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