title: Effect of site-directed mutations in multidrug efflux pump AcrB examined by quantitative efflux assays.
abstract: BACKGROUND
  The Resistance-Nodulation-Division (RND) family transporter AcrB plays a major role in the intrinsic and increased resistance of Escherichia coli to a large number of antibiotics. The distal binding pocket within this multidrug efflux transporter is very large, but the effort to define the roles of various residues facing this pocket through site-directed mutagenesis so far involved only the determination of minimal inhibitory concentrations of drugs in mutants.
  
  
  METHODS
  We measured in intact E. coli cells the kinetics of efflux of two substrates, nitrocefin (a cephalosporin) that is predicted mainly to bind to the upper, "groove" domain of the pocket, and L-alanyl-β-naphthylamide (Ala-Naph) that is likely to bind to the lower, "cave" domain, in a number of site-directed mutants of AcrB, where a hydrophobic or aromatic residue was changed into alanine.
  
  
  RESULTS
  The efflux of nitrocefin became attenuated by some mutations in the groove domain, such as I278A and F178A, but in some experiments a mutation in the cave domain, F628A produced a similar result. In some cases an increased value of KMwas detected. The efflux of Ala-Naph was increased by mutations in the cave domain, such as F136A and I626A, but also by those in the groove domain (I277A, I278A, F178A). In most cases the increased Vmaxvalues appeared to be responsible. F610A mutation had a profound effect on the efflux of both substrates, as reported earlier.
  
  
  CONCLUSIONS
  Our data show for the first time effects of various substrate-binding pocket mutations on the kinetics of efflux of two substrates by the AcrB pump. They also confirm interactions between substrates and drugs predicted by MD simulation studies, and also reveal areas that need future research.
authors: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
authors: Alfred D
authors: AD
authors: Kinana
authors: Department of Physics, University of Cagliari, 09042, Monserrato, Italy.
authors: Attilio V
authors: AV
authors: Vargiu
authors: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA. Electronic address: nhiroshi@berkeley.edu.
authors: Hiroshi
authors: H
authors: Nikaido
mesh_terms: D000577:Amides / Q000737:chemistry*; D001665:Binding Sites; D002511:Cephalosporins / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics*; D062105:Molecular Docking Simulation; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D013329:Structure-Activity Relationship
chemical_list: D000577:Amides; D002511:Cephalosporins; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083496:AcrB protein, E coli; C021720:nitrocefin
keywords: Efflux kinetics; Multidrug efflux; RND pump; Site-directed mutagenesis; Substrate-binding site