The RING-between-RING (RBR) E3 ubiquitin ligase family in humans consists of 14 enzymes which regulate a diversity of important biological processes in health and disease. RBR E3 ligases conjugate ubiquitin to substrate molecules via a unique two-step mechanism in which ubiquitin is first transferred from an E2-conjugating enzyme to the E3 active site and then to the substrate.
Since the first description of this ‘RING-HECT hybrid’ mechanism in 2011, the field has made considerable advances in understanding the molecular details of the RBR E3 catalytic mechanism, including structural determinants of auto-inhibition and ubiquitin-chain type specificity. A limited focus on only a handful of well characterised family members, however, leaves a gap in our understanding as to which features are conserved within the entire family and which are specific adaptions of individual enzymes for specialised function.
To extend our understanding of RBR structure and function, we have structurally and biochemically characterised the less well studied RBR E3 ligases RNF216 and HOIL-1. Crystal structures of both enzymes captured in ‘transthiolation’ complexes with E2-ubiquitin conjugates provides a snapshot of the first catalytic step and suggests a structurally conserved reaction intermediate. We additionally identify a conserved allosteric mechanism, whereby ubiquitin chains with distinct linkage types enhance RBR E3 ligase activity, hinting toward a feed-forward mechanism that promotes amplification of ubiquitination by these enzymes at their sites of activity. Despite an evolutionarily conserved general mechanism of catalysis, our structural data reveals a number of striking structural adaptations in both RNF216 and HOIL-1 that confer specialised functionality to these previously over-looked enzymes.