title: Blocking dynamics of the SMR transporter EmrE impairs efflux activity.
abstract: EmrE is a small multidrug resistance transporter that has been well studied as a model for secondary active transport. Because transport requires the protein to convert between at least two states open to opposite sides of the membrane, it is expected that blocking these conformational transitions will prevent transport activity. We have previously shown that NMR can quantitatively measure the transition between the open-in and open-out states of EmrE in bicelles. Now, we have used the antiparallel EmrE crystal structure to design a cross-link to inhibit this conformational exchange process. We probed the structural, dynamic, and functional effects of this cross-link with NMR and in vivo efflux assays. Our NMR results show that our antiparallel cross-link performs as predicted: dramatically reducing conformational exchange while minimally perturbing the overall structure of EmrE and essentially trapping EmrE in a single state. The same cross-link also impairs ethidium efflux activity by EmrE in Escherichia coli. This confirms the hypothesis that transport can be inhibited simply by blocking conformational transitions in a properly folded transporter. The success of our cross-linker design also provides further evidence that the antiparallel crystal structure provides a good model for functional EmrE.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Supratik
authors.initials: S
authors.lastname: Dutta
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Emma A
authors.initials: EA
authors.lastname: Morrison
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri. Electronic address: khenzler@biochem.wustl.edu.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Henzler-Wildman
mesh_terms: D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism; D001692:Biological Transport; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry / Q000494:pharmacology; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; C075436:EmrE protein, E coli; D004996:Ethidium
references.citation: J Mol Biol. 2006 Nov 17;364(1):54-67
references.pmid: 17005200
references.citation: J Biomol NMR. 1994 Sep;4(5):603-14
references.pmid: 22911360
references.citation: Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15141-6
references.pmid: 21876140
references.citation: J Biol Chem. 2008 Feb 8;283(6):3281-3288
references.pmid: 18042544
references.citation: J Biomol NMR. 1995 Nov;6(3):277-93
references.pmid: 8520220
references.citation: J Mol Biol. 2008 Apr 4;377(4):1094-103
references.pmid: 18295794
references.citation: Nature. 1966 Aug 27;211(5052):969-70
references.pmid: 5968307
references.citation: J Biol Chem. 2010 Aug 20;285(34):26710-8
references.pmid: 20551331
references.citation: J Am Chem Soc. 2014 Jun 4;136(22):8072-80
references.pmid: 24856154
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20659-65
references.pmid: 18093952
references.citation: Biol Chem. 2013 May;394(5):641-8
references.pmid: 23362203
references.citation: J Biol Chem. 2010 May 14;285(20):15234-15244
references.pmid: 20308069
references.citation: J Biol Chem. 2009 Dec 4;284(49):33807-14
references.pmid: 19837674
references.citation: J Mol Biol. 2013 Nov 15;425(22):4642-51
references.pmid: 23920359
references.citation: Methods Mol Biol. 2010;634:343-54
references.pmid: 20676995
references.citation: J Am Chem Soc. 2013 Oct 23;135(42):15754-62
references.pmid: 24047229
references.citation: J Biol Chem. 2012 Jul 27;287(31):26052-9
references.pmid: 22700980
references.citation: J Biomol NMR. 2009 Dec;45(4):357-60
references.pmid: 19890725
references.citation: J Biochem. 2007 Nov;142(5):621-5
references.pmid: 18006522
references.citation: Nature. 2014 Jan 23;505(7484):569-73
references.pmid: 24317697
references.citation: Biochim Biophys Acta. 2012 Mar;1818(3):814-20
references.pmid: 22226849
references.citation: Biochemistry. 1984 Jan 3;23(1):166-76
references.pmid: 6197994
references.citation: Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):18999-9004
references.pmid: 18024586
references.citation: Nature. 2011 Dec 18;481(7379):45-50
references.pmid: 22178925
references.citation: J Biol Chem. 2012 Dec 28;287(53):44036-45
references.pmid: 23150661
references.citation: Biochim Biophys Acta. 2007 Dec;1768(12):3036-43
references.pmid: 17976529
references.citation: Biochim Biophys Acta. 2011 Feb;1807(2):167-88
references.pmid: 21029721
references.citation: J Biol Chem. 1995 Mar 24;270(12):6856-63
references.pmid: 7896833
references.citation: EMBO J. 2003 Dec 1;22(23):6175-81
references.pmid: 14633977
references.citation: Nature. 2013 Jul 4;499(7456):107-10
references.pmid: 23685453
references.citation: Curr Opin Struct Biol. 2008 Dec;18(6):726-33
references.pmid: 18948194
references.citation: Angew Chem Int Ed Engl. 2013 Sep 23;52(39):10321-4
references.pmid: 23939862
references.citation: J Biol Chem. 2014 Mar 7;289(10):6825-6836
references.pmid: 24448799