it is
not surprising to find that ZnFs from Nup358 behave similarly. The ITC data for Nup358-ZnF12
is best fit to a single site model, with two molecules of RanGDP binding per molecule of
Chapter 2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
Nup358-ZnF1 2. This corresponds to each ZnF binding independently, similar to Nup1 53,
however differs from Nup1 53 because both ZnFs of Nup358 lack the Glu/Gln8 residue needed
to H-bond with Gln1ORanGDP. To summarize the data from our calorimetric experiments, we
compared binding of the entire ZnF regions of Nup1 53 to RanGDP or RanQ69LGTP (Fig 2.8).
Nup153 binds RanGDP in a 2-site model with affinities of 1 pM and 8 pM, with two molecules of
RanGDP binding per site. This accounts for each ZnF binding with RanGDP independently for
a total of four molecules of RanGDP binding with the ZnF domain of Nup1 53, in agreement with
published data (Higa et al., 2007). RanQ69LGTP binds with lower affinity to Nup1 53, with one
site showing negligible binding and the other exhibiting a KD of 20 pM in an independent 2-site
model. The Nup358-ZnF cassette binds both GDP- and GTP-bound Ran, and again, the
interaction with RanGDP is measurably stronger (Fig. 2.9).
Conservation of RanBP2-type ZnF cassettes in nucleoporins
We have performed a phylogenetic analysis to put our structural and biochemical data in
an evolutionary context. The RanBP2-type ZnF domain can be readily recognized by its
characteristic signature sequence motif (Fig. 2.2). The ZnF domain is found in all Nup153 and
Nup358 homologs, which are exclusively present in animals, but absent in plants and fungi.
RanBP2-type ZnF cassettes usually contain 4 repeats in Nup1 53, and 8 repeats in Nup358.
Exceptions are however not infrequent. The number in sequenced Nupl53 homologs varies
between 2 and 6, while in Nup358 we found between 2 and 8 ZnFs (data not shown). These
variations match our observation that the ZnF modules function largely independently and not in
a cooperative fashion. Similarly, we fail to find a specific signature that would define an order in
which the ZnF modules are arranged within the ZnF region. We also asked whether there is a
distinction between ZnFs present in Nup153 compared to those in Nup358. Given the
asymmetric distribution of Nup1 53 and Nup358 at the nucleoplasmic and cytoplasmic side of
the NPC, respectively, one could hypothesize that the ZnFs exhibit locus-specific tasks.
45
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
However, we do not find such differences. The sequence conservation of the Ran-binding ZnF
matches well among structurally and functionally important residues. The structurally important
residues Trp7, Cys9, Cys12, Asn16, Cys23 and Cys26 are strictly conserved (Fig. 2.10). The
residues involved in hydrophobic interactions with Ran Leu13, Val14, and Ala25, are also quite
well conserved across all animals and the functional significance has been recognized even
before structural data was available (Higa et al., 2007). Comparing just the linker between
individual ZnFs shows no sequence conservation, consistent with it not having a significant role
in protein-protein interactions.
Comparison of bindinginteractionsamong RanBP2-type zinc fingers
RanBP2-type zinc fingers are recognized by the conserved sequence W-X(2)-C-X(3)-NX(6)-C-X(2)-C as shown in Fig. 2.2. This sequence signature only defines the structural
scaffold, but excludes the residues important for binding interactions. Currently three different
binding partners are known. The Nupl53/Nup358 class binds Ran, the Npl4 class binds
ubiquitin and the ZRANB2 class binds single-stranded RNA (ssRNA) (Schrader et al., 2008a;
Wang et al., 2003). Phylogenetic analysis of these RanBP2-ZnF classes readily reveals that
each type of ZnF has additional conserved residues (Fig. 2.10). For Nupl53/Nup358, these are
the discussed residues Leu13, Val14 and Ala25 (Higa et al., 2007). In the Npl4-type, the same
ZnF surface binds ubiquitin involving the conserved residues Thrl3, Phel4 and Met25. By
switching these residues the Nup153 ZnF can be converted into a ubiquitin-binding moiety (Higa
et al., 2007). The ssRNA-binding splicing factor ZRANB2 is the best conserved of the three
known RanBP2-ZnF classes. The publication of the ssRNA-bound ZRANB2 structure (PDB
code 3G9Y) details ssRNA bound at a different surface of the scaffold via the highly conserved
residues between residues 14 and 20 (Loughlin et al., 2009). Considering the sequence
signature of the Nup1 53/Nup358-ZnF class it is unlikely that it can bind nucleic acids, as was
reported when Nupl53 was first described (Sukegawa and Blobel, 1993). In summary, the
Chapter 2: CrystallographicandBiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
RanBP2-type zinc finger emerges as a scaffold for various binding partners and it will be
interesting to learn about the interacting partners of the additional, less characterized classes,
including Sharpin, Mdm2, and Mdm4 (Lim et al., 2001; Yu et al., 2006).
Discussion
Here, we present a comprehensive analysis of the interaction of the Nup1 53/Nup358
ZnF class with Ran. We identify individual residues within the four ZnFs of mammalian Nup1 53
that modulate the binding affinity to Ran. The G protein is preferentially bound in the GDPbound form, because switch I residues are involved in critical hydrogen bond interactions with
ZnF and because switch I contributes about one-third of the total interaction surface.
Nonetheless, binding to RanGTP is also observed and the local RanGTP to RanGDP ratio likely
determines the nucleotide-binding state of ZnF-bound Ran. We cannot detect a significant
difference between the binding behavior of Nup358 and that of Nup1 53 fingers, arguing for a
common function for both proteins. Neither Nup358 nor Nup153 are universally conserved
nucleoporins, but are specific to animals. We suggest that the ZnF moieties are used to
increase the local concentration of Ran at both the nucleoplasmic and cytoplasmic face of the
NPC. This may accelerate nucleocytoplasmic protein transport by keeping Ran close to the
NPC, a sophistication that may be dispensable in unicellular eukaryotes, or potentially replaced
by a separate mechanism, as proposed for plants (Meier et al., 2008). Our phylogenetic
analysis suggests that other RanBP2-type ZnFs do not bind Ran, since they share only the
structurally important residues and use distinct binding interfaces for their respective protein and
nucleic acid interactions.
Materials and Methods
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
Protein purification
Bacterial expression constructs for Nup1 53 domains from Rattus norvegicus and
Nup358 from Homo sapiens were cloned as glutathione-S-transferase (GST) fusion proteins in
the pGEX-6P1 vector (GE Healthcare) (Table 2.1). Ran was expressed as a 6xHis-tag fusion
protein from a pET-28a vector, engineered to contain a protease 3C site after the N-terminal
affinity tag. All proteins were expressed in Escherichia coli strain BL21 (DE3) RIL (Stratagene).
Bacterial cell pellets harboring GST fusion proteins were suspended in 20 mM
potassium phosphate pH 7.0, 150 mM NaCl, 2 mM DTT and lysed using a french press. The
crude lysate was supplemented with 200 pM phenylmethanesulfonyl fluoride (PMSF) and
centrifuged at 15 000 g for 15 minutes. Soluble protein was mixed with 0.5ml of glutathione
Sepharose beads (4 Fast Flow, GE Healthcare) per 1000 OD of cells for 2 hrs at 277 K. After
three batch washes in resuspension buffer, resin was washed in ZnCl 2 buffer (10 mM Tris/HCI
pH 8.0, 100 mM NaCl, 10 pM ZnCl 2, 2 mM DTT), and Nup153 proteins were eluted directly from
the resin by incubating with protease overnight at 277 K. The eluted protein was purified by
anion exchange chromatography on a HiTrapQ column (GE Healthcare) via a linear NaCl
gradient and size exclusion chromatography using a Superdex S75 26/60 column (GE
Healthcare) run in 15 mM Tris/HCI pH 8.0, 150 mM NaCl 1 mM MgCl 2 , 5 pM ZnCl 2, 1.5 mM
DTT. All truncations of the full-length Nup153-ZnF domain, as well as point mutations, were
generated with PCR mutagenesis and purified as described above.
Bacterial cell pellets harboring His-tagged Ran were suspended in 20 mM Tris/HCI
pH 8.0, 200 mM NaCl, 5 mM imidazole, 3 mM p-mercaptoethanol (p-ME), and lysed using a
french press. Crude lysate was supplemented with 200 pM phenylmethanesulfonyl fluoride and
clarified by centrifugation at 15 000 g for 15 minutes. The soluble fraction was then incubated
with 1ml Ni-NTA per 1000 ODs for 1 hour at 277 K and loaded onto a disposable column
(Pierce). The column was washed with 4 bed volumes of 10 mM Tris/HCI pH 8.0, 400 mM
NaCl, 10 mM imidazole, 3 mM p-ME, and eluted with 6 bed volumes of 10 mM Tris/HCI pH 8.0,
Chapter 2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
50 mM NaCl, 150 mM imidazole, 3 mM p-ME. Eluted protein was dialyzed against 10 mM
Tris/HCI pH 8.0, 100 mM NaCl, 1 mM DTT, for 1 hour before the 6xHis-tag was cleaved.
RanGDP was separated from RanGTP using anion exchange chromatography on a HiTrapQ
column (GE Healthcare) via a linear NaCI gradient, followed by purification with size exclusion
chromatography using a Superdex S200 26/60 column (GE Healthcare) in 15 mM Tris/HCI pH
8.0, 150 mM NaCl 1 mM MgCl 2, 5 pM ZnC12, 1.5 mM DTT. RanQ69L and RanF35S were
generated with PCR mutagenesis and purified as described above. The nucleotide-bound state
of Ran was confirmed by HPLC, on a analytical C18 column. The nucleotide was released from
Ran by heat denaturation at 369 K and centrifugation to separate coagulated protein from
soluble nucleotide. The soluble fraction was loaded on a HPLC and GTP was separated from
GDP with a linear acetonitrile gradient in 50 mM triethanolamine/HCI pH 7.5, 10 mM
tetrabutylammonium-sulfamate (TBA).
Crystallization
Ran was concentrated to 15 mg/ml using Vivaspin 20 concentrators (Sartorius) and
mixed with Nup1 53-ZnF protein at equimolar concentrations. All Nup1 53-ZnF fragments in
complex with RanGDP were crystallized in the same condition (0.1 M BisTris/HCI pH 6.5, 1820% PEG3350) using the hanging-drop method and mixing 1 pL protein with 1 pL of reservoir
solution at 291 K. Crystals of wt-RanGDP in complex with ZnF2 grew in 5-7 days forming
birefringent plates of 400 x 200 x 30 pm. RanGDP crystals formed on the edges of Nup153RanGDP complex crystals after two weeks, as small 150 x 150 x 150 pm bipyramidal crystals.
The crystallization of tandem zinc finger constructs Nup1 53ZnF1 2 and Nup1 53ZnF34 was
confirmed by fishing crystals from crystallization drops, washing the crystals with mother liquor,
dissolving the crystals in SDS loading buffer, and analyzing protein composition of the crystal
with SDS-PAGE. Crystals of NupI 53 ZnF constructs in complex with RanF35S-GDP grew in 1-2
days. All crystals were cryoprotected by adding 12% (v/v) glycerol to the crystallization solution
Chapter2: Crystallographicand Biochemical Analysis of the Ran-Binding Zinc Finger Domain
before flash freezing in liquid nitrogen. All datasets were collected at the NE-CAT beamlines
241D-C and 241D-E at Argonne National Laboratory. Crystal parameters and data collection
statistics are listed in Table 2.1.
Structure determination
Data reduction was carried out using HKL2000 (Minor, 1997). The initial Nup1 53ZnF2-RanGDP structure was solved by molecular replacement, using RanGDP as the search
model (PDB code 1BYU). All other structures were solved by molecular replacement using our
1.48 A RanGDP structure as the search model. Model building was carried out using COOT
(Emsley and Cowtan, 2004). PHENIX (Adams et al., 2002) was used for all refinement steps.
For the tandem ZnF constructs only one zinc finger domain was visible. The second ZnF is
present in each tandem ZnF construct as determined by analysis of the difference maps and
refinement statistics with the respective ZnF sequences. Proper assignment of the correct ZnF
correlates with better refinement statistics, judged by lower R and Rfree values. Data collection
and refinement statistics are summarized in Table 2.1. All figures were made using PyMOL
(DeLano, 2002).
Isothermal titration calorimetry
RanGDP and all ZnF constructs were purified and dialyzed into the same buffer
containing 15 mM Tris/HCI pH 8.0, 150 mM NaCl 1 mM MgCl 2 , 5 pM ZnC12, 1.5 mM DTT, and
either 2.5 pM GDP or 2.5 pM GTP as indicated, prior to ITC. Protein concentrations were
determined spectrophotometrically at 280 nm immediately before the experiment. ITC was
performed using a VP-ITC microcalorimeter (MicroCal, Northhampton, MA). Titrations were
performed at 293 K, or 278 K, by injecting 14 pL aliquots of RanGDP, or RanQ69LGTP (Table
2.2), into the ITC cell containing 1.43 ml of Nup1 53 or Nup358 ZnF proteins (Table 2.2).
Binding stoichiometry, enthalpy and entropy, as well as the equilibrium binding dissociation
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
constant was determined using a "one site model" for individual zinc fingers and Nup358 ZnF1 2,
or a "two sets of independent sites" model for all other experiments, to define molecular
association in the software suite, MicroCal Origin 2.9 (MicroCal).
Sequence analysis
Sequences were aligned using MUSCLE (Edgar, 2004) and edited in JaIVIEW
(Waterhouse et al., 2009). Sequence logos were made using WebLogo (Crooks et al., 2004;
Schneider and Stephens, 1990).
Protein Data Bank accession numbers
Coordinates and structure factors for all crystal structures have been deposited in the
PDB (IDs 3GJO, 3GJ3, 3GJ4, 3GJ5, 3GJ6, 3GJ7, 3GJ8).
Acknowledgements
We thank the staff at beamlines 24-ID-C/-E at Argonne National Laboratory and X6A at
the National Synchrotron Light Source for assistance and data collection, past and present
members of the Schwartz lab for discussions and editing, especially T. Boehmer for early
experimental advice and constructs. We would like to thank Katie Ullman for the kind gift of
Nup358. The Biophysical Instrumentation Facility for the Study of Complex Macromolecular
Systems (NSF-0070319 and NIH-GM68762) provided instrumentation. Supported by a Pew
Scholarship (T.U.S.) and the Herman Eisen Fellowship in Biology (J.R.P.).
...........
I
Chapter2: CrystallographicandBiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
Figures - Chapter 2
Zn Finger Domain
NETD
1
1475
880
850
Figure 2. 1 - Domain architecture of Nup153
Nup153 is a large nucleoporin with a diverse set of functions. The N-terminal domain between residues 1
and 650 contains three functional regions: the nuclear envelope targeting domain (NETD), the nuclear
pore associating region and the RNA binding domain. At the C-terminus is a large unstructured FG-repeat
domain between residues 880 and 1475. Between residues 650 and 880 is a Ran-binding zinc finger
domain consisting of four individual RanBP2-type zinc finger modules. Each zinc finger module contains
30 residues and between each module is an unstructured stretch of-35 residues.
1
5
Nupl53 ZnF1 656 LOAGS
PAl
Nup153 ZnF2 719
Nup153 ZnF3 787
PV
Nupl53 ZnF4 844
PE
10
T
T
PV
15
20
LT
N
AV
S
30
25
I
V
PL
S
I
ST
E C
W-x-C-x-x-C-x-x-x-N-x-x-x-x-x-x-C-x-x-C
TP
L
T
Ran-binding Zinc Finger
Figure 2. 2 - Alignment of the four Ran-binding zinc fingers of Nup153 from R. norvegicus
The numbering used for individual zinc fingers is listed above. Identical residues are highlighted in dark
blue, with decreasing levels of conservation highlighted in lighter shades of blue. Below is the consensus
sequence for the"RanBP2" family of zinc finger proteins.
Chapter 2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
Nupl53
Zinc Finger
s3
N
Cys9
N
Cys23
Zn 2
2
Cys1 2
"RanGDP
Figure 2. 3 - Ribbon diagram representation of the Nup153-ZnF2-RanGDP complex
RanGDP f-sheets are colored dark blue with a-helices colored light blue. The Nupl53-ZnF molecule is
colored orange. Both the switch I and switch II regions of Ran are highlighted. Representative of the
"RanBP2" family of protein-binding zinc fingers, the Nup153 ZnF contains two orthogonal p-hairpins
with four cysteine residues, colored yellow, to coordinate a single Zn 2+ ion colored grey. The zinc finger
binds near the switch I region of RanGDP, indicated here by the secondary structural element $2. The
GDP nucleotide is shown in the center of Ran with a bound Mg 2+ ion.
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
Nup1 53-ZnF-RanGDP Crystal Lattice
Figure 2. 4 - Crystallographic lattice formed in the crystallization of a NupI53ZnF-RanGDP complex
Residues highlighted in red indicate contacts made between neighboring Ran molecules down a single
plane of the P21 lattice. This plane represents the smallest crystallographic interface and was chosen for
crystal engineering to stabilize packing, in hopes of improving diffraction. The enlarged 3D image on the
right depicts residues binding at the interface. Thr32' and His53 make a single hydrogen bond, mirrored
across a 2-fold symmetrical face as indicated. A clash between Phe35' and Pro58, though tolerated, was
theorized to partially counteract the strong interaction between Thr32' and His53. A point-mutation of
Phe35' to Serine stabilizes the crystal contact and increases the resolution of our crystallographic
experiments by 1 A.
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
Nup1 53-Zinc Fingers
Overlay
Conserved H-Bond
w/ Thr42
\
Conserved H-Bond
w/ Lys38 -
F97 Variable H-Bond
w/ GIn1O""
.
N
Conserved Hydrophobic
Interactions
RanGDP
b
VaflIe2a
Cys26z~'
Leuiy3aI
Pro49
Thr420"TrO
pos10OM
Lys38"
Conserved Hypdrophobic Interactions
Figure 2. 5 - Comparison of the four individual zinc fingers with RanGDP
Zinc fingers (ZnF), colored orange, in complex with RanGDP, colored blue. The four individual ZnFs
are overlaid to highlight differences and similarities in binding with Ran. Residues that make
intermolecular hydrogen bonds are colored in green, while residues that facilitate hydrophobic
Chapter 2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
interactions are colored violet. (a) A overview of the Nup153-ZnFoRanGDP complex showing that each
ZnF binds independently and at the same location with RanGDP. Hydrogen bonds conserved among the
four ZnFs are labeled. The variable hydrogen bond at residue 8 ZnF with Gln1ORan is only formed between
Ran and ZnF 1 or ZnF3. (b) An enlarged view of all residues responsible for the hydrophobic interaction
at the Nup153-ZnFeRanGDP interface. (c) An enlarged view of hydrogen bonds, in green, that facilitate
the Nupl53-ZnFeRanGDP interaction. Conserved H-bonds are made between the carbonyl of Cys26ZnF
with the side chain hydroxyl group of Thr42Ran, and Lys3 8 Ran with the carbonyl at residue 10ZnF. A
variable H-bond is present at residue 8 ZnF binding with Gln1ORan. A stable bond is formed between
Gln8ZnF1 and GlnORan , as well as between Glu8ZnF3 and Glnl0Ran. Residue 8 of ZnF2 and ZnF4, colored
dark-blue, contain an Asp residue, too short to make the H-bond with Gln1ORan.
Chapter2: CrystallographicandBiochemical Analysis of the Ran-Binding Zinc FingerDomain
Nup1 53-ZnF-RanGDP
b
Thr42R
-
Asp8z02
oo
*
[b& Asp8-F
W
Lys38Ran
/
Gin82hF1
& Glu82nF3
Gin10F"
I,
[N',
4
4
4
Nup153-ZnF.RanGDP
Figure 2. 6 - Conserved water network and non-conserved hydrogen bond
(a) Conserved water network at the interface between Nup1 53-ZnF and RanGDP. Residues facilitating
these contacts are labeled and highlighted in green, with water molecules colored violet. (b) A more
detailed view of the non-conserved hydrogen bond made with GlnORan by Gln8z 1 and Glu8ZnF3 , colored
green. Asp 8ZnF 4 and Asp 8ZF4, colored blue, are unable to bridge the distance necessary to make the Hbond with Gln10Ra, a calculated distance of-5.0 A.
57
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
a
Lys388""
/01-
ZnF
ZThr42""
Nup153-ZnF-RanGDP
b
,.Lys38-i"
Thr42 "
RanGDP
Lys38a
Thr42 "f
RanGTP
Figure 2. 7 - Surface representation of RanGDP I RanGTP with or without Nupl53-ZnF
RanGDP or RanGTP, grey, with or without Nupl53-ZnF,blue ribbon-diagram. Residues involved in
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
intermolecular contact are colored orange. (a) ZnF*RanGDP complex. Lys38Ran and Thr42Ran, located
in the switch I region, form conserved H-bonds with the ZnF, and are, known to undergo significant
conformational changes upon binding of GTP. Both residues are positioned to interact with ZnF in this
conformation. (b) RanGDP interaction surface in the absence of ZnF. (c) RanGTP in absence of modeled
ZnF. Lys38Ran and Thr42Ran are displaced up to 30 A, and are no longer able to interact with the
Nup153-ZnF in this conformation.
Chapter 2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
>
(a)-0 0 1020 30 40---50 (mn
007 000 IO110 120
-1b 0 10 20 30 40 50
00?0
90 100110120
-10 0 10 20
30
40 50
00
70 0 SO100110120
05
I11~
2+RanGDP
-- NuptI 52
RanGDP
DOQ+
-Ntpl S3ZnF2
03
-020
--
- -ft-r-ON p 3Z l
02
3201 + RanGDP
up1S3ZnF1Q08 + RanGDP
-- Nupl 5
--
9
0I1
00
I-048
12
42
0*
04:
-o
Z00
1.2
14N-
I0
#
Molar Rado
Molar Rato
(d)
0
N-I
Tne (m)
10 20 30 40 50 O 70 80
0
O110120
(e)
-10
Mohr ReIo
Tune (m)
0
10 20
30
40
50 OD 10 8D 0o
045
040
-- upIt 53Znf4+ RanGDP
-Nup1532nF4000 +RanGP.
036
030
02
0.0
025
010
102
010
04
-006
0
-0-10
-Nup 1532f1234 + RanGDP
-Np153ZnF1234
+ RanQ69L
'0
1.
Mhdet SOO~ Sft
K.
45
00 05
10
Is
20 25
Moi Ralo
N,-
ZI4
14,. 2
K2
K,*20 jid
#
30 35
-
d5000501520253035404,550556065
Molr Rato
Figure 2. 8 - Isothermal titration calorimetry (ITC) for each individual ZnF binding
RanGDP
Point-mutations are used to probe the variable H-bond interaction with Gln10R", determined to be the
interaction responsible for discrepancies in affinities measured between ZnFs and RanGDP. Data for
independent ZnFs is fit to a single site model with N=1. (a) WT-ZnF1 is shown in red compared with
mutated ZnF1 in black. Mutating Gln8 to Asp is shown to have a minor effect on binding with Gln1O'a
(b) WT-ZnF2 is shown in red with the mutant protein shown in black. WT-ZnF2 has low affinity binding
measured at 47 pM, however by mutating Asp8 to Gln high affinity binding is measured at 4 piM. Note
that the two titrations were performed at different temperatures, 278K (red, WT) 293K (black, mutant).
No signal was recorded for WT at room temperature (data not shown). (c) WT-ZnF3 is shown in red and
mutant ZnF3 is shown in black. Mutating Glu8 to Asp is enough to decreased the measured affinity by
disrupting a single H-bond with Gln1O'. (d) WT-ZnF4 is shown in red and has a measured binding
constant of 47 pM. However, mutating Asp6 to Gln introduces a new H-bond with Gln1ORa and we
subsequently measure higher affinity binding. Note that the two titrations were performed at different
temperatures, 278K (red, WT) 293K (black, mutant). No signal was recorded for WT at room temperature
(data not shown). (e) Binding of the WT Nup153-ZnF domain to GDP (in red) is compared to binding
Chapter2: Crystallographicand Biochemical Analysis of the Ran-Binding Zinc FingerDomain
with RanGTP (in black). In agreement with our structural observations, RanGDP binds ZnFs with strong
and weak sites, while RanGTP binds with the same stoichiometry, but only with weak affinity.
Experimental values for N, Kd, enthalpy, AH, and entropy, TAS, are listed in Table 2.2. Constructs are
described in Table 2.3.
Chapter2: Crystallographicand Biochemical Analysis of the Ran-Binding Zinc FingerDomain
(a)
T
-10 0
*'e
(min)
10 20 30 4D 50 60 70
01
80 90
100 110
Ti"m (mi)
(1
0 10 20 30 40 50 80 70 80 90 100 110
10
0 10*
00
0 06
-01
o00
-(02
Nup15 n 1
I
025
-06
-0301
-07
-036.
-10
0
Ti'e(mn)9
10 20 3D 40 50 Do 70 9D 90
100110
1005U111
-10
03
(c)
-NuPISOZnF34.960009
0.
ModelTwoSte
1
ModelTwoSite
N,- 1
N,=I
N,= 1
K,= 57 pM
Ke=59 pM
K,=4 pM
5000
510
1 20 2!>3 0 354 4 5 55
Molar Ratio
00
0
1M0
15
20
Molar Ratio
2
30
0
2
4
6
A
10
12
Molar Ratio
Figure 2. 9 - ITC illustrating low micro-molar affinity between RanGDP and two tandem
ZnF pairs from the Nup153- and Nup358-ZnF domain
(a) Interaction between RanGDP and ZnF 12. The data is best fit to a two-site model suggesting each ZnF
binds independently, but with different affinities. (b) Interaction between ZnF34 and RanGDP, again
suggesting two independent sites with different affinities. (c) Interaction between Nup3 58-ZnF 12 and
RanGDP. As with Nup153, the ZnFs of Nup358 bind with low pM affinity to RanGDP. The data is best
fit to a single site model in accordance with both ZnFs from Nup358 lacking the residue necessary at pos8
to H-bond with Gln1ORan. Experimental values for N, Kd, enthalpy, AH, and entropy, TAS, are listed in
Table 2.2. Constructs are described in Table 2.3.
62
Chapter 2: Crystallographicand Biochemical Analysis of the Ran-Binding Zinc FingerDomain
a
Nupl53 and Nup358
4
30
b
1SLC~X~VJLL_ 1cA 5?.P
10
N 5
15
20
25
C
15
20
25
C
Np4
4
31
a
10
N 5
C ZRANB2
430
1K
N5
W11CNCVWALcICL P
10
15
20
25
c
iny
w.bbgo bufty
Figure 2. 10 - Weblogos from members of the RanBP2 class of zinc fingers known to
have unique binding partners
The logos highlight similarities and between the three groups of zinc fingers: the Ran binding
Nupl53/Nup358 group, the ubiquitin binding Npl4 group, and the single-stranded RNA binding
ZRANB2 group. The logos demonstrate an overall conservation of the canonical RanBP2 type zinc
finger sequence W-X-C-X(2,4)-C-X(3)-N-X(6)-C-X(2)-C, while highlighting regions important for
facilitating binding with a unique binding partner. (a) ZnFs from Nup153 and Nup358 that bind with Ran.
Only the first finger from each protein has been used in the alignment. The previously described "LVA,"
in positions 13, 14, and 25, have low information content, although position 25 has been shown influence
Ran binding (Higa et al., 2007). (b) The logo for Npl4 again demonstrates the elevated conservation of
positions 13, 14, and 15, previously shown to modulate interaction with ubiquitin. (c) The ZRANB2 logo
highlights the strong conservation this sequence in eukaryotes. A structure of ZRANB2 (PDB codes
3G9Y and 2KlP), demonstrates the strict conservation and the preponderance of positive charges at
positions 14-20 that regulate binding of single-stranded RNA (Loughlin et al., 2009). Each logo is based
on alignments of sequences from a diverse group of eukaryotes.
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
a
Nup358ZnF1 1343
Nup358ZnF2 1407
Nup358ZnF3 1471
Nup358ZnF4 1535
Nup358ZnF5 1595
Nup358ZnF6 1657
Nup358ZnF7 1716
Nup358ZnFO 1774
b
Nupl53ZF 1
Nup15321nF2
Ntop1532nF3
Nup153ZnF4
650
714
785
843
R
FQVA
LVTP
RSV5
S
S
I
K
STAK
PTVS
A
7G
AK
CC
CC
E 5 L- DKL,KPV
CDK KRP
L Gt EK KKP
I
NAT
EA
NLNSNKEL ------------VCPPLAETVFTPKT5PENVQDTKSANK5G ---------- SSFVHQ
K FQGDLPKP INSQSL -------Q5
PAI IPTP
FK
LTL TT
xP SP --------STSVPAP
fK FGT E
APK--
INO
- -
ft
I
t 55 5 L
T
5S
EL
KVTON
KPEA1
NAEN
KADST
I
------
QN- - - --SKQNQ --------
T TAV
- - -- T T AVS
TT-AI5
AEAPSAFTLG-
|(APK$APiS
C-I APG
AS
HKPI --------------MKLID5 AAKLSPRDT - - - - - - - - - - - AKQTGIETPNKSCKTTLSASGT
ETP
TCVK- - - -RALTLTVVSESAETMTAS SISCTVTTCTL
--SE 55VP - - - - - - - -A5555TVPV LPS5G
ESA
TKSGFKGFDT55SSNSAA55 FKPFV SSSSC-
Figure 2. 11 - Sequence alignment of the human Nup358/RanBP2 and Nup153 ZnFs,
including the respective C-terminal linker regions
(a) Conservation of the "RanBP2-like" ZnF motif (residues 1343-1833) and C-terminal linker (residues
650-913) between Nup358 and Nup 153 from human. Strict conservation is shown in dark-blue, while
lighter shades of blue depict decreasing levels of conservation. The linker regions between these ZnFs is
neither conserved in sequence or length. (b) Weblogo of the same alignment showing conservation in the
ZnF motif, and lack thereof in the linker region.
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-Binding Zinc FingerDomain
MW
35kDa
27
O
6
Ran
20
14
6.5
Figure 2. 12 - Nupl53ZnF12-Ran crystal contains Ran and intact Nupl53ZnF12
In the electron density map for Nupl53ZnF12-Ran we can only detect one visible zinc finger. To ensure
that this is not the result of protein degradation the crystals used for data collection were analyzed by
SDS-PAGE to confirm that the Nupl53ZnF12 polypeptide has not been degraded. Here we show three
different crystals that have been fished from a crystallization drop and run on a 17% poly-acrylamide gel,
Crystal 1, Crystal 2, and Crystal 3. In addition, a gradient of the Nup 153ZnF12-Ran protein solution used
for crystallization has been included for reference. This data shows that the crystals with tandem
Nup 153ZnF12 polypeptide (13.5 kDa) contain an intact Nup 153 polypeptide in addition to Ran.
Chapter2: Crystallographicand BiochemicalAnalysis of the Ran-BindingZinc FingerDomain
Table 2.1 -X-ray crystallographic data collection and refinement statistics
Data collection and refinement statistics
Nupl53ZnF12
RanGDP
Data Set
RanF35S-GDP
PDB Code
Data Collection*
Space group
Cell dimensions:
a,b,c (A)
0(*)
Unique reflections
Resolution (A)
RG8
Completeness
Redundancy
I/o
2
Wilson B factor (A
)
Refinement
Resolution (A)
Nonhydrogen Atoms
Water molecules
R..b / R.*.j(%)
R.m.s. deviations
Bond lengths (A)
Bond angles (0 )
Bactorsj2
ZnF
Ran
GDP
Water
Ramachandran Plot (%)d
Most Favored
Allowed
Generously Allowed
Disallowed
P41212
3GJ7
C
P21
Nup163ZnF34
RanF35S-GDP
3GJ8
B
P21
Nup153ZnF1
RanF35S-GDP
3GJ6
A
P212 12
Nupl53ZnF2
RanGDP
3GJ3
A
P21212
Nupl53ZnF3
RanF35S-GDP
3GJ4
B
P21
Nupl53ZnF4
RanF35S-GDP
3GJ5
B
P21
81.5, 81.5, 130.5
58.5, 61.1, 80.2
74.3, 61.7, 70.6
60.1, 80.3, 54.6
59.7, 80.2, 58.0
68.4, 61.6, 72.1
70.4, 61.3, 74.0
90
93.7
112.3
90
90
110.0
112.6
71,145
50-1.48
(1.53-1.48)
7.5 (99.9)
95.6 (93.0)
9.7 (7.7)
32.3 (1.8)
19.6
39,366
50-1.95
(2.02-1.95)
9.6(82.7)
93.3 (90.1)
2.3 (1.7)
12.9 (1.2)
37.0
52,824
50-1.82
(1.89-1.82)
6.9 (51.6)
99.3 (99.3)
3.2 (3.0)
19.2 (2.0)
24.3
18,561
50-2.40
(2.46-2.40)
6.5 (44.7)
94.7 (76.2)
2.0 (2.0)
22.9 (2.3)
53.3
26,894
50-1.80
(1.86-1.80)
7.2 (73.3)
99.6(99.4)
7.8 (7.7)
30.2 (2.9)
26.4
29,511
50-2.15
(2.23-2.15)
9.4(45.8)
95.3 (92.4)
2.7 (2.8)
15.2 (2.5)
34.9
53,894
50-1.79
(1.85-1.79)
6.3 (33.3)
98.1 (97.2)
2.7(2.7)
18.6 (3.0)
23.2
30-1.48
3264
478
18.0/20.3
30-1.93
3689
294
18.6 /22.9
30-1.82
3714
548
17.0/20.3
30-2.40
1851
46
19.0/26.6
30-1.78
1854
250
16.1 /19.4
30-2.15
3652
206
20.5 /25.7
30-1.79
3654
458
19.9/23.3
0.008
1.24
0.007
1.06
0.005
0.943
0.008
1.12
0.007
1.11
0.007
1.02
0.007
1.07
24.8
18.9
37.2
86.0
51.1
40.1
54.8
58.2
32.9
24.1
45.7
132.5
54.7
46.6
48.5
50.7
33.4
24.4
43.8
71.5
48.5
39.1
46.8
67.0
40.5
34.0
53.1
93.3
6.7
0
0
89.0
10.5
0.3
0.3
90.1
9.1
0.7
0
83.6
16.4
0
0
88.9
11.1
0
0
88.7
10.8
0.5
0
90.3
9.3
0.5
0
3GJO
66
Chapter2: Crystallographicand Biochemical Analysis of the Ran-Binding Zinc FingerDomain
= Ell, - <1>l/Ii, where I is the intensity of the ith observation and
<Ii> is the mean intensity of the reflection.
a
b Rwoa =
(lFabi - IFcaic
I/ I
Fbs1)
aRfrae
= R value for
a randomly selected subset (5%) of the data that were
not used for minimization of the crystallographic residual.
dCalculated with the program PROCHECK (Laskowski et aL, 1996).
Highest resolution shell (10% of data) shown in parenthesis.
*Related crystal groups
Table 2.2 - Thermodynamic parameters for ZnF-Ran binding in ITC experiments
Islthermal Titration Calorimetry Data
Titrand
j [Titrand]
Titrant
T
(K)
293
Kd1
N
RanGDP
[Titrant]
(mM)
0.31
(pM"
1
RanQ69L
0.40
293
20
15
23
GTP
RanGDP
RanGDP
0.29
0.35
293
293
Nupl53-ZnF1
Nup1 53-ZnF1 Q8D
Nup153-ZnF2
Nupl53-ZnF2D8Q
Nupl53-ZnF3
13
20
32
18
29
RanGDP
RanGDP
RanGDP
RanGDP
RanGDP
0.30
0.36
1.25
Nup1 53-ZnF3E8D
17
Nupl53-ZnF4
Nupl53-ZnF4D8Q
Nup358-ZnF12
50
19
25
(pM)
12
12
Nup153-ZnF12
Nupl53-ZnF34
Nup153-ZnF1234
N2
2
Kd2
(yM)
8
2
AH, / AH2
(kJ/mol)
-40.8/3.9
2
-
-
-39.3/-
4
3
1
1
57
59
1
1
-41.0/20.4
-17.2/-2.2
-10.3/44.2
14.0/21.6
6
8
49
4
7
1
1
1
1
1
-
-
-7.8
-11.2
21.4
17.6
39.8
0.30
293
293
278
293
293
RanGDP
0.34
293
-
-
-
--
RanGDP
RanGDP
RanGDP
0.95
0.36
1.25
278
293
278
47
3
15
1
2
-
-
0.34
-
16.8
TAS, I TAS 2
(kJ/mol)
-7.38/32.7
-12.9/-
-5.5
-3.1
24.9
12.6
-6.6
-3.4
35.7
24.3
22.5
26.0
Chapter 2: Crystallographicand Biochemical Analysis of the Ran-BindingZinc FingerDomain
Table 2.3 - Experimental constructs, abbreviations, and descriptions
$q$eryejenta Constructs
at Protein Residues # ZnFs
Description
Ran
RAN
1:216
0
Full-length WT Ran
RanQ69L
RAN
1:216
0
Full-length Ran with Gin69 mutated to Leu to lock Ran in a GTP nucleotide bound
RanF35S
RAN
1:216
0
Full-length WT Ran with Phe35 mutated to Ser to increase the strength of the
Nup1 53-
Nup153
658:876
4
ZnF12
Nup153
658:750
2
ZnF34
Nup153
790:876
2
ZnF1
Nup1 53
658:686
1
Zinc finger domain (ZnF) of Nup1 53 shortened to include only the first two ZnFs of
the domain, ZnF1 and ZnF2. The WT linker between the two ZnFs remains.
Zinc finger domain (ZnF) of Nup1 53 shortened to include only the first two ZnFs of
the domain, ZnF3 and ZnF4. The WT linker between the two ZnFs remains.
Zinc finger domain of Nup1 53 shortened to include only ZnF1 with no linker
ZnF2
Nupl53
723:750
1
Zinc finger domain of Nupl53 shortened to include only ZnF2 with no linker
ZnF3
Nup1 53
790:817
1
Zinc finger domain of Nup1 53 shortened to include only ZnF3 with no linker
ZnF4
Nup1 53
848:876
1
Zinc finger domain of Nup1 53 shortened to include only ZnF4 with no linker
Nup358-
Nup358
1390:1489
2
Zinc finger domain of Nup358. Includes 2 ZnFs with WT linkers between the ZnFs.
ZnF1234
ZnF12
state
RaneZnF crystal lattice
Entire zinc finger domain (ZnF) of Nup1 53. The WT linker between each ZnF
remains intact.
Chapter3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
Chapter 3: Biophysical Analysis of the Tetrameric Assembly
Between Nic96 and the Trimeric Nspl/Nup57/Nup49 Complex
Experimental contributions: James Partridge and Thomas Schwartz have conducted all
experiments.
Chapter 3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
Introduction
The nuclear pore complex (NPC) is one of the largest protein assemblies inside the
eukaryotic cell and is the sole conduit by which all nucleocytoplasmic transport proceeds. Cryoelectron microscopy/tomography studies have demonstrated that the NPC exists as a protein
scaffold embedded in the nuclear membrane, exhibiting 8-fold rotational symmetry about a
central channel through which cargo can pass between cytoplasm and nucleus (Hinshaw et al.,
1992). Proteomic studies that account for the NPC's 8-fold rotational symmetry and 2-fold
symmetry across the nuclear envelope demonstrate the entire NPC weighs -44 MDa in S.
cerevisiae and -66 MDa in rat (Alber et al., 2007b; Cronshaw et al., 2002; Rout et al., 2000).
Still the NPC is composed of only about 30 proteins called nucleoporins or Nups, which
assemble into biochemically distinct subcomplexes. These subcomplexes exist within the NPC
in accordance to the symmetry described above and represent stable building blocks from
which the structural scaffold of the NPC is built. Each subcomplex is composed of between 2
and 10 Nups, and these subcomplexes are known to also exist in mitotic extracts following
breakdown of the NPC in higher eukaryotes (Matsuoka et al., 1999). Due to the high degree of
internal symmetry, multiple copies of each Nup are present throughout an intact NPC. The
general architecture of the NPC is conserved between yeast and vertebrates and improved
methods in cryo-electron tomography, field-emission in-lens scanning electron microscopy
(FEISEM), 4pi confocal microscopy, and cell fixation techniques, serve as the best method for
such sweeping observations (Beck et al., 2007; Lim et al., 2008a; Maco et al., 2006; Stoffler et
al., 2003). Based on structural data and increasingly powerful methods of structural modeling, it
is clear that only a small number of structural domains make up the NPC: a-helical stacked
domains, P propellers, and coiled-coil domains (Berke et al., 2004; Cronshaw et al., 2002;
Devos et al., 2004; Weirich et al., 2004). The fourth distinct domain, found to compose at least
one-third of total NPC mass, is the phenylalanine-glycine-repeat domain. This flexible,
Chapter3: BiophysicalAnalysis ofthe TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
unstructured Phe-Gly rich domain harbors repeats of FG dipeptides separated by polar spacers
of varying lengths. It is believed that highly disordered and hydrophilic FG-repeat regions
represent the primary interaction site between nuclear transport receptors (NTRs) and the NPC,
thereby playing a direct role in the transport of import- and export-complexes through the NPC
(Ben-Efraim and Gerace, 2001). Only a small subset of Nups, including (in S. cerevisiae)
Pom152, Pom34, and Ndcl have transmembrane domains and are the Nups responsible for
anchoring the NPC scaffold into the nuclear envelope (Hetzer and Wente, 2009).
Although the NPC is built around a rigid scaffold responsible for structural integrity, the
NPC remains a dynamic macromolecular machine. Evidence is emerging to suggest that the
cytoplasmic ring, the nuclear basket, and the luminal spoke ring of NPC undergo significant
conformational rearrangements to adjust for translocation events (Beck et al., 2007). Aside
from itself being a dynamic and flexible machine, some nucleoporins are dynamic and can
shuttle on and off the NPC (Rabut et al., 2004). While some nucleoporin subcomplexes
represent rigid scaffolds from which the NPC is framed, including the structurally wellcharacterized Y-complex (Brohawn et al., 2009; Kampmann and Blobel, 2009; Lutzmann et al.,
2002), other nucleoporin subcomplexes remain something of an enigma, with less structural and
biochemical data available to specifically define their functional role in the NPC.
As with many Nups, the Nup62 complex was originally identified in extracts of
fractionated rat liver nuclei as a biochemically stable subcomplex composed primarily of three
proteins (Davis and Blobel, 1986; Rout and Blobel, 1993; Snow et al., 1987). In mammals the
homologous proteins are: Nup62, Nup54, and Nup58 respectively. In several instances this
manuscript will compare the two trimeric complexes and we will invoke the yeast nomenclature
for generalizations made between the two complexes. The size and sequence of Nspl, Nup57,
and Nup49, differ among different eukaryotes, however the overall structural characteristics
remain concordant. Nspl, Nup57, and Nup49 are symmetrically localized within the NPC as
Chapter3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
judged by immunogold electron microscopy (Rout et al., 2000). Each member of the Nspl
complex is an essential component for both function and structure of the NPC, as determined
with deletions and point-mutants (Grandi et al., 1995; Hurt, 1988; Wente et al., 1992); (Strawn et
al., 2004). In addition, in vitro experiments such as immunodepletion of the Nspl complex
during reconstitution of the NPC with rat nuclei and Xenopus egg extracts, disrupts transport of
NLS tagged cargo and even the formation of NPCs as revealed by scanning EM (Finlay et al.,
1991; Mutvei et al., 1992). In addition to the three major components of the Nspl complex a
fourth protein, Nic96 (yeast) / Nup93 (metazoa), is present as a partially stable component of
the Nspl complex (Grandi et al., 1993; Grandi et al., 1995; Guan et al., 1995). It is believed that
Nic96 serves as a bridging molecule, linking together the heterotrimeric Nspl complex with the
heteropentameric Nic96 subcomplex.
As the first crystal structures of karyopherins and their complexes with: Ran, NLS-/NEScargos, and the FG-repeats of Nups, propelled our knowledge of the mobile phase of
nucleocytoplasmic transport, the answers to most questions regarding assembly, structure, and
function of the NPC will ultimately depend on architectural determination of Nups and NPC
subcomplexes at atomic resolution. As a cornerstone of NPC architecture and function, the
trimeric Nspl complex is a primary target for structural studies. The Nspl complex occupies the
central channel of the NPC and plays a key regulatory role in the transport of cargo across the
nuclear membrane (Fahrenkrog et al., 1998). Domain architecture of each component from the
Nspl complex maintains a conserved arrangement of secondary structure consisting of a
coiled-coil domain, approximately 200 residues in length, flanked by fiber-like extensions of
unstructured amino acid sequence containing Phe-Gly-rich (FG) domains (Hu et al., 1996;
Schwarz-Herion et al., 2007). FG-repeat domains are found in a total of -11 different Nups and
remarkably account for almost 13% of NPC's total mass to fill the central channel of the NPC
(Brohawn et al., 2009). It is well established that FG-repeats are the primary interaction sites
Chapter3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric NspJ/Nup57/Nup49
Complex
between the NPC and nuclear transport receptors (NTRs) that shuttle cargo during
nucleocytoplasmic transport (Denning et al., 2003; Isgro and Schulten, 2007a, b; Macara, 2001;
Peters, 2005). However it is disputed how FG-repeats interact to form a transport barrier that
prohibits molecules greater then
-
40 kDa from passing through the NPC by diffusion, unless
bound to a karyopherin (KAP) (Frey and Gorlich, 2007; Lim et al., 2007; Peters, 2009; Rout et
al., 2003). The coiled-coil domains from each member of the Nspl complex interact to form a
trimeric assembly that lines the inner-channel of the NPC, while the FG-repeat regions are
largely unstructured and as such are nonessential for formation of the Nspl complex (Bailer et
al., 2001; Finlay and Forbes, 1990; Hu et al., 1996; Strawn et al., 2004).
Nic96 is one of the most abundant nucleoporins inside the eukaryotic cell, contributing to
nearly 10% of the total NPC mass with an estimated 32-48 copies per intact NPC (Cronshaw et
al., 2002; Rout et al., 2000). The Nic96 subcomplex is a key structural module of the NPC's core
scaffold and bridges together the Nspl complex with the heteroheptameric Y-complex. Although
an essential component of the NPC scaffold, little biochemical data has been gathered to detail
the molecular workings of the Nic96 subcomplex. Like members of the Nspl complex Nic96 is
essential for both NPC assembly and function, as shown in a variety of eukaryotes (Allende et
al., 1996; Galy et al., 2003; Grandi et al., 1993; Osmani et al., 2006). Unlike members of the
Nspl complex Nic96 is not an FG-repeat Nup, but does have two structural domains common to
other Nups, a 150-residue long coiled-coil domain at the N-terminus followed by a large ahelical domain roughly -600 residues in length (PDB codes 2QX5, 2RFO) (Jeudy and Schwartz,
2007; Schrader et al., 2008b). The helical domain of Nic96 contains 30 helices arranged in a Jlike fashion with the N-terminus starting in the middle of the elongated helical stack. The Calpha backbone of Nic96 traverses up one side of the molecule, makes a U-turn, and then
continues past the N-terminus to the other end of the elongated molecule. Three other a-helical
scaffold nucleoporins (Nup84, Nup85, and Nup145C) have since been structurally characterized
Chapter 3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
and shown to adopt the same unique fold, pointing to a common ancestor (Brohawn et al.,
2008). These four scaffold nucleoporins share a common a-helical domain that to date, outside
of the NPC, has only been identified in Sec3l, an essential component of the COPII vesicle
coat. The similarity between these five proteins was not expected, as sequence conservation is
so low that no structural specific relationship was previously inferred (Alber et al., 2007a; Hsia et
al., 2007). This unique structural domain has been termed, the Ancestral Coatomer Element
(ACE1), provides structural evidence that the nuclear pore complex and vesicle coats, notably
COPII, share a common origin (Devos et al., 2004).
The N-terminal coiled-coil domain of Nic96 mediates a direct interaction between Nic96
and the Nspl trimeric complex (Grandi et al., 1995). In addition to the strong interaction with
Nspl, Nic96 has been reported to bind with other members of the Nic96 complex, including:
Nup53, Nup1 88, and Nup1 92. These interactions however appear to be significantly weaker
then the interaction with Nspl as shown in multiple pull-down experiments (Alber et al., 2007a;
Kosova et al., 1999; Nehrbass et al., 1996; Zabel et al., 1996). In addition to using the Cterminal domain for localization to the NPC it appears that the N-terminal coiled-coil domain of
Nic96 is equally important for assembling the nuclear pore complex (Schrader et al., 2008b).
To understand the interaction between members of the Nspl trimeric complex and
Nic96, we have reassembled the tetrameric complex made between the coil-coiled core of the
Nspl trimeric complex and full-length Nic96. Using liquid chromatography, and biophysical
characterization we have dissected the interactions responsible for assembly of this large
coiled-coil complex. In addition to analysis of the yNspl complex from yeast, we have also
reassembled the homologous complex from Rat, the rNup62 complex. Based on this analysis
and previous data we can now see that not only is the N-terminal coiled-coil domain of Nic96
essential for interaction with the Nspl triple complex, but also is essential for incorporation of
Nic96 into the nuclear pore complex.
Chapter 3: Biophysical Analysis of the Tetrameric Assembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
Results
Reassembly of the trimericyNsp1 / rNup62 subcomplex
A biophysical and crystallographic study of the yNspl complex requires not only the
reassembly of the triple complex in vitro, but milligram amounts of recombinant protein for
crystallization trials and biophysical characterization experiments. This is difficult because,
although the coiled-coil domains of yNspl, yNup57, and yNup49 can individually be expressed
as N-terminal His-tag fusion proteins in E. coli, the proteins are largely insoluble. Under
denaturing conditions, the proteins can be resolubilized with urea, purified via Ni-affinity
chromatography, and mixed in stoichiometric amounts to reassemble the triple-complex.
However, binding by the coiled-coil domains is not entirely specific, resulting in assemblies other
than the desired triple-complex being formed. For instance, a hetero-dimeric complex between
yNup57 and yNspl is a standard impurity in this assembly procedure. In this study the core of
the yNspl complex has been reassembled in vitro using only the coiled-coil domains, expressed
from a modified polycistronic vector. Polycistronic vectors have become a critical tool for
expression studies of large complexes and are especially useful when components are
insoluble if expressed alone (Selleck and Tan, 2008; Tan, 2001). For our experiments a
bicistronic pET-Duet vector was modified to contain a third ribosome binding for coexpression of
all three components with a 6xHis-tag fused to the N-terminus of yNup49 in the first cassette of
the vector, followed by yNup57 in the second cassette, and yNspl in the final and third cassette
(Fig. 3.1). Mapping the interaction between members of the yNspl complex was done in a
systematic fashion. By removing one predicted helix at a time with N- and C-terminal deletion
constructs of yNup49, and yNup57, yNspl, we could identify a recombinantly expressed trimeric
complex with optimal solubility (Fig. 3.1 and Fig 3.3). Placing the His-tag on yNup49 is done to
purify away the hetero-dimeric yNup57eyNspl complex. Mutants lacking one or more helices
demonstrate a range in stability and solubility, however one exemplary construct that includes
Chapter 3: BiophysicalAnalysis of the Tetrameric Assembly Between Nic96 and the TrimericNspl/Nup57/Nup49
Complex
yNup49 3 o-4
66 , yNup57 35 453 4 , and yNsp1 579-824 (Fig. 3.2) yields about 20 mg of trimeric complex
purified from 4 L of liquid E. coli culture, sufficient quantities for crystallographic and biophysical
experiments.
The purification procedure described above has the inherent flaw of purifying soluble,
but unbound yNup49, in addition to the entire Nspl complex. Because the yNspl complex
binds to other components of the NPC, and is composed entirely of coiled-coils, there may exist
regions on the outer surface conducive to auxiliary protein-protein interactions. Left
unoccupied, these typically hydrophobic patches can bind additional non-stoichiometric
amounts of yNup49, resulting in the heterogeneous purification of the yNspl trimeric complex
and yNup49. To identify hydrophobic patches that mediate coiled-coil contacts, the
hydrophobicity of helices was analyzed by hydrophobic cluster analysis (HCA) using the
program Drawhca and the mammalian rNup62 complex (Callebaut et al., 1997). After
identifying hydrophobic patches, specific amino acids from each helical region were mutated to
charged residues using site-directed mutagenesis. With this method we were able to
significantly increase solubility of the rNup62 complex with mutations in the first and third helix
of rNup62 (Fig. 3.2). Our HCA based mutagenesis experiments also uncovered four critical
hydrophobic patches, necessary for stable complex formation. Two mutations of rNup58
completely abolish purification of trimeric complex. Mutations to the fifth helix of rNup54 abolish
interaction between rNup54 and rNup62, as rNup54 and rNup62 no longer copurify in the
presence of excess rNup58. Further mapping experiments described below have confirmed
that the C-terminal helix of rNup54/yNup57 is responsible for binding directly with
rNup62/yNspl.
Not surprisingly, the trimeric complex from R. norvegicus (rNup62 complex) behaves
quite differently from the trimeric complex reassembled using S. cerevisiae genes (the yNspl
complex). The yNspl complex is more soluble then the rNup62 complex and does not require
Chapter3: Biophysical Analysis of the TetramericAssembly Between Nic96 and the Trimeric NspJ/Nup57/Nup49
Complex
solubility-based point mutations. For purification of soluble protein, using the full coiled-coil
domain from yNspl and yNup57 is tolerable, but where the rNup62 complex was optimized with
a longer version of rNup58 (Fig. 3.1), the yNspl complex will not tolerate using a yNup49
construct that includes all four helices of the coiled-coil domain. As the solubility of yNup49
drops so does the solubility of the trimeric complex. In addition to solubility differences, the
yNspl trimeric complex has two auto-proteolytic sites in yNup57. These sites are not present in
rNup54 (Fig. 3.1). The two proteolytic sites have been mapped to a loop between the predicted
fourth and fifth helix of the coiled-coil domain. Between amino acids 480 and 498 is an
unstructured loop, non-conserved among yNup57 orthologs. With N-terminal sequencing and
mass spectroscopy the primary proteolytic site of yNup57 was mapped to residue 482. A
proteolytic site no doubt leads to a mixed population of yNup57, with a low percentage of fulllength protein intact after several days at room temperature (Fig. 3.3). A second construct was
modified to remove the loop entirely, as well as remove C-terminal helix 5. In this process we
reconfirmed our HCA analysis on rNup54 and found out that yNup57-helix-5 is critical for
binding directly to yNspl. If removed, yNspl dissociates from the triple complex during gel
filtration and ion exchange purifications, leaving a core heterodimeric complex between yNup49
and yNup57. This allowed us to map the helical interactions between coiled-coil domains of the
Nspl trimeric complex (Fig. 3.3).
Mapping interactionswithin the Nspl complex
Based on a series of helical truncations at the N- and C-terminus of yNup57 we believe
the heterodimeric interaction between yNup49 and yNup57 constitutes a core scaffold of the
trimeric yNspl complex. The presence of consecutive heptad repeats in a peptide sequence,
denoted [abcdefg]n, serves as a powerful tool for the prediction of coiled-coil domains that can
be evaluated with several different prediction programs (Lupas et al., 1991; McDonnell et al.,
Chapter 3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
2006; Wolf et al., 1997). Although the identification of coiled-coil domains can be fairly simple,
designating interacting helices within large coiled-coil assemblies is not trivial. Predicting the
structure of coiled-coil assemblies is made difficult due to the two orientations that each helix
can make (coiled-coils can be parallel or anti-parallel), the assembly into coiled-coils composed
of two, three, four or five helices, and the potential axial shifts in heptad interactions. There are
however studies demonstrating that it is possible to discriminate between two-stranded versus
three-stranded assemblies, providing insight into assembly order and plausible binding pairs
(Apgar et al., 2008; Wolf et al., 1997; Woolfson and Alber, 1995). Using hydrophobic cluster
analysis, coupled with the mutation of residues from 11 hydrophobic clusters to charged,
discrete point mutations can have a drastic effect on solubility as well as assembly. As
demonstrated with mutations 5 and 6 to rNup58 in one-step affinity tag purifications, disruption
of either rNup58 helix results in complete disassembly of the trimeric complex (Fig. 3.2).
Mutations 1, 8, and 11 are similar to wild-type and produce similar levels of soluble trimeric
complex (Fig. 3.2). We rationalize these hydrophobic patches likely have little effect on trimeric
complex assembly. Mutations 2, 7, 9, and 10, appear to increase solubility of the trimeric
complex, while maintaining stable complex assembly. As a result of this analysis, mutations 7
and 9 were specifically used to increase solubility of the rNup62 trimeric complex for all further
biophysical experiments described herein. Interestingly, constructs containing mutation 3 or 4 in
the fifth helix of Nup54 have the most aberrant trimeric assembly. Only in these constructs is the
solubility of Nup58 much higher than wild-type, however there is far less rNup54 and rNup62
present after the one-step Ni-affinity purification. Because all proteins are stably expressed, the
purification of excess rNup58 likely indicates that these helices play a critical role in the full
trimeric assembly. This data is in agreement with additional mapping studies done using
rNup54. As described above, the scissile site between helices four and five of rNup54 led us to
truncation at the C-terminus of rNup54. Upon removal of the fifth and most C-terminal helix of
Chapter 3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
yNup57 at residue 512, purification of two distinct species is evident during gel filtration
experiments (Fig. 3.3). It is clear that after removing the fifth helix, yNspl dissociates from the
trimeric complex. This results in the purification of two distinct species, full trimeric assembly
and a smaller heterodimeric "core" assembly between yNup49 and yNup57. Further truncations
at the C-terminus of yNup57 to residue 474, in addition to removal of the first two helices at the
N-terminus, results in a stable dimeric "core" interaction between yNup57 and yNup49. This
"core" interaction represents the smallest interaction possible between members of the Nspl
trimeric complex, as further truncations disrupt dimer assembly. This core heterodimeric
assembly between yNup49 and yNup57 has been used in further biophysical experiments,
alongside the larger trimeric, and tetrameric assembly with full-length Nic96.
Biophysicalcharacterization of the Nspl complex
Biophysical characterization of the purified Nspl/Nup62 trimeric complex and smaller
components is critical for understanding the structure and biochemical nature of such a large
coiled-coil protein assembly. This analysis gives us information regarding size, stoichiometry,
and homogeneity of all purified assemblies. Size exclusion chromatography (SEC) provides a
rough approximation of size and stoichiometry for macromolecular protein complexes. Protein
from the yNsp1/rNup62 complex elute as a single stable peak at a volume roughly equivalent to
twice the calculated mass, as compared with globular protein standards. However, the shape of
macromolecules can dramatically affect the elution volumes obtained with SEC. In the
purification of rod shaped assemblies it is difficult to distinguish size differences with SEC
because the stokes radius can remain constant, even upon addition of more components.
Based on SEC alone, it is unclear if the yNsp1/rNup62 complex exists as a heterotrimer or a
heterohexamer. In the purification of a tetrameric complex including Nic96 it is possible that the
extended Nic96 molecule with the N-terminal coiled-coil domain exhibits the same stokes radius
Chapter 3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
as the yNspl .yNic96 tetrameric complex. Because of these limitations a more appropriate and
advanced method for measuring mass, stoichiometry, and shape is necessary.
Analytical ultracentrifugation
Analytical ultracentrifugation (AUC) allows for the measurement of molecular weight of a
soluble protein independent of shape. Two types of experiments can be performed:
sedimentation velocity and sedimentation equilibrium. Data collected using sedimentation
velocity is influenced by both size and shape of the protein being studied. However, AUC data
obtained using sedimentation equilibrium is unaffected by shape of the molecule and is
sensitive only to the mass of the protein in native buffer conditions. Used in combination, these
methods can provide precise measurements of molecular mass under native conditions, and
even provide information regarding stoichiometry of the macromolecule based on fluctuations of
thermodynamic parameters for self-associating systems (Hansen et al., 1994; Lebowitz et al.,
2002).
The only known partial structure of any yNspl/rNup62 complex member is an antiparallel hairpin, homo-dimeric assembly of rNup58 327-4 11 (PDB code 2OSZ) (Melc k et al.,
2007). The crystal structure rNup58 327-411 forms a dimer of dimers, with the core interaction
being a tight dimer between two a-helices connected by a short loop (Melchk et al., 2007). The
crystal structure suggests that the core homodimer of rNup58 is a stable component of the
entire rNup62 complex. Numerous van der Waals interactions exist between the two helices
with a total buried surface area of 1,262 A2 . With a contact area of this size and
complementarity it seems likely that the rNup58 dimer is representative of the rNup58 state
found within the full rNup62 trimeric assembly. To test this observation and determine the
stoichiometry of components within the yNspl/rNup62 trimeric assembly, several variants of the
assembly were tested using an analytical ultracentrifuge. With sedimentation velocity
experiments we determined that the rNup62/yNspl complex exists in a 1:1:1 ratio between
Chapter 3: Biophysical Analysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
components in both rat and yeast versions with only one molecule per assembly (Table 3.1). In
addition to the trimeric assembly, dimeric complexes between rNup54erNup62 and
yNup49eyNup57 were tested and shown to exist in a 1:1 ratio with only one molecule each per
assembly. However, in agreement with the crystal structure, a preparation of the rNup58
orthologue yNup49 was shown to self-associate into a higher order species in sedimentation
velocity experiments. This higher-order species appears to be a homotetramer. Under
physiological conditions in the cell, our results suggest that the yNsp1/rNup62 complex exists as
a stable assembly with only one copy of each component, with 1:1:1 stoichiometry. Selfassociation of yNup49/rNup58, likely a non-physiological interaction, is only observed when the
two other binding partners are limiting and can self-associate to form a unique higher order
assembly in absence of the trimeric complex partners.
Assembly of a tetrameric assembly between the Nspl complex and Nic96
Previous studies have demonstrated that the Nspl complex interacts directly with the
Nic96 complex via the coiled-coil domain of Nspl and the N-terminal coiled-coil domain of Nic96
(Grandi et al., 1993; Grandi et al., 1995; Guan et al., 1995). Although more recent work
demonstrates this interaction with careful pulldown experiments (Alber et al., 2007a), complete
reassembly in vitro has not previously been described. The N-terminal domain of Nic96 is a
predicted coiled-coil domain that spans approximately 160 residues prior to a 30 residue long
loop, followed by the ACE1 domain, spanning residue 200 to the C-terminus. Interaction
between Nic96 and Nspl does not depend on the C-terminal ACE1 domain (Grandi et al.,
1995). The N-terminal coiled-coil domain is insoluble unless expressed with the highly charged
and soluble C-terminal fragment of yNic96. For this reason, reconstitution of a tetrameric
yNspl eyNic96 complex was only achieved in the presence of full-length yNic92 with the yNspl
trimeric complex described above, yNup49 360 .466 , yNup57 3 4.53 4, and yNspl579-824. For reassembly
Chapter3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric NspJ/Nup57/Nup49
Complex
of this large complex a two-vector system was used, with the Nspl complex expressed from a
pET-Duet-1 (ampicillin resistance) based tricistronic vector and full-length yNic96 expressed
from pET-28a (kanamycin resistance). Both yNic96 and yNspl are fused to a 6x His tag at the
N-terminus. Following copurification with Ni-NTA resin the tetrameric complex is purified away
from smaller assemblies and other contaminants by ion-exchange chromatography, followed by
size exclusion chromatography (SEC). With SEC, the yNsp1/rNup62 trimeric complex elutes at
a volume equivalent to twice the calculated mass and at roughly the same position as the
tetrameric yNspl eyNic96 complex (Fig. 3.5). From our analytical ultracentrifugation data we
calculate a high frictional ratio (f/fO) above 1.5, highly indicative of a rod-like conformation for
each protein assembly (Table 3.1). yNup49 purified alone is the only sample to have a f/fO
below 1.5, which suggests that the protein adopts shorter, more spherical assemblies in the
absence of yNup57 and yNspl. The yNsp1/rNup62 trimeric complex exists as a hetero-trimer in
a 1:1:1 ratio, with one copy of each component per assembly, and an early elution volume with
SEC is due to the large stokes radius of the elongated coiled-coil assembly. Surprisingly,
binding extended rod-shaped Nic96 (Jeudy and Schwartz, 2007; Schrader et al., 2008b), to the
Nspl trimeric assembly does not affect the elution volume obtained via SEC (Fig. 3.5),
indicating that the two elongated structures may collapse into a more compact structure or
maintain an equivalent stokes radius.
Discussion
Reconstitution of a tetrameric protein assembly between the Nspl trimeric complex and
FL-Nic96 represents the first reported assembly between proteins of the Nspl and the Nic96
nuclear pore subcomplexes. The stable trimeric yNsp1/rNup62 complex has been reconstituted
from S. cerevisiae and R. norvegicus respectively and exhibits the shape of a long rod, as
definitively shown by analytical ultracentrifugation and size exclusion chromatography. Data
from analytical ultracentrifugation also demonstrates that the trimeric assembly exhibits a stable
Chapter 3: Biophysical Analysis of the Tetrameric Assembly Between Nic96 and the Trimeric NspJ/Nup57/Nup49
Complex
1:1:1 stoichiometry between the three coiled-coil domains of yNspl, yNup57, and yNup49.
Mapping experiments reveal a defined interaction between yNup57 and yNspl mediated by
helices 1, 2, and 5 of yNup57 and the coiled-coil domain of yNspl. In addition to the trimeric
assembly between yNup49, yNup57, and yNspl, a stable dimeric "core" has been isolated
between yNup49 and yNup57. The yNup49-yNup57 core complex also exists in a stable
stoichiometric 1:1 ratio, as seen with SEC and analytical ultracentrifugation. Although this study
provides considerable insight into the assembly and interaction between the yNspl and yNic96
subcomplexes, further experiments will probe the molecular details of such a large structural
assembly, namely by crystallographic analysis.
In this study we have focused on the coiled-coil domains that mediate contact between
Nic96 and the Nspl trimeric complex, as Nic96 tethers the dynamic Nspl complex to the
structural scaffold of the NPC. The structural scaffold is made up of the Y-complex, composed
of seven proteins that serve as a scaffold lattice for the more dynamic Nups that reside on either
face of the NPC and that line the inner channel. The Nic96 complex probably contains at least
five stable components and most likely has a similarly important architectural role as the Ycomplex. The ACE1 domain of Nic96 is a stable rod-like domain, and other proteins from the
Nic96 complex, such as Nup188 and Nup192, are also large helical units. Nic96 is related to
three of the five helical proteins that build the Y-complex. Nup1 57/Nup1 70 of the Nic96 complex
is structurally related to Nup1 33 of the Y-complex (Whittle and Schwartz, 2009). The next stage
of NPC structural studies will likely focus on the Nic96 complex, such that the lattice scaffold
can be completely understood. The anchor points for the Nspl complex will then become more
apparent and will likely bring us closer to a comprehensive functional characterization.
Methods
Chapter3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric NspJ/Nup57/Nup49
Complex
Protein purification
A bacterial expression construct containing rNup58 328 -4 15 , rNup54 348 .495 , and rNup62 322-5 23
from Rattus norvegicus was constructed for reassembly of the rNup62 subcomplex. The genes
were cloned in a pET28a vector (EMD Biosciences), modified to be a tricistronic vector
containing three independent ribosome binding sites for each ORF and expressed as a 6xHistag fusion protein engineered to contain a protease 3C site after the N-terminal affinity tag. The
rNup62 subcomplex construct containing an extended rNup58 239-415 was similarly cloned using
the original rNup62 subcomplex vector as a template. The yNspl subcomplex, yNup49 36 0 -46 6 ,
yNup57 3
4-5 34 ,
and yNsp157 9 -8 2 4 , was cloned into pET-Duet 1, modified to include a protease 3C
site after the N-terminal 6xHis-tag fused to yNup49 and to contain a third RBS site for
expression of a third ORF. Full-length Nic96 was cloned as a 6xHis-tag fusion protein in
pET28a, engineered to include a 3C site after the N-terminal affinity tag. All proteins were
expressed in Escherichia coli strain BL21 (DE3) RIL (Stratagene). Cells were grown at 300C in
Luria-Bertani broth supplemented with 0.4% glucose to OD600= 0.8 and induced with 0.2 mM IPTG at
180C for 18 hours. Bacterial cell pellets harboring His-tagged proteins were suspended in 50 mM
potassium phosphate pH 8.0, 500 mM NaCl, 20 mM imidazole, 3 mM p-mercaptoethanol (pME), and lysed using a french press. Crude lysate was supplemented with 200 pM
phenylmethanesulfonyl fluoride and clarified by centrifugation at 9,000 g for 20 minutes. The
soluble fraction was then incubated with 1ml Ni-NTA per 1000 ODs for 1 hour at 277 K and
loaded onto a disposable column (Pierce). The column was washed with 4 bed volumes of 50
mM potassium phosphate pH 8.0, 500 mM NaCl, 20 mM imidazole, 3 mM p-ME, and eluted with
6 bed volumes of 10 mM Tris/HCI pH 8.0, 50 mM NaCl, 150 mM imidazole, 3 mM p-ME. Eluted
protein was dialyzed against 10 mM Tris/HCI pH 8.0, 250 mM NaCl, 0.1 mM EDTA, 1 mM DTT,
for 1 hour before the 6xHis-tag was cleaved with 3C protease. The rNup62 trimeric assembly
was purified using anion exchange chromatography on a HiTrapQ column (GE Healthcare) via a
Chapter 3: Biophysical Analysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
linear NaCl gradient, followed by purification with size exclusion chromatography using a
Superdex S200 26/60 column (GE Healthcare) in 10 mM Tris/HCI pH 8.0, 200 mM NaCl 1.0 mM
DTT, 0.1 mM EDTA. The Nspl trimeric complex alone does not bind tightly to a Q column and
as such was not purified with a Q column. The tetrameric assembly between full-length Nic96
and the Nspl complex was assembled by coexpression on the pET28a and pET-Duet-1 vectors
respectively in Escherichia coli strain BL21 (DE3) RIL (Stratagene), and purified with Ni-NTA
resin as described above. The tetrameric complex between FL-yNic96 and the yNspl trimeric
complex does bind to a Q column and elutes at 200 mM NaCl. The tetrameric assembly was
purified on a Q column prior to further purification using a Superdex S200 26/60 column (GE
Healthcare) equilibrated in 10mM Tris/HCL pH 8.0, 250mM NaCl, 0.1 mM EDTA, 1 mM DTT.
Hydrophobic clusteranalysis
Hydrophobic cluster analysis (HCA) was conducted using Mobyle@RPBS (Callebaut et
al., 1997; Gaboriaud et al., 1987). Hydrophobic patches were identified with HCA and two
residues within each patch were mutated using an updated QuickChange Site-Directed
Mutagenesis System (Stratagene) protocol (Zheng et al., 2004). Hydrophobic residues were
mutated to large hydrophilic residues and the resultant construct was tested for trimeric complex
assembly and solubility with the one-step Ni-NTA purification protocol described above.
Mapping helical interactions
Using N-terminal sequencing via Edman degradation and mass spectroscopy we were
able to identify the two degradation products described in Fig. 3.1 as yNup57, truncated in a
loop just prior and within helix 5. In an attempt to trim yNup57 past the proteolytic site we
identified several key interacting domains between yNup57 and yNspl. By utilizing PCR
techniques to truncate helices at the N- and C-terminus of yNup57 we were able to map
Chapter 3: Biophysical Analysis of the TetramericAssembly Between Nic96 and the Trimeric NspJ/Nup57/Nup49
Complex
interactions among members of the yNspl complex. To construct a yNup49-yNup57 dimeric
complex, helices 1, 2, and 5, were removed from yNup57, using the yNspl construct described
above as template. Although removal of these helices does abolish interaction between
yNup57 and yNspl, yNspl was removed from the tricistronic vector to increase overall purity in
purification of a dimeric yNup49-yNup57 complex.
Analytical ultracentrifugation
Several purified rNup62 and yNspl complex assemblies were gel-filtered in 10 mM
Tris/HCI pH 8.0, 200 mM NaCl, 0.5 mM TCEP, and 0.1 mM EDTA immediately prior to the
velocity sedimentation experiments. Analytical ultracentrifugation experiments were carried out
with an Optima XL-A centrifuge using An50Ti (6 hole, equilibrium runs) or An60Ti (4 hole,
velocity runs) rotors. Samples for sedimentation velocity (440 pL sample or 450 pL buffer) were
loaded into Epon-charcoal filled 2 channel centerpieces, fit with sapphire windows, and spun at
42,000 rpm. Sedimentation velocity data was analyzed globally to generate a c(s) distribution
using the continuous distribution model in SEDFIT (Schuck, 2000). The data was fit from 0.5 to
10 s-1 3 with an estimated vbar = 0.7300 cm3/g, n = 1.0182 cP, and p = 1.002 g/ cm3.
86
Chapter 3: Biophysical Analysis of the TetramericAssembly Between Nic96 and the Trimeric NspJ/Nup57/Nup49
Complex
Figures - Chapter 3
Nsp 1Trimeric Complex
N
Nspl
I
823
600
Nsp1
Nup57
1
271
541
Nup57 4
* 40
Nup49
Nup49 4Wy
1
472
269
Nup62 Trimeric Complex_
Nup62
3
342
5
525
Nup62
Nup54
1
1
310
510
*
a L-Nup58
Nup54
4o
-u
Nup58
E( IIZ
S-Nup58 ob
410
585
234
Figure 3. 1 - Domain architecture of both the Nspl and Nup62 trimeric complex
The domain architecture of the Nup62 trimeric complex from rat and the homologous Nspl trimeric
complex from yeast are represented in cartoon form with the largely unstructured FG-repeat domain in
grey and the predicted coiled-coil domain of each protein in blue. The mammalian trimeric Nup62
complex is composed of Nup62, Nup54, and Nup58, the yeast orthologues are Nspl, Nup57, and Nup49
respectively. Orange boxes above each coiled-coil domain represent the minimal sequence of each
component necessary to assemble the trimeric complex shown to right. The coomassie-stained-SDS
polyacrylamide gels shown to the right demonstrate the results of electrophoresis of each purified trimeric
assembly. Each component is labeled, and the two stars below Nup57 in the Nsp 1 trimeric assembly
represent two degradation products that result from proteolytic cleavage at the N-terminus of helix 5 of
Nup57. Two versions of the Nup62 assembly have been used in this study, with a short and long form of
Nup58. This difference is noted with an orange and yellow box above the Nup58 coiled-coil domain, and
is shown with the SDS gels at the right.
Chapter3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric NspJ/Nup57/Nup49
Complex
10
20
30
40
50
70
60
s0
90
100
120
110
1
130
1
1
140
Nup54
10
20
Nup58
D
30
,D
40
30
70
f0
N'((DK Q A
90
go
'-
AA
Q
AR
10
20
30
40
50
60
70
so
90
100
1
1O10
A
=GU
20
I1
130
1
A
R
vA
6
5
140
150
160
I1
Hf
110
I
180
190
I1
Nup62
MW
(kDa) 1
2 3 4 5 6 7 8 91011WT
34
27 20
14
7
Nup62
,40-
- -
Nup54
Nup58
Figure 3. 2 - Hydrophobic cluster analysis of the Nup62 trimeric assembly
As typically seen with coiled-coil assemblies, hydrophobic interactions form the key interactions for the
88
2(
Chapter 3: Biophysical Analysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
large coiled-coil Nup62 trimeric assembly. Hydrophobic cluster analysis (HCA) was used to identify
hydrophobic patches that serve as potential interaction sites between helices of Nup62 trimeric assembly.
In the diagrams shown above hydrophobic patches are outlined and hydrophobic residues are shown in
green. In addition to abolishing interactions among helices, HCA was used to identify hydrophobic
patches that could potentially impair solubility of the trimeric assembly. In both instances two
hydrophobic residues, typically isoleucine/leucine and denoted by an arrow and number inside of the
identified hydrophobic patches, were mutated to aspartic acid residues except for mutation 5 which was
mutated to arginine. (b) The wild-type trimeric complex, as well as all mutations listed above, are
visualized with this coomassie-stained-SDS polyacrylamide gel following one-step affinity tag
purifications. The mutations are listed by number and have drastically different effects on the overall
solubility and stability of the trimeric complex. Notably, mutations 3, 4, and 10 appear to disrupt the
trimeric assembly, as evident in the increase in Nup58 purification but less than stoichiometric amounts
of Nup62, and Nup54. Mutations 2, 7, and 9 appear to increase the overall solubility of the trimeric
complex while maintaining stability among all members. Both mutations to Nup58, mutations 5 and 6,
completely disrupt interaction within the known hairpin structure of Nup58 and leads to insoluble Nup58
and complete lack of a Nup62 trimeric assembly.
Chapter 3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric NspJ/Nup57/Nup49
Complex
a
Napi Triple Complex
-
66 kDa
I
I
Nap1
~~Nup57
e*auvow mn Nup49
1. Load Material
Elution volume (mL)
-
Nepi Triple Complex
Nup57 - res. 512
66 koa
Nspl
Nup57
res 512
Nup49
o
--
75
'13
22
Ehution volume (mL)
MW
hap49 - N0p57 Core
66 kDa
27 vMM
*
75
ISO
22s
Elution volume (mL)
3
-3Sewwws=
d
Nspl
H
H2 4
Nup57
Nup49
Figure 3. 3 - Systematic deletion of helices from Nup57 map an interaction between
Nup57 and Nsp1
Nup49
NuO57
. ................
.
..............
.......................................
Chapter3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the TrimericNspJ/Nup57/Nup49
Complex
The helical domains of Nup57 were systematically removed at both the N- and C-terminus to identify a
minimal binding domain between Nup49 and Nup57, as well as identify the helical interactions between
Nup57 and Nspl. (a) A trace of the purification peak of the Nsp1 trimeric complex demonstrates that
Nspl, Nup57, and Nup49 are purified in stoichiometric amounts. (b) By removing the 5h helix of Nup57
a critical interaction between Nup57 and Nspl is lost. This is made evident in the gel filtration trace on
the left where two obvious species elute at different volumes. To the right is the coomassie-stained-SDS
polyacrylamide gel showing that the two species separated are the Nsp1 trimeric assembly, as well as a
small assembly between Nup49 and Nup57. (c) In addition to the 5h helix, the 1 st and 2 "dhelices also play
a critical role in binding Nup57 with Nspl. Upon removal of these three helices, interaction between
Nup57 and Nspl is abolished leaving the core dimeric interaction between Nup49 and Nup57.
Nup62 trimeric complex
Nup62 trimeric complex with extended Nup58
i~4I2~J-2
Dimeric Nup54 - Nup62 complex
1
Nup49 alone
Nspl trmeric complex
Dimeric Nup49 - Nup57 complex
0-177______
I
II
Figure 3. 4 - Analytical ultracentrifugation of Nsp1 I Nup62 assemblies
Analytical ultracentrifugation is a valuable tool for assessing the stoichiometry of the yNspl/rNup62
NO-
Chapter3: Biophysical Analysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
assemblies presented in this manuscript. It had been previously reported that rNup58 exists as a large
homo-tetramer assembly suggesting that assembly of the yNsp l/rNup62 complex would proceed with
multiple copies of each component per full assembly (Melcik et al., 2007). This does not seem to be the
case as based on our results recorded in Table 3.1. For every assembly only one copy of each molecule is
present, expect for our study of yNup49 alone. yNup49 is the yeast orthologue of rNup58, and in our
study yNup49 exists as a tetramer in solution. Here the data and fits, in addition to residuals in the form of
a bitmap are shown for all of our analytical ultracentrifugation experiments. The peak for a calculated
sedimentation coefficient value is shown to demonstrate homogeneity of each purified assembly.
Chapter 3: Biophysical Analysis of the TetramericAssembly Between Nic96 and the Trimeric Nspl/Nup57/Nup49
Complex
MWm
4Np
01
111:7M flP
FL-Nic96
6 kDa
-FL-At96
{
4
-c-oorms
_
2D
Nup7
Nup49
e t14se
EBud=o Votin
(MO
b
MW A
+ NFIcB.
-
mil.
(2son4
FL-McgS+ NI
(260 nia
ame
B_
_
FL-Nic96
%rM
Caqnx
5
42
34
Np
27
20
is
a
Emimn V o
75
In
Nup57
14
lw
Nup49
A.load material
B.0 column flow through
C
FL-Nic96
+Nt, Como"x
Im
Nspl
Nup57
75
.M5 aaNup49
12 1 175 25* 2k 2k 276
EiuM=n Voahns (u
Figure 3. 5 - Purification of a tetrameric assembly between full-length Nic96 and the
trimeric Nsp1 complex
The purification of a tetrameric assembly is possible by co-expressing both full-length Nic96 and the
Nspl trimeric assembly in E. coli. (a) Analytical gel filtration experiments demonstrate that full-length
Nic96 and the Nspl coiled-coil trimeric assembly elute at roughly the same volume. When co-expressed
and analyzed with gel filtration, the elution volume of the tetrameric assembly is not significantly
different from the elution volume of full-length Nic96 alone. (b) Although not significantly observed with
analytical gel filtration, analytical studies with anion exchange chromatography show a dramatic shift
elution conditions on a Hi-Trap Q column between full-length Nic96, the Nspl trimeric complex, and the
Chapter 3: BiophysicalAnalysis of the TetramericAssembly Between Nic96 and the Trimeric NspJ/Nup57/Nup49
Complex
combined tetrameric assembly. The tetrameric assembly elutes as stable peak at 200 mM NaCl, while the
Nsp1 trimeric complex does not bind tightly to a Q column and elutes below 100 mM NaCl. (c)
Following purification by anion exchange chromatography, the tetrameric assembly between full-length
Nic96 and the Nsp1 trimeric complex elutes as a stable peak as shown with gel filtration and coomassiestained SDS-polyacrylamide gel analysis.
Table 3. 1 - AUC analysis data
Nup62 Triple
Extended Nup58
61.92
60.9
0.0059
1.68
3.23
Nup62 Triple
51.92
58.3
0.0092
1.56
3.45
Dimeric Nup54 -
41.17
43.9
0.0045
1.70
2.64
12.47
60.63
34.56
47
57.94
34.5
0.0145
0.0051
0.0234
1.41
1.70
1.60
3.33
3.16
2.38
Nup62
Nup49
Nspl Triple
Dimeric Nup49Nup57
Chapter4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
Chapter 4: A Structural Element Within The HUWE1
HECT Domain Modulates Self- and Substrate
Ubiquitination Activities
The material presented in this chapter was adapted, with permission, from the following
publication:
Partridge, J.R. , Pandya, R.K. , Love, K.R., Schwartz, T.U., and Ploegh, H.L. (2010). A
Structural Element within the HUWE1 HECT Domain Modulates Self-ubiquitination and Substrate
,Ubiquitination Activities. J Biol Chem 285, 5664-5673.
equal contribution
Experimental contributions: James R. Partridge and Renuka Pandya conducted all
experiments.
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Chapter4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
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Introduction
Targeted modification of proteins with ubiquitin satisfies several essential
functions inside the eukaryotic cell. Many processes such as, cell-cycle regulation,
receptor trafficking, signal transduction, epigenetic regulation of gene expression, DNA
repair, regulated proteolysis, and proteasome targeting are all under partial control by
ubiquitin (Ub) labeling (Bernassola et al., 2008; Glickman and Ciechanover, 2002). The
ability of ubiquitin to function in such a diverse array of biological functions is explained
by the existence of distinct ubiquitin labeling signals recognized by an equally diverse
set of ubiquitin-binding domains found in several regulatory proteins (Hicke et al., 2005;
Hurley et al., 2006). Ubiquitin is linked to target proteins in three steps via a series of
enzymes that act in a defined sequence. The Ub cascade begins with an activating El
enzyme that requires ATP to generate a high-energy thioester intermediate. An E2
ubiquitin-conjugating enzyme then transfers the activated Ub molecule, via a highenergy thioester intermediate, to a substrate that has previously been bound to a
member of the ubiquitin ligase family of enzymes, or E3 enzyme (Christensen et al.,
2007; Pickart, 2001). E3 enzymes serve as the primary determinant for substrate
recognition and come from one of two families. The RING (really interesting new gene)
family of E3 enzymes function primarily as a rigid scaffold to facilitate interaction
between the E2-Ub complex and a substrate targeted for ubiquitination (Zheng et al.,
2002; Zheng et al., 2000). The second class of E3 enzymes is the HECT (homologous
to E6AP C-terminus) domain family, all of which possess a highly conserved ~ 350
residue long domain at the C-terminus, responsible for catalyzing transfer of Ub from E2
to substrate. In contrast to the RING E3s, the HECT domain forms a direct thioester
intermediate with Ub prior to active transfer of Ub to substrate in a highly mechanistic
fashion. Each HECT domain contains an absolutely conserved cysteine near the C-
Chapter 4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
terminus that is responsible for formation of the Ub-E3 thioester intermediate
(Huibregtse et al., 1995; Scheffner et al., 1995).
Mechanism of ubiquitinationmediated by E3 HECT domains
Approximately 600 E3s exist in the human proteome and 28 of those belong to
the HECT domain family (Li et al., 2008). HECT domains generally share a conserved
structural architecture consisting of a stable N-lobe and a mobile C-lobe responsible for
transferring charged Ub from E2 to substrate. The need for a mobile and flexible enzyme
is made apparent in recent structural and mechanistic studies. Early crystal structures
between the E3 HECT domain E6AP and an E2, UbcH7, mapped the distance between
the E3 catalytic cysteine, present on the C-lobe, and the E2 binding site, from the Nlobe, at 41 A (PDB accession code 1D5F and 1C4Z) (Huang et al., 1999). Further
crystallographic studies on the E3 HECT domain WWP1 confirmed movement of the Clobe, and that a flexible hinge between the N- and C- lobes of the HECT domain allows
for movement of the C-lobe to transfer Ub from E2 to substrate (PDB accession code
1ND7) (Verdecia et al., 2003). Modeling an E2 enzyme docked at the E2 binding site of
the WWP1 structure demonstrates that the gap between the E3 catalytic cysteine and
E2 is only 16 A. Despite this detailed structural knowledge of E2 and E3 HECT domains,
little is known regarding the molecular details of Ub exchange between E2-E3-substrate.
In a study of two different E3 HECT domains it was shown that E3 enzymes could adopt
distinct methods for ubiquitinating substrate. E6A6 uses a mechanism termed
indexation, where a full poly-Ub chain is built on the E6AP catalytic cysteine prior to
substrate transfer. Another E3 HECT domain, KIAA1 0, utilizes a sequential addition
method where one Ub molecule at a time is transferred to substrate to form a poly-Ub
chain (Wang and Pickart, 2005). These differences highlight the characteristically
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modular nature of the E3 HECT domain, an enzyme that requires high-level flexibility
while maintaining a level of specificity critical for governing ubiquitination of target
substrate.
Structural characteristicsof the E3 HECT domain
The E3 HECT domain is particularly flexible for mechanistic reasons. It is likely
that this flexibility enables the E3-ligase HECT domain to adopt variable mechanisms for
poly-Ub labeling of substrate. Based on crystallographic data from previously solved
structures we know that the HECT domain contains two major lobes referred to as the Nand C- lobe (Fig. 3.2). The large N-lobe contains the E2 binding region, while the C-lobe
contains the catalytic cysteine responsible for binding Ub during transfer of Ub from E2
to target substrate. While the crystallographic structure of the E6AP HECT domain
bound to Ub demonstrates a gap of 41 A between E2 active site and the catalytic
cysteine, the WWP1 structure shows that the C-lobe rotates -1000 to close this gap.
Further experiments to inhibit rotation at the unstructured hinge between the N- and Clobes leaves little doubt that a flexible hinge is an functional feature of HECT domains
that enables rotation of the C-lobe to facilitate substrate labeling (Verdecia et al., 2003).
Some HECT domains require adaptor proteins to catalyze E3 activity. In a study that
describes the structure of another E3 HECT domain Smurf2 (PDB accession code
1ZVD), the authors describe a phenomenon by which catalytic activity of Smurf2 is
greatly enhanced by the addition of Smad7, a protein that belongs to the TGFp family of
proteins involved in multiple cell signaling pathways as a regulatory transcription factor
(Varelas et al., 2008). Addition of the N-terminal domain (NTD) domain of Smad7 is
shown to link the E2 (Ubch7) and Smurf2, as Smad7 can bind individually to either
component alone. Additionally, Smad7 appears to have a regulatory role. E2 enzymes
Chapter4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- andSubstrate
UbiquitinationActivities
such as UbcH6 and Ubc10 show decreased activity in the presence of Smad7. Although
not entirely necessary for the entire family of E3 HECT domains, it appears that some
E3s require a secondary regulatory protein to facilitate cooperation between E2 and E3
to increase specificity of substrate labeling. A recent paper from Brenda Schulman's
group describes a trimeric crystallographic structure between E2, E3, and ubiquitin
(Kamadurai et al., 2009). In this structure (PDB codes 3JVZ and 3JWO), an E3 HECT
domain NEDD4L and E2 enzyme, UbcH5B, are docked at the HECT domain E2 binding
site, with a single ubiquitin molecule covalently bound to UbcH5B. To suppress
conjugation between E3 and ubiquitin, the catalytic cysteine of NEDD4L was modified to
either alanine or serine. In this stunning crystallographic complex, the ubiquitin molecule
is positioned directly between E2 and E3 enzymes, primed for transfer to E3.
Conserved hydrophobic patches on the E3 C-lobe appear to stabilize a non-covalent
interaction with ubiquitin, effectively positioning ubiquitin against E3 so that the thioester
transfer reaction can take place between catalytic cysteines of E2 and E3 enzymes.
Overall the structure of NEDD4L in the NEDD4L-UbcH5B-ubiquitin complex is similar to
the HUWE1 HECT domain with fluctuations to the N-lobe at the E2 binding site,
significant for E2 specificity. The authors further elaborate that modification to substrate
specificity can likely be made based on changes to the HECT domain C-lobe, as was
seen by swapping the entire C-lobe for another from a second HECT domain (Kim and
Huibregtse, 2009). This also stems from the observation that modifying hydrophobic
residues on the C-lobe, distant from the both the catalytic cysteine and non-covalent
ubiquitin-interaction site modulate substrate-labeling activity (Salvat et al., 2004). These
observations make it clear that the E3 HECT domain has developed a variety of
mechanistic methods to regulate E2 and substrate specificity for accurate and
processive regulation of the ubiquitin pathway.
Chapter4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- andSubstrate
UbiquitinationActivities
An important question regarding HECT domain function is the control of ligase
activity and specificity. Here we present a functional analysis of the HECT domain of the
E3 ligase HUWE1, based on crystal structures, and show that a single, N-terminal helix
significantly stabilizes the HECT domain. We observe that this element modulates
HECT domain activity, as measured by self-ubiquitination induced in the absence of this
helix, as distinct from its effects on Ub conjugation of substrate Mcl-1. Such subtle
changes to the protein may be at the heart of the vast spectrum of substrate specificities
displayed by HECT domain E3 ligases.
HUWE1 (also called ARF-BP1, Mule, Lasul, Ureb1, E3 histone, and HectH9) is
a large 482-kDa HECT domain E3 Ub ligase implicated in the regulation of cell
proliferation, apoptosis, and DNA damage response (Adhikary et al., 2005; Chen et al.,
2005; Hall et al., 2007; Herold et al., 2008; Zhao et al., 2008; Zhong et al., 2005). We
recovered this enzyme with immunoprecipitation using Ub C-terminal electrophilic
probes (Love et al., 2009). Following an initial biochemical characterization (Love et al.,
2009), we completed a structural and biophysical analysis of the HECT domain to
understand modulation of its robust in vitro activity. Here we present crystal structures
of the HUWE1 HECT domain and characterize a structural element that both stabilizes
this domain and modulates its activity. This structural element, the al helix, is an
important component of the HECT domain that largely restricts its autoubiquitination
activity, while only nominally affecting Mcl-1 ubiquitination activity.
Results
Structure of the HUWEI HECT domain
We first attempted to crystallize the HUWE1 HECT domain using a fragment
defined by the founding member of the HECT domain E3 ligase family, E6AP (PDB
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Chapter 4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
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codes 1C4Z and ID5F) (Huang et al., 1999). The crystals diffracted to only 3.5 A (data
not shown), with fairly high temperature factors indicating vibrational disorder within the
protein crystals. By sequence comparison, we noted the significance of a conserved Nterminal helix that seals the hydrophobic core of the N-lobe in the structures of WWP1,
Smurf2, and Nedd4-like (residues 546-560 in WWP1 and 371-387 in Smurf2) (Ogunjimi
et al., 2005; Verdecia et al., 2003). The presence of this helix is conserved in over 13
HECT domain E3 ligases based on sequence comparison (Fig. 4.1a) and (Verdecia et
al., 2003), highlighting its structural importance. The al helix has been previously
described as a critical element for structural stability, yet an element dispensable for
HECT domain function (Huang et al., 1999; Verdecia et al., 2003). Initial model-building
into the 3.5 A electron density showed a noticeable hydrophobic groove on the surface
of helices 5, 11, 12, and 13, possibly indicating an additional helix being bound here. As
sequence conservation drops off N-terminal of this al helix (Fig. 4.1 b), we hypothesized
that this element is an important part of the HECT domain. We note that expression of
the HECT domain of Nedd4 yielded soluble folded product when the homologous helical
segment was included in the expression construct, but was not successful in its absence
(E. Maspero and S. Polo, personal communication). We therefore asked whether
addition of helix al would not only assist in our crystallographic efforts but also affect the
catalytic activity of the HECT domain.
Addition of helix al greatly stabilizes the HECT domain, as made evident in
thermal denaturation experiments (Fig. 4.1c), shifting the transition midpoint by 160C
from 44 *C to 60 0C. Although thermal stability differs between the two versions of the
HECT domain, the level of secondary structure remains the same (Fig. 4.1d), indicating
that the absence of helix al does not lead to unfolding, but to less rigidity of the domain.
We solved the structure of the helix-extended HECT domain by molecular replacement
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using the E3 ligase WWP1 (Verdecia et al., 2003) as a search model. The final model
(R/Rree 19.3%/24.5%) was built and refined to 1.9 A resolution (Table 1). The structure
of HUWE1 HECT domain closely resembles that of WWP1, with which it shares 41.3%
sequence identity (Fig. 4.2a). The HUWE1 HECT domain contains two distinct lobes
similar to previously determined HECT domain structures (E6AP, Smurf2, WWP1). The
larger N lobe (residues 3993-4252) contains the E2 binding region, and the smaller C
lobe (residues 4259-4374) contains the conserved catalytic cysteine (C4341). The N
lobe is composed of 13 a-helices and 7 p-strands, and the C lobe is composed of 4 ahelices and 4 p-strands. Residues 4253-4258 form the hinge that connects the two
lobes. A rotary movement about this linker likely repositions the N and C lobes to bring
the catalytic cysteine of the cognate Ub-loaded E2 in proximity to its E3 counterpart
(Verdecia et al., 2003). Like WWP1, HUWE1 is oriented in an inverted T shape (I), in
which the C lobe is positioned over the middle of the N lobe, with approximately 800 A2
of contact surface area (Fig. 4.2b). Hydrogen bonds between Glu4248 (N lobe) and
Ser4304 (C lobe), as well as Gln4245 (N lobe) and Gln4298 (C lobe), and a salt bridge
between Glu4246 and Lys4295, stabilize the I conformation. The I conformation is
further stabilized by water-mediated hydrogen bonds between the two lobes, involving
residues Arg4130, Glu4147, Ser4148, and Glu4244 from the N lobe and Gln4298,
Thr4340, Gly4302, and Lys4295 in the C lobe. The orientation of the N and C lobes of
the HUWE1 HECT domain differs from the more open conformation observed in the
crystal structures of E6AP and Smurf2 (Fig. 4.2c) (Huang et al., 1999; Ogunjimi et al.,
2005), although we cannot exclude the possibility that crystal contacts influence the
observed orientation of the C lobe. The stabilizing nature of helix al is apparent from
the extended structure, as it closes the hydrophobic core of the N-lobe (Fig. 4.2a, d).
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The most notable difference between HUWE1 HECT domain and previously
solved structures concerns the E2 binding region (residues 4150-4200). Most of the
hydrophobic residues in WWP1 that mediate contact with the E2 are similar to those in
HUWE1, obvious from the alignment between HECT E3 ligases (Fig. 4.2e). The
HUWE1 E2 binding region differs from that of WWP1, in that it contains additional
structured elements - mainly ordered p-strands not previously identified. The wellordered p-strands in the E2 binding region of the HUWE1 HECT domain extend farther
from the helical core of the protein than seen in the structure of WWP1, and the loop is
folded back on itself to complete the P3 strand and form the a8 helix (Fig. 4.2a). It is
possible that HUWE1 uses its unique E2 binding region to interact with a specific set of
E2 enzymes in vivo that differ from WWP1.
A comparison of our two HUWE1 HECT domain structures shows that they are
nearly identical with respect to the positioning of the N and C lobes (Fig. 4.7a, b). For
the structure lacking the al helix, the additional p-strands and a-helix seen in the E2
binding region remain unresolved, likely due to the low resolution data and high
temperature factors.
Catalytic activityof the HECT domain
As addition of the al helix to the HECT domain stabilized the protein, we asked
whether the presence of this structural element affected HECT domain catalytic activity.
We hypothesized its addition might confer altered catalytic properties to the HECT
domain compared to its helix-lacking counterpart. We therefore examined the ability of
the HECT domain to catalyze self-ubiquitination in the presence of El and E2 (UBE2L3)
enzymes, an ATP regenerating system, and [32 P]-Ub (Fig. 4.3). The use of [3 2 P]-Ub
allowed us to quantify the amount of Ub adducts formed and calculate initial rates of
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product formation in HECT-domain limiting conditions. In this assay, the HECT domain
catalyzes the formation of a complex mixture of self-ubiquitinated species (Fig. 4.3a, b)
that are not observed in absence of the HECT domain (lane marked "N").
Immunoblotting using an anti-His antibody confirmed that these species are
ubiquitinated E3 enzyme, as it is the only species in this reaction that contains a
polyhistidine tag (data not shown). The pattern of autoubiquitination observed is similar
regardless of the presence of the al helix - both versions form multi- and
polyubiquitinated species (Fig. 4.3a, b). Although the pattern of product formation is
similar, the presence of the al helix suppresses the autoubiquitination activity of the
HECT domain by more than 25-fold (Fig. 4.3c). As autoubiquitination is observed for
many Ub ligases and is often used as a criterion of E3 Ub ligase activity, we sought to
further characterize the reasons for its modulation.
The autoubiquitination reaction described above produces a complex mixture of
products. We examined HECT domain activity in a single-turnover reaction to monitor
the first round of Ub addition to the HECT domain. This assay encompasses two steps.
In the first step, E2-Ub thioester is generated by incubating El, E2, an ATP
regenerating system, and Ub. After the E2-Ub thioester has formed, this reaction is
quenched by the addition of EDTA to prevent further El-catalyzed activation of Ub. In
the second step, the HUWE1 HECT domain is added, and Ub is chased from the
E2-thioester onto the HECT domain (Eletr et al., 2005). The use of a mutant version of
Ub, in which all lysines are mutated to arginine (KO Ub), prevents polyubiquitin chain
formation on the HECT domain. Ub-conjugated HECT domain is visualized using antiUb immunoblot (Fig. 4.3d). We find that the HUWE1 A al HECT domain shows
increased activity under single turnover conditions compared to the HECT domain
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containing helix al (Fig. 4.3d), confirming the rate differences observed in the
autoubiquitination assay.
Thioester formation in the HECT domain
Catalysis by HECT domain E3 enzymes is a multi-step process. The E3 enzyme
binds Ub-loaded E2 and substrate, followed by Ub transfer between the E2 and E3
catalytic cysteines. The E3 then catalyzes isopeptide bond formation between Ub and a
lysine residue on the substrate, which may be the E3 itself, Ub, or another protein. We
next determined whether the presence of the al helix affects this upstream step, in
which the catalytic cysteine of the E3 enzyme forms a thioester bond with ubiquitin.
We first attempted to assay thioester formation using the wild-type HECT
domain, but the enzyme efficiently catalyzes formation of the isopeptide bond on a time
scale too fast to measure (Singer et al., 2008; Wang and Pickart, 2005). Instead, we
analyzed thioester formation using a four-amino acid, C-terminal truncation of the HECT
domain (Salvat et al., 2004). This truncation removes a crucial determinant for
isopeptide bond formation, a conserved phenylalanine located four amino acids from the
C terminus of most HECT E3s (Salvat et al., 2004). HUWE1 A4 displays a diminished
rate of isopeptide bond formation, allowing us to monitor formation of thioester-linked Ub
to the enzyme. After incubation of the HECT domain with El, the E2 UBE2L3, Ub, and
an ATP regenerating system, the reaction is quenched with SDS-PAGE loading buffer
with or without p-mercaptoethanol, and following electrophoretic resolution, is analyzed
by anti-Ub immunoblot. The presence of the al helix greatly reduces the rate of
thioester formation (Fig. 4.4), proportional to its suppression of autoubiquitination
activity. The al helix, located on the back surface of the N lobe, is clearly not sufficiently
close to interact with the E2 binding region of the HECT domain (Fig. 4.2a), suggesting
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that the vibrational disorder in this protein contributes to the HECT domain-E2
interaction.
Substrate ubiquitinationcatalyzed by the HECT domain
Having seen that removal of helix al destabilizes the HECT domain and
increases its autoubiquitination activity; we asked whether this effect is also observed
during substrate ubiquitination. The anti-apoptotic Bcl-2 family member Mcl-1 is an in
vivo target of HUWE1 (Zhong et al., 2005). HUWE1 recruits McI-1 via its BH3-domain,
while the HECT domain presented here catalyzes Mcl-1 ubiquitination. Although Mcl-1
is a substrate of the full-length HUWE1, we use this assay with the isolated HECT
domain here as a measure of non-self ubiquitination activity with an in vivo verified
substrate of HUWE1. We examined initial rates of product formation, under HECTdomain limiting conditions, to determine whether the intrinsic activity of the HECT
domain toward substrate is altered by the destabilizing effect of removing the al helix
(Fig. 4.5a, b). We find that the HECT domain lacking al helix is -5-fold more active in
catalyzing McI-1 ubiquitination than the more stable HECT domain containing helix al
(Fig. 4.5c). These results also suggest that autoubiquitination of the HECT domain does
not impair catalytic activity toward substrate. The two versions of the HUWE1 HECT
domain, which differ 25-fold in autoubiquitination rates, show only a 5-fold difference in
their Mcl-1 ubiquitination rates. A similar observation was made for the heterodimeric
complex of the minimal catalytic domains of Ringi a/Bmil, in which autoubiquitination of
the Ring Ib protein did not affect E3 ligase activity toward its substrate, histone H2A, in
an in vitro reconstituted system (Buchwald et al., 2006). We also observe similar
differences in autoubiquitination and Mcl-I ubiquitination activity between the two
versions of the HECT domain at 370 C (Fig. 4.6).
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Catalytic activityof the C4341A mutants
Mutation of the conserved catalytic cysteine to alanine (C4341A) abolishes
activity of the HECT domain (Fig. 4.8). In the case of the helix-lacking HECT domain,
we consistently observed that the C4341A mutant is capable of transferring a single
ubiquitin to self (Fig. 4.8) or Mci-I (Fig. 4.8). These species are not generated when the
HECT domain is omitted from the reaction (lane marked "N"). Although the failure of
mutation of the catalytic cysteine to abolish activity has been previously observed
(Adhikary et al., 2005), quantification of the monoubiquitinated species shows that this
activity represents at best a minor fraction of wild-type activity (Fig. 4.8).
Discussion
We present here crystal structures of the HUWE1 HECT domain and identify a
conserved structural element, helix al, which stabilizes the HECT domain and tightly
modulates its activity. Helix al is present in the structure of the WWP1 HECT domain
(referred to in the WWP1 structure as HI') (Verdecia et al., 2003), where the authors
note that it plays an obvious role in contributing to HECT domain stability. As the HI'
helix is oriented between the C lobe and domains N-terminal to the HECT domain that
presumably mediate protein-protein interactions, the authors suggested that HI' helix
contributes to target protein specificity in reactions catalyzed by the HECT domain. We
confirm that the al helix is indeed crucial for stability and identify a role for this structural
element in modulating HECT domain activity, as judged by autoubiquitination and Mcl-I
ubiquitination assays. Further experiments will determine whether this conserved helix
modulates activity of other members of the HECT domain family.
In the absence of the N-terminal al helix, the HUWE1 HECT domain gains
activity relative to its helix-extended counterpart. What could be the reason for this
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unexpected behavior? Deletion of helix al might expose hydrophobic residues that
trigger assembly of HUWE1 HECT domains into oligomers. Such behavior has been
observed in the crystals of E6AP (PDB codes 1C4Z and 1D5F) (Huang et al., 1999).
However, HUWE1 HECT Aal behaves as a monomer and is properly folded in solution,
as judged by several criteria (Fig. 4.1c and data not shown).
Our structural data on HUWE1 shows that the HECT domain adopts the same
conformation regardless of the presence of helix al, and both variants contain the same
helical content (Fig. 4.1c). HUWE1 A al, however, is far more active in catalyzing selfubiquitination and in single-turnover assays. It also accepts Ub from the E2 UBE2L3
more readily than the helix-extended HECT domain. Furthermore, we observed
elevated temperature factors, indicative of conformational flexibility, in the crystal
structure of HUWE1 HECT Aal; a similar observation was made in the crystals of the
E3 Ub ligase lpaH from the bacterial pathogen Shigella flexneri (Singer et al., 2008). We
favor the interpretation that removal of helix al destabilizes the HECT domain to
produce a more relaxed version of the enzyme that exhibits greater intra-domain
flexibility. This increased flexibility allows the enzyme to sample more conformational
states, thereby increasing its level of activity. Some of these conformational states may
resemble the extended HECT domain structures observed in the crystal structures of
Smurf2 and E6AP, in which the C-lobe has rotated about the flexible linker that connects
the two subdomains of the HECT domain. In this scenario, removal of the al helix is
analogous to the linker-extension mutations made in WWP1 (Verdecia et al., 2003). The
removal of helix al may also shift the conformational equilibrium of the HECT domain
into an orientation that facilitates the E2-HECT interaction or product release. This
possibility is supported by evidence that enzymes exist in a dynamic range of
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conformations, and the equilibrium between these different conformers can be shifted by
mutation (Eisenmesser et al., 2005).
We did not anticipate that destabilization of the HECT domain would increase
enzymatic activity. The Ub transfer reaction involves defined regions including the
ordered p-strands that describe the E2 binding region and the catalytic site surrounding
residue C4341. However, other steps, such as product release, may contribute to
catalytic rate and may be influenced by increased conformational flexibility (Tokuriki and
Tawfik, 2009). A correlation between conformational flexibility and promiscuous activity
has been observed for several other proteins (Tokuriki and Tawfik, 2009). An example
of a flexible enzyme is cytochrome P450, which can adopt a range of different
conformations that allow it to act upon a variety of substrates. Among the P450 family of
enzymes, the rigid CYP2A6 enzyme exhibits limited substrate specificity, whereas the
highly flexible CYP3A4 is far more promiscuous (Tokuriki and Tawfik, 2009). In the case
of HUWE1 HECT domain, the al helix may serve to impose a constraint on the inherent
flexibility of the catalytic domain, thus fine-tuning enzymatic activity.
Autoubiquitination is often used as a criterion of E3 Ub ligase activity and, for
some ligases, has been proposed as a mechanism of self-regulation of stability and
downstream signaling functions (Varfolomeev et al., 2007; Wiesner et al., 2007). Our
data show that this type of activity can be largely suppressed by minor extensions of
what has been considered the core catalytic domain. We note, however, that our study
focuses on the HECT domain of a multi-domain protein, and there may exist other
structural elements in the 482-kD Huwel protein that affect its activity. This has been
observed for the E3 Ub ligases Smurf2 (Wiesner et al., 2007), IpaH9.8 (Singer et al.,
2008), and SspH2 (Quezada et al., 2009), in which domains N-terminal to the catalytic
domain suppress autoubiquitination activity. While autoubiquitination is clear evidence of
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catalytic activity of Ub ligases, the functional relevance of this reaction remains to be
established for many E3s, including HUWE1. The increase in activity seen in the
absence of helix al appears to stem from increased conformational flexibility in the
enzyme. It is difficult to rationalize this behavior from static crystal structures, yet
increased thermal motion observed in the Ahelix al structure is at least an indirect
indicator. The significance of thermal motion within a protein with respect to reaction
parameters is an emerging theme (Lange et al., 2008). HECT domains may have
diverged to arrive at their extant spectrum of substrates by modulating flexibility (Tokuriki
and Tawfik, 2009), in addition to more tractable changes of surface properties. We
conclude that the activity of the HUWE1 HECT domain is tightly modulated, through
restriction of conformational space rather than steric considerations, by the presence of
a 19-residue helix al.
Methods
Plasmids
HECT domain constructs of HUWE1 (amino acids 3993-4374 or amino acids
4012-4374) were cloned into a modified pET-28a plasmid (Novagen) containing a
human rhinovirus 3C (HRV3C) protease site to generate an N-terminal His6 fusion
protein for use in biochemical assays. The catalytic mutants C4341A and
C4099A/C4184A/C4367A were generated using site-directed mutagenesis (Stratagene).
For biochemical assays with radiolabeled substrate, Flag-McI-1 (amino acids 1-327) and
Ub were both cloned with an N-terminal protein kinase A (PKA) site for [3 2P]-labeling into
pET-16b and pET-28a with the HRV3C site, respectively, as previously reported (Love et
al., 2009). UBE2L3 was cloned into the pET-28a plasmid (Novagen).
110
Chapter 4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
Bacterial protein expression and purification
All versions of the HUWE1 HECT domain were expressed and purified as
previously reported (Love et al., 2009). [32 P]-labeled proteins were purified and labeled
as previously described (Love et al., 2009). Native UBE2L3 was expressed in Rosetta
(DE3) cells (Novagen). UBE2L3 was precipitated from bacterial lysate by addition of
saturated ammonium sulfate to 90%. The precipitated protein was resuspended in 50
mM HEPES pH 7.4, 200 mM NaCl and purified by gel filtration (Superdex 75 PC 3.2/30,
GE Healthcare).
Circular dichroism
HUWE1 Aal and HUWE1 + al HECT domains were dialyzed into 5 mM HEPES pH
7.5, 100 mM NaCl immediately prior to the scanning and melting CD experiments using
an AVIV model 202 CD spectrometer. HUWE1 Aal HECT domain at 2.4 jAM and
HUWE1 + al HECT domain at 2.8 [M were used for scanning experiments between
195 and 280 nm at 250C. CD signal at 222 nm of 4.8 [M HUWEI Aal and 5.6 [M
HUWE1 + al was recorded every 2 degrees over a 20-94 *C temperature ramp with 2
minutes of equilibration time at every step.
Biochemicalassays
Reaction mixtures (10 pl) for HUWEI autoubiquitination assay contained 100 nM
human El (Ubel, Boston Biochem), 5.6 pM E2 (UBE2L3), HECT domain, and 60 pM
[32P]-Ub with an ATP regenerating system (50 mM Tris [pH 7.6], 5mM MgCl 2 , 5 mM ATP,
10 mM creatine phosphate, 3.5 U/mI creatine kinase). Reactions were incubated at
room temperature and aliquots were removed after the indicated amount of time and
terminated in reducing SDS-PAGE sample buffer. Samples were boiled 10 min. and
111
Chapter 4: A StructuralElement Within The HUWE] HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
separated on 8% Tris-tricine SDS-PAGE. Dried gels were exposed to a phosphor
screen. [3 2 P]-Ub bands were visualized by autoradiography and quantification of data
was performed using a phosphorimager. Background correction for each sample was
performed by subtracting the counts from an equivalent area of the gel from a lane
containing all reaction components except E3 enzyme (lane marked "N"). The [32P]-Ub
signal from this lane was used to convert the observed sample counts to a concentration
value. The concentrations of HUWE1-[ 3 2 P]-Ub were measured and rates of product
formation determined by fitting the initial linear data points to a least squares regression
line.
Mci-1 ubiquitinationassay
Reaction mixtures (10 pl) for the Mcl-1 ubiquitination assay were set up as above
except with the addition of 5 pM [3 2 P]-Flag-Mcl-1 and 100 pM Ub (Sigma). Reactions
were quenched with reducing sample buffer and separated on 10% SDS-PAGE. Bands
from dried gels were analyzed as above.
Thioester assay
Reaction mixtures for the thioester assay (10 pl) contained 100 nM human El
(Ubel, Boston Biochem), 5.6 pM E2 (UBE2L3), 2 pM HUWE1 A4 HECT domain, and 60
pM Ub (Sigma) with an ATP regenerating system described above. Reactions were
incubated at room temperature and aliquots were removed after the indicated amount of
time and terminated in 4M urea and incubated 15 min. at 30*C, or terminated in reducing
SDS-PAGE sample buffer. Samples were boiled 10 min., separated on 10% Tris-glycine
SDS-PAGE, and analyzed by immunoblot using anti-Ub antibody (Sigma).
112
Chapter4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
Single-turnoverassay
For the single-turnover assay, the E2-Ub thioester was generated in a 20 [I
reaction containing 200 nM El (Boston Biochem), 8 [M E2, ATP regenerating system
described above, 60 [M mutant Ub in which all lysines are mutated to arginine (KO Ub)
(Boston Biochem), and 1 tg/ i BSA incubated 25 min. at room temperature. Formation
of the E2-Ub thioester was quenched with 50 mM EDTA on ice for 5 min. The E2-Ub
thioester was diluted into chase mixture containing 2 [M HECT domain, 100 mM NaCl,
50 mM EDTA, and 1 [tg/Rl BSA, or the same reaction components lacking the HECT
domain (labeled "N"). Reactions were incubated at room temperature and aliquots were
removed after the indicated amount of time and terminated in either 4 M urea and
incubated 15 min. at 30*C, or in reducing SDS-PAGE sample buffer. Samples were
boiled 10 min., separated on 10% Tris-glycine SDS-PAGE, and analyzed by immunoblot
using anti-Ub antibody (Sigma).
Crystallizationof HUWE1 HECT domain
Crystallization experiments with purified HUWE1 HECT domain including the Nterminal His 6 tag, HRV3C protease site and with C4099A, C4184A, and C4341A
mutations, were set up in 96-well sifting drop trays using commercially available sparsematrix screens (Hampton Research, Qiagen). The initial crystals were improved in
hanging-drop vapor diffusion setups. The HUWE1 + al HECT domain crystallized by
mixing 1 pl of protein sample concentrated to 17 mg/ml with a 1 pl solution containing
0.1 M citric acid (pH 5.2) and 1.8 M (NH4)2SO 4 . Birefringent crystals in the shape of thick
rods with dimensions of approximately 80 x 40 x 40 [im grew within two days of
incubation at 18 0C. The HUWE1 Aal HECT domain crystallized by mixing a 1 pl
113
Chapter4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
solution containing (Na/K) 2PO4 (pH 6.5) and 1.4 M (Na/K) 2PO4. Thin rod-shaped
crystals grew within 10 days at 230C.
Data collectionand processing
For native X-ray diffraction studies, crystals were cryoprotected by soaking in 0.1
M citric acid (pH 5.2), 1.8 M (NH4) 2 SO4 , 12% glycerol for 30 sec prior to vitrifying in liquid
nitrogen. X-ray diffraction data were collected on a single cryogenized crystal at
beamline 241D-E, Advanced Photon Source (Argonne, IL, USA), summarized in Table
4.1. Data were processed using DENZO and SCALEPACK (Otwinowski, 1993). The
crystals belong to the monoclinic space group C2 and diffracted to 1.9 A. Initial phases
were obtained by molecular replacement using PHASER from the CCP4 crystallographic
program suite (Collaborative Computational Project, 1994; McCoy, 2007), with the
coordinates of the E3 ligase WWP1 (PDB accession code 1ND7) as search model. The
final model was refined at resolution of 1.9 A using PHENIX (Verdecia et al., 2003).
Details of refinement are given in Table 4.1. Figures were made using PyMol (DeLano,
2002).
Acknowledgements
We thank Christian Schlieker for helpful discussions and critical reading of the
manuscript, and Fenghe Du and Xiaodong Wang at UT Southwestern Medical Center for
providing cDNA plasmids for HUWE1 (Mule) and Mcl-1. RKP is supported by a US
Department of Defense Breast Cancer Research Program award (W81XWH-06-1-0789).
KRL was supported by an NIH postdoctoral fellowship (F32 A163854).
114
Chapter 4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- andSubstrate
UbiquitinationActivities
The abbreviations used are: HECT, homologous to E6AP C-terminus; Ub, ubiquitin;
HUWE1, HECT, UBA and WWE domain containing 1. Protein coordinates have been
deposited in the RCSB Protein Data Bank (PDB code 3H1 D).
115
Chapter4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
Figures
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-Huwel
1V~
+ 01
A al
.seesm~vn
. se.wm"
4i'e is
is
-s
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A
- 1-
Figure 4. 1 - The al helix stabilizes the HUWEI HECT domain
(a) Multiple sequence alignment of helix al with a diverse set of human HECT E3 ligases.
Residue conservation is indicated by degree of shading ranging from orange (most conserved) to
light yellow (least conserved). Secondary structure is illustrated with a-helices as cylinders and
b-sheets as arrows. (b) Multiple sequence alignment with a diverse set of human HECT E3
ligases indicating that sequence conservation drops off N-terminal to the al helix. The N116
11
0
-
-
1
00v:, 1
-
7
0 .:*::,
....
. .....
..- -
-
.
. ..........
...........................
Chapter 4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
terminus of the HECT domain + al helix is indicated. (c) Thermostability of the HUWE1 HECT
domain was measured in a CD melting experiment. HUWE1 HECT domain, +/D al helix, wildtype or with cysteine mutations, was heated in a circular dichroism cuvette and unfolding
measured at 222 nm as a loss of helical content. Deletion of helix al results in reduction of
thermostability. (d) A CD scan demonstrates structural similarities of the +/D al helix versions
of the HECT domain.
Huwel
C-lobe
P"
N-lobe
Ala4291
PR3994,
Ptie242
Arg3998
1194230
FLe"250
-
VaM238
Pro4253
Lu40050
0 UtB.'a
Val4118
Asn4121
Ph.4230
114234
Tyr4119
Asp4009
Lys4043
Lys4014
Figure 4. 2 - Structure of the HUWEI HECT domain
(a) Stereo view of HUWE1 HECT domain (residues 3993-4374) showing the N- and C-lobes
connected by the hinge loop. Helix al is colored green. The N-lobe contains the E2 binding
region and the C-lobe contains the catalytic cysteine (C4341). (b) Overlay of HUWE1 (blue) and
WWPl (orange; PDB 1ND7) crystal structures. (c) Overlay of HUWEl (blue) and Smurf2
(orange; PDB 1ZVD) crystal structures. (d) Helix al plays a significant role in mediating
117
Chapter 4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
hydrophobic contacts that maintain the core of the HUWE1 HECT domain. Hydrophobic
residues in the al helix, Phe3994 and Phe4001, pack into hydrophobic pockets in the N lobe.
Arg3998 and Asp4009 form hydrogen bonds stabilizing the N-lobe. Lys4014 and Tyr4l19, Cterminal to the al helix, orient the al helix to further stabilize the N-lobe. (e) Multiple sequence
alignment of E2 binding region with a diverse set of human HECT E3 ligases. Residues
important for E2 binding are indicated with blue circles.
118
Chapter4: A StructuralElement Within The HUWEJ HECT DomainModulates Self- and Substrate
UbiquitinationActivities
b
a
WT HECT Aa1
3040506070 N
WT HECT + al
N
2 2.5 3 4 5
sec.
250150-
250150Huwel -Ub
170-
Huwel-Ubn
I-
50 -
50 -
25-.*
25 20-
20 10-
C
min.
is-
Ub
-5Ub
10-
Autoubiquitination Activity
Sample
WTHECTA1
WT HECT + (l
d
WT HECT al
1 3 5 7 9 12
U ation rate (min)
11.475±2.19
0.454 ±0.053
WT HEC+
l
1 3 5 7 9 12 min.
15075
Reducing
-
50
HUWEl -Ub,
37N
1 3 5 7 9 12 min.
25150-
150-
75-
75
-HUWEl-Ub,
Non-reducing 50 -
37-
-
50 -
37-
- UBE2L3-Ub 25-
25-
Figure 4. 3 - E3 Ubiquitin ligase activity of HUEWI HECT domain
-UBE2L3-Ub
The autoubiquitination activity of HUWEl HECT domain was tested using 60 pM [3 2P]-Ub as
substrate and (a) 2 pM WT D al or (b) 2.9 pM WT + al HECT domains incubated with UBEl,
UBE2L3, and an ATP regenerating system. (Note the different time scale for the two variants of
the HECT domain). HECT domain is omitted in the lane marked "N". Asterisk denotes a likely
ubiquitin polymer; double asterisk denotes likely mono-ubiquitinated UBE2L3. Concentrations
of HECT domain were chosen to obtain initial rate conditions. (c) Ligation activity of the WT
D al and WT + al HECT domains in the autoubiquitination assay. Activity is given as the ratio
between initial velocity (pmol total [32 P]-Ub product/min) and total enzyme concentration E
(pmol). Errors are the standard deviations calculated from three independent experiments, shown
in parenthesis. (d) Single turnover assay monitoring transfer of Ub from the UBE2L3-Ub
thioester to a lysine in the WT D al and WT + al HECT domains. The UBE2L3-Ub thioester is
generated in a pulse reaction containing E1, UBE2L3, ATP regenerating system, and a Ub mutant
in which all lysines are mutated to arginine (KO Ub). Ub is chased from the E2 enzyme to HECT
domain added to the reaction. Ub-conjugated HECT domain is visualized by anti-Ub
119
Chapter4: A StructuralElement Within The HUWE] HECT DomainModulates Self- and Substrate
UbiquitinationActivities
immunoblot. Samples were terminated in reducing or non-reducing sample buffer as indicated.
Panel marked "N" is a chase reaction performed in absence of HECT domain and terminated in
non-reducing sample buffer.
WT HECT
al
15 30 45 60 120 180
WT HECT + al
15 30 45 60 120 180 sece
150 -
10075 -
Non-reducin
- HECT-Ub,
50 -
37-
25
-
-
UBE2L3-Ub,
150 100 75 -
Redudng
50 37-
25 -
Figure 4. 4 - Detection of Ub~thioesters in the HUWE1 HECT domains
Ub-thioester assay with the indicated HUWE1 HECT domain proteins. Purified HECT domains
containing a C-terminal truncation of the last four amino acids were incubated with El, UBE2L3,
an ATP regenerating system, and Ub for the indicated amount of time. Reactions were stopped
with 4M urea and non-reducing sample buffer (upper panel) or reducing sample buffer (lower
panel), separated by SDS-PAGE, and analyzed by immunoblot with anti-Ub antibody.
120
Chapter 4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
a
WT HECA al
22.53 4 5 N
b
min.
u.
5037-
3 Mc-i-Ub,
- Mci-I
C
WT HECT+al
2 2.5 3 4 5 N
min.
50-
z MdI-1-Ub,
37-
- Mci-I
Mci-i Ubiquitination Activity
I
Sampe
WT HECT Aal
WT HECT + al
d
ation rate min')
0.635 ±0.184
0.130± 0.033
e
WT HECT a1
WTHECT+ al
4.5
4
3.5
3
2.5
2
1.5
1
o.s
n
0
2
4
6
0
2
4
6
Time (min.)
Time (min.)
Figure 4. 5 - Substrate ubiquitination activity of the HUWEI HECT domains
(a,b) The Mcl-I ubiquitination activity of HUWE1 HECT domain was tested using 5 piM [32P]Mcl-i as substrate and (a) 100 nM WT D al or (b) 300 nM WT + al HECT domains incubated
with UBEl, UBE2L3, Ub, and an ATP regenerating system. HECT domain was omitted from
the lane marked "N". Concentrations of HECT domain were chosen to obtain initial rate
conditions. (c) Ligation activity of the HECT domains in the Mcl-I ubiquitination assay. Activity
is given as the ratio between initial velocity (pmol total [32P]-Ub product/min) and total enzyme
concentration E (pmol). Errors are the standard deviations calculated from three independent
experiments, shown in parenthesis. (d. e) Graph showing percent ubiquitinated Mcl- 1 as a
function of time in the reactions shown in panels (a) and (b) catalyzed by HUWEl D al (d) or
HUWEl + al (e) HECT domains.
121
Chapter 4: A StructuralElement Within The HUWE] HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
a
WT HECT A a I
30 40 SO AO 7n
WT HECT + alI
-n 40 S1fn 70
sec.
250150100Huwel-Ub,
7550-
b
WT HECT A c
2 2.5 3 4 5
WT HECT + ail
2 2.5 3 4 5
min.
2501501007550-
McI-Ub
37-
Figure 4. 6 - Ubiquitination activity of the HUWEI HECT domains at 37*C
(a) The autoubiquitination activity of HUWE1 HECT domain at 37'C was tested using 60 pM
Ub as substrate and 2 pM WT HECT domains incubated with UBE1, UBE2L3, and an ATP
regenerating system. Reaction mixtures were separated on SDS-PAGE and analyzed by anti-Ub
immunoblot. (b) The Mcl-i ubiquitination activity of HUWE1 HECT domain at 37'C was tested
using 5 pM Flag Mcl-I as substrate and 100 nM WT HECT domains incubated with UBE1,
UBE2L3, Ub, and an ATP regenerating system. Reaction mixtures were separated on SDSPAGE and analyzed by anti-Flag immunoblot.
122
............
Chapter 4: A StructuralElement Within The HUWEJ HECT Domain ModulatesSelf- andSubstrate
UbiquitinationActivities
a
b
Figure 4. 7 - Both HUWEI Aal and +al HECT domains maintain the .L
conformation
(a) Overlay of the Ca backbone of the HUWEl + al HECT domain (orange) and HUWEl D al
HECT domain (blue). (b) The Ca backbone of the HUWEl + al HECT domain fit into electron
density of the HUWEl D al structure demonstrates the similarity of these structures.
123
Chapter 4: A Structural Element Within The HUWE] HECT Domain Modulates Self- and Substrate
Ubiquitination Activities
a
b
C4341A HECT domain A al
time (min.)
2.5
5
250-
8
16 20 4060
N
time (min.)
~
Arf-bpl-Ubi
25
C4341A HECT domain
8
14
25 36 45 60
N
4060
N
-Ub
~6____4&____M__
C4341 A HECT domain + al
time (min.)
2.5 5
8
14
28
40.5 60.5 N
75-
50-
e
20
5037-
d
A a1
75-
37-
16
2015100
Ub
4
8
25-
*
20
15
10__
2.5
5
75-
5037-
time (min.)
2.5
250_
1000
C
C4341A HECT domain + al
McI-1-Ub 1
f
Autoubiquitination Activity
Sample
I
C4341 AAfl
C4341 A + l
50
-__
Md-1*______
37_-
_____Md-1
I
Uqation rate (min')
Mc-i Ubiquitination Activity
Sample
I Uation rate (min')
0.0007 (7.85e-5)
C4341A A u1
0.0009 (0.0002)
ND*
C4341A + al
ND*
Product formation above background not detected.
Figure 4. 8 - Mutation of the catalytic cysteine (C4341) to alanine abolishes activity
of the HECT domain
(a, b) The autoubiquitination activity of C4341A HECT domain was tested using 60 pM [32P]-Ub
as substrate and (a) 11.6 iM C4341A D al or (b) 12.1 ptM C4341A + al HECT domains
incubated with UBE1, UBE2L3, and an ATP regenerating system. Aliquots of the reaction mix
were removed after the indicated amount of time, quenched in reducing SDS sample buffer, and
separated on SDS-PAGE. HECT domain is omitted in the lane marked "N". Asterisk denotes a
likely ubiquitin polymer. (c, d) Substrate ubiquitination activity was measured using 5 pM [32P]Mel-i as substrate and (a) 11.6 pM C4341A D al or (b) 12.1 pM C4341A + al HECT domains
incubated with UBE1, UBE2L3, Ub, and an ATP regenerating system. HECT domain is omitted
in the lane marked "N". (e, f) Ligation activity of the HECT domains in the
(e) autoubiquitination or (f) Mel-I ubiquitination assay. Activity is given as the ratio between
initial velocity (pmol total [32P]-Ub product/min) and total enzyme concentration E (pmol).
Errors are the standard deviations calculated from three independent experiments, shown in
parenthesis.
124
Chapter4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- andSubstrate
UbiquitinationActivities
Table 4. 1 - Crystallographic data collection and refinement statistics
Data Set
HUWE1 HECT + al
Data Collection
Wavelength
Space group
Cell dimensions:
a,b,c (A)
P (0)
Unique reflections
Resolution (A)
Rsyma
Rr.i.m.
Rp.i.m.
Completeness
Redundancy
I/or
Wilson B factor (A2)
Refinement
Resolution (A)
Nonhydrogen Atoms
Water Molecules
Rworkh
Rfreoc
R.m.s. deviations
Bond lengths (A)
Bond angles
(0)
B factors (A2)
Protein
Water
Coordinate error (A)
Ramachandran plotd
Most favored (%)
Allowed (%)
Generously allowed (%)
Disallowed (%)
0.9793
C2
119.6, 56.6, 69.6
122.5
30,847
30-1.9 (1.93-1.9)
0.069 (0.392)
0.084 (0.493)
0.048 (0.295)
98.3 (96.3)
3.2 (2.5)
17.2 (2.1)
25
30-1.9
3190
357
16.6
22.9
0.01
1.20
30.0
39.4
0.68
93.1
6.9
0
0
= EII - <II>|/XI, where I, is the intensity of the ith observation and
<I,> is the mean intensity of the reflection.
a Rsym
125
Chapter 4: A StructuralElement Within The HUWEJ HECT Domain Modulates Self- and Substrate
UbiquitinationActivities
1)]112 i|Ii(hkl) - <I(hkl) >I/Zbkl~I I;(hkO, where I,(hkl) is the observed
intensity and <I(hkl)> is the average intensity of multiple observations of symmetry-related
reflections.
c Rp.i.m. = hkIl[1/(N - 1)]1/12 Ei Ii(hkl) - <I(hk) >I/hkl Zi I;(hkl), where I;(hk) is the observed
intensity and <I(hkl)> is the average intensity of multiple observations of symmetry-related
reflections.
d Rwork = (I|IFobs| - |FcalcI| / ZIFobsi)
e Rfree = R value for a randomly selected subset (5%) of the data that were
not used for minimization of the crystallographic residual.
Highest resolution shell is shown in parenthesis.
f Calculated with the program MolProbity (Davis et al., 2007).
bRri.m. = )jhkl[N/(N--
126
Chapter5: Conclusion
Chapter 5: Conclusion
127
Chapter 5: Conclusion
Summary
The body of work presented in this dissertation comes from a collection of three
distinct projects that utilize a crystallographic and biophysical approach towards
understanding protein-protein interactions. Two studies have focused on
characterization of reassembled nucleoporin subcomplexes and the third project
concerns the structure and function of an E3 HECT domain ubiquitin ligase. Details and
results for each project have been summarized below, with some thoughts on what
future studies might entail.
Ran-binding zinc fingers from Nupl53 bind weakly at the switch I region of
RanGDP. The strongest binding constant is measured at -5 pM for two out of four
Nup1 53 zinc fingers and ~ 50 pM for the other two. RanGTP also binds to Nup1 53 zinc
fingers albeit with lower affinity. Considering the weak binding constant and symmetric
localization across NPC, the Ran-binding zinc finger domain helps to facilitate proper
regulation of nucleocytoplasmic transport by sequestering RanGTP and RanGDP in
close proximity to the NPC. These interactions are likely to be fast on/off interactions that
play a subsidiary role in the Ran regulated transport through the NPC.
Reconstitution of a tetrameric complex between the Nspl trimeric subcomplex
(Nspl, Nup57, Nup49) and full-length Nic96 marks a significant step in elucidating
structural properties that govern assembly of the NPC. The coiled-coil domains of the
Nspl complex are sufficient to assemble a trimeric complex that interacts with full-length
Nic96. Helical deletions at the N- and C-terminus of Nup57 confirm that Nup57 interacts
directly with Nspl and that this interaction is necessary for copurification of Nspl with
Nup49 and Nup57. We have also demonstrated that a core interaction domain exists
between Nup57 and Nup49. Biophysical analysis with ultracentrifugation demonstrates
that the Nspl trimeric complex contains one molecule of Nspl, Nup57, and Nup49 per
128
Chapter5: Conclusion
assembly. Purification of Nup49 alone forms a tetrameric assembly in the absence of
Nup57 and Nspl, in agreement with previous structural data for the Nup49 homologue
Nup58. Assembly of a tetrameric species between the Nspl trimeric complex and Nic96
has been characterized with gel filtration and further experiments are necessary to fully
characterize this large coiled-coil assembly.
Structural characterization of the HECT domain from the E3 ubiquitin ligase
HUWE1 is a significant deviation from our studies on the NPC. This difference is only
topical however, as much of the same methods and scientific principles that were used
throughout our studies of the NPC have been used to study HUWE1. During
crystallization experiments with HUWE1 we found that a small conserved helical element
(the al-helix) at the N-terminus strongly influences substrate- and self-ubiquitination
activity. Although the al-helix strongly enhances thermal stability of HUWE1, the
absence of the al-helix does not disrupt the secondary structure of HUWE1.
Structurally, the HUWE1 HECT domain is similar to previously solved HECT domains,
exhibiting a conformation of the N- and C-lobes most similar to WWP1. Upon analysis of
our structure and other similar HECT domains, it is surprising that a small helix would
have such a drastic effect on thermal stability and ubiquitination activity. The al-helix
most likely contributes to stabilizing the N-lobe hydrophobic core and removal is enough
to destabilize regulation of self- and substrate-labeling mediated by rotation of the C-lobe
about the N-lobe.
Future Directions
A role for Nup 153 zinc fingers in nucleocytoplasmictransport
Several research groups have now demonstrated that both Nup153 and Nup358
bind to RanGDP with low micro-molar affinity via the RanBP2-type zinc finger domain.
Crystallographic studies have provided detailed information regarding the interaction
129
Chapter5: Conclusion
between RanBP2-type zinc fingers and RanGDP that exists in a 1:1 stoichiometric ratio
with one molecule of each component per complex (Partridge and Schwartz, 2009;
Schrader et al., 2008a). Although we understand the structural details of how RanBP2type zinc fingers bind preferentially to RanGDP over RanGTP, we do not yet fully
comprehend why Nup1 53 and Nup358 bind RanGDP at either face of the NPC. Several
live imaging studies done with microinjected wild-type Ran, labeled with dye, have
described accumulation of Ran at either side of the nuclear envelope, and with
fluorescence imaging provide evidence that Ran exists at a surprisingly high
concentration around the nuclear rim (Hinkle et al., 2002; Paradise et al., 2007; Smith et
al., 2002). More recent analysis using EYFP-tagged wild-type Ran in HELA cells
describe Ran accumulating at the nuclear envelope with an estimated 800 molecules of
Ran/pm 2 nuclear envelope (Abu-Arish et al., 2009). The authors further explain that
although Ran accumulates at the nuclear envelope, Ran is not evenly dispersed across
the envelope but rather focused as discrete puncta around nuclear pores. With this in
mind the authors calculate that nearly 200 wild-type Ran molecules surround one NPC
at any given time (Abu-Arish et al., 2009). Our research supports the notion that a
population of Ran binds to the NPC via low pM interactions with RanBP2-like zinc
fingers of Nup1 53 and Nup358. It remains unclear if these interactions are intermediary
steps of nucleocytoplasmic transport within the RanGDP-RanGTP cycle, or perhaps
these interactions are simply responsible for localizing Ran near the NPC for interactions
with transport factors. It is conceivable that Nupl53 at the nuclear basket is a
designated site for nucleotide exchange mediated by RCC1. RanGTP does bind to
RanBP2-type zinc fingers with 50 pM affinity and following nucleotide exchange,
RanGTP might remain at the nuclear basket and bind tightly with importin-p-cargo
complexes to terminate import and release cargo into the nucleoplasm. These details
130
Chapter5: Conclusion
remain unknown and future transport experiments will decipher the molecular basis for
direct NPC interaction with RanGDP mediated by RanBP2-type zinc fingers.
From a structural perspective the interaction between these two proteins is clear.
A protein engineering approach allowed us to achieve very high-resolution data for each
zinc finger in question, including tandem zinc finger pairs. What remains to be
understood is the functional significance behind much of our binding data. We have
shown with both isothermal calorimetry and crystallography that individual Nup1 53 zinc
fingers bind favorably with RanGDP versus RanGTP. Nup1 53 and Nup358 are
respectively localized to the nucleoplasmic and cytoplasmic face of the NPC. They each
contain a varying number of Ran binding zinc fingers and based on our data Nup358
binds RanGDP with a slightly weaker affinity then Nup1 53. It is possible that both
Nupl53 and Nup358 play intermediary roles in the Ran cycle, such as recycling
RanGDP back into the nucleus via NTF2 and the binding of RanGDP by RCC1 to
mediate nucleotide exchange. In vitro experiments to monitor GTPase activity,
localization, and nucleotide exchange could address some of these lingering questions.
It is possible that the zinc fingers from Nupl53 and Nup358 play a role in nucleotide
exchange and GTPase activity, respectively. These questions could be addressed using
radiolabeled nucleotides in the presence of components from the Ran pathway,
including RCC1 and RanGAP1.
The tetrameric assembly between Nic96 and the Nsp1 subcomplex
The tetrameric assembly between the Nspl trimeric subcomplex and Nic96 has
been successfully purified and crystallographic studies are ongoing. All Nspl complex
assemblies have been screened for crystals in 96-well format, however no crystals of an
assembled complex have been identified. We crystallized Nup49 in setups with the
131
Chapter5: Conclusion
trimeric complex, indicating that our purification scheme results in excess Nup49, or
Nup49 falls off the trimeric complex. We are currently in the process of designing a
series of constructs that utilize a dual-tag purification scheme to eliminate excess
Nup49. In addition to removal of Nup49, we have designed a series of dual-tag
constructs to increase purity and favor crystallization of the tetrameric coiled-coil
assembly. We will also attempt to crystallize some of the Nspl assemblies as fusion
proteins, fused to smaller proteins known to readily crystallize. We hope that this method
will coax the coiled-coil domains into forming a stronger crystallographic lattice and
crystals suitable for data collection. In addition to crystallographic studies the tetrameric
complex with full-length Nic96 still requires biophysical analysis with analytical
ultracentrifugation.
The HUWE1 HECT domain
A balance between conformationalflexibilityand enzyme promiscuity
A flexible hinge between the N- and C- lobes of the E3 HECT domain regulates
conformational flexibility to facilitate rotation of the C-lobe for Ub transfer from E2 to
substrate. The C-lobe pivots on this unstructured hinge and rotates over 1000 to transfer
Ub from docked E2 over 41 A to substrate. It is an amazing structural accomplishment
for the E3 HECT domain to develop such an active mechanism and adopt a range of
conformations to regulate ubiquitination. Any modifications towards increased
promiscuity by the E3 HECT domain could no doubt prove costly to the cell, as the Ub
pathway regulates a range of vital cellular functions. We have focused our studies on a
highly conserved helix at the N-terminus of the HUWE1 HECT domain, shown to
regulate enzymatic activity (Pandya et al., 2010). Removal of the al helix did not
detrimentally affect the overall fold of the domain, as measured with circular-dichroism
132
Chapter5: Conclusion
(CD) scans and shown with crystallographic data, however stability of the enzyme was
substantially decreased when measured with CD melting curves. There is a disparate
increase in activity between auto-ubiquitination and substrate ubiquitination, potentially
demonstrating that the two mechanisms are differentially regulated by the al helix.
A case can be made for the importance of enzymes having some potential for
conformational variability, given multiple selective pressures from the cellular
environment. A recent review describes the significance of conformational variability in
several enzyme families that can adopt multiple structural conformations under selective
pressure to facilitate a host of cellular functions (Tokuriki and Tawfik, 2009). In the case
of the E3 HECT domain, the N- and C-lobes have evolved to employ a mechanism of
incredible flexibility that allows the C-lobe to sample a diverse conformational landscape
for controlled transfer of ubiquitination between multiple E2 enzymes and an even
greater number of substrates. Removal of the N-terminal helix does not necessarily
result in a conspicuous change of function. Rather, the N-terminal helix serves as means
to restrict conformational space sampled by the C-lobe and potentially directs activity
towards specific E2 enzymes and substrates. Where we have used a sledgehammer a
surgeon could use a fine scalpel. The helix in question is roughly twenty amino acids
long, and within those twenty residues, there are three obvious hydrophobic residues
that appear to be critical for maintaining stability of hydrophobic core of the N-lobe and
two residues that make hydrogen bonds with the N-lobe. An interesting question is to
examine mutations of these residues and observe potential effects on the Ub pathway,
or what phenotypes would result in organisms harboring these mutations. It is entirely
possible that only a single point mutation can push the balance between regulated
activity and stability of this enzyme towards over activity and unregulated promiscuity.
133
Chapter 5: Conclusion
Our structural studies on the HECT domain from HUWE1 unexpectedly
uncovered a key element responsible for regulating activity of both self- and substratelabeling with ubiquitin. From our structural data it is clear which residues from the alhelix mediate key interactions with the N-lobe of the HECT domain. These residues
could be mutated in vivo to monitor ubiquitination of model substrates, such as Mcl-1, or
self-ubiquitination by HUWE1. To further assess contribution of the al-helix in vitro,
stability and activity could be monitored upon removal or mutation of specific residues
from the al-helix with labeling assays described in this manuscript. From a structural
and functional perspective, there is still minimal molecular information regarding how the
HECT domain facilitates transfer of ubiquitin from E2 to substrate. A recent crystal
structure from the Schulman lab describes a trimeric complex between, E2, E3 HECT
domain, and Ub, representing the most detailed "image" of the ubiquitin cascade to date.
However we still know very little about how: 1) substrate is bound to the HECT domain
and, 2) how ubiquitin is transferred between the HECT domain C-lobe and substrate.
These questions will likely soon be answered with both structural and biochemical data,
greatly advancing our knowledge of the ubiquitin pathway.
134
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