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

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.

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

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  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? -Localizes 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 *Starting 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 Daniela Damjanovic Faisal Uddin Jack Iwanczyk University of Utah, UT, USA Martin Rechsteiner LSBR. NIAMS. NIH, MD, USA Alasdair C. Steven Bernard Heymann

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

  17. Side Views Fill 3D Space Side & Top Views Side Views Only

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