Structural basis for substrate recruitment and catalytic ubiquitin transfer by the E2/E3 hybrid enzyme UBE2O

UBE2O is a promiscuous ubiquitin ligase involved in cellular quality control pathways. Along with BIRC6, UBE2O is one of only two E2 enzymes that can ubiquitinate substrates in an E3-independent manner. The E2/E3 hybrid targets and multi-monoubiquitinates a multitude of orphan proteins; however, the mechanisms underlying substrate specificity and ubiquitin transfer remain poorly understood. By combining structural and biochemical approaches, we show that substrate binding by UBE2O occurs through a conserved acidic pocket formed by the N-terminal SH3-like domains and that this platform allows the recruitment of a broad range of proteins. Furthermore, we identified specific residues in the catalytic UBC domain that position ubiquitin in a closed state, confirming its confirmation, and priming it for nucleophilic attack by the incoming substrate. Importantly, the activated E2∼Ub conjugate is protected by a tryptophan residue, avoiding premature hydrolysis. By incorporating these findings into the UBC domain of BIRC6 our data provide the molecular basis of how specialized E2/E3 hybrid proteins function as potent ubiquitination enzymes reminiscent of the catalytic principle of RING E3 ligases.