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 RING 1 domain that binds E2 Ub‐conjugating enzyme and a RING 2 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 RBR s 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 RBR s utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING 1 domains of HHARI and RNF 144 promote open E2~Ubs. Second, we identify a Ub‐binding site on HHARI RING 2 important for its recruitment to RING 1‐bound E2~Ub. Mutations that ablate Ub binding to HHARI RING 2 also decrease RBR ligase activity, consistent with RING 2 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 RBR s.