title: Mutations in MexB that affect the efflux of antibiotics with cytoplasmic targets.
abstract: Drug efflux pumps such as MexAB-OprM from Pseudomonas aeruginosa confer resistance to a wide range of chemically different compounds. Within the tripartite assembly, the inner membrane protein MexB is mainly responsible for substrate recognition. Recently, considerable advances have been made in elucidating the drug efflux pathway through the large periplasmic domains of resistance-nodulation-division (RND) transporters. However, little is known about the role of amino acids in other parts of the protein. We have investigated the role of two conserved phenylalanine residues that are aligned around the cytoplasmic side of the central cavity of MexB. The two conserved phenylalanine residues have been mutated to alanine residues (FAFA MexB). The interaction of the wild-type and mutant proteins with a variety of drugs from different classes was investigated by assays of cytotoxicity and drug transport. The FAFA mutation affected the efflux of compounds that have targets inside the cell, but antibiotics that act on cell wall synthesis and membrane probes were unaffected. Combined, our results indicate the presence of a hitherto unidentified cytoplasmic-binding site in RND drug transporters and enhance our understanding of the molecular mechanisms that govern drug resistance in Gram-negative pathogens.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK.
authors.forename: Thelma
authors.initials: T
authors.lastname: Ohene-Agyei
authors.forename: Jon D
authors.initials: JD
authors.lastname: Lea
authors.forename: Henrietta
authors.initials: H
authors.lastname: Venter
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000493:pharmacokinetics*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects* / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial; D004917:Erythromycin / Q000493:pharmacokinetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; C118597:MexB protein, Pseudomonas aeruginosa; D010649:Phenylalanine; D004917:Erythromycin; D000409:Alanine