Structural insights into ubiquitin recognition by USP15 revealed through a covalent activity-based probe.

Ubiquitin-specific protease 15 (USP15) catalyzes deubiquitination of multiple substrates involved in immune responses and cancer through diverse biological processes. However, its ubiquitin recognition and conformational transition mechanisms remain poorly understood. Here, we report the 2.3 Å crystal structure of the USP15 catalytic domain covalently bound to ubiquitin-propargylamine. This structure reveals that ubiquitin binding induces a conformational switch from an inactive-closed to an active-open state, driven by realignment of the catalytic triad and a hinge-like outward swing of the fingers subdomain. Importantly, we characterize two conserved structural features-an α8 helix and an α3-α4 loop-that are essential for maintaining the functional architecture of USP15 for ubiquitin engagement. Deletion of α8 destabilizes USP15 and abrogates its catalytic activity, while cancer-associated mutation P331S destabilizes the α3-α4 loop, weakens ubiquitin binding, and reduces catalytic efficiency. These previously less-characterized structural elements are critical for the enzymatic activity and structural integrity of USP15 and its paralogues, providing potential targets for selective inhibition of deubiquitinases.