Three-dimensional Reconstruction of the PA200-20S Proteasome Complex by Cryo-EM

1
Three-dimensional Reconstruction of the PA200-20S
Proteasome Complex by Cryo-EM Joaquin Ortega.
Ph.D. Biochemistry Biomedical
Sciences Mcmaster University. Hamilton, ON.
Canada
2
Protein Quality Control System
From Wickner et al. Science (1999). v. 286,
1888-1893
3
Current Projects in the lab
20S Proteasome Major cellular protease HtrA
(Deg P) Periplasmic chaperone / protease
4
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

5
Structure of 20S Proteasome
Top view
Side view
From Baumeister et al. (1998) Cell 92, 367-380
6
Inside the Barrel
Catalytic Chamber
Channel Gate
7
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
8
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? -Localiz
es in nucleus -After g-irradiation, PA200 forms
intranuclear foci
9
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?
10
Electron Microscopy of the PA200-20S Proteasome
Complex
11
Data collection and image processing for the
cryo-images
CM200 FEG 120 kV Magnification x 38000
21,091 particles Software -Bsoft -EMAN Sta
rting models -Atomic structure of 20S
-Featureless barrel
12
3D Reconstruction of the Singly Bound PA200-20S
Proteasome Complex.
13
Visualization of the Axial Channel Gate Upon
Binding of PA200
14
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.
15
McMaster University, Hamilton, ON, Canada
Jack Iwanczyk
Faisal Uddin
Daniela Damjanovic
University of Utah, UT, USA Martin Rechsteiner
LSBR. NIAMS. NIH, MD, USA Alasdair C.
Steven Bernard Heymann
16
(No Transcript)
17
Orientation of the 20S proteasome in the
PA200-20S complex
18
Side Views Fill 3D Space
Side Top Views
Side Views Only