Three-dimensional Reconstruction of the

PA200-20S Proteasome Complex by Cryo-EM

Joaquin Ortega. Ph.D.

Biochemistry & Biomedical SciencesMcmaster University. Hamilton, ON. Canada

Protein Quality Control SystemProtein Quality Control System

From: Wickner et al. Science (1999). v. 286, 1888-1893

*20S Proteasome Major cellular protease

*HtrA (Deg P)Periplasmic chaperone / protease

Current Projects in the lab

Major Functions of the Major Functions of the ProteasomeProteasome

*Regulation of protein functions*Regulation of protein functionsTerminate lifespan of regulatory proteinsTerminate lifespan of regulatory proteins

*Protein quality control*Protein quality controlDegrade damaged/misfolded proteinsDegrade damaged/misfolded proteins

*Immunocompetent peptide generation*Immunocompetent peptide generationDegradation of foreign antigensDegradation of foreign antigens

Structure of Structure of 20S Proteasome20S Proteasome

Top view Side viewFrom: Baumeister et al. (1998) Cell 92, 367-380

Inside the BarrelInside the Barrel

Channel Gate

Catalytic Chamber

Proteasome Regulatory Complexes

The 11S/PA28 activators: An ATP-independent activator

The 19S activator: An ATP-dependent activator

From Baumeister et al. (1998)Cell 92, 367-380

PA200: Novel Proteasome PA200: Novel Proteasome Activator FamilyActivator Family

*200 kDa, 1843 aa.*200 kDa, 1843 aa.

*Subject to phosphorylation.*Subject to phosphorylation.

*Functionally more like PA28 than 19S.*Functionally more like PA28 than 19S.-ATP-independent-ATP-independent-Activate peptide hydrolysis only-Activate peptide hydrolysis only

*DNA Repair Involvement?*DNA Repair Involvement?-Localizes in nucleus-Localizes in nucleus-After -After -irradiation, PA200 forms intranuclear foci-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:Questions Revisited:*What is the structure of the PA200 activator?

PA200 is a highly asymmetric molecule organized in HEAT-like repeats, probably as -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 -ring of the 20S particle.

Jack IwanczykFaisal UddinDaniela Damjanovic

LSBR. NIAMS. NIH, MD, USA Alasdair C. StevenBernard Heymann

University of Utah, UT, USAMartin Rechsteiner

McMaster University, Hamilton, ON, Canada

Orientation of the 20S proteasome in the PA200-20S complex

Side Views Fill 3D SpaceSide Views Fill 3D Space

Side & Top Views Side Views Only