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J. Biol. Chem., Vol. 283, Issue 6, 3281-3288, February 8, 2008

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The Key Residue for Substrate Transport (Glu¹⁴) in the EmrE Dimer Is Asymmetric^*

Ines Lehner, Daniel Basting, Bjoern Meyer, Winfried Haase^¶, Theofanis Manolikas, Christoph Kaiser, Michael Karas, and Clemens Glaubitz¹

From the Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance and Institute of Pharmaceutical Chemistry, J. W. Goethe University, 60438 Frankfurt, Germany and the ^¶Max Planck Institute of Biophysics, 60438 Frankfurt, Germany

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report ¹³C chemical shifts of the essential residue Glu¹⁴ within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered ¹³C solid state NMR. For an unambiguous assignment of Glu¹⁴, an E25A mutation was introduced to create a single glutamate mutant. Glu¹⁴ was ¹³C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu¹⁴, two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu¹⁴ in the dimer.

Received for publication, September 20, 2007 , and in revised form, November 21, 2007.

^* This work was supported by the Collaborative Research Centre 628 "Functional Membrane Proteomics," the Federal Ministry of Education and Research network ProAMP, and EU-NMR. 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–S3.

¹ To whom correspondence should be addressed: Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. Fax: 49-69-798-29929; E-mail: glaubitz{at}em.uni-frankfurt.de.

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