LAUSR

Repair of DNA damage is critical to genomic integrity and frequently disrupted in cancers. USP1, a nucleus-localized deubiquitinase, lies at the interface of multiple DNA repair pathways and is a promising drug target for certain cancers. Although multiple inhibitors of this enzyme, including one in phase I clinical trials, have been established, their binding mode is unknown. Here we use cryo-Electron Microscopy to study an assembled enzyme-substrate-inhibitor complex of USP1 and the well-established inhibitor, ML323. Achieving 2.5 Å resolution, we discover an unusual binding mode in which the inhibitor displaces part of the hydrophobic core of USP1. The consequent conformational changes in the secondary structure lead to subtle rearrangements in the active site that underlie the mechanism of inhibition. These structures provide a platform for structure-based drug design targeting USP1. One Sentence Summary USP1, a cancer target, is inhibited by ML323 displacing part of the protein fold, allosterically disrupting the active site.