Conformational exchange in the mechanism of multidrug efflux by EmrE (470.3)

Abstract

EmrE is a small multidrug resistance efflux pump found in the inner membrane of E. coli. As a secondary active transporter, it imports two protons down their electrochemical gradient to drive the export of one polyaromatic cation substrate. By pumping polyaromatic cations out of the cell, it thus confers resistance to this broad class of substrates and contributes to antibiotic resistance.As a multidrug transporter, EmrE transports a broad range of substrates with widely varying affinities. How does such a small transporter with a single small active site bind and transport this broad array of ligands? We have studied a series of tetrahedral compounds, as well as several planar substrates, to examine multidrug recognition and transport by EmrE. Using ZZ‐exchange NMR spectroscopy and lineshape analysis we find that the rate of conformational exchange between inward‐ and outward‐facing states varies over several orders of magnitude even within this limited set of substrates. Thus, the identity of the bound ligand controls the rate of this critical step in the transport process. The binding affinity also varies over a similar range and our data indicate that ligand off‐rate and conformational exchange rate are of similar magnitude for most ligands, indicating both processes may influence the rate of overall drug efflux. Our results suggest that tight binding ligands may stabilize the ground state sufficiently to block conformational exchange, and thus efflux. Assay in E. coli support this hypothesis, demonstrating that tight binding substrates reduce the rate of ethidium efflux by EmrE in its native environment.