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Structure of the EmrE multidrug transporter and its use for inhibitor peptide design

Significance Bacterial pathogens are developing resistance to antibiotic compounds at an alarming rate. We use computer simulations to design inhibitors of the Escherichia coli multidrug resistance protein EmrE (efflux-multidrug resistance E) from the small multidrug family. Starting with low-resolution X-ray data, we obtain an atomic structure of EmrE using extensive molecular simulations. Based on the structure, we design hydrocarbon-stapled peptide inhibitors of EmrE, which are synthesized and shown to be effective in vivo. The rational drug design approach described here holds promise for combating efflux-mediated drug resistance in microbes and, more generally, in cancer. 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 Escherichia coli efflux-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 the EmrE active site occupied by two different ligands and in the absence of a ligand, illustrating how EmrE 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 EmrE dimerization. Guided by the atomic details of this interface, we design proteolysis-resistant stapled peptides that bind to helix H4 of an EmrE 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 EmrE. 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.