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J. Biol. Chem., Vol. 280, Issue 38, 32849-32855, September 23, 2005

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Exploring the Binding Domain of EmrE, the Smallest Multidrug Transporter^*

From the Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu¹⁴) from both EmrE monomers. Previous studies implied that other residues in the vicinity of Glu¹⁴ are part of the binding domain. Alkylation of Cys replacements in the same transmembrane domain inhibits the activity of the protein and this inhibition is fully prevented by substrates of EmrE. To monitor directly the reaction we tested also the extent of modification using fluorescein-5-maleimide. While most residues are not accessible or only partially accessible, four, Y4C, I5C, L7C, and A10C, were modified at least 80%. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of two of these residues by up to 80%. To study other essential residues we generated functional hetero-oligomers and challenged them with various methane thiosulfonates. Taken together the findings imply the existence of a binding cavity accessible to alkylating reagents where at least three residues from TM1, Tyr⁴⁰ from TM2, and Trp⁶³ in TM3 are involved in substrate binding.

Received for publication, May 4, 2005 , and in revised form, July 26, 2005.

^* This work was supported in part by National Institutes of Health Grant NS16708 and Grant 119/04 from the Israel Science Foundation. 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 Mathilda Marks-Kennedy Professor of Biochemistry at the Hebrew University of Jerusalem. To whom correspondence should be addressed. Tel.: 972-2-6585992; Fax: 972-2-5634625; E-mail: Shimon.Schuldiner{at}huji.ac.il.

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