Structural Insight into the Mechanisms of Activation and Substrate Specificity of Human Deubiquitinating Enzyme USP7

The major part of this thesis describes studies of human ubiquitin-specific protease 7 (USP7), a deubiquitinating enzyme that regulates cellular levels of key oncoproteins and tumor suppressors. Inactivation of USP7 has recently emerged as a new approach to treatment of malignancies. However, design of potent and specific small-molecule compounds requires detailed understanding of the molecular mechanisms of USP7 substrate recognition and regulation of its catalytic activity. The goal of this work was to explore these mechanisms using solution nuclear magnetic resonance spectroscopy in combination with other methods. In our studies of USP7 substrate recognition, we structurally characterized its interaction with ICP0 protein from Herpes Simplex virus 1 and identified a novel USP7 substrate-binding site harbored within its C-terminal region. To address the question of USP7 activity regulation, we investigated its interaction with ubiquitin, which was believed to cause structural rearrangement of USP7 active site from an unproductive to a catalytically competent conformation. Surprisingly, we showed that in solution USP7 – ubiquitin interaction alone is not sufficient for activation of the enzyme as was previously postulated. Finally, we uncovered a previously unknown mechanism of USP7 inactivation by two of its known inhibitors. We found that these compounds bind to the active site of USP7 and inactivate the enzyme via covalent modification of a catalytic cysteine residue. The efficacy of the inhibitors was confirmed in cells. Altogether, these results advance our understanding of the mechanisms of substrate specificity, activation and inhibition of USP7 and open new strategies for rational structure-based drug design. Chapter 4 describes another example of using NMR spectroscopy in studies of complex biological systems. REV1 is a translesion synthesis (TLS) DNA polymerase that plays a key role in replication across the sites of DNA damage. Beyond its catalytic activity, its major role is to recruit other TLS polymerases to DNA lesions. The goal of this work was to structurally characterize protein – protein interactions mediated by the C-terminal region of REV1 (REV1-CT). We determined a solution NMR structure of REV1-CT alone and in complex with TLS polymerase η, and unraveled the molecular mechanism by which REV1 recognizes other Y-family TLS polymerases