title: Molecular basis of polyspecificity of the Small Multidrug Resistance Efflux Pump AbeS from Acinetobacter baumannii.
abstract: Secondary multidrug efflux transporters play a key role in the bacterial resistance phenotype. One of the major questions concerns the polyspecific recognition of substrates by these efflux pumps. To understand the molecular basis of this promiscuous recognition, we compared the substrate specificity of the well-studied Escherichia coli small multidrug resistance protein EmrE with that of the poorly studied Acinetobacter baumannii homologue AbeS. The latter drug/H(+) antiporter is a 109-amino-acid membrane protein with predicted four transmembrane helices. It effectively confers resistance toward ethidium, acriflavine and benzalkonium in an E. coli ΔemrEΔmdfA background. Purified AbeS and the substrate-specific hyperactive variant A16G bind tetraphenylphosphonium with nanomolar affinity and exhibit electrogenic transport of 1-methyl-4-phenylpyridinium after reconstitution into liposomes. A16G hyperactivity was apparent toward acriflavine and ethidium, resulting in 7- to 10-fold higher normalized IC50 values, respectively, but not toward substrates 1-methyl-4-phenylpyridinium and benzalkonium. Substitution of Y3 and A42 with Ala or Ser, respectively, also displayed a substrate-dependent phenotype, as these variants were strongly affected in their properties to confer resistance against acriflavine and ethidium, but not against benzalkonium. The size and planarity of the conjugated aromatic moieties appear to be a critical and subtle criterion for substrate recognition by these transporters. Rather moderate changes in the property of side chains postulated to be part of the substrate binding site result in a large phenotypical difference. These observations provide indications for the molecular basis of specificity within the binding pocket of polyspecific transporters.
authors.affiliation: Institute of Biochemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany.
authors.forename: Iryna
authors.initials: I
authors.lastname: Lytvynenko
authors.affiliation: Alexander A. Silbermann Institute of Life Science, Hebrew University of Jerusalem, Israel.
authors.forename: Shlomo
authors.initials: S
authors.lastname: Brill
authors.affiliation: Institute of Biochemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Oswald
authors.affiliation: Institute of Biochemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany. Electronic address: pos@em.uni-frankfurt.de.
authors.forename: Klaas M
authors.initials: KM
authors.lastname: Pos
mesh_terms: D000151:Acinetobacter Infections / Q000188:drug therapy / Q000382:microbiology; D040981:Acinetobacter baumannii / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000900:Anti-Bacterial Agents; D017920:Antiporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C075436:EmrE protein, E coli
keywords: EmrE; antibiotic resistance; membrane transport; reconstitution; substrate promiscuity