The key residue for substrate transport (Glu14) in the EmrE dimer is asymmetric.
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.
Lehner
Ines
I
Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, J. W. Goethe University, 60438 Frankfurt, Germany.
Basting
Daniel
D
Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, J. W. Goethe University, 60438 Frankfurt, Germany.
Meyer
Bjoern
B
Institute of Pharmaceutical Chemistry, J. W. Goethe University, 60438 Frankfurt, Germany.
Haase
Winfried
W
Max Planck Institute of Biophysics, 60438 Frankfurt, Germany.
Manolikas
Theofanis
T
Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, J. W. Goethe University, 60438 Frankfurt, Germany.
Kaiser
Christoph
C
Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, J. W. Goethe University, 60438 Frankfurt, Germany.
Karas
Michael
M
Institute of Pharmaceutical Chemistry, J. W. Goethe University, 60438 Frankfurt, Germany.
Glaubitz
Clemens
C
Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, J. W. Goethe University, 60438 Frankfurt, Germany. Electronic address: glaubitz@em.uni-frankfurt.de.
D017920:Antiporters / Q000737:chemistry*; D001692:Biological Transport; D002474:Cell-Free System; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005614:Freeze Fracturing; D018698:Glutamic Acid / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017920:Antiporters; D029968:Escherichia coli Proteins; D008055:Lipids; C075436:EmrE protein, E coli; D018698:Glutamic Acid