Structure of the substrate binding pocket of the multidrug transporter EmrE: site-directed spin labeling of transmembrane segment 1.
Site-directed spin labeling (SDSL) was used to explore the structural framework responsible for the obligatory drug-proton exchange in the Escherichia coli multidrug transporter, EmrE. For this purpose, a nitroxide scan was carried out along a stretch of 26 residues that include transmembrane segment 1 (TMS1). This segment has been implicated in the catalytic mechanism of EmrE due to the presence of the highly conserved glutamate 14, a residue absolutely required for ligand binding. Sequence-specific variation in the accessibilities of the introduced nitroxides to molecular oxygen reveals a transmembrane helical conformation along TMS1. One face of the helix is in contact with the hydrocarbon interior of the detergent micelle while the other face appears to be solvated by an aqueous environment, resulting in significant exposure of the nitroxides along this face to NiEDDA. TMS1 from two different subunits are in close proximity near a 2-fold axis of symmetry as revealed by the analysis of spin-spin interactions at sites 14 and 18. The limited extent of spin-spin interactions is consistent with a scissor-like packing of the two TMS1. This results in a V-shaped chamber which is in contact with the aqueous phase near the N-terminus. The spatial organization of TMS1, particularly the close proximity of E14, is consistent with a proposed mechanistic model of EmrE [Yerushalmi, H., and Schuldiner, S. (2000) Biochemistry 39, 14711-14719] where substrate extrusion is coupled to proton influx through electrostatic interactions and shifts of the glutamate 14 pK(a) during the cycle.
Koteiche
Hanane A
HA
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
Reeves
Matthew D
MD
McHaourab
Hassane S
HS
D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013113:Spin Labels; D055672:Static Electricity
D017920:Antiporters; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D013113:Spin Labels; C075436:EmrE protein, E coli