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The key residue for substrate transport (Glu14) in the EmrE dimer is asymmetric.

Authors
  • Lehner, Ines
  • Basting, Daniel
  • Meyer, Bjoern
  • Haase, Winfried
  • Manolikas, Theofanis
  • Kaiser, Christoph
  • Karas, Michael
  • Glaubitz, Clemens
Type
Published Article
Journal
Journal of Biological Chemistry
Publisher
American Society for Biochemistry & Molecular Biology (ASBMB)
Publication Date
Feb 08, 2008
Volume
283
Issue
6
Pages
3281–3288
Identifiers
PMID: 18042544
Source
Medline
License
Unknown

Abstract

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 (13)C chemical shifts of the essential residue Glu(14) 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 (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-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 Glu(14), 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 Glu(14) in the dimer.

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