Structure of theEmrEmultidrug 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 theEscherichia coliefflux-multidrug resistance E (EmrE) 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 (KD) of the tetraphenylphosphonium+cation. The refined structures show the arrangement of residue side chains in theEmrEactive site occupied by two different ligands and in the absence of a ligand, illustrating howEmrEcan 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 forEmrEdimerization. Guided by the atomic details of this interface, we design proteolysis-resistant stapled peptides that bind to helix H4 of anEmrEmonomer. 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 monomericEmrE. 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.