Determinants of chemoselectivity in ubiquitination by the J2 family of ubiquitin-conjugating enzymes

Ubiquitin-conjugating enzymes (E2) play a crucial role in the attachment of ubiquitin to proteins. Together with ubiquitin ligases (E3), they catalyze the transfer of ubiquitin (Ub) onto lysines with high chemoselectivity. A subfamily of E2s, including yeast Ubc6 and human Ube2J2, also mediates noncanonical modification of serines, but the structural determinants for this chemical versatility remain unknown. Using a combination of X-ray crystallography, molecular dynamics (MD) simulations, and reconstitution approaches, we have uncovered a two-layered mechanism that underlies this unique reactivity. A rearrangement of the Ubc6/Ube2J2 active site enhances the reactivity of the E2-Ub thioester, facilitating attack by weaker nucleophiles. Moreover, a conserved histidine in Ubc6/Ube2J2 activates a substrate serine by general base catalysis. Binding of RING-type E3 ligases further increases the serine selectivity inherent to Ubc6/Ube2J2, via an allosteric mechanism that requires specific positioning of the ubiquitin tail at the E2 active site. Our results elucidate how subtle structural modifications to the highly conserved E2 fold yield distinct enzymatic activity. The mechanistic basis of non-canonical ubiquitination of serine and threonine residues is not well understood. This work demonstrates how subtle rearrangements in the active site of the ubiquitin-conjugating enzyme (E2) Ubc6/Ube2J2 lead to preferential activity toward hydroxyl groups.X-ray crystallography and MD simulations reveal structural rearrangements in the Ubc6/Ube2J2 active site that enhance E2-Ub thioester reactivity to facilitate nucleophilic attack.A conserved histidine in Ubc6/Ube2J2 activates substrate serine residues through general base catalysis.Binding of RING-type E3 ligases enhances serine selectivity through an allosteric mechanism requiring precise positioning of the ubiquitin tail at the E2 active site. Structural and modeling work shows how subtle active-site rearrangements in the E2s Ubc6/Ube2J2 lead to preferential activity toward hydroxyl groups of substrate serines.