Structure of an H+-coupled, Substrate-bound MATE Transporter Yields Mechanistic Insights into Multidrug Extrusion

Multidrug and toxic compound extrusion (MATE) proteins constitute a ubiquitous family of multidrug transporters and couple the efflux of structurally dissimilar drugs to the influx of either Na + or H + . The ~900 MATE transporters identified thus far can be classified into the NorM, DinF (DNA‐damage‐inducible protein F) and eukaryotic subfamilies based on amino‐acid sequence similarity. Structures of Na + ‐coupled, extracellular‐facing NorM transporters had been determined, which revealed twelve membrane‐spanning segments that are related by a quasi‐twofold rotational symmetry as well as a multidrug‐binding cavity situated near the membrane‐periplasm interface. Here we report the crystal structures of an H + ‐coupled MATE transporter from the DinF subfamily, with and without substrate, unveiling a surprisingly asymmetric arrangement of twelve transmembrane helices and a largely hydrophobic multidrug‐binding chamber located in the middle of the lipid bilayer. Combining structural and biochemical analyses, we confirmed the biological relevance of the substrate‐binding site and suggested a direct competition between H + and substrate during DinF‐mediated drug transport. Our findings provided fundamental brushstrokes to the molecular picture depicting how a MATE transporter works and laid the groundwork for future experimental efforts aimed at overcoming multidrug resistance.