A single carboxyl mutant of the multidrug transporter EmrE is fully functional.
EmrE, a multidrug transporter from Escherichia coli removes toxic compounds from the cell in exchange with protons. Glu-14 is the only charged residue in the putative membrane domains and is fully conserved in more than 50 homologues of the protein. This residue was shown to be an essential part of the binding site, common to protons and substrate. EmrE bearing a single carboxylic residue, Glu-14, shows uptake and binding properties similar to those of the wild type. This suggests that a small protein bearing only 110 amino acids with a single carboxyl in position 14 is the most basic structure that shows ion-coupled transport activity. The role of Glu-14 in substrate binding was examined by using dicyclohexylcarbodiimide, a hydrophobic carbodiimide that is known to react with carboxyls. Tetraphenylphosphonium binding to both wild type and the single carboxyl mutant is inhibited by dicyclohexylcarbodiimide in a dose-dependent manner. Ethidium and other substrates of EmrE prevent this inhibition with an order of potency in accord with their apparent affinities. This suggests that dicyclohexylcarbodiimide binding is sterically prevented by the substrate, supporting the contention that Glu-14, the reactive residue, is part of the substrate-binding site.
Yerushalmi
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H
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Mordoch
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SS
Schuldiner
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S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009861:Onium Compounds / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010269:Paraquat / Q000493:pharmacokinetics; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism
D017920:Antiporters; D029968:Escherichia coli Proteins; D008081:Liposomes; D008565:Membrane Proteins; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; C075436:EmrE protein, E coli; D018698:Glutamic Acid; D004024:Dicyclohexylcarbodiimide; D010269:Paraquat; C013289:tetraphenylphosphonium