Investigating the Interaction Mechanism of ClpP Protease with Small Molecule Antimicrobials Using H/D Exchange Mass Spectrometry



Brozdnychenko, Dmytro

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University of Guelph


The caseinolytic protease P (ClpP) is a cylindrical serine protease that works together with energy-dependent unfoldases to degrade target proteins. In prokaryotes, the ClpP degradation machinery plays an essential role in maintaining proteostasis and is essential for many human pathogens. As such, ClpP is a prime target for the development of novel antimicrobials. Emerging antibiotics such as acyldepsipeptides (ADEPs), activators of compartmentalizing proteases (ACPs), dioctatin, and boron-based active site inhibitors bind ClpP and dysregulate its activity. Despite the availability of high-resolution structures of ClpP bound to these small molecules, their mechanism of action remains poorly characterized. Here I use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe the interactions of a diverse set of small-molecule antimicrobials with N. meningitidis ClpP (NmClpP). These measurements provide a dynamic molecular framework for the mechanism by which antimicrobials dysregulate ClpP function and highlight the complementarity of HDX-MS to X-ray crystallography and electron cryomicroscopy.



Structural biology, Hydrogen-deuterium exchange mass spectrometry, ClpP protease, Small-molecule antibiotics, Conformational dynamics