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J. Biol. Chem., Vol. 281, Issue 47, 36205-36212, November 24, 2006

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On Parallel and Antiparallel Topology of a Homodimeric Multidrug Transporter^*

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

The recently suggested antiparallel topology of EmrE has intriguing implications for many aspects of the biology of ioncoupled transporters. However, it is at odds with biochemical data that demonstrated the same topology for all protomers in the intact cell and with extensive cross-linking studies. To examine this apparent contradiction we chemically cross-linked dimers with a rigid bifunctional maleimide using Cys replacements at positions not permissible by an antiparallel topology. A purified cross-linked dimer binds substrate and transports it in proteoliposomes with kinetic constants similar to those of the non-cross-linked dimer. The cross-linked dimers do not interact with non-cross-linked dimers as judged from the fact that inactive mutants do not affect their activity (negative dominance). The results support the contention that EmrE with parallel topology is fully functional. We show that the detergents used in crystallization increase the fraction of monomers in solution. We suggest that the antiparallel orientation observed is a result of the arrangement of the monomers in the crystal. Functionality of EmrE with the suggested antiparallel orientation of the monomers remains to be characterized.

Received for publication, July 28, 2006 , and in revised form, September 15, 2006.

^* This work was supported by Grant NS16708 from the National Institutes of Health 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 The Mathilda Marks-Kennedy Professor of Biochemistry at the Hebrew University of Jerusalem. To whom correspondence should be addressed: Dept. of Biological Chemistry, Alexander Silberman Inst. of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Tel.: 972-2-6585992; Fax: 972-2-5634625; E-mail: Shimon.Schuldiner{at}huji.ac.il.

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