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

Abstract

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.