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

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 H-N-F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphonium (F-4-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. EmrE transporter effluxes cationic substrates across lipid membranes in a pH-coupled manner. Here, the authors solve the structure of ligand-bound EmrE at high pH by NMR, with insights into the transport mechanism.

Automated summary

The structure of EmrE protein in different pH conditions has been solved using solid-state NMR, providing insights into the alternating-access model of EmrE. The study reveals that the conformation of EmrE at high pH exposes the substrate to both sides of the membrane, while the conformation at low pH asymmetically exposes the substrate to one side. These findings are crucial for understanding the substrate transport mechanism.