Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using F-19 and H-1 solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F-4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F-4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance. The small proton-coupled transporter EmrE confers multidrug resistance in bacteria. The structure of drug-bound EmrE in phospholipid bilayers is now determined using solid-state NMR. The structure provides detailed insights into the molecular mechanism of substrate recognition by this transporter.