Antiparallel EmrE exports drugs by exchanging between asymmetric structures

Abstract

Small multidrug resistance (SMR) transporters provide an ideal system to study the minimal requirements for active transport. EmrE is an E. coli SMR transporter that exports a broad class of polyaromatic cation substrates, thus conferring resistance to drug compounds matching this chemical description. However, a great deal of controversy has surrounded the topology of the EmrE homodimer. Here we show that asymmetric antiparallel EmrE exchanges between inward- and outward-facing states that are identical except that they have opposite orientation in the membrane. We quantitatively measure the global conformational exchange between these two states for substrate-bound EmrE in bicelles using solution NMR dynamics experiments. FRET reveals that the monomers within each dimer are antiparallel, and paramagnetic relaxation enhancement NMR experiments demonstrate differential water accessibility of the two monomers within each dimer. Our experiments reveal a “dynamic symmetry” that reconciles the asymmetric EmrE structure with the functional symmetry of residues in the active site.

Small Multidrug Resistance Protein EmrE Reduces Host pH and Osmotic Tolerance to Metabolic Quaternary Cation Osmoprotectants

“…These transporters are present on a variety of mobile genetic elements, primarily in the 3= conserved regions of class 1 to 3 integrons and multidrug resistance plasmids, in addition to the host chromosome (as reviewed in references 4 and 29). In general, members of the SMR family form a 4-TM ␣-helix monomer (as reviewed in reference 4) within the plasma membrane that functions as a homo-oligomer, where the minimum oligomeric subunit is a homodimer (23). SMR transport activity and ligand binding are known to involve a highly conserved (98%) (5) active site Glu residue within the first TM strand, and mutations to this residue eliminate host resistance to QCCs (16,24,37).…”

Asymmetric perturbations of signalling oligomers

“…An elegant example of this is a study of the homodimeric multidrug efflux protein EmrE, where bulk and single-molecule FRET measurements were used as additional evidence for an asymmetric antiparallel arrangement of the two subunits (Morrison et al, 2012). Single-molecule FRET in general has proven to be a highly valuable biophysical tool for distinguishing between asymmetric and symmetric protein complexes (Takei et al, 2012) and revealing directionality in macromolecular interactions (Cristóvao et al, 2012).…”

“…Additional NMR techniques are available to address motions in the intermediate and slow motional regime (Kleckner and Foster, 2011). Among them, ZZ-exchange has played a key role recently in a structural and dynamic investigation of the multidrug efflux transporter, EmrE, confirming an asymmetric antiparallel arrangement with two exchanging subunits (Morrison, 2012).…”

“…The structure and topology of the homodimeric carrier protein remained controversial (Ninio et al, 2004;McHaourab et al, 2008;Steiner-Mordoch et al, 2008;Korkhov et al, 2009;Henzler-Wildman, 2012) until a joint NMR and single-molecule FRET investigation has recently revealed an asymmetric antiparallel arrangement with inward-and outward-facing subunits related by a pseudo-twofold symmetry axis parallel to the membrane plane (Morrison et al, 2012). Furthermore, solid state NMR and cryo-EM have demonstrated asymmetry at the ligand-binding pocket (Lehner et al, 2008), whereas spin labelling and EPR have revealed asymmetry in the TM helices .…”

Drug Efflux by a Small Multidrug Resistance Protein Is Inhibited by a Transmembrane Peptide

“…The minimal functional unit of SMRs has been characterized as a dimer, although higherorder oligomerization has also been proposed (4,9,33,36,37,39,40,42). EmrE from E. coli is the most extensively studied SMR family member and has been characterized by both crystallographic and nuclear magnetic resonance (NMR) methods as an antiparallel homodimer bound to the substrate tetraphenylphosphonium (6,17,21). This model shows that TM helices 1 (TM1) through 3 of each monomer surround the substrate, forming a six-helix binding pocket within the membrane bilayer, and utilize the conserved negatively charged residue Glu14 in TM1 to coordinate the protons and/or cationic portions of substrates (6,23,36).…”