An essential glutamyl residue in EmrE, a multidrug antiporter from Escherichia coli.
EmrE is an Escherichia coli 12-kDa protein that confers resistance to toxic compounds, by actively removing them in exchange with protons. The protein includes eight charged residues. Seven of these residues are located in the hydrophilic loops and can be replaced with either Cys or another amino acid bearing the same charge, without impairing transport activity. Glu-14 is the only charged residue in the membrane domain and is conserved in all the proteins of the family. We show here that this residue is the site of action of dicyclohexylcarbodiimide, a carbodiimide known to act in hydrophobic environments. When Glu-14 was replaced with either Cys or Asp, resistance was abolished. Whereas the E14C mutant displays no transport activity, the E14D protein shows efflux and exchange at rates about 30-50% that of the wild type. The maximal DeltapH-driven uptake rate of E14D is only 10% that of the wild type. The mutant shows a different pH profile in all the transport modes. Our results support the notion that Glu-14 is an essential part of a binding domain shared by substrates and protons but mutually exclusive in time. This notion provides the molecular basis for the obligatory exchange catalyzed by EmrE.
Yerushalmi
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Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Schuldiner
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D000167:Acriflavine / Q000494:pharmacology; D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000493:pharmacokinetics; D001426:Bacterial Proteins / Q000737:chemistry*; D002234:Carbodiimides / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D004996:Ethidium / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry* / Q000493:pharmacokinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010269:Paraquat / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000378:metabolism; D013997:Time Factors
D017920:Antiporters; D001426:Bacterial Proteins; D002234:Carbodiimides; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; C075436:EmrE protein, E coli; D000167:Acriflavine; D018698:Glutamic Acid; D004024:Dicyclohexylcarbodiimide; D004996:Ethidium; D010269:Paraquat