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Abstract The multidrug efflux transporter EmrE fromEscherichia colirequires anionic residues in the substrate binding pocket for coupling drug transport with the proton motive force. Here, we show how protonation of a single membrane embedded glutamate residue (Glu14) within the homodimer of EmrE modulates the structure and dynamics in an allosteric manner using NMR spectroscopy. The structure of EmrE in the Glu14 protonated state displays a partially occluded conformation that is inaccessible for drug binding by the presence of aromatic residues in the binding pocket. Deprotonation of a single Glu14 residue in one monomer induces an equilibrium shift toward the open state by altering its side chain position and that of a nearby tryptophan residue. This structural change promotes an open conformation that facilitates drug binding through a conformational selection mechanism and increases the binding affinity by approximately 2000-fold. The prevalence of proton-coupled exchange in efflux systems suggests a mechanism that may be shared in other antiporters where acid/base chemistry modulates access of drugs to the substrate binding pocket. 

Abstract Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of theStaphylococcus aureusefflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a “belt and suspenders” protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.