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 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 7A 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 alpha-helix.
Tate
Christopher G
CG
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. cgt@mrc-lmb.cam.ac.uk
Ubarretxena-Belandia
Iban
I
Baldwin
Joyce M
JM
D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D003902:Detergents / Q000737:chemistry; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009861:Onium Compounds / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
D017920:Antiporters; D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009861:Onium Compounds; D009943:Organophosphorus Compounds; C075436:EmrE protein, E coli; C013289:tetraphenylphosphonium