Chimeric deubiquitinase engineering reveals structural basis for specific inhibition of USP30 and a framework for DUB ligandability

The mitochondrial deubiquitinase USP30 negatively regulates Pink1/Parkin-driven mitophagy. Whether enhanced mitochondrial quality control through inhibition of USP30 can protect dopaminergic neurons is currently explored in a clinical trial for Parkinson’s disease. However, the molecular basis for specific inhibition of USP30 by small molecules has remained elusive. Here, we report the crystal structure of human USP30 in complex with a specific inhibitor, enabled by chimeric protein engineering. Our study uncovers how the inhibitor extends into a cryptic pocket facilitated by a compound-induced conformation of the USP30 switching loop. Our work underscores the potential of exploring induced pockets and conformational dynamics to obtain specific deubiquitinase inhibitors and identifies underlying USP30-specific residues. More broadly, we delineate a conceptual framework for specific USP deubiquitinase inhibition based on a common ligandability hotspot in the Leu73-Ubiquitin binding site and on diverse compound extensions. Collectively, our work establishes a generalizable chimeric protein engineering strategy to aid deubiquitinase crystallization and enables structure-based drug design with relevance to neurodegeneration.