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J. Biol. Chem., Vol. 281, Issue 22, 15546-15553, June 2, 2006

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Evidence for Assembly of Small Multidrug Resistance Proteins by a "Two-faced" Transmembrane Helix^*

Arianna Rath¹, Roman A. Melnyk², and Charles M. Deber³

From the Division of Structural Biology & Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8 and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the assembly of at least two inactive monomers by intermolecular association of their four transmembrane helices. Here, we have used peptides that correspond to each of the four wild type transmembrane helices of the Halobacterium salinarum protein Hsmr and a corresponding library of mutant peptides to determine the interactive surfaces that likely contribute to protein oligomerization. Hsmr peptides were examined for strong (sodium dodecyl sulfate-resistant) and weaker (perfluorooctanoate-resistant) helix-helix interactions, in conjunction with circular dichroism, fluorescence energy transfer measurements, and molecular modeling. The results are compatible with a scheme in which two faces of helix four permit self-assembly via a higher affinity asymmetric pairing and a lower affinity symmetric interaction, resulting in a discrete tetramer. Our finding that two surfaces of helix four can contribute to the stability of small multidrug resistance protein assembly provides a molecular basis for the design of therapeutics that target this antibiotic resistance mechanism.

Received for publication, January 17, 2006 , and in revised form, March 21, 2006.

^* This work was supported in part by a grant from the Canadian Institutes of HealthResearch (CIHR) (to C. M. D.). 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.

¹ Recipient of a post-doctoral award from the CIHR Training Program in Protein Folding:Principles and Diseases.

² Recipient of a CIHR doctoral award. Present address: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.

³ To whom correspondence should be addressed: Div. of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8. Tel.: 416-813-5924; Fax: 416-813-5005; E-mail: deber{at}sickkids.ca.

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