The mechanism of OTUB1-mediated inhibition of ubiquitination

Abstract

SUMMARYHistones are ubiquitinated in response to DNA double strand breaks (DSB), promoting recruitment of repair proteins to chromatin1. UBC13 (UBE2N) is an ubiquitin conjugating enzyme (E2) that heterodimerizes with UEV1a2 and synthesizes K63–linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF1683. K63Ub synthesis is regulated in a noncanonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13~Ub thiolester4. Residues N-terminal to the OTU domain, which had been implicated in ubiquitin binding5, are required for binding to UBC13~Ub and inhibition of K63Ub synthesis5. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13~Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13~Ub, while at the same time disrupting interactions with UEV1a in a manner that depends upon the OTUB1 N-terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13~Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N-terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13~Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer6,7. The N-terminal helix of OTUB1 is positioned to interfere with UEV1a binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes4 in a non-catalytic manner.

Activated Protein C Ameliorates Renal Ischemia-Reperfusion Injury by Restricting Y-Box Binding Protein-1 Ubiquitination

“…39,40 Second, OTUB1 can segregate E2-E3 complexes and inhibit the transfer of ubiquitin molecules from E2 conjugating enzymes to E3 ligases independent of its catalytic activity. [41][42][43] Although our immunoprecipitation experiments demonstrate that OTUB1 and YB-1 act in close proximity to each other, we currently cannot differentiate which of these two alternative mechanisms inhibit ubiquitination of YB-1. Interestingly, OTUB1 has been proposed to preferentially inhibit K48-linked ubiquitination, 40,44 which may specifically avert proteasomal degradation of target proteins, such as YB-1, while still allowing other ubiquitinationdependent processes.…”

Deubiquitylases From Genes to Organism

“…This binding induces conformational changes in the catalytic domain, which allow simultaneous binding to the E2-ubiquitin, such that both ubiquitins together mimic the configuration of a cleaved Lys48 di-ubiquitin. Thus OTUB1 is proposed to utilize a mechanism akin to product inhibition to inhibit the activity of associated E2 enzymes (111,224,273).…”

PTEN regulates RPA1 and protects DNA replication forks

“…Five DUB families have been identified including ovarian tumor proteases (OTUs) [15]. OTUB1 is an OTU family DUB cysteine protease highly specific for cleaving Lys48-linked polyubiquitin chains, which targets proteins for proteasomal degradation [16][17][18][19].…”