18-Crown-6-ether Utilizes Distinct Allosteric Interactions to Uncouple Transport by the Multidrug Efflux Pump EmrE

The recent discovery that the model multidrug efflux pump from <i>Escherichia coli</i>, EmrE, can perform multiple types of transport suggests that this may be a compelling target for therapeutic intervention. Initial studies have identified several small-molecule substrates capable of inducing transporter-dependent susceptibility rather than the well-known antibiotic resistance phenotype. However, many questions regarding the underlying mechanism and regulation of this transporter still remain. Prior studies identified lysine 22 as well as threonine 56 as important residues for regulating the formation of an occluded state critical to the prevention of an uncoupled leak in the WT transporter. Here, we use NMR chemical shift perturbations and <i>in vivo</i> EC₅₀ assays to confirm that 18-crown-6-ether binds at lysine 22, while liposomal leak assays verify that this substrate triggers uncoupled proton leak. In addition to characterizing the mechanism of action of another susceptibility substrate for EmrE, the characterization of K22 mutants herein solidifies the importance of this residue, as well as the nearby residue T56, in the allosteric regulation of the C-terminal tail. With a high degree of familial conservation in addition to a suggested role in transporter evolution, mechanistic insight into the transport regulation of EmrE may be broadly applicable across small multidrug-resistant efflux pumps.