title: Intrinsic conformational plasticity of native EmrE provides a pathway for multidrug resistance.
abstract: EmrE is a multidrug resistance efflux pump with specificity to a wide range of antibiotics and antiseptics. To obtain atomic-scale insight into the attributes of the native state that encodes the broad specificity, we used a hybrid of solution and solid-state NMR methods in lipid bilayers and bicelles. Our results indicate that the native EmrE dimer oscillates between inward and outward facing structural conformations at an exchange rate (k(ex)) of ~300 s(-1) at 37 °C (millisecond motions), which is ~50-fold faster relative to the tetraphenylphosphonium (TPP(+)) substrate-bound form of the protein. These observables provide quantitative evidence that the rate-limiting step in the TPP(+) transport cycle is not the outward-inward conformational change in the absence of drug. In addition, using differential scanning calorimetry, we found that the width of the gel-to-liquid crystalline phase transition was 2 °C broader in the absence of the TPP(+) substrate versus its presence, which suggested that changes in transporter dynamics can impact the phase properties of the membrane. Interestingly, experiments with cross-linked EmrE showed that the millisecond inward-open to outward-open dynamics was not the culprit of the broadening. Instead, the calorimetry and NMR data supported the conclusion that faster time scale structural dynamics (nanosecond-microsecond) were the source and therefore impart the conformationally plastic character of native EmrE capable of binding structurally diverse substrates. These findings provide a clear example how differences in membrane protein transporter structural dynamics between drug-free and bound states can have a direct impact on the physical properties of the lipid bilayer in an allosteric fashion.
authors.affiliation: Department of Chemistry, New York University , New York, New York 10003, United States.
authors.forename: Min-Kyu
authors.initials: MK
authors.lastname: Cho
authors.forename: Anindita
authors.initials: A
authors.lastname: Gayen
authors.forename: James R
authors.initials: JR
authors.lastname: Banigan
authors.forename: Maureen
authors.initials: M
authors.lastname: Leninger
authors.forename: Nathaniel J
authors.initials: NJ
authors.lastname: Traaseth
mesh_terms: D017920:Antiporters / Q000737:chemistry* / Q000235:genetics*; D003432:Cross-Linking Reagents; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics*; D007202:Indicators and Reagents; D008051:Lipid Bilayers; D009682:Magnetic Resonance Spectroscopy; D009861:Onium Compounds / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D011487:Protein Conformation
chemical_list: D017920:Antiporters; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D008051:Lipid Bilayers; D009861:Onium Compounds; D009943:Organophosphorus Compounds; C075436:EmrE protein, E coli; C013289:tetraphenylphosphonium