Kazi, Nafizul Haque2025-04-042025-04-042025http://hdl.handle.net/2003/4359210.17877/DE290R-25425The mitochondrial deubiquitinase USP30 is a key antagonistic regulator of mitophagy. The potential of USP30 inhibition as a therapeutic strategy for Parkinson’s disease is currently being explored in clinical trials. However, the molecular basis for specific inhibition of USP30 by small molecules has remained elusive because of the limited crystallizability of USP30. This dissertation reports the crystal structure of human USP30 in complex with a specific inhibitor, enabled by chimeric protein engineering. The thesis uncovers a unique mode of deubiquitinase inhibition in which the inhibitor extends into a cryptic pocket facilitated by a compound-induced conformation of the USP30 switching loop. The thesis underscores the potential of exploring induced pockets and conformational dynamics to obtain specific deubiquitinase inhibitors and identifies underlying USP30-specific residues. More broadly, the obtained structure helped to 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, this dissertation establishes a generalizable chimeric protein engineering strategy to aid deubiquitinase crystallization and enables structure-based drug design with relevance to neurodegeneration.enStructural biologyDrug discoveryUbiquitinDeubiquitinaseProtein engineeringMitochondriaParkinsons's570540PhDThesisStrukturbiologieParkinson-Krankheit