title: Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, USA CoValence Laboratories, Chandler, Arizona, USA.
authors.forename: Ketaki
authors.initials: K
authors.lastname: Soparkar
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
authors.forename: Alfred D
authors.initials: AD
authors.lastname: Kinana
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, USA Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA.
authors.forename: Jon W
authors.initials: JW
authors.lastname: Weeks
authors.affiliation: School of Earth & Space Exploration, Arizona State University, Tempe, Arizona, USA.
authors.forename: Keith D
authors.initials: KD
authors.lastname: Morrison
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nikaido
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, Arizona, USA rajeev.misra@asu.edu.
authors.forename: Rajeev
authors.initials: R
authors.lastname: Misra
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D002511:Cephalosporins / Q000378:metabolism; D019295:Computational Biology; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083496:AcrB protein, E coli; C021720:nitrocefin