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 (where ∼ denotes a thioester bond) complex to catalyze Ub transfer. Noncovalent 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 noncovalently 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 noncovalent 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 noncovalent 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 noncovalent Ub binding further stabilizes the cIAP1-UbcH5B∼Ub complex in the active conformation to stimulate Ub transfer.
Highlights
Ubiquitin (Ub)-conjugating enzymes and Ub ligases control protein degradation and regulate many cellular processes in eukaryotes
To investigate how dimeric Cellular inhibitor of apoptosis protein-1 (cIAP1) RING activates E2ϳUb for Ub transfer and what role noncovalently 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 noncovalent Ub to 1.7 Å resolution
UbcH5B variants were precharged with equimolar concentrations of 32P-Ub and chased by the addition of cIAP1 RING domain (cIAP1R) alone and in the presence of Ub lacking the C-terminal diglycine motif (Ub⌬GG), which cannot be charged by E1 but can still bind to the backside of UbcH5B WT
Summary
We showed that the addition of Ub⌬GG (lacking the C-terminal diglycine motif) can serve as UbB and bind to UbcH5B’s backside to stimulate UbcH5BϳUb discharge catalyzed by the monomeric RING E3 RNF38 and dimeric RING E3 XIAP. To assess whether UbB can exert similar effects on cIAP1R-catalyzed Ub transfer, we performed single-turnover lysine discharge assays using WT and S22R UbcH5B. The addition of 300 M Ub⌬GG stimulated the discharge of UbcH5BϳUb but had no effect on UbcH5B S22RϳUb (Fig. 1A), indicating that UbB stimulates cIAP1R-catalyzed Ub transfer. Our prior study showed that UbB stimulates RNF38 and XIAP-catalyzed Ub transfer by enhancing RING E3 affinity for UbcH5B–Ub by ϳ5–10-fold [25]. To determine whether UbB functions in a similar manner to stimulate cIAP1R-catalyzed Ub transfer, we performed surface plasmon resonance (SPR) experiments to investigate the effects of UbB on cIAP1R’s affin-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
