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Structural insights into non-covalent ubiquitin activation of the cIAP1-UbcH5B~ubiquitin complex

DOI: 10.1074/jbc.RA118.006045 DOI Help

Authors: Amrita Patel (Cancer Research UK Beatson Institute) , Gary J. Sibbet (Beatson Institute for Cancer Research) , Danny T. Huang (Cancer Research UK Beatson Institute)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: December 2018
Diamond Proposal Number(s): 8659

Abstract: Ubiquitin (Ub)-conjugating enzymes and Ub ligases control protein degradation and regulate many cellular processes in eukaryotes. Cellular inhibitor of apoptosis protein-1 (cIAP1) plays a central role in apoptosis and tumor necrosis factor signaling. It harbors a C-terminal RING domain that homodimerizes to recruit E2~Ub (~ denotes a thioester bond) complex to catalyze Ub transfer. Non-covalent Ub binding to the backside of the E2 Ub-conjugating enzyme UbcH5 has previously been shown to enhance RING domain activity, but the molecular basis for this enhancement is unclear. To investigate how dimeric cIAP1 RING activates E2~Ub for Ub transfer and what role non-covalently bound Ub has in Ub transfer, here we determined the crystal structure of the cIAP1 RING dimer bound to both UbcH5B covalently-linked to Ub (UbcH5B–Ub) and a non-covalent Ub to 1.7 Å resolution. The structure along with biochemical analyses revealed that the cIAP1 RING domain interacts with UbcH5B–Ub and thereby promotes the formation of a closed UbcH5B–Ub conformation that primes the thioester bond for Ub transfer. We observed that the non-covalent Ub binds to the backside of UbcH5B and abuts UbcH5B’s α1β1-loop, which, in turn, stabilizes the closed UbcH5B–Ub conformation. Our results disclose the mechanism by which cIAP1 RING dimer activates UbcH5B~Ub and indicate that non-covalent Ub binding further stabilizes the cIAP1-UbcH5B~Ub complex in the active conformation to stimulate Ub transfer.

Journal Keywords: cIAP1; UbcH5B; activation; non-covalent; RING E3; structural biology; ubiquitylation (ubiquitination); allosteric regulation; ubiquitin; E3 ubiquitin ligase; ubiquitin ligase; ubiquitin-conjugating enzyme (E2 enzyme)

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography