The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel

Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor β (TGF-β) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ∼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail–binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1′ region likely to confer specificity, whereas the S1 ubiquitin–binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro. A USP15–mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1′ binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development.