Searching For the Hinge of E3 Ubiquitin Ligase Machinery with MD Simulations

Abstract

In Cullin-RING E3 ubiquitin ligases, substrate binding proteins, such as VHL-box, SOCS-box or the F-box proteins, recruit substrates for ubiquitination, accurately positioning and orienting the substrates for ubiquitin transfer. Yet, how the E3 machinery precisely positions the substrate is unclear. We performed molecular dynamics simulations for seven substrate binding proteins: Skp2, Fbw7, β-TrCP1, Cdc4, pVHL, SOCS2, and SOCS4, in the unbound form and bound to Skp1 or Elongin C. All seven proteins have two domains: one binds to the substrate; the other to E3 ligase modules Skp1/Elongin C. In all seven cases, the flexible inter-domain linker serves as a hinge rotating the substrate binding domain, optimally and accurately positioning it for ubiquitin transfer. A conserved proline is noticed in the linker of all seven proteins. The prolines pucker substantially and the pucker is associated with the backbone rotation toward the E2/ubiquitin. We further observed that the linker flexibility could be regulated allosterically by binding events associated with either domain. Thus searching for the allosteric sites to regulate the flexibility could provide a new strategy for drug discovery targeting the ubiquitin system. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under contract number NO1-CO-12400.