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A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study.

ABSTRACT: The Escherichia coli AcrB multidrug efflux pump is a membrane protein that recognizes many structurally dissimilar toxic compounds. We previously reported the X-ray structures of four AcrB-ligand complexes in which the ligands were bound to the wall of the extremely large central cavity in the transmembrane domain of the pump. Genetic studies, however, suggested that discrimination between the substrates occurs mainly in the periplasmic domain rather than the transmembrane domain of the pump. We here describe the crystal structures of the AcrB mutant in which Asn109 was replaced by Ala, with five structurally diverse ligands, ethidium, rhodamine 6G, ciprofloxacin, nafcillin, and Phe-Arg-beta-naphthylamide. The ligands bind not only to the wall of central cavity but also to a new periplasmic site within the deep external depression formed by the C-terminal periplasmic loop. This depression also includes residues identified earlier as being important in the specificity. We show here that conversion into alanine of the Phe664, Phe666, or Glu673 residue in the periplasmic binding site produced significant decreases in the MIC of most agents in the N109A background. Furthermore, decreased MICs were also observed when these residues were mutated in the wild-type AcrB background, although the effects were more modest. The MIC data were also confirmed by assays of ethidium influx rates in intact cells, and our results suggest that the periplasmic binding site plays a role in the physiological process of drug efflux.

SUBMITTER: Yu EW

PROVIDER: S-EPMC1251581 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study.

Yu Edward W EW Aires Julio R JR McDermott Gerry G Nikaido Hiroshi H

Journal of bacteriology 20051001 19

The Escherichia coli AcrB multidrug efflux pump is a membrane protein that recognizes many structurally dissimilar toxic compounds. We previously reported the X-ray structures of four AcrB-ligand complexes in which the ligands were bound to the wall of the extremely large central cavity in the transmembrane domain of the pump. Genetic studies, however, suggested that discrimination between the substrates occurs mainly in the periplasmic domain rather than the transmembrane domain of the pump. We ...[more]

PMID: 16166543

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Project description:The continued challenges of the COVID-19 pandemic combined with the growing problem of antimicrobial-resistant bacterial infections has severely impacted global health. Specifically, the Gram-negative pathogen Klebsiella pneumoniae is one of the most prevalent causes of secondary bacterial infection in COVID-19 patients, with approximately an 83% mortality rate observed among COVID-19 patients with these bacterial coinfections. K. pneumoniae belongs to the ESKAPE group of pathogens, a group that commonly gives rise to severe infections that are often life-threatening. Recently, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae has drawn wide public attention, as the mortality rate for this infection can be as high as 71%. The most predominant and clinically important multidrug efflux system in K. pneumoniae is the acriflavine resistance B (AcrB) multidrug efflux pump. This pump mediates resistance to different classes of structurally diverse antimicrobial agents, including quinolones, β-lactams, tetracyclines, macrolides, aminoglycosides, and chloramphenicol. We here report single-particle cryo-electron microscopy (cryo-EM) structures of K. pneumoniae AcrB, in both the absence and the presence of the antibiotic erythromycin. These structures allow us to elucidate specific pump-drug interactions and pinpoint exactly how this pump recognizes antibiotics. IMPORTANCE Klebsiella pneumoniae has emerged as one of the most problematic and highly antibiotic-resistant pathogens worldwide. It is the second most common causative agent involved in secondary bacterial infection in COVID-19 patients. K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae is a major concern in global public health because of the high mortality rate of this infection. Its drug resistance is due, in a significant part, to active efflux of these bactericides, a major mechanism that K. pneumoniae uses to resist to the action of multiple classes of antibiotics. Here, we report cryo-electron microscopy (cryo-EM) structures of the prevalent and clinically important K. pneumoniae AcrB multidrug efflux pump, in both the absence and the presence of the erythromycin antibiotic. These structures allow us to understand the action mechanism for drug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

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.

Project description:Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly understood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regulators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlorpromazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by interfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors.IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.

Dataset Information

A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study.

Publications

A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study.

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