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Symmetrically Asymmetric: EmrE Seen from the NMR Perspective

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Membrane Transport Mechanism

Part of the book series: Springer Series in Biophysics ((BIOPHYSICS,volume 17))

Abstract

Secondary active transporters couple substrate with ion translocation across the membrane. The actual transport mechanism must involve a number of distinct steps with alternatingly accessible substrate/ion binding sites and potentially occurring occluded states. To understand their functional mechanism, it is necessary to link transport kinetics to conformational dynamics. Furthermore, various biophysical studies indicate high conformational flexibility in secondary transporters, which raises the question whether transport requires switching between well-defined conformational states or is merely based on shifted conformational equilibriums. Addressing this question requires biophysical approaches sensitive to different timescales and length scales such as solid-state NMR, liquid-state NMR, EPR, optical- and infrared spectroscopy in close combination with mutagenesis-based functional studies. In this chapter, we demonstrate and discuss for the case of EmrE, how liquid- and solid-state NMR spectroscopy can contribute to resolving the mechanism of secondary transporters. EmrE, a homo-dimeric multidrug antiporter from the SMR family, has been suggested to offer a convenient paradigm for secondary transporters due to its small size. It has attracted almost all available NMR methods making it a good case for demonstrating advanced methodology by which fundamental properties of secondary transporters can be obtained.

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Abbreviations

CCCP:

Carbonyl cyanide 3-chlorophenylhydrazone

CODEX:

Centerband only detection of exchange

CP:

Cross polarisation

CSA:

Chemical shift anisotropy

DMPC:

1,2-dimyristoyl-sn-glycero-3-phosphocholine

DHPC:

1,2-dihexanoyl-sn-glycero-3-phosphocholine

DNP:

Dynamic nuclear polarisation

EM:

Electron microscopy

EPR:

Electron paramagnetic resonance

FRET:

Förster resonance energy transfer

GPCR:

G-protein coupled receptor

MAS:

Magic angle spinning

MTP:

Methyltriphenylphosphonium

NMR:

Nuclear magnetic resonance

NOE:

Nuclear Overhauser effect

O-SSNMR:

Oriented solid-state NMR

PISA wheel:

Polarity index slant angle

PISEMA:

Polarisation inversion spin exchange at magic angle

SMR protein:

Small multidrug resistance protein

TMH:

Transmembrane helix

TPP:

Tetraphenylphosphonium

TROSY:

Transverse relaxation optimised spectroscopy

References

  • Adam Y, Tayer N, Rotem D, Schreiber G, Schuldiner S (2007) The fast release of sticky protons: kinetics of substrate binding and proton release in a multidrug transporter. Proc Natl Acad Sci U S A 104:17989–17994

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Agarwal V, Fink U, Schuldiner S, Reif B (2007) MAS solid-state NMR studies on the multidrug transporter EmrE. Biochim Biophys Acta Biomembr 1768:3036–3043

    Article  CAS  Google Scholar 

  • Amadi ST, Koteiche HA, Mishra S, Mchaourab HS (2010) Structure, dynamics, and substrate-induced conformational changes of the multidrug transporter EmrE in liposomes. J Biol Chem 285:26710–26718

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bak M, Rasmussen JT, Nielsen NC (2000) SIMPSON: a general simulation program for solid-state NMR spectroscopy. J Magn Reson 147:296–330

    Article  CAS  PubMed  Google Scholar 

  • Bak M, Schultz R, Vosegaard T, Nielsen NC (2002) Specification and visualization of anisotropic interaction tensors in polypeptides and numerical simulations in biological solid-state NMR. J Magn Reson 154:28–45

    Article  CAS  PubMed  Google Scholar 

  • Banigan JR, Gayen A, Traaseth NJ (2013) Combination of N-15 reverse labeling and afterglow spectroscopy for assigning membrane protein spectra by magic-angle-spinning solid-state NMR: application to the multidrug resistance protein EmrE. J Biomol NMR 55:391–399

    Article  CAS  PubMed  Google Scholar 

  • Basting D, Lorch M, Lehner I, Glaubitz C (2008) Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence. FASEB J 22:365–373

    Article  CAS  PubMed  Google Scholar 

  • Bayrhuber M, Meins T, Habeck M, Becker S, Giller K, Villinger S, Vonrhein C, Griesinger C, Zweckstetter M, Zeth K (2008) Structure of the human voltage-dependent anion channel. Proc Natl Acad Sci U S A 105:15370–15375

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Castellani F, van Rossum B, Diehl A, Schubert M, Rehbein K, Oschkinat H (2002) Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy. Nature 420:98–102

    Article  CAS  PubMed  Google Scholar 

  • Chen YJ, Pornillos O, Lieu S, Ma C, Chen AP, Chang G (2007) X-ray structure of EmrE supports dual topology model. Proc Natl Acad Sci U S A 104:18999–19004

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • De Angelis AA, Opella SJ (2007) Bicelle samples for solid-state NMR of membrane proteins. Nat Protoc 2:2332–2338

    Article  PubMed  Google Scholar 

  • deAzevedo ER, Hu WG, Bonagamba TJ, Schmidt-Rohr K (2000) Principles of centerband-only detection of exchange in solid-state nuclear magnetic resonance, and extension to four-time centerband-only detection of exchange. J Chem Phys 112:8988–9001

    Article  CAS  Google Scholar 

  • Fleishman SJ, Harrington SE, Enosh A, Halperin D, Tate CG, Ben-Tal N (2006) Quasi-symmetry in the cryo-EM structure of EmrE provides the key to modeling its transmembrane domain. J Mol Biol 364:54–67

    Article  CAS  PubMed  Google Scholar 

  • Franks WT, Wylie BJ, Schmidt HLF, Nieuwkoop AJ, Mayrhofer R-M, Shah GJ, Graesser DT, Rienstra CM (2008) Dipole tensor-based atomic-resolution structure determination of a nanocrystalline protein by solid-state NMR. Proc Natl Acad Sci U S A 105:4621–4626

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gautier A, Mott HR, Bostock MJ, Kirkpatrick JP, Nietlispach D (2010) Structure determination of the seven-helix transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy. Nat Struct Mol Biol 17:768–U147

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gayen A, Banigan JR, Traaseth NJ (2013) Ligand-induced conformational changes of the multidrug resistance transporter EmrE probed by oriented solid-state NMR spectroscopy. Angew Chem Int Ed Engl 52(39):10321–10324

    Article  CAS  PubMed  Google Scholar 

  • Glover KJ, Whiles JA, Wu GH, Yu NJ, Deems R, Struppe JO, Stark RE, Komives EA, Vold RR (2001) Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules. Biophys J 81:2163–2171

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hellmich UA, Glaubitz C (2009) NMR and EPR studies of membrane transporters. Biol Chem 390:815–834

    Article  CAS  PubMed  Google Scholar 

  • Hong M, Zhang Y, Hu F (2012) Membrane protein structure and dynamics from NMR spectroscopy. In: Johnson MA, Martinez TJ (eds) Annual review of physical chemistry, vol 63. Annual Reviews, Palo Alto, CA, pp 1–24

    Google Scholar 

  • Lakatos A, Mors K, Glaubitz C (2012) How to investigate interactions between membrane proteins and ligands by solid-state NMR. Meth Mol Biol 914:65–86

    CAS  Google Scholar 

  • Lange A, Giller K, Hornig S, Martin-Eauclaire MF, Pongs O, Becker S, Baldus M (2006) Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR. Nature 440:959–962

    Article  CAS  PubMed  Google Scholar 

  • Lehner I, Basting D, Meyer B, Haase W, Manolikas T, Kaiser C, Karas M, Glaubitz C (2008) The key residue for substrate transport (Glu(14)) in the EmrE dimer is asymmetric. J Biol Chem 283:3281–3288

    Article  CAS  PubMed  Google Scholar 

  • Li Y, Palmer AG III (2009) TROSY-selected ZZ-exchange experiment for characterizing slow chemical exchange in large proteins. J Biomol NMR 45:357–360

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lloris-Garcera P, Bianchi F, Slusky JS, Seppala S, Daley DO, von Heijne G (2012) Antiparallel dimers of the small multidrug resistance protein EmrE are more stable than parallel dimers. J Biol Chem 287:26052–26059

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lorch M, Lehner I, Siarheyeva A, Basting D, Pfleger N, Manolikas T, Glaubitz C (2005) NMR and fluorescence spectroscopy approaches to secondary and primary active multidrug efflux pumps. Biochem Soc Trans 33:873–877

    Article  CAS  PubMed  Google Scholar 

  • Maly T, Debelouchina GT, Bajaj VS, Hu KN, Joo CG, Mak-Jurkauskas ML, Sirigiri JR, van der Wel PCA, Herzfeld J, Temkin RJ, Griffin RG (2008) Dynamic nuclear polarization at high magnetic fields. J Chem Phys 128:052211–052219

    Article  PubMed Central  PubMed  Google Scholar 

  • Marassi FM, Opella SJ (2000) A solid-state NMR index of helical membrane protein structure and topology. J Magn Reson 144:150–155

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Miller D, Charalambous K, Rotem D, Schuldiner S, Curnow P, Booth PJ (2009) In vitro unfolding and refolding of the small multidrug transporter EmrE. J Mol Biol 393:815–832

    Article  CAS  PubMed  Google Scholar 

  • Morrison EA, Henzler-Wildman KA (2012) Reconstitution of integral membrane proteins into isotropic bicelles with improved sample stability and expanded lipid composition profile. Biochim Biophys Acta Biomembr 1818:814–820

    Article  CAS  Google Scholar 

  • Morrison EA, DeKoster GT, Dutta S, Vafabakhsh R, Clarkson MW, Bahl A, Kern D, Ha T, Henzler-Wildman KA (2012) Antiparallel EmrE exports drugs by exchanging between asymmetric structures. Nature 481:45–U50

    Article  CAS  Google Scholar 

  • Mors K, Hellmich UA, Basting D, Marchand P, Wurm JP, Haase W, Glaubitz C (2013) A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein TBsmr. Biochim Biophys Acta 1828:561–567

    Article  PubMed  Google Scholar 

  • Muth TR, Schuldiner S (2000) A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE. EMBO J 19:234–240

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ong YS, Lakatos A, Becker-Baldus J, Pos KM, Glaubitz C (2013) Detecting substrates bound to the secondary multidrug efflux pump EmrE by DNP-enhanced solid-state NMR. J Am Chem Soc 135:15754–15762

    Article  CAS  PubMed  Google Scholar 

  • Park SH, Das BB, Casagrande F, Tian Y, Nothnagel HJ, Chu M, Kiefer H, Maier K, De Angelis AA, Marassi FM, Opella SJ (2012) Structure of the chemokine receptor CXCR1 in phospholipid bilayers. Nature 491:779–783

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Poget SF, Cahill SM, Girvin ME (2007) Isotropic bicelles stabilize the functional form of a small multidrug-resistance pump for NMR structural studies. J Am Chem Soc 129:2432–2433

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Prosser RS, Hunt SA, DiNatale JA, Vold RR (1996) Magnetically aligned membrane model systems with positive order parameter: Switching the sign of S-zz with paramagnetic ions. J Am Chem Soc 118:269–270

    Article  CAS  Google Scholar 

  • Rapp M, Granseth E, Seppala S, von Heijne G (2006) Identification and evolution of dual-topology membrane proteins. Nat Struct Mol Biol 13:112–116

    Article  CAS  PubMed  Google Scholar 

  • Raschle T, Hiller S, Etzkorn M, Wagner G (2010) Nonmicellar systems for solution NMR spectroscopy of membrane proteins. Curr Opin Struct Biol 20:471–479

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Reckel S, Gottstein D, Stehle J, Lohr F, Verhoefen MK, Takeda M, Silvers R, Kainosho M, Glaubitz C, Wachtveitl J, Bernhard F, Schwalbe H, Guntert P, Dotsch V (2011) Solution NMR structure of proteorhodopsin. Angew Chem Int Ed Engl 50:11942–11946

    Article  CAS  PubMed  Google Scholar 

  • Renault M, Cukkemane A, Baldus M (2010) Solid-state NMR spectroscopy on complex biomolecules. Angew Chem Int Ed 49:8346–8357

    Article  CAS  Google Scholar 

  • Rotem D, Schuldiner S (2004) EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry. J Biol Chem 279:48787–48793

    Article  CAS  PubMed  Google Scholar 

  • Rotem D, Sal-man N, Schuldiner S (2001) In vitro monomer swapping in EmrE, a multidrug transporter from Escherichia coli, reveals that the oligomer is the functional unit. J Biol Chem 276:48243–48249

    CAS  PubMed  Google Scholar 

  • Sauvee C, Rosay M, Casano G, Aussenac F, Weber RT, Ouari O, Tordo P (2013) Highly efficient, water-soluble polarizing agents for dynamic nuclear polarization at high frequency. Angew Chem Int Ed Engl 125(41):11058–11061

    Article  Google Scholar 

  • Schuldiner S (2007) When biochemistry meets structural biology: the cautionary tale of EmrE (vol 32, pg 252, 2007). Trends Biochem Sci 32:300

    Article  Google Scholar 

  • Schuldiner S, Lebendiker M, Yerushalmi H (1997) EmrE, the smallest ion-coupled transporter, provides a unique paradigm for structure-function studies. J Exp Biol 200:335–341

    CAS  PubMed  Google Scholar 

  • Schwaiger M, Lebendiker M, Yerushalmi H, Coles M, Groger A, Schwarz C, Schuldiner S, Kessler H (1998) NMR investigation of the multidrug transporter EmrE, an integral membrane protein. Eur J Biochem 254:610–619

    Article  CAS  PubMed  Google Scholar 

  • Shi L, Ahmed MA, Zhang W, Whited G, Brown LS, Ladizhansky V (2009) Three-dimensional solid-state NMR study of a seven-helical integral membrane proton pump–structural insights. J Mol Biol 386:1078–1093

    Article  CAS  PubMed  Google Scholar 

  • Song CS, Hu KN, Joo CG, Swager TM, Griffin RG (2006) TOTAPOL: A biradical polarizing agent for dynamic nuclear polarization experiments in aqueous media. J Am Chem Soc 128:11385–11390

    Article  CAS  PubMed  Google Scholar 

  • Spooner PJR, Rutherford NG, Watts A, Henderson PJF (1994) NMR observation of substrate in the binding-site of an active sugar-h + symport protein in native membranes. Proc Natl Acad Sci U S A 91:3877–3881

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Steiner-Mordoch S, Soskine M, Solomon D, Rotem D, Gold A, Yechieli M, Adam Y, Schuldiner S (2008) Parallel topology of genetically fused EmrE homodimers. EMBO J 27:17–26

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tate CG (2010) Practical considerations of membrane protein instability during purification and crystallisation. Meth Mol Biol 601:187–203

    Article  CAS  Google Scholar 

  • Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114

    Article  CAS  PubMed  Google Scholar 

  • Ubarretxena-Belandia I, Baldwin JM, Schuldiner S, Tate CG (2003) Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer. EMBO J 22:6175–6181

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Vold RR, Prosser RS, Deese AJ (1997) Isotropic solutions of phospholipid bicelles: A new membrane mimetic for high-resolution NMR studies of polypeptides. J Biomol NMR 9:329–335

    Article  CAS  PubMed  Google Scholar 

  • Wang J, Denny J, Tian C, Kim S, Mo Y, Kovacs F, Song Z, Nishimura K, Gan Z, Fu R, Quine JR, Cross TA (2000) Imaging membrane protein helical wheels. J Magn Reson 144:162–167

    Article  CAS  PubMed  Google Scholar 

  • Wang S, Munro RA, Shi L, Kawamura I, Okitsu T, Wada A, Kim SY, Jung KH, Brown LS, Ladizhansky V (2013) Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein. Nat Methods 10(10):1007–1012

    Article  PubMed  Google Scholar 

  • Wu CH, Ramamoorthy A, Opella SJ (1994) High-resolution heteronuclear dipolar solid-state NMR-spectroscopy. J Magn Reson A 109:270–272

    Article  Google Scholar 

  • Yerushalmi H, Lebendiker M, Schuldiner S (1996) Negative dominance studies demonstrate the oligomeric structure of EmrE, a multidrug antiporter from Escherichia coli. J Biol Chem 271:31044–31048

    Article  CAS  PubMed  Google Scholar 

  • Yerushalmi H, Mordoch SS, Schuldiner S (2001) A single carboxyl mutant of the multidrug transporter EmrE is fully functional. J Biol Chem 276:12744–12748

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Doherty T, Li J, Lu W, Barinka C, Lubkowski J, Hong M (2010) Resonance assignment and three-dimensional structure determination of a human alpha-Defensin, HNP-1, by solid-state NMR. J Mol Biol 397:408–422

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgements

We are grateful to Andrea Lakatos and Yean-Sin Ong for helpful discussions and comments to the manuscript.

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Authors and Affiliations

  1. Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, Goethe Universität Frankfurt, Frankfurt am Main, Germany

    Johanna Becker-Baldus & Clemens Glaubitz

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  1. Johanna Becker-Baldus
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  2. Clemens Glaubitz
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Correspondence to Clemens Glaubitz .

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Editors and Affiliations

  1. Department Chemistry Institute of Biochemistry, University of Cologne, Köln, Germany

    Reinhard Krämer

  2. Faculty for Biology and Preclinical Medicine, University of Regensburg, Regensburg, Germany

    Christine Ziegler

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Becker-Baldus, J., Glaubitz, C. (2014). Symmetrically Asymmetric: EmrE Seen from the NMR Perspective. In: Krämer, R., Ziegler, C. (eds) Membrane Transport Mechanism. Springer Series in Biophysics, vol 17. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-53839-1_11

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  • DOI: https://doi.org/10.1007/978-3-642-53839-1_11

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