Our studies of the ubiquitin-ligase E3, called gp78, are being conducted in collaboration with Drs. Allan Weissman and Xinhua Ji, 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. These first studies 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. 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 studies have been extended in both the Weissman and Byrd labs. We have 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 are continuing our examination of the detailed interactions and mechanism of ubiquitin transfer, including the investigation of potential antagonists in collaboration with Dr. Terry Burke. IN conjunction with Dr. Burke's lab, we have designed and synthesized a mimetic that is a partial antagonist for the G2BR region of gp78. Further structural and optimization studies are in progress. Another aspect of this system is the role of dynamics in determining chain linking specificity. We are employing NMR methods to directly correlate molecular motions/dynamics of regions of the Ube2g2 enzyme with the pertinent interactions to domains of gp78. These data will be critical to developing an overview of the reaction cycle for ubiquitin loading, transfer, and chain specificity.
