High-pH structure of EmrE reveals the mechanism of proton-coupled substrate transport

Abstract

The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand HN–F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphonium (F4-TPP+), using solid-state NMR. This structure was solved at low pH where one of the two proton-binding Glu14 residues is protonated. Here, to understand how substrate transport depends on pH, we determine the structure of the EmrE-TPP complex at high pH, where both Glu14 residues are deprotonated. The high-pH complex exhibits an elongated and hydrated binding pocket in which the substrate is similarly exposed to the two sides of the membrane. In contrast, the low-pH complex asymmetrically exposes the substrate to one side of the membrane. These pH-dependent EmrE conformations provide detailed insights into the alternating-access model, and suggest that the high-pH conformation may facilitate proton binding in the presence of the substrate, thus accelerating the conformational change of EmrE to export the substrate.

Charge neutralization of the active site glutamates does not limit substrate binding and transport by EmrE

“…The ability of this binding site to accommodate a net positive charge suggests that +3 drug-substrates also have the potential to bind and transport of such molecules should be tested in the future. This result is perhaps not entirely unexpected given recent crystal structures that show the plasticity of the EmrE binding pocket bound to different substrates (19) and NMR studies confirm the dynamic behavior of the substrate within the binding pocket (11). Indeed the NMR data we have reported previously for +1 substrates (8) and here for +2 substrates (Fig.…”

“…This line broadening is likely due to the motion of the planar PP 2+ causing fluctuating ring currents. TPP + is highly dynamic even when bound tightly and asymmetrically within the transport pore (5, 11, 12), but the symmetric nature of this substrate limits the impact of this motion on the observed NMR resonances. Despite these challenges, pH-dependent CSPs of PP 2+ -bound EmrE (Fig.…”

Solid-State NMR 19F–1H–15N Correlation Experiments for Resonance Assignment and Distance Measurements of Multifluorinated Proteins

“…The 19 F DP spectrum of F 4 -TPP + bound to EmrE in the lipid membrane shows four peaks at isotropic chemical shifts of −96.5, −98.8, −100.6, and −101.4 ppm. This distribution indicates that the four chemically equivalent fluorines of the ligand are magnetically inequivalent due to their interactions with different protein side chains …”

“…EmrE is a dimeric membrane protein that effluxes polyaromatic cationic substrates across the inner membrane of Gram-negative bacteria to cause multidrug resistance. − Substrate export against the concentration gradient is driven by coupling to proton import from the acidic periplasm to the neutral cytoplasm. Using 19 F– 1 H REDOR NMR, we recently determined two high-resolution structures of EmrE bound to a tetrafluorinated substrate, F 4 -TPP + . , The two structures were solved at pH 5.8 and pH 8.0 to understand how the protonation state of the proton-selective residue E14 affects the substrate-bound structures of the protein.…”

“…CDN-labeled S64 V-EmrE was expressed and purified as described previously . The protein was bound to DMPC- d 54 (DMPC = dimyristoylphosphatidylcholine) bilayers at pH 8.0.…”