Structural Basis for Substrate Recruitment and Catalytic Ubiquitin Transfer by the E2/E3 Hybrid Enzyme UBE2O

ABSTRACT

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 confirmation, 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.