Using hydrogen/deuterium exchange mass spectrometry to understand bacterial membrane efflux proteins

Bacterial multidrug efflux pumps play major roles in antibiotic and multidrug resistance as well as fulfilling many important physiological functions. These molecular machines are highly dynamic, with structural malleability at the core of their action, and elaborating their conformational features can provide insight into the mechanisms underpinning their activities and responses to transport substrates and inhibitors. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a structural biology assay used to monitor protein backbone dynamics and is capable of elucidating conformational signatures that define function and fold. In recent years, HDX-MS methods have advanced, making its use more amenable to the study of membrane proteins, yet experimental challenges remain. In this chapter, we provide background on HDX-MS, its limitations, and discussions around experimental design and optimization for studying efflux pump proteins, including its application to study membrane proteins in lipid environments. To demonstrate its utility, we provide an original case study on AcrB inhibition by an efflux pump inhibitor (MBX-3756), employing membrane-scaffold protein lipid nanodiscs, revealing perturbation of backbone motions distinguishing of hydrophobic-trap targeting inhibitory action. This chapter can serve as a guide for designing HDX-MS investigations on efflux pump proteins and membrane proteins in general.