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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 KM was 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 Vmax values 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.

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 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.

Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a well-known inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket.

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. IMPORTANCE 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.