Allosteric zinc inhibition and interdomain regulation govern the catalytic mechanism of the E3-independent ubiquitin-conjugating enzyme hUBE2O.

UBE2O, an E3-independent E2 ubiquitin-conjugating 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. Specifically, hUBE2O lacks dedicated catalytic cysteines for E3 ligase activity. Instead, its E3-independent ubiquitination function relies on the coordination of multiple domains: the catalytically essential UBC domain requires the flanking coiled-coil (CC) and C-terminal regulatory (CTR) domains to maintain its full enzymatic competence, whereas the N-terminal region imposes an autoinhibitory constraint on the enzyme's activity. Interestingly, hUBE2O's activity is refractory to its phosphorylation and lysine-mediated self-ubiquitination state. Instead, specific non-cysteine residues (H939, T995, S1042, T1046, S1060 and H1130) in the UBC domain emerge as critical regulators of 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.