A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE.
EmrE is an Escherichia coli multidrug transporter that confers resistance to a variety of toxins by removing them in exchange for hydrogen ions. The detergent-solubilized protein binds tetraphenylphosphonium (TPP(+)) with a K(D) of 10 nM. One mole of ligand is bound per approximately 3 mol of EmrE, suggesting that there is one binding site per trimer. The steep pH dependence of binding suggests that one or more residues, with an apparent pK of approximately 7.5, release protons prior to ligand binding. A conservative Asp replacement (E14D) at position 14 of the only membrane-embedded charged residue shows little transport activity, but binds TPP(+) at levels similar to those of the wild-type protein. The apparent pK of the Asp shifts to <5.0. The data are consistent with a mechanism requiring Glu14 for both substrate and proton recognition. We propose a model in which two of the three Glu14s in the postulated trimeric EmrE homooligomer deprotonate upon ligand binding. The ligand is released on the other face of the membrane after binding of protons to Glu14.
Muth
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Institute of Life Sciences, Givat Ram, Hebrew University, Jerusalem 91904, Israel.
Schuldiner
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D000595:Amino Acid Sequence; D017920:Antiporters; D001224:Aspartic Acid; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D017124:Conserved Sequence; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009861:Onium Compounds / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D008565:Membrane Proteins; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C075436:EmrE protein, E coli; D001224:Aspartic Acid; D018698:Glutamic Acid; C013289:tetraphenylphosphonium