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Journal of Bacteriology, August 2007, p. 5550-5558, Vol. 189, No. 15
0021-9193/07/$08.00+0     doi:10.1128/JB.00471-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Vishakha Dastidar,1 Weimin Mao,2,{dagger} Olga Lomovskaya,3 and Helen I. Zgurskaya1*

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 (AcrBHI). Similar to other RND transporters AcrBHI associates with AcrAHI, the periplasmic membrane fusion protein, and the outer membrane channel TolCHI. Here, we report that AcrABHI confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolCEC) protein. To investigate the intracellular dynamics of AcrABHI, single cysteine mutations were constructed in AcrBHI 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 AcrABHI and in the presence or absence of AcrAHI and TolCEC. 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 AcrBHI.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, 620 Parrington Oval, Norman, OK 73019. Phone: (405) 325-1678. Fax: (405) 325-6111. E-mail: elenaz{at}ou.edu

{triangledown} Published ahead of print on 25 May 2007.

{dagger} Present address: Anacor Pharmaceuticals, Palo Alto, CA 94303.

Journal of Bacteriology, August 2007, p. 5550-5558, Vol. 189, No. 15
0021-9193/07/$08.00+0     doi:10.1128/JB.00471-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Krishnamoorthy, G., Tikhonova, E. B., Zgurskaya, H. I. (2008). Fitting Periplasmic Membrane Fusion Proteins to Inner Membrane Transporters: Mutations That Enable Escherichia coli AcrA To Function with Pseudomonas aeruginosa MexB. J. Bacteriol. 190: 691-698 [Abstract] [Full Text]  


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