Reversal of the drug binding pocket defects of the AcrB multidrug efflux pump protein of Escherichia coli

Ketaki Soparkar, Alfred D. Kinana, Jon W. Weeks, Keith D. Morrison, Hiroshi Nikaido, Rajeev Misra

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3255-3264
Number of pages10
JournalJournal of Bacteriology
Volume197
Issue number20
DOIs
StatePublished - 2015

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Escherichia coli Proteins
N-phenyl-1-naphthylamine
Anti-Bacterial Agents
Mutation
Pharmaceutical Preparations
Drug Substitution
Amino Acid Substitution
Phenotype
nitrocefin

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Reversal of the drug binding pocket defects of the AcrB multidrug efflux pump protein of Escherichia coli. / Soparkar, Ketaki; Kinana, Alfred D.; Weeks, Jon W.; Morrison, Keith D.; Nikaido, Hiroshi; Misra, Rajeev.

In: Journal of Bacteriology, Vol. 197, No. 20, 2015, p. 3255-3264.

Research output: Contribution to journalArticle

Soparkar, Ketaki ; Kinana, Alfred D. ; Weeks, Jon W. ; Morrison, Keith D. ; Nikaido, Hiroshi ; Misra, Rajeev. / Reversal of the drug binding pocket defects of the AcrB multidrug efflux pump protein of Escherichia coli. In: Journal of Bacteriology. 2015 ; Vol. 197, No. 20. pp. 3255-3264.
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