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Driving Biological Project. What Defines Substrate Specificity of Deubiquitinating Enzyme USP7?. Irina Bezsonova, University of Connecticut Health Center, Department of Molecular Biology and Biophysics. Normal homeostasis DNA damage. USP7. USP7. mdm2. p53. P. P. p53. mdm2.
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Driving Biological Project What Defines Substrate Specificity of Deubiquitinating Enzyme USP7? Irina Bezsonova, University of Connecticut Health Center, Department of Molecular Biology and Biophysics
Normal homeostasis DNA damage USP7 USP7 mdm2 p53 P P p53 mdm2 26S USP7 • USP7 – Ubiquitin Specific Protease 7 • Cleaves Ubiquitin off ubiquitinated substrates • Rescues substrates from proteasomal degradation • Regulates stability of tumor suppressor p53 in the cell
TRAF-like 1 2 3 4 5 CATALYTIC USP7 Structure p53 Mdm2 EBNA1 Ubiquitin E2 p53, Mdm2, Icp0, FOX(O)4, PRC1, GMPS, UHRF1, …
TRAF-like 1 2 3 4 5 CATALYTIC Project Goal • The goal of this projects is to • define USP7 domains responsible for binding to: • p53 • Hdm2 • FOX(O)4 • Icp0 • map the substrate binding sites on the surface of USP7 • determine 3D structures of the complexes.
TRAF-like 1 2 3 4 5 CATALYTIC Preliminary data: NMR of USP7
Typical workflow for NMR structure determination: Collect NMR data Convert and process data (nmrPipe) Pick and annotate peaks, create peak lists (Sparky) Assign peaks in the spectra to specific amino acid residue/atom in the protein (ABACUS) Generate dihedral angle restraints based on chemical shift assignment (TALOS/TALOS+) Calculate structure based on NOE-derived distance restraints and dihedral angle restraints (CYANA) Water-refine structure (CNS)
