Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

RING‐in‐between‐RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub‐conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT‐type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING‐type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub‐binding site on HHARI RING2 important for its recruitment to RING1‐bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs. Synopsis This study shows that RING‐in‐between‐RING (RBR) RING1 domains oppose the function of canonical RING domains, and that this opposing function fulfills an important role in RBR E3 Ub transfer mechanisms. Despite structural similarity to canonical RINGs that promote closed E2˜Ub states, RING1 domains of HHARI and RNF144 actively promote open E2˜Ub states. By favoring open E2˜Ub states, RBR E3s ensure Ub transfer through the E3 active site cysteine by inhibiting off‐target transfer of Ub to lysines by the E2˜Ub. The Ub hydrophobic patch binds to HHARI RING2 and is required for Ub transfer from the E2 active site to the E3 active site. The Ub transfer mechanism defined here is utilized by a variety of RBR E3s. Graphical Abstract This study shows that RING‐in‐between‐RING (RBR) RING1 domains oppose the function of canonical RING domains, and that this opposing function fulfills an important role in RBR E3 Ub transfer mechanisms.