Regulation of the catalytic activity of HECT domain-containing ubiquitin-protein ligases studied by NMR spectroscopy

• Applicant: Privatdozentin Dr. Silke Wiesner
• Subject Area: Biochemistry
• Term: from 2004 to 2008
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5430095

Attachment of ubiquitin (Ub) to proteins (ubiquitination) serves as a marker for protein degradation thereby regulating the abundance of many key regulatory proteins in the cell. Ubiquitination is catalyzed by a cascade of three enzymes: the Ub-activating enzyme (E1), the Up-conjugating enzyme (E2) and the specificity determining Ub-protein ligase (E3). Members of the Nedd4 family of E3s share a common domain architecture comprising an N-terminal C2 domain, multiple WW domains and a C-terminal catalytic HECT domain. Two recent crystal structures have shown that HECT domains consist of two lobes. While the individual folds of the lobes are virtually identical in the two structures, their relative orientations differ substantially suggesting that conformational flexibility unterlies HECT domain activity. To gain insight into the regulation of HECT domain activity we propose to determine the time-scales of backbone and side-chain motions of the HECT domain of the Nedd4 family member Smurf2 from NMR relaxation experiments. Moreover, structural information about the relative orientations of the Smurf2 HECT domain lobes will be gained from orientations dependent NMR restraints, such as residual dipolar couplings. We also plan to investigate potential modulations of conformational rearrangements and dynamics by intra- and intermolecular interactions. Results from this study should significantly enhance our understanding of the regulation of the enzymatic activity of HECT domains.

• DFG Programme: Emmy Noether International Fellowships
• Participating Person: Professorin Dr. Julie Forman-Kay