STRUCTURAL BIOLOGY OF HUMAN UBIQUITIN E3 LIGASES AND INTERACTING FACTORS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ubiquitylation regulates a variety of processes, including DNA repair, signal transduction, cell cycle progression, endocytosis and protein degradation, amongst others, and has been implicated in disease. The substrate specificity of the ubiquitylation system is carried out by the E3 protein ligases, which belong to two major families: the RING E3 ligases and the HECT E3 ligases. The HECT E3 ligases each contain an approximately 350 amino acid C-terminal catalytic domain with a conserved active site cysteine that forms a thiol-intermediate with ubiquitin, transferring it from an E2 ligase to the protein substrate. The highly variable N-terminal regions of the HECT E3s interact with specific protein substrates while the catalytic domain interacts with its cognate E2 and carries out the catalytic reaction. A variety of domains (WW domain, C2 domain, armadillo like-repeats, BH3, UBA, amongst others) have been observed in the N-terminus of the HECT domain proteins and these have been used to further classify these proteins. Unlike the HECT E3 ligases, the RING E3 ligases do not directly carry out the transfer of ubiquitin to substrate, but instead bring together the activated E2 and its substrate thus allowing catalysis to occur. We propose to determine the high-resolution structures of human E3 ligases and their complexes with cognate E2s, substrates and regulating factors in order to further elucidate their catalytic mechanisms, specificity and regulation. As part of the Structural Genomics Consortium, we have the capability to use high-throughput techniques to express and purify proteins, crystallize and characterize these crystals. However we require the brilliant X-rays available at the APS in order to collect data from weakly diffracting crystals and to collect anomalous data from heavy-atom derivatives.