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

Alexander A Shcherbakov,Aurelio J Dregni,Mei Hong,Katherine A Henzler-Wildman,Peyton J Spreacker

doi:10.1038/s41467-022-28556-6

Alexander A Shcherbakov, Aurelio J Dregni + Show 3 more

Open Access

https://doi.org/10.1038/s41467-022-28556-6

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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.

Highlights

The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance
We previously carried out extensive pH titrations of TPP+-bound EmrE in bicelles using solution NMR and found that the WT transporter had a single pKa of 6.8 ± 0.117, while S64V-EmrE had a single pKa of 7.0 ± 0.17
We prepared a sample of S64V-EmrE bound to F4-TPP+ in DMPC bilayers at pH 8.0 to determine the structure of the unprotonated complex, to compare with the protonated complex previously determined at pH 5.8 in DMPC bilayers

Summary

Introduction

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 Glu[14] residues is protonated. 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, accelerating the conformational change of EmrE to export the substrate. Elucidating the mechanism of membrane transport by EmrE requires atomicresolution structural information for multiple states of the protein, as well as dynamics information about the protein and the ligands throughout the transport cycle

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