Our studies of the ubiquitin-ligase E3, called gp78, are being conducted in collaboration with Dr. Allan Weissman, CCR. The gp78 protein has a role in sarcoma metastasis, and it is an excellent target for a combined structural and molecular biological investigation of mechanism and intervention. We have reported our first major findings from this project, which was an interdisciplinary effort involving molecular biology in the Weissman lab, NMR structural biology and biophysics in the Byrd lab, and X-ray crystallograpny in the Ji lab. We have uncovered a new aspect of the E2:E3 interaction, wherein the action of a newly identified E2-binding site in gp78 results in an allosteric effect on the E2 Ube2g2 increasing the affinity for the gp78-RING finger domain by 50-fold. This results in increased levels of ubiquitination by this E2:E3 pair. We also showed that the effect of the binding region (G2BR) is effective for other RING finger containing E3s. The generalization of this phenomenon is leading to a broader understanding of ubiquitination and shifing in the paradigm for the molecular mechanism of ubiquitin transfer. These results were published in Molecular Cell and were the subject of a perspective in Structure. These studies are being rapidly extended in both the Weissman and Byrd labs. We have initial structural information on the ternary complex of the gp78-RING:Ube2g2:G2BR system from both NMR and crystallographic studies. We have also examined the solution structure of the CUE domain, which is a distinct functional domain within gp78, and its interactions with ubiquitin, di-ubiquitin, and tetra-ubiquitin. We will continue our examination of the detailed interactions and mechanism of ubiquitin transfer in the coming year, including the investigation of potential antagonists in collaboration with Dr. Terry Burke.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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Chakrabarti, Kalyan S; Li, Jess; Das, Ranabir et al. (2017) Conformational Dynamics and Allostery in E2:E3 Interactions Drive Ubiquitination: gp78 and Ube2g2. Structure 25:794-805.e5
Gill, Michelle L; Byrd, R Andrew (2014) Dynamic activation of apoptosis: conformational ensembles of cIAP1 are linked to a spring-loaded mechanism. Nat Struct Mol Biol 21:1022-3
Huang, Tao; Li, Jess; Byrd, R Andrew (2014) Solution structure of lysine-free (K0) ubiquitin. Protein Sci 23:662-7
Das, Ranabir; Liang, Yu-He; Mariano, Jennifer et al. (2013) Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine. EMBO J 32:2504-16
Byrd, R Andrew; Weissman, Allan M (2013) Compact Parkin only: insights into the structure of an autoinhibited ubiquitin ligase. EMBO J 32:2087-9
Metzger, Meredith B; Liang, Yu-He; Das, Ranabir et al. (2013) A structurally unique E2-binding domain activates ubiquitination by the ERAD E2, Ubc7p, through multiple mechanisms. Mol Cell 50:516-27
Liu, Shan; Chen, Yinghua; Li, Jess et al. (2012) Promiscuous interactions of gp78 E3 ligase CUE domain with polyubiquitin chains. Structure 20:2138-50