Conformational Control of Ubiquitination in the Cullin-Ring E3 Ligase Machinery

Abstract

Tagging proteins by polyubiquitin is a key step in protein degradation. Cullin-RING E3 ubiquitin ligases (CRLs) facilitate ubiquitination by transfer ubiquitin from ubiquitin-conjugating enzyme E2 to the target protein. Neddylation by conjugation of ubiquitin-like protein NEDD8 to cullin can stimulate ubiquitination process. However, crystallography indicates a 25-35 A distance between neddylation activate sites and a 50-60 A distance between ubiquitination active sites, raising questions of how NEDD8 and ubiquitin are transfered, and how neddylation stimulates ubiquitination. Here we performed molecular dynamics simulations to address these questions. CRLs have cullin as scaffold holding two arms. One arm, substrate binding protein, binds to substrates; the other arm, Rbx protein, binds to E2. In our simulations, we observed big conformational changes on both arms. The flexible linker on the arm of Rbx1, serving as a hinge to rotate the RING domain, thus brings E2 toward substrate to shorten the 50-60 A distance gap to a minimum of 13 A, while the flexible linkers on the other arm of the substrate binding protein could also serve as hinges to rotate the substrate binding domain1,2 and bring substrate toward E2 thus further shorten the distance by 7∼12 A to bridge the ubiquitin transfer distance gap. The distance gap for neddylation could also be shortened due to the conformational change during the simulations. We therefore propose that a large ensemble of conformations could provide the possible conformation to bridge the distance gap for ubiquitin transfer and NEDD8 transfer, and that neddylation stimulates the ubiquitination by stimulating conformational change of CRLs and generating a larger conformational ensemble. This project is funded by NCI contract HHSN261200800001E.1. Liu J, Nussinov R. Proc Natl Acad Sci USA 105:901-6, 20082. Liu J, Nussinov R. PLoS Comput Biol 5:e1000527, 2009