Three-dimensional
Reconstruction of the
PA200-20S Proteasome
Complex by Cryo-EM
Joaquin Ortega. Ph.D.
Biochemistry & Biomedical Sciences
Mcmaster University.
Hamilton, ON. Canada
Protein Quality Control System
From: Wickner et al.
Science (1999). v.
286, 1888-1893
Current Projects in the lab
*20S Proteasome
Major cellular protease
*HtrA (Deg P)
Periplasmic chaperone / protease
Major Functions of the
Proteasome
*Regulation of protein functions
Terminate lifespan of regulatory proteins
*Protein quality control
Degrade damaged/misfolded proteins
*Immunocompetent peptide generation
Degradation of foreign antigens
Structure of 20S Proteasome
Top view
Side view
From: Baumeister et al.
(1998) Cell 92, 367-380
Inside the Barrel
Catalytic
Chamber
Channel
Gate
Proteasome Regulatory Complexes
The 19S activator: An ATP-dependent activator
The 11S/PA28 activators: An ATP-independent activator
From Baumeister et al. (1998)
Cell 92, 367-380
PA200: Novel Proteasome
Activator Family
*200 kDa, 1843 aa.
*Subject to phosphorylation.
*Functionally more like PA28 than 19S.
-ATP-independent
-Activate peptide hydrolysis only
*DNA Repair Involvement?
-Localizes in nucleus
-After g-irradiation, PA200 forms intranuclear foci
Questions
*What is the structure of the PA200 activator?
*Where does PA200 bind to the 20S proteasome?
*Does PA200 binding induce structural changes in the
20S proteasome that correlate with its activation?
Electron Microscopy of the PA200-20S
Proteasome Complex
Data collection and image
processing for the cryo-images
*CM200 FEG 120 kV
Magnification x 38000
21,091 particles
*Software:
-Bsoft
-EMAN
*Starting models:
-Atomic structure of 20S
-Featureless barrel
3D Reconstruction of the Singly Bound PA200-20S
Proteasome Complex.
Visualization of the Axial Channel
Gate Upon Binding of PA200
Questions Revisited:
*What is the structure of the PA200 activator?
PA200 is a highly asymmetric molecule organized in
HEAT-like repeats, probably as a-solenoid.
*Where does PA200 bind to the 20S proteasome?
A monomer of PA200 binds to one or both ends of 20S.
*Does PA200 binding induce structural changes in the
20S proteasome that correlate with its activation?
PA200 opens the axial channel in the proximal a-ring of
the 20S particle.
McMaster University, Hamilton, ON, Canada
Daniela Damjanovic
University of Utah, UT, USA
Martin Rechsteiner
Faisal Uddin Jack Iwanczyk
LSBR. NIAMS. NIH, MD, USA
Alasdair C. Steven
Bernard Heymann
Orientation of the 20S proteasome in the
PA200-20S complex
Side Views Fill 3D Space
Side & Top Views
Side Views Only