The unifying catalytic mechanism of the RING-between-RING E3 ligase family

The RING-between-RING (RBR) E3 ubiquitin ligase family in humans comprises 14 members and is defined by a two-step catalytic mechanism in which ubiquitin is first transferred from an E2 to the RBR active site and then to the substrate. To define the core features of this catalytic mechanism, we structurally and biochemically characterise the two RBRs HOIL-1 and RNF216. Crystal structures of both enzymes in their RBR/E2-Ub/Ub transthiolation complexes capturing the first catalytic step, in combination with complementary functional experiments, reveal the defining features of the RBR catalytic mechanism. RBRs catalyse ubiquitination via a conserved transthiolation complex structure that enables efficient E2-to-RBR ubiquitin transfer. Our data also highlight a conserved RBR allosteric mechanism by distinct ubiquitin linkages that suggests that RBRs employ a feed-forward mechanism accelerating their signalling pathways. We finally identify that the HOIL-1 RING2 domain contains an unusual Zn2/Cys6 bivalent zinc cluster that may represent a specific adaptation enabling HOIL-1’s atypical substrate specificity. ### Competing Interest Statement The authors have declared no competing interest.