Structural insights into H+-coupled multidrug extrusion by a MATE transporter

Abstract

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.

Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux

“…The formation of such disulfide bonds likely prevented complete unfolding of the protein under nonreducing and yet denaturing conditions and thus substantially altered the electrophoretic mobility of the transporter (13,16). Significantly, under our experimental conditions, the five detergent-purified dicysteine mutants could be converted into the faster migrating species almost completely, further attesting to the validity of our experimental design and the intracellular facing model (9).…”

“…Our studies further predicted that in the intracellular facing NorM-NG, the extracellular portions of TM1 and TM2 are in close proximity to those of TM8 and TM7, respectively (9). If our prediction is correct, once we replace the amino acids envisioned to be close enough in the intracellular facing NorM-NG with cysteines, we may be able to employ disulfide cross-linking to "stitch" together the extracellular portions of TM1 and TM8, TM2 and TM7, or TM2 and TM8.…”

“…adopting the intracellular facing conformation, to acquire a new substrate. In prior studies, we suggested that the N and C domains of NorM-NG rotate relative toward each other during these structural changes (9). The observation that T42C and L289C, A45C and V285C (TM1 and TM8), L58C and F270C, A62C and S266C (TM2 and TM7), and A57C and Q284C (TM2 and TM8) formed disulfide bonds places their C␣ atoms within 6 Å of each other in the intracellular facing NorM-NG.…”

“…1). The intracellular facing model of NorM-NG was constructed by rotating the C domain as a rigid body in the extracellular facing structure by 20°rela-tive to the N domain (9). Among the 14 pairs of amino acids, 8 are located within TM1 and TM8, 5 are within TM2 and TM7, and 1 is within TM2 and TM8.…”

“…Previously we deduced the intracellular facing models of MATE transporters and posited how they undergo conformational changes during drug export (9). Here we utilized mutational, cross-linking, and biochemical methods to elucidate such structural changes in NorM-NG.…”