Structure of the <i>EmrE</i> multidrug transporter and its use for inhibitor peptide design.

Abstract

Small multidrug resistance (SMR) pumps represent a minimal paradigm of proton-coupled membrane transport in bacteria, yet no high-resolution structure of an SMR protein is available. Here, atomic-resolution structures of the <i>Escherichia coli</i> efflux-multidrug resistance E (<i>EmrE</i>) multidrug transporter in ligand-bound form are refined using microsecond molecular dynamics simulations biased using low-resolution data from X-ray crystallography. The structures are compatible with existing mutagenesis data as well as NMR and biochemical experiments, including pKas of the catalytic glutamate residues and the dissociation constant ([Formula: see text]) of the tetraphenylphosphonium⁺ cation. The refined structures show the arrangement of residue side chains in the <i>EmrE</i> active site occupied by two different ligands and in the absence of a ligand, illustrating how <i>EmrE</i> can adopt structurally diverse active site configurations. The structures also show a stable, well-packed binding interface between the helices H4 of the two monomers, which is believed to be crucial for <i>EmrE</i> dimerization. Guided by the atomic details of this interface, we design proteolysis-resistant stapled peptides that bind to helix H4 of an <i>EmrE</i> monomer. The peptides are expected to interfere with the dimerization and thereby inhibit drug transport. Optimal positions of the peptide staple were determined using free-energy simulations of peptide binding to monomeric <i>EmrE</i> Three of the four top-scoring peptides selected for experimental testing resulted in significant inhibition of proton-driven ethidium efflux in live cells without nonspecific toxicity. The approach described here is expected to be of general use for the design of peptide therapeutics.