Role of a conserved membrane-embedded acidic residue in the multidrug transporter MdfA.
According to the current topology model of the Escherichia coli multidrug transporter MdfA, it contains a membrane-embedded negatively charged residue, Glu26, which was shown to play an important role in substrate recognition. To further elucidate the role of this substrate recognition determinant, various Glu26 replacements were characterized. Surprisingly, studies with neutral MdfA substrates showed that, unlike many enzymatic systems where the size and chemical properties of binding site residues are relatively defined, MdfA tolerates a variety of changes at position 26, including size, hydrophobicity, and charge. Moreover, although efficient transport of positively charged substrates requires a negative charge at position 26 (Glu or Asp), neutralization of this charge does not always abrogate the interaction of MdfA with cationic drugs, thus demonstrating that the negative charge does not play an essential role in the multidrug transport mechanism. Collectively, these results suggest a link between the broad substrate specificity profile of multidrug transporters and the structural and chemical promiscuity at their substrate recognition pockets.
Adler
Julia
J
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Lewinson
Oded
O
Bibi
Eitan
E
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000668:Ampicillin Resistance / Q000235:genetics; D001548:Benzalkonium Compounds / Q000737:chemistry; D002412:Cations; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics; D017124:Conserved Sequence; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004996:Ethidium / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D007613:Kanamycin Resistance / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009861:Onium Compounds / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D011691:Puromycin / Q000737:chemistry; D013379:Substrate Specificity
D001548:Benzalkonium Compounds; D002412:Cations; D029968:Escherichia coli Proteins; C118438:Mdfa protein, E coli; D026901:Membrane Transport Proteins; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D018698:Glutamic Acid; D011691:Puromycin; D004996:Ethidium; C013289:tetraphenylphosphonium