title: Protonation-dependent conformational dynamics of the multidrug transporter EmrE.
abstract: The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1-3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232;
authors.forename: Reza
authors.initials: R
authors.lastname: Dastvan
authors.affiliation: Department of Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN 37232.
authors.forename: Axel W
authors.identifier: http://orcid.org/0000-0003-0237-7365
authors.initials: AW
authors.lastname: Fischer
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232;
authors.forename: Smriti
authors.initials: S
authors.lastname: Mishra
authors.affiliation: Department of Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN 37232.
authors.forename: Jens
authors.initials: J
authors.lastname: Meiler
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232; hassane.mchaourab@vanderbilt.edu.
authors.forename: Hassane S
authors.initials: HS
authors.lastname: Mchaourab
mesh_terms: D017920:Antiporters / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D011522:Protons; D014961:X-Ray Diffraction
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; D011522:Protons; C075436:EmrE protein, E coli
keywords: DEER; EPR; EmrE; SMR; multidrug transport