RING Finger Protein 216 (RNF216, also TRIAD3) is an E3 ubiquitin ligase of the RING-between-RING (RBR) family of E3 ligases. These enzymes catalyse the specific attachment of the protein ubiquitin or polyubiquitin chains to substrate proteins, and in doing so regulate the stability, signalling interactions and localisation of these targets. Polyubiquitin chains can exist in various forms depending on the position of lysine-linkage, and the type of attached chain typically influences the fate of the ubiquitinated substrate. RNF216 appears to be a multifunctional regulator of innate immunity signalling (through modulation of Toll-like receptors and their adaptors) and autophagy (via BECN1). Additionally, RNF216 is known to regulate the neural protein ARC, and consequently, deleterious mutations in RNF216 have been implicated in the neurodegenerative disease Gordon-Holmes syndrome (GDHS).
Unlike the more well characterised members of the RBR family (PARKIN, HOIP, HHARI), the catalytic mechanism of substrate ubiquitination by RNF216 and the molecular determinants of its specificity and regulation remain largely unknown. In this work, we sought to carry out a comprehensive biochemical and structural characterisation of RNF216, in order to expand our understanding of the RBR E3 ligases as potential therapeutic targets or facilitators of targeted ubiquitination.
We have shown that RNF216 specifically generates lysine 63 (K63)-linked ubiquitin chains and that the RING1-IBR-RING2 catalytic core (including short flanking helical regions) is required for full E3 ligase activity in vitro. Unlike other RBR E3 ligases, RNF216 does not appear to be auto-inhibited by domains outside of the catalytic core. Furthermore, we have identified K63-linked ubiquitin chains as putative allosteric activators of RNF216 activity in vitro. Future work will focus on illuminating the structural basis of specificity for K63-linked chain formation, and the pathobiology of RNF216 dependent ubiquitination.