Mechanism of ubiquitin transfer by RING E3 ligases

R Medicine (General)

Dou, Hao ; Huang, Danny T

University:University of Glasgow

Department:School of Medicine, Dentistry & Nursing

关键词: ubiquitin, RING E3, CBL, BIRC7;   

Others  :  http://theses.gla.ac.uk/4660/6/phd%20coversheet.pdf

来源: University of Glasgow

【 摘 要 】

In ubiquitin (Ub) system, E3 ligases have a central role in mediating substrate ubiquitination and triggering substrate degradation or other signalling transductions. RING E3s are the largest family of E3s and they promote direct Ub transfer from E2’s active site to substrate. Some RING E3s require dimerization for their activity, but structural evidence on the importance of dimerization and the mechanism of Ub transfer are lacking. I report the structure of the human dimeric RING domain from BIRC7 (also called MLIAP or Livin) in complex with the E2 UbcH5B covalently linked to Ub (UbcH5B~Ub; ~ refers to thioester or oxyester linkage). This 2.18 Å complex structure reveals extensive Ub interactions with UbcH5B and both subunits of the RING domain dimer that stabilize the globular body and C-terminal tail of Ub. Mutations that disrupt these noncovalent interactions or RING dimerization reduce E2~Ub binding affinity and ubiquitination activity. My results provide structural insights into how dimeric RING E3s recruit E2~Ub and optimize the donor Ub configuration for transfer. CBL family proteins (CBLs) are monomeric RING E3s that negatively regulate receptor tyrosine kinase (RTK) signalling pathway. Precise control of CBLs E3 activity is crucial for many cellular functions. My structural and biochemical data show that the RING domain of c-CBL adopts an autoinhibited conformation, where the E2-binding surface of the RING domain is blocked by the tyrosine kinase-bindingdomain (TKBD). CBLs can be activated by phosphorylation of a conserved tyrosine residue on the linker helix region (LHR; Tyr371 in c-CBL and Tyr363 in CBL-B). This activation is required for RTK ubiquitination. I report a crystal structure of Tyr363-phosphorylated human CBL-B, bound to a stabilized Ub-linked E2 (UbcH5B–Ub; – is used to separate componets of a complex, in this case, also refers to isopeptide linkage). This 2.21 Å complex structure reveals that the pTyr363 of CBL-B contacts Ub’s Ile36 surface. Using kinetic analyses, I demonstrate that this interaction is crucial in Ub transfer. Together, my results suggest that both monomeric and dimeric RING E3s use a similar mechanism in optimizing the Ub conformation and activating E2~Ub thioester for transfer, although the Ub-stabilizing components may vary in different RING E3s.