HDX-MS reveals structurally dynamic mechanisms of drug action: Insights from deubiquitinase inhibitors

Abstract

Abstract Protein conformational heterogeneity presents a major challenge for structure determination and structure-based inhibitor design, particularly for enzymes containing flexible or intrinsically disordered regions. X-ray crystallography and cryo-EM often capture only static conformations and frequently rely on truncated constructs that omit dynamic regions. Here, we integrate hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular docking and available crystal structures to investigate the solution-state conformational dynamics of the mitochondrial deubiquitinase USP30. HDX-MS reveals dynamic features not resolved in crystal structures and shows that both covalent and non-covalent inhibitors bind the thumb-palm cleft of the catalytic domain through distinct binding modes. These interactions stabilize different conformational states of the regions surrounding switching (SL1) and blocking (BL1 and BL2) loops, providing mechanistic insight into inhibitor recognition and highlighting the value of HDX-MS for guiding DUB-targeted drug discovery.