Structural Dynamics of the Small Multidrug Transporter EMRE

Abstract

The small multidrug transporter from E. coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient (1-2). While the principal elements of the antiport mechanism have been revealed (3), the structural record is limited to the crystal structure of the substrate-bound state (4). Here we utilize spin labeling and Q-band Double Electron Electron Resonance (DEER; 5) to uncover the conformational changes that are subsequent to protonation of critical acidic residues, a central and missing link in the structure/mechanism relationship. We find that protonation of E14 leads to extensive helix rotation and tilt in conjunction with repacking of loops, conformational changes which alter the orientation of side chains that coordinate substrate binding and modulate substrate access from the lipid bilayer. The 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 isoenergetic alternating access between inward- and outward facing conformations of the substrate-loaded transporter (6), thus enabling antiport without dissipating the proton gradient.(1) Schuldiner S (2009) Biochim Biophys Acta 1794(5): 748-762.(2) Amadi ST, Koteiche HA, Mishra S, Mchaourab HS (2010) J Biol Chem 285(34): 26710-26718.(3) Yerushalmi H, Schuldiner S (2000) FEBS Lett 476(1-2): 93-97.(4) Chen YJ, et al. (2007) Proc Natl Acad Sci USA 104(48): 18999-19004.(5) Jeschke G (2012) Annu Rev Phys Chem 63: 419-446.(6) Morrison EA, et al. (2012) Nature 481(7379): 45-50.