Unveiling the biochemical catalysis and regulatory mechanisms of the E3-independent E2 enzyme hUBE2O

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Abstract UBE2O, an E3-independent E2 ubiquitin-conjugation enzyme, directly engages substrates to mediate Ubiquitin conjugation and ligation. Despite its critical role in ubiquitination of multiple substrates, the catalytic and regulatory mechanisms of Homo sapiens UBE2O (hUBE2O) remain incompletely understood. Here, combining domain truncation, systematic mutagenesis and well-designed biochemical approaches, we demonstrate that hUBE2O mediates both mono- and polyubiquitination through a unique catalytic architecture. While none cysteines function for hUBE2O’s E3 activity, the coiled-coil (CC) and C-terminal regulatory (CTR) domains maintain catalytic competence, with the N-terminal regions impose activity constraints. Interestingly, hUBE2O activity is refractory to its self-ubiquitination and phosphorylation state. Instead, specific non-cysteine residues (H939, T995, S1042A, S1046A, S1060A and H1130) emerge as critical regulators of substrate selectivity and catalytic optimization. Surprisingly, zinc ions emerge as potent allosteric inhibitors that bind cysteines of hUBE2O, sterically occluding the access of catalytic site C1040 to Ubiquitin. Our findings reveal that hUBE2O-mediated E3-independent ubiquitination is governed by dynamic interdomain cooperation and allosteric modulation, establishing a mechanistic framework for understanding non-canonical ubiquitination and informing the development of targeted hUBE2O modulators.

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