The mechanism of ubiquitination in the cullin-RING E3 ligase machinery: conformational control of substrate orientation.
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 unknown. Here, we simulated nine substrate binding proteins: Skp2, Fbw7, beta-TrCP1, Cdc4, Fbs1, TIR1, pVHL, SOCS2, and SOCS4, in the unbound form and bound to Skp1, ASK1 or Elongin C. All nine proteins have two domains: one binds to the substrate; the other to E3 ligase modules Skp1/ASK1/Elongin C. We discovered that in all cases the flexible inter-domain linker serves as a hinge, rotating the substrate binding domain, optimally and accurately positioning it for ubiquitin transfer. We observed a conserved proline in the linker of all nine proteins. In all cases, 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. We conclude that the flexible linker in the substrate binding proteins orients the substrate for the ubiquitin transfer. Our findings provide a mechanism for ubiquitination and polyubiquitination, illustrating that these processes are under conformational control.
Liu
Jin
J
Basic Science Program, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, NCI-Frederick, Frederick, Maryland, USA.
Nussinov
Ruth
R
D001665:Binding Sites; D003198:Computer Simulation; D044842:Cullin Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000648:ultrastructure*; D054875:Ubiquitination; D014452:Ubiquitins / Q000737:chemistry* / Q000648:ultrastructure*
D044842:Cullin Proteins; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases