Promiscuity in the geometry of electrostatic interactions between the Escherichia coli multidrug resistance transporter MdfA and cationic substrates.
The Escherichia coli multidrug transporter MdfA contains a single membrane-embedded charged residue (Glu-26) that plays a critical role in the recognition of cationic substrates (Edgar, R., and Bibi, E. (1999) EMBO J. 18, 822-832). Using an inactive mutant (MdfA-E26T), we isolated a spontaneous second-site mutation (MdfA-E26T/V335E) that re-established the recognition of cationic drugs by the transporter. Only a negative charge at position 335 was able to restore the functioning of the inactive mutant MdfA-E26T. Intriguingly, the two genetically interacting residues are located at remote and distinct regions along the secondary structure of MdfA. Glu-26 is located in the periplasmic half of transmembrane helix 1, and as shown here, the complementing charge at position 335 resides within the cytoplasmic loop connecting transmembrane helices 10 and 11. The spatial relation between the two residues was investigated by cross-linking. A functional split version of MdfA devoid of cysteines was constructed and introduced with a cysteine pair at positions 26 and 335. Strikingly, the results indicate that residues 26 and 335 are spatially adjacent, suggesting that they both constitute parts of the multidrug recognition pocket of MdfA. The fact that electrostatic interactions are preserved with cationic substrates even if the critical acidic residue is placed on another face of the pocket reveals an additional dimension of promiscuity in multidrug recognition and transport.
Adler
Julia
J
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Bibi
Eitan
E
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001692:Biological Transport; D015153:Blotting, Western; D002412:Cations; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D004996:Ethidium / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013379:Substrate Specificity
D002412:Cations; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C118438:Mdfa protein, E coli; D026901:Membrane Transport Proteins; D001120:Arginine; D004996:Ethidium; D003545:Cysteine