Journal
BIOCHEMISTRY
Volume 42, Issue 20, Pages 6099-6105Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi0342867
Keywords
-
Categories
Ask authors/readers for more resources
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 C purpose, a nitroxide scan was carried out along a stretch of 26 residues that include transmembrane segment 1 (TMS 1). 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. TMSI 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 TMSI, 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available