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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. 

Abstract Eurasia, home to ~70% of global population, is characterized by (semi-)arid climate. Water scarcity in the mid-latitude Eurasia (MLE) has been exacerbated by a consistent decline in terrestrial water storage (TWS), attributed primarily to human activities. However, the atmospheric mechanisms behind such TWS decline remain unclear. Here, we investigate teleconnections between drying in low-latitude North Atlantic Ocean (LNATO) and TWS depletions across MLE. We elucidate mechanistic linkages and detecte high correlations between decreased TWS in MLE and the decreased precipitation-minus-evapotranspiration (PME) in LNATO. TWS in MLE declines by ~257% during 2003-2017 due to northeastward propagation of PME deficit following two distinct seasonal landfalling routes during January-May and June-January. The same mechanism reduces TWS during 2031-2050 by ~107% and ~447% under scenarios SSP245 and SSP585, respectively. Our findings highlight the risk of increased future water scarcity across MLE caused by large-scale climatic drivers, compounding the impacts of human activities. 

EmrE is anEscherichia colimultidrug efflux pump and member of the small multidrug resistance (SMR) family that transports drugs as a homodimer by harnessing energy from the proton motive force. SMR family transporters contain a conserved glutamate residue in transmembrane 1 (Glu14 in EmrE) that is required for binding protons and drugs. Yet the mechanism underlying proton-coupled transport by the two glutamate residues in the dimer remains unresolved. Here, we used NMR spectroscopy to determine acid dissociation constants (pK_a) for wild-type EmrE and heterodimers containing one or two Glu14 residues in the dimer. For wild-type EmrE, we measured chemical shifts of the carboxyl side chain of Glu14 using solid-state NMR in lipid bilayers and obtained unambiguous evidence on the existence of asymmetric protonation states. Subsequent measurements of pK_avalues for heterodimers with a single Glu14 residue showed no significant differences from heterodimers with two Glu14 residues, supporting a model where the two Glu14 residues have independent pK_avalues and are not electrostatically coupled. These insights support a transport pathway with well-defined protonation states in each monomer of the dimer, including a preferred cytoplasmic-facing state where Glu14 is deprotonated in monomer A and protonated in monomer B under pH conditions in the cytoplasm ofE. coli. Our findings also lead to a model, hop-free exchange, which proposes how exchangers with conformation-dependent pK_avalues reduce proton leakage. This model is relevant to the SMR family and transporters comprised of inverted repeat domains.