Probing the Secondary Active Transport Mechanism of the Bacterial Efflux Pump EmrE

Abstract

Secondary active transporters are able to confer bacterial multidrug resistance to both antibiotics and antiseptics. While the energy source for the transport mechanism is derived from the electrochemical potential across the membrane, this thermodynamic view does not provide a mechanistic understanding regarding how the energy is converted into useful work. To gain insight into the ion-coupled transport process, we applied a plethora of biophysical techniques including solid-state and solution NMR spectroscopy in conjunction with resistance assays to the drug transporter EmrE from E. coli. EmrE is an anti-parallel homodimer from the small multidrug resistance (SMR) family that effluxes a wide variety of quaternary cationic compounds and has served as a model for ion-coupled transport. We carried out pH titrations using NMR spectroscopy to reveal an elevated acid dissociation constant for a conserved and membrane embedded glutamate residue that is responsible for conferring drug resistance in E. coli. Furthermore, we found that the acid/base chemistry at this anionic residue was responsible for regulating the global dynamics of the transporter based on the protonation state. The implication of this result is that an open conformation of EmrE in the presence of a pH gradient would be oriented toward the higher pH side of the membrane. Indeed, tryptophan fluorescence experiments acquired in lipid bilayers strongly validate this interpretation. Finally, protonation at E14 showed long-range chemical shift perturbations far removed from the drug-binding pocket, which is evidence of an intricate allosteric network involving both concerted conformational changes to the transmembrane helices and loops that are involved in regulating the inward-open to outward-open structural switch.