Protein ubiquitination is a post-translational modification that regulates nearly all eukaryotic cellular signalling pathways by modifying protein stability, activity, localisation, and interactions. Accordingly, misregulation of the ubiquitin system is at the root of many human pathologies, including cancers and neurodegenerative diseases.
E3 ubiquitin ligases are the final effector enzymes of the ubiquitination machinery and catalyse conjugation of ubiquitin to the substrate protein. Loss of function mutations in the E3 ligase RNF216 (TRIAD3) cause Gordon-Holmes syndrome (GHS) and related neurodegenerative diseases. RNF216 belongs to the family of RING-between-RING (RBR) E3 ubiquitin ligases, which are characterised by a highly regulated multi-step catalytic mechanism. RNF216 assembles Lys63-linked (K63) polyubiquitin chains and has been implicated in regulation of innate immune signalling pathways and synaptic plasticity. However, little is known about bona fide RNF216 substrates in the cell, and the molecular mechanisms that define RNF216 activity, regulation and K63 chain-type specificity are ill-defined.
We employed a structural, biochemical, and biophysical approach to understand the molecular principles of RNF216 catalysis and the pathology of RNF216 GHS mutations. Determining structures mimicking three key reaction intermediates of RNF216 in complex with its substrate and product allowed us to understand how unique structural features within RNF216 confer its chain-type specificity. We further visualise how a phosphorylation site in RNF216 enhances its activity and, surprisingly, chain-type specificity, highlighting how two post-translational modification machineries cooperate to fine-tune cellular signalling.
Our molecular insights expand our understanding of RNF216 function and regulation, its role in disease and provide a foundation for future cell and in vivo studies. Our findings also further refine the catalytic mechanism and regulation of the RBR E3 ligase family.