title: Evidence for simultaneous binding of dissimilar substrates by the Escherichia coli multidrug transporter MdfA.
abstract: The mechanism by which multidrug transporters interact with structurally unrelated substrates remains enigmatic. Based on transport competition experiments, photoaffinity labeling, and effects on enzymatic activities, it was proposed in the past that multidrug transporters can interact simultaneously with a number of dissimilar substrate molecules. To study this phenomenon, we applied a direct binding approach and transport assays using the Escherichia coli multidrug transporter MdfA, which exports both positively charged (e.g., tetraphenylphosphonium, TPP(+)), zwitterionic (e.g., ciprofloxacin), and neutral (e.g., chloramphenicol) drugs. The interaction of MdfA with various substrates was examined by direct binding assays with the purified transporter. The immobilized MdfA binds TPP(+) in a specific manner, and all the tested positively charged substrates inhibit TPP(+) binding. Surprisingly, although TPP(+) binding is not affected by zwitterionic substrates, the neutral substrate chloramphenicol stimulates TPP(+) binding by enhancing its affinity to MdfA. In contrast, transport competition assays show inhibition of TPP(+) transport by chloramphenicol. We suggest that MdfA binds TPP(+) and chloramphenicol simultaneously to distinct but interacting binding sites, and the interaction between these two substrates during transport is discussed.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: O
authors.initials: O
authors.lastname: Lewinson
authors.forename: E
authors.initials: E
authors.lastname: Bibi
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001548:Benzalkonium Compounds / Q000494:pharmacology; D001562:Benzimidazoles / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002701:Chloramphenicol / Q000378:metabolism / Q000494:pharmacology; D003630:Daunorubicin / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D004996:Ethidium / Q000494:pharmacology; D026901:Membrane Transport Proteins; D009861:Onium Compounds / Q000037:antagonists & inhibitors / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014316:Tritium / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001548:Benzalkonium Compounds; D001562:Benzimidazoles; D029968:Escherichia coli Proteins; C118438:Mdfa protein, E coli; D026901:Membrane Transport Proteins; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D014316:Tritium; D002701:Chloramphenicol; D004996:Ethidium; C017807:bisbenzimide ethoxide trihydrochloride; C013289:tetraphenylphosphonium; D003630:Daunorubicin