Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na + or H + gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na + -coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs + (a Na + congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-π interaction in the Na + -binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate-and cationbinding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na + -induced drug export. Based on our structural and functional analyses, we suggest that Na + triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.cation coordination | substrate recognition | membrane protein | multidrug resistance | monobody

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by coupling the efflux of drugs to the influx of Na+ or H+. Known structures of Na+-coupled, extracellular-facing MATE transporters from the NorM subfamily revealed twelve membrane-spanning segments related by a quasi-twofold rotational symmetry and a multidrug-binding cavity situated near the membrane surface. Here we report the crystal structure of an H+-coupled MATE transporter from Bacillus halodurans and the DinF subfamily at 3.2 Å-resolution, unveiling a surprisingly asymmetric arrangement of twelve transmembrane helices. We also identified a membrane-embedded substrate-binding chamber by combining crystallographic and biochemical analyses. Our studies further suggested a direct competition between H+ and substrate during DinF-mediated transport, and how a MATE transporter alternates between its extracellular- and intracellular-facing conformations to propel multidrug extrusion. Collectively, our results demonstrated hitherto unrecognized mechanistic diversity among MATE transporters.

P II proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. They play a major role in coordinating nitrogen metabolism by interacting with, and regulating the activities of, a variety of enzymes, transcription factors, and membrane transport proteins. The regulatory properties of P II proteins derive from their ability to bind three effectors: ATP, ADP, and 2-oxoglutarate. However, a clear model to integrate physiological changes with the consequential structural changes that mediate P II interaction with a target protein has so far not been developed. In this study, we analyzed the fluctuations in intracellular effector pools in Escherichia coli during association and dissociation of the P II protein GlnK with the ammonia channel AmtB. We determined that key features promoting AmtB-GlnK complex formation are the rapid drop in the 2-oxoglutarate pool upon ammonium influx and a simultaneous, but transient, change in the ATP/ADP ratio. We were also able to replicate AmtB-GlnK interactions in vitro using the same effector combinations that we observed in vivo. This comprehensive data set allows us to propose a model that explains the way in which interactions between GlnK and its effectors influence the conformation of GlnK and thereby regulate its interaction with AmtB.

Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.

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