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J. Biol. Chem., Vol. 283, Issue 14, 9369-9376, April 4, 2008

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Interactions between Phosphatidylethanolamine Headgroup and LmrP, a Multidrug Transporter

A CONSERVED MECHANISM FOR PROTON GRADIENT SENSING?^*

From the Department of Structure and Function of Biological Membranes, Structural Biology and Bioinformatics Center, Université Libre de Bruxelles, CP206/2, Boulevard du Triomphe, Brussels 1050, Belgium

In a number of cases, the function of membrane proteins appears to require the presence of specific lipid species in the bilayer. We have shown that the secondary multidrug transporter LmrP requires the presence of phosphatidylethanolamine (PE), as its replacement by phosphatidylcholine (PC) inhibits transport activity and directly affects its structure, although the underlying mechanism was unknown. Here, we show that the effect of PE on the structure and the function of LmrP is mediated by interactions between the lipid headgroup and the protein. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by PC, including energy transfer between the protein tryptophan residues and the lipid headgroups. We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.

Received for publication, October 10, 2007 , and in revised form, January 29, 2008.

^* This work was supported in part by the Actions de Recherche Concertées de la Communauté Française, Belgium. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Recipients of financial support from Fonds pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture (Belgium).

³ A Chargé de Recherches from the Fonds National de la Recherche Scientifique and supported by a return grant of the Belgian Federal Science Policy.

² To whom correspondence should be addressed: Tel.: 32-2-650-53-77; Fax: 32-2-650-53-82; E-mail: jmruyss{at}ulb.ac.be.

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