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

EmrE is an Escherichia coli multidrug transporter that confers resistance t o a variety of toxins by removing them in exchange for hydrogen ions. The d etergent-solubilized protein binds tetraphenylphosphonium (TPP+) with a K-D of 10 nM. One mole of ligand is bound per similar to 3 mol of EmrE, sugges ting that there is one binding site per trimer. The steep pH dependence of binding suggests that one or more residues, with an apparent pK of similar to 7.5, release protons prior to ligand binding. A conservative Asp replace ment (E14D) at position 14 of the only membrane-embedded charged residue sh ows little transport activity, but binds TPP+ at levels similar to those of the wild-type protein. The apparent pK of the Asp shifts to <5.0. The data are consistent with a mechanism requiring Glu14 for both substrate and pro ton 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 prot ons to Glu14.