Conformational Changes in the Multidrug Transporter EmrE Associated with Substrate Binding

Abstract

EmrE is a bacterial multidrug transporter of the small multidrug resistance family, which extrudes large hydrophobic cations such as tetraphenylphosphonium (TPP +) out of the cell by a proton antiport mechanism. Binding measurements were performed on purified EmrE solubilized in dodecylmaltoside to determine the stoichiometry of TPP + binding; the data showed that one TPP + molecule bound per EmrE dimer. Reconstitution of purified EmrE at low lipid:protein ratios in either the presence or the absence of TPP + 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 Å resolution. An average native EmrE projection structure was calculated from the c222 and p222 1 crystals, which was subsequently subtracted from the average of two independent p2 projection maps of EmrE with TPP + bound. The interpretation of the difference density image most consistent with biochemical data suggested that TPP + bound at the monomer–monomer interface in the centre of the EmrE dimer, and resulted in the movement of at least one transmembrane α-helix.