Ubiquitination plays an important role in regulating many key proteins including cyclins, cyclin-dependent kinase inhibitors, histones, oncogenes, p53, c-JUN, C-FOS and transcription factors, and is involved in many cellular processes, including cell-cycle progression and apoptosis, transcription regulation and antigen presentation. This proposal is aimed at elucidating the poorly characterized protein-protein interactions in the ubiquitination pathway. NMR methods, such as chemical shift perturbation, are extremely sensitive to detect protein interactions. Using NMR methods, we will investigate the interactions of a ubiquitin conjugation enzyme UBC9 (E2) with UBL-1 (a ubiquitin homologue) and target proteins Rad51, Rad52 and p53. We will characterize the UBL1- UBC9 conjugate and its interactions with target proteins. These studies should provide a better understanding of the molecular details of substrate recognition and the role of ubiquitin in this process. In addition, conformational flexibility and self-association of UBC9 will be studied. Since we have made the first NMR resonance assignments of an E2 enzyme, it is now highly feasible to address these important and timely issues in ubiquitination. Mutants of UBC9 will be designed and created based on the NMR studies to further test the functional roles of individual residues in collaboration with Dr. David J. Chen at Los Alamos National Laboratories. With an increased understanding of the molecular basis of this biological process, it should be possible to design pharmacological intervention strategies in several human diseases via manipulation of ubiquitination pathways.
