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A membrane‐embedded glutamate is required for ligand binding to the multidrug transporter EmrE
Author(s) -
Muth T.R.,
Schuldiner S.
Publication year - 2000
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/19.2.234
Subject(s) - biology , ligand (biochemistry) , binding site , trimer , atp binding cassette transporter , biophysics , transporter , membrane , transport protein , plasma protein binding , stereochemistry , integral membrane protein , membrane protein , biochemistry , dimer , chemistry , receptor , organic chemistry , gene
EmrE is an Escherichia coli multidrug transporter that confers resistance to a variety of toxins by removing them in exchange for hydrogen ions. The detergent‐solubilized protein binds tetraphenylphosphonium (TPP + ) with a K D of 10 nM. One mole of ligand is bound per ∼3 mol of EmrE, suggesting that there is one binding site per trimer. The steep pH dependence of binding suggests that one or more residues, with an apparent p K of ∼7.5, release protons prior to ligand binding. A conservative Asp replacement (E14D) at position 14 of the only membrane‐embedded charged residue shows little transport activity, but binds TPP + at levels similar to those of the wild‐type protein. The apparent p K of the Asp shifts to <5.0. The data are consistent with a mechanism requiring Glu14 for both substrate and proton recognition. We propose a model in which two of the three Glu14s in the postulated trimeric EmrE homooligomer deprotonate upon ligand binding. The ligand is released on the other face of the membrane after binding of protons to Glu14.