Unique gluing effect of ASXL1 K351 monoubiquitination stimulates the PR-DUB activity

Protein ubiquitination critically regulates biological processes through both proteolytic and nonproteolytic mechanisms. While classically known for protein degradation, ubiquitination also modulates enzymatic activity. However, current mechanisms of ubiquitination-mediated enzymatic modulation are spatially constrained near enzyme-substrate interfaces. Here, we report a unique ubiquitination-mediated regulatory paradigm that activates the Polycomb repressive deubiquitinase (PR-DUB) complex from a site distal to the enzyme-substrate interface. We found that ASXL1 K351 monoubiquitination promotes nucleosomal H2AK119Ub deubiquitination by stabilizing the PR-DUB catalytic pocket, thereby increasing catalytic velocity (V_max) without affecting substrate affinity (K_m). Structurally, ubiquitin at ASXL1 K351 bridges the BAP1 and ASXL1 subunits, functioning as a cross-bracing 'glue' that constrains their conformational dynamics without altering the nucleosome-binding interface. Molecular dynamics and hydrogen-deuterium exchange mass spectrometry revealed that this modification locks PR-DUB in a catalytic state poised for substrate cleavage. This study reveals a unique ubiquitin function of intersubunit fastening through a molecular glue effect and clarifies the mechanism of PR-DUB activation.