Certain RING ubiquitin ligases (E3s) dimerize to facilitate ubiquitin (Ub) transfer from ubiquitin-conjugating enzyme (E2) to substrate, but structural evidence on how this process promotes Ub transfer is lacking. Here we report the structure of the human dimeric RING domain from BIRC7 in complex with the E2 UbcH5B covalently linked to Ub (UbcH5B∼Ub). The structure reveals extensive noncovalent donor 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 UbcH5B∼Ub binding affinity and ubiquitination activity. Moreover, NMR analyses demonstrate that BIRC7 binding to UbcH5B∼Ub induces peak-shift perturbations in the donor Ub consistent with the crystallographically-observed Ub interactions. Our results provide structural insights into how dimeric RING E3s recruit E2∼Ub and optimize the donor Ub configuration for transfer.

Covalent attachment of one or more ubiquitin molecules to protein substrates governs numerous eukaryotic cellular processes including apoptosis, cell division and immune response. Ubiquitylated proteins can be targeted for degradation but ubiquitylation also mediates processes such as protein-protein interactions and cell signalling, depending on the type of ubiquitin conjugation. Ubiquitin ligases (E3s) catalyze the final step of ubiquitin conjugation by transferring ubiquitin from ubiquitinconjugating enzymes (E2s) to substrates. In humans, over 600 E3s contribute to determining the fates of thousands of substrates; hence E3s are tightly regulated to ensure accurate substrate ubiquitylation. Recent findings illustrate how E3s are selfregulated and how they coordinate with E2s and substrates to meticulously conjugate ubiquitin.

Cbls are RING ubiquitin ligases that attenuate receptor tyrosine kinase (RTK) signal transduction. Cbl ubiquitination activity is stimulated by phosphorylation of a linker helix region (LHR) tyrosine residue. To elucidate the mechanism of activation, we determined the structures of human CBL, a CBL-substrate peptide complex and a phosphorylated-Tyr371-CBL-E2-substrate peptide complex, and we compared them with the known structure of a CBL-E2-substrate peptide complex. Structural and biochemical analyses show that CBL adopts an autoinhibited RING conformation, where the RING's E2-binding surface associates with CBL to reduce E2 affinity. Tyr371 phosphorylation activates CBL by inducing LHR conformational changes that eliminate autoinhibition, flip the RING domain and E2 into proximity of the substrate-binding site and transform the RING domain into an enhanced E2-binding module. This activation is required for RTK ubiquitination. Our results present a mechanism for regulation of c-Cbl's activity by autoinhibition and phosphorylation-induced activation.

RING ubiquitin ligases (E3) recruit ubiquitin-conjugate enzymes (E2) charged with ubiquitin (Ub) to catalyze ubiquitination. Non-covalent Ub binding to the backside of certain E2s promotes processive polyUb formation, but the mechanism remains elusive. Here, we show that backside bound Ub (Ub(B)) enhances both RING-independent and RING-dependent UbcH5B-catalyzed donor Ub (Ub(D)) transfer, but with a more prominent effect in RING-dependent transfer. Ub(B) enhances RING E3s' affinities for UbcH5B-Ub, and RING E3-UbcH5B-Ub complex improves Ub(B)'s affinity for UbcH5B. A comparison of the crystal structures of a RING E3, RNF38, bound to UbcH5B-Ub in the absence and presence of Ub(B), together with molecular dynamics simulation and biochemical analyses, suggests Ub(B) restricts the flexibility of UbcH5B's α1 and α1β1 loop. Ub(B) supports E3 function by stabilizing the RING E3-UbcH5B-Ub complex, thereby improving the catalytic efficiency of Ub transfer. Thus, Ub(B) serves as an allosteric activator of RING E3-mediated Ub transfer.