A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE

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