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

Abstract

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 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-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. F4-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.

Molecular mechanism of proton-coupled ligand translocation by the bacterial efflux pump EmrE

“…Simultaneously, backbone H-bonding in a region of transmembrane helix 3 (TM3) stretching from G65 A to I71 A undergoes rearrangement due to formation of a kink in the exceptionally elastic 65GVG67 region. This kinking was indeed deemed important for ligand binding and the rate of conformational change, as concluded from past mutagenesis, NMR, EPR and cryo-EM studies [26,44,45,46], and might provide an easy way of storing excess free energy for the subsequent ligand release. Finally, minor rearrangements occur at the entry site -S75 B switching from an intermolecular H-bond with S105 A to an intramolecular one with the backbone of a neighboring I75 B -reflecting a more open conformation of the binding channel in the bound than in the apo state.…”

A combination of solid-state NMR and MD simulations reveals the binding mode of a rhomboid protease inhibitor

“…So far, there are few examples where the power of solid-state NMR has been demonstrated in determining the exact binding mode of drug-like molecules with membrane proteins. 41,42 Especially, we could show in this study that H/D exchange patterns enable mapping of the interaction site, while CSPs give information on both binding interactions and allosteric effects. Furthermore, it is oen difficult to obtain crystal structures of small molecules bound to membrane embedded proteins and we have shown here that solid-state NMR experiments of GlpG-inhibitor complexes are straight-forward for both reversible (SBO) and irreversible (JLK6) inhibitors.…”

“…The combination of solid-state NMR and MD simulations constitutes a promising approach to study substrate binding to membrane proteins. 41,42 We employed this combined strategy here to investigate the structural and dynamic effects caused by inhibitor binding to a membrane-embedded enzyme on the atomic level. There are many examples of membrane proteins with excellent solid-state NMR spectral features and for those systems the applied approach should be similarly useful.…”

Activating alternative transport modes in a multidrug resistance efflux pump to confer chemical susceptibility

“…1). To better understand how EmrE confers susceptibility to harmane, we turned to a solid supported membrane electrophysiology (SSME) assay recently developed in our lab to characterize the ion-coupling behavior of secondary active transporters (Extended Data Figure 3) 23,24 . SSME allows the detection of net charge movement in proteoliposomes adsorbed onto a gold electrode sensor upon buffer perfusion and is ideal for measuring small transport currents produced by moderate-flux transporters such as EmrE 25 .…”