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Drug-Induced Conformational Changes in Multidrug Efflux Transporter AcrB from Haemophilus influenzae

Vishakha Dastidar,¹ Weimin Mao,^2, Olga Lomovskaya,³ and Helen I. Zgurskaya^1*

Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019,¹ Microcide Pharmaceuticals, Mountain View, California,² Mpex Pharmaceuticals, San Diego, California 92109³

Received 28 March 2007/ Accepted 11 May 2007

In gram-negative bacteria, transporters belonging to the 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 the multidrug efflux transporter AcrB (AcrB_HI). Similar to other RND transporters AcrB_HI associates with AcrA_HI, the periplasmic membrane fusion protein, and the outer membrane channel TolC_HI. Here, we report that AcrAB_HI confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolC_EC) protein. To investigate the intracellular dynamics of AcrAB_HI, single cysteine mutations were constructed in AcrB_HI in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrAB_HI and in the presence or absence of AcrA_HI and TolC_EC. 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 AcrB_HI.

Published ahead of print on 25 May 2007.

Present address: Anacor Pharmaceuticals, Palo Alto, CA 94303.

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