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J. Biol. Chem., Vol. 278, Issue 50, 50474-50482, December 12, 2003

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pH-induced Conformational Changes of AcrA, the Membrane Fusion Protein of Escherichia coli Multidrug Efflux System^*

Hermia Ip, Kelly Stratton, Helen Zgurskaya, and Jun Liu¶

From the Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and the Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019

The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates.

Received for publication, May 16, 2003 , and in revised form, September 30, 2003.

^* This work was supported by a grant from Canadian Foundation for Innovation (to J. L.) and by a Scientist Development grant from the American Heart Association (to H. I. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ A Canadian Institutes of Health Research (CIHR) New Investigator. To whom correspondence should be addressed: 4382 Medical Sciences Building, Dept. of Medical Genetics and Microbiology, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Tel.: 416-946-5067; Fax: 416-978-6885; E-mail: jun.liu{at}utoronto.ca.

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