Conformational changes in the multidrug transporter EmrE associated with substrate binding

EmrE is a bacterial multidrug transporter of the small multidrug resistance family, which extrudes large hydrophobic cations such as tetra-phenylphosphonium (TPPþ) out of the cell by a proton antiport mechanism. Binding measurements were performed on purified EmrE solubilized in dodecylmaltoside to determine the stoichiometry of TPPþ binding; the data showed that one TPPþ molecule bound per EmrE dimer. Reconstitution of purified EmrE at low lipid:protein ratios in either the presence or the absence of TPPþ produced well ordered two-dimensional crystals. Electron cryo-microscopy was used to collect images of frozen hydrated EmrE crystals and projection maps were determined by image processing to 7 A ̊ resolution. An average native EmrE projection structure was calculated from the c222 and p2221 crystals, which was subsequently subtracted from the average of two independent p2 projec-tion maps of EmrE with TPPþ bound. The interpretation of the difference density image most consistent with biochemical data suggested that TPPþ bound at the monomer–monomer interface in the centre of the EmrE dimer, and resulted in the movement of at least one transmembrane a-helix