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J. Bacteriol. doi:10.1128/JB.00471-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Drug-induced conformational changes in multidrug efflux transporter AcrB from Haemophilus influenzae

Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019; Mpex Pharmaceuticals, San Diego, CA 92109

	Abstract

In Gram-negative bacteria, transporters belonging to Resistance-Nodulation-cell Division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance. Haemophilus influenzae, a Gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces multidrug efflux transporter AcrB. Similar to other RND transporters H. influenzae AcrB associates with AcrA, the periplasmic membrane fusion protein (MFP), and the outer membrane channel TolC. Here, we report that H. influenzae AcrAB (HIAcrAB) confers multidrug resistance when expressed in E. coli and requires for its activity the E. coli TolC protein. To investigate the intracellular dynamics of HIAcrAB, single cysteine mutations were constructed in HIAcrB in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of HIAcrAB and in the presence or absence of HIAcrA and TolC. We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in HIAcrB.

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