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Some ligands enhance the efflux of other ligands by the Escherichia coli multidrug pump AcrB.

ABSTRACT: By measuring quantitatively the active efflux of cephalosporins by the RND (resistance-nodulation-division) family efflux pump AcrB in intact cells of Escherichia coli, we found that the simultaneous presence of another substrate, such as chloramphenicol, benzene, cyclohexane, or Arg ?-naphthilamide, significantly enhanced the extrusion of cephalosporins. The stimulation occurred also in a strain expressing the covalently linked trimer of AcrB, and thus cannot be ascribed to the enhanced assembly of the trimer from AcrB monomers. When Val139 of AcrB was changed into Phe, the stimulation by benzene was found to occur at much lower concentration of the solvent. A plausible explanation of these observations is that the AcrB pump is constructed to pump out very rapidly the solvent or chloramphenicol molecules, and thus the efflux of cephalosporins, which presumably bind to a different subsite within the large binding pocket of AcrB, can become facilitated. Computer simulations of ligand binding to AcrB, both by docking and by molecular dynamics simulations, produced results supporting and extending this hypothesis. Benzene and the cephalosporin nitrocefin can bind simultaneously to the distal binding pocket of AcrB, both in the wild type and in the V139F variant. Interestingly, while the binding position and strength of benzene are almost unaffected by the presence of nitrocefin, this latter substrate is significantly displaced toward the exit gate in both wild type and mutant transporter in the presence of benzene. Additionally, the cephalosporin efflux may be enhanced by the binding of solvents (sometimes to the cephalosporin-free protomer), which could accelerate AcrB conformational changes necessary for substrate extrusion.

SUBMITTER: Kinana AD

PROVIDER: S-EPMC3951271 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Some ligands enhance the efflux of other ligands by the Escherichia coli multidrug pump AcrB.

Kinana Alfred D AD Vargiu Attilio V AV Nikaido Hiroshi H

Biochemistry 20131111 46

By measuring quantitatively the active efflux of cephalosporins by the RND (resistance-nodulation-division) family efflux pump AcrB in intact cells of Escherichia coli, we found that the simultaneous presence of another substrate, such as chloramphenicol, benzene, cyclohexane, or Arg β-naphthilamide, significantly enhanced the extrusion of cephalosporins. The stimulation occurred also in a strain expressing the covalently linked trimer of AcrB, and thus cannot be ascribed to the enhanced assembl ...[more]

PMID: 24205856

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Project description:The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions--Y49S, V127A, V127G, D153E, and G288C--mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions--F453C and L486W--were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain.The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.

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Some ligands enhance the efflux of other ligands by the Escherichia coli multidrug pump AcrB.

Publications

Some ligands enhance the efflux of other ligands by the Escherichia coli multidrug pump AcrB.

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