Post-translational modification of protein substrates by mono-ubiquitin, or poly-ubiquitin chains is a fundamental regulatory process in eukaryotic biology. Far beyond the well-established role of marking proteins for proteasomal degradation, ubiquitin chains also perform critical non-degradative functions in signalling pathways and regulate protein activity, interactions, and localisation. The nature of the ubiquitin modification, particularly the ubiquitin chain linkage type, dictates the biological function of that modification, and therefore the fate of the ubiquitinated substrate. For this reason, it is of great interest to understand how ubiquitin ligases recognise substrates, and how they generate distinct poly-ubiquitin chains.
RNF216 (also TRIAD3) is an E3 ubiquitin ligase of the RING-between-RING (RBR) family of E3 ligases, which modifies target substrates with lysine 63(K63)-linked ubiquitin chains and acts as a regulator of innate immunity, autophagy and synaptic plasticity. Loss of function mutations in RNF216, and subsequent de-regulation of target proteins, results in neurological and endocrine pathologies including Gordon-Holmes Syndrome (GDHS) and Huntington-like disorders.
Using a structural and biochemical approach, we have uncovered the molecular basis for specific K63-linked ubiquitin chain formation by RNF216. By structurally capturing several important reaction intermediates, we have gained rare insight into how an E3-ubiquitin ligase orients ubiquitin molecules to specifically generate the K63-linked chains that modify its substrates. These structures allow visualisation of GDHS patient mutations and provide an explanation for their effect on RNF216 activity. Furthermore, we have identified a novel mechanism of allosteric regulation of RNF216 by ubiquitin chains. Together these findings contribute to our understanding of RBR E3 ligase catalytic mechanism, with important implications for the RBR family as a whole.