title: Exploring the binding domain of EmrE, the smallest multidrug transporter.
abstract: EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu14) from both EmrE monomers. Previous studies implied that other residues in the vicinity of Glu14 are part of the binding domain. Alkylation of Cys replacements in the same transmembrane domain inhibits the activity of the protein and this inhibition is fully prevented by substrates of EmrE. To monitor directly the reaction we tested also the extent of modification using fluorescein-5-maleimide. While most residues are not accessible or only partially accessible, four, Y4C, I5C, L7C, and A10C, were modified at least 80%. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of two of these residues by up to 80%. To study other essential residues we generated functional hetero-oligomers and challenged them with various methane thiosulfonates. Taken together the findings imply the existence of a binding cavity accessible to alkylating reagents where at least three residues from TM1, Tyr40 from TM2, and Trp63 in TM3 are involved in substrate binding.
authors.affiliation: Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Sharoni
authors.forename: Sonia
authors.initials: S
authors.lastname: Steiner-Mordoch
authors.forename: Shimon
authors.initials: S
authors.lastname: Schuldiner
mesh_terms: D017920:Antiporters / Q000737:chemistry*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry; D008698:Mesylates / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009861:Onium Compounds / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D013379:Substrate Specificity
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D011522:Protons; C075436:EmrE protein, E coli; D018698:Glutamic Acid; C074502:methanethiosulfonate; C037041:fluorescein 5-maleimide; D003545:Cysteine; C013289:tetraphenylphosphonium