Bacterial Filamentous Appendages Investigated by Solid-State NMR Spectroscopy

  • Birgit Habenstein
  • Antoine Loquet
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)


The assembly of filamentous appendages at the surface of bacteria is essential in many infection mechanisms. The extent of mechanical, dynamical, and functional properties of such appendages is very diverse, ranging from a structural scaffold of the pathogen–host cell interaction to cell motility, surface adhesion, or the export of virulence effectors. In particular, the architectures of several bacterial secretion systems have revealed the presence of filamentous architectures, known as pili, fimbriae, andneedles. At the macroscopic level, filamentous bacterial appendages appear as thin extracellular filaments of several nanometers in diameter and up to several microns in length. The structural characterization of these appendages at atomic-scale resolution represents an extremely challenging task because of their inherent noncrystallinity and very poor solubility. Here, we describe protocols based on recent advances in solid-state NMR spectroscopy to investigate the secondary structure, subunit–subunit protein interactions, symmetry parameters, and atomic architecture of bacterial filaments.

Key words

Solid-state nuclear magnetic resonance Structure determination Pilus Needle Protein assembly Protein complex Helical symmetry 



The authors thank their past and present colleagues, in particular Prof. Adam Lange at the Leibniz-Institut für Molekulare Pharmakologie for his guidance during the author postdoctoral periods and his main intellectual contribution for the T3SS needle and type I pilus projects. This work was further supported by the Fondation pour la Recherche Médicale (FRM-AJE20140630090 to A.L.), the ANR (13-PDOC-0017-01 to B.H. and ANR-14-CE09-0020-01 to A.L.), the FP7 program (FP7-PEOPLE-2013-CIG to A.L.), the IdEx Bordeaux University (Chaire d’Installation to B.H.)and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC Starting Grant to A.L., agreement 105945). Erick Dufourc is acknowledged for his continuous support.


  1. 1.
    Loquet A, Sgourakis NG, Gupta R, Giller K, Riedel D, Goosmann C et al (2012) Atomic model of the type III secretion system needle. Nature 486:276–279CrossRefGoogle Scholar
  2. 2.
    Fujii T, Cheung M, Blanco A, Kato T, Blocker AJ, Namba K (2012) Structure of a type III secretion needle at 7-A resolution provides insights into its assembly and signaling mechanisms. Proc Natl Acad Sci U S A 109:4461–4466CrossRefGoogle Scholar
  3. 3.
    Blocker AJ, Deane JE, Veenendaal AK, Roversi P, Hodgkinson JL, Johnson S et al (2008) What’s the point of the type III secretion system needle? Proc Natl Acad Sci U S A 105:6507–6513CrossRefGoogle Scholar
  4. 4.
    Sauer FG, Futterer K, Pinkner JS, Dodson KW, Hultgren SJ, Waksman G (1999) Structural basis of chaperone function and pilus biogenesis. Science 285:1058–1061CrossRefGoogle Scholar
  5. 5.
    Habenstein B, Loquet A, Hwang S, Giller K, Vasa SK, Becker S et al (2015) Hybrid structure of the type 1 pilus of Uropathogenic Escherichia coli. Angew Chem Int Ed Engl 54:11691–11695CrossRefGoogle Scholar
  6. 6.
    Hospenthal MK, Redzej A, Dodson K, Ukleja M, Frenz B, Rodrigues C et al (2016) Structure of a chaperone-usher pilus reveals the molecular basis of rod uncoiling. Cell 164:269–278CrossRefGoogle Scholar
  7. 7.
    Geibel S, Waksman G (2014) The molecular dissection of the chaperone-usher pathway. Biochim Biophys Acta 1843:1559–1567CrossRefGoogle Scholar
  8. 8.
    Chandran Darbari V, Waksman G (2015) Structural biology of bacterial type IV secretion systems. Annu Rev Biochem 84:603–629CrossRefGoogle Scholar
  9. 9.
    Melville S, Craig L (2013) Type IV pili in Gram-positive bacteria. Microbiol Mol Biol Rev 77:323–341CrossRefGoogle Scholar
  10. 10.
    Stones DH, Krachler AM (2015) Fatal attraction: how bacterial adhesins affect host signaling and what we can learn from them. Int J Mol Sci 16:2626–2640CrossRefGoogle Scholar
  11. 11.
    Cheung M, Shen DK, Makino F, Kato T, Roehrich AD, Martinez-Argudo I et al (2015) Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex. Mol Microbiol 95:31–50CrossRefGoogle Scholar
  12. 12.
    Rathinavelan T, Lara-Tejero M, Lefebre M, Chatterjee S, McShan AC, Guo DC et al (2014) NMR model of PrgI-SipD interaction and its implications in the needle-tip assembly of the Salmonella type III secretion system. J Mol Biol 426:2958–2969CrossRefGoogle Scholar
  13. 13.
    Jones CH, Pinkner JS, Roth R, Heuser J, Nicholes AV, Abraham SN et al (1995) FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae. Proc Natl Acad Sci U S A 92:2081–2085CrossRefGoogle Scholar
  14. 14.
    Campos M, Nilges M, Cisneros DA, Francetic O (2010) Detailed structural and assembly model of the type II secretion pilus from sparse data. Proc Natl Acad Sci U S A 107:13081–13086CrossRefGoogle Scholar
  15. 15.
    Habenstein B, Loquet A. (2015). Solid-state NMR: an emerging technique in structural biology of self-assemblies. Biophys Chem.Google Scholar
  16. 16.
    Meier BH, Bockmann A (2015) The structure of fibrils from ‘misfolded’ proteins. Curr Opin Struct Biol 30:43–49CrossRefGoogle Scholar
  17. 17.
    Miao Y, Cross TA (2013) Solid state NMR and protein-protein interactions in membranes. Curr Opin Struct Biol 23:919–928CrossRefGoogle Scholar
  18. 18.
    Tang M, Comellas G, Rienstra CM (2013) Advanced solid-state NMR approaches for structure determination of membrane proteins and amyloid fibrils. Acc Chem Res 46:2080–2088CrossRefGoogle Scholar
  19. 19.
    Weingarth M, Baldus M (2013) Solid-state NMR-based approaches for supramolecular structure elucidation. Acc Chem Res 46:2037–2046CrossRefGoogle Scholar
  20. 20.
    Loquet A, Habenstein B, Lange A (2013) Structural investigations of molecular machines by solid-state NMR. Acc Chem Res 46:2070–2079CrossRefGoogle Scholar
  21. 21.
    Yan S, Suiter CL, Hou G, Zhang H, Polenova T (2013) Probing structure and dynamics of protein assemblies by magic angle spinning NMR spectroscopy. Acc Chem Res 46:2047–2058CrossRefGoogle Scholar
  22. 22.
    Tycko R, Wickner RB (2013) Molecular structures of amyloid and prion fibrils: consensus versus controversy. Acc Chem Res 46:1487–1496CrossRefGoogle Scholar
  23. 23.
    Hong M, Zhang Y, Hu F (2012) Membrane protein structure and dynamics from NMR spectroscopy. Annu Rev Phys Chem 63:1–24CrossRefGoogle Scholar
  24. 24.
    Egelman EH (2015) Three-dimensional reconstruction of helical polymers. Arch Biochem Biophys 581:54–58CrossRefGoogle Scholar
  25. 25.
    Egelman EH (2010) Reducing irreducible complexity: divergence of quaternary structure and function in macromolecular assemblies. Curr Opin Cell Biol 22:68–74CrossRefGoogle Scholar
  26. 26.
    Demers JP, Habenstein B, Loquet A, Kumar Vasa S, Giller K, Becker S et al (2014) High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy. Nat Commun 5:4976CrossRefGoogle Scholar
  27. 27.
    Hong M (1999) Determination of multiple ***φ***-torsion angles in proteins by selective and extensive (13)C labeling and two-dimensional solid-state NMR. J Magn Reson 139:389–401CrossRefGoogle Scholar
  28. 28.
    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 420:98–102CrossRefGoogle Scholar
  29. 29.
    Lundstrom P, Teilum K, Carstensen T, Bezsonova I, Wiesner S, Hansen DF et al (2007) Fractional 13C enrichment of isolated carbons using [1-13C]- or [2-13C]-glucose facilitates the accurate measurement of dynamics at backbone Calpha and side-chain methyl positions in proteins. J Biomol NMR 38:199–212CrossRefGoogle Scholar
  30. 30.
    Loquet A, Giller K, Becker S, Lange A (2010) Supramolecular interactions probed by 13C-13C solid-state NMR spectroscopy. J Am Chem Soc 132:15164–15166CrossRefGoogle Scholar
  31. 31.
    Bockmann A, Gardiennet C, Verel R, Hunkeler A, Loquet A, Pintacuda G et al (2009) Characterization of different water pools in solid-state NMR protein samples. J Biomol NMR 45:319–327CrossRefGoogle Scholar
  32. 32.
    Bertini I, Engelke F, Luchinat C, Parigi G, Ravera E, Rosa C et al (2012) NMR properties of sedimented solutes. Phys Chem Chem Phys 14:439–447CrossRefGoogle Scholar
  33. 33.
    Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  34. 34.
    Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M et al (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59:687–696CrossRefGoogle Scholar
  35. 35.
    Ulrich EL, Akutsu H, Doreleijers JF, Harano Y, Ioannidis YE, Lin J et al (2008) BioMagResBank. Nucleic Acids Res 36:D402–D408CrossRefGoogle Scholar
  36. 36.
    Wang Y, Jardetzky O (2002) Probability-based protein secondary structure identification using combined NMR chemical-shift data. Protein Sci 11:852–861CrossRefGoogle Scholar
  37. 37.
    Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44:213–223CrossRefGoogle Scholar
  38. 38.
    Berjanskii MV, Neal S, Wishart DS (2006) PREDITOR: a web server for predicting protein torsion angle restraints. Nucleic Acids Res 34:W63–W69CrossRefGoogle Scholar
  39. 39.
    Shen Y, Bax A (2010) SPARTA+: a modest improvement in empirical NMR chemical shift prediction by means of an artificial neural network. J Biomol NMR 48:13–22CrossRefGoogle Scholar
  40. 40.
    Han B, Liu Y, Ginzinger SW, Wishart DS (2011) SHIFTX2: significantly improved protein chemical shift prediction. J Biomol NMR 50:43–57CrossRefGoogle Scholar
  41. 41.
    Kohlhoff KJ, Robustelli P, Cavalli A, Salvatella X, Vendruscolo M (2009) Fast and accurate predictions of protein NMR chemical shifts from interatomic distances. J Am Chem Soc 131:13894–13895CrossRefGoogle Scholar
  42. 42.
    Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM (2003) The Xplor-NIH NMR molecular structure determination package. J Magn Reson 160:65–73CrossRefGoogle Scholar
  43. 43.
    Brunger AT (2007) Version 1.2 of the crystallography and NMR system. Nat Protoc 2:2728–2733CrossRefGoogle Scholar
  44. 44.
    Rieping W, Habeck M, Bardiaux B, Bernard A, Malliavin TE, Nilges M (2007) ARIA2: automated NOE assignment and data integration in NMR structure calculation. Bioinformatics 23:381–382CrossRefGoogle Scholar
  45. 45.
    Shen Y, Vernon R, Baker D, Bax A (2009) De novo protein structure generation from incomplete chemical shift assignments. J Biomol NMR 43:63–78CrossRefGoogle Scholar
  46. 46.
    Dominguez C, Boelens R, Bonvin AM (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125:1731–1737CrossRefGoogle Scholar
  47. 47.
    Bhattacharya A, Tejero R, Montelione GT (2007) Evaluating protein structures determined by structural genomics consortia. Proteins 66:778–795CrossRefGoogle Scholar
  48. 48.
    Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8:477–486CrossRefGoogle Scholar
  49. 49.
    Loquet A, Habenstein B, Chevelkov V, Vasa SK, Giller K, Becker S et al (2013) Atomic structure and handedness of the building block of a biological assembly. J Am Chem Soc 135:19135–19138CrossRefGoogle Scholar
  50. 50.
    Higman VA, Flinders J, Hiller M, Jehle S, Markovic S, Fiedler S et al (2009) Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins. J Biomol NMR 44:245–260CrossRefGoogle Scholar
  51. 51.
    Etzkorn M, Bockmann A, Lange A, Baldus M (2004) Probing molecular interfaces using 2D magic-angle-spinning NMR on protein mixtures with different uniform labeling. J Am Chem Soc 126:14746–14751CrossRefGoogle Scholar
  52. 52.
    Hartmann SR, Hahn EL (1962) Nuclear double resonance in rotating frame. Phys Rev 128:2042CrossRefGoogle Scholar
  53. 53.
    Baldus M, Petkova AT, Herzfeld J, Griffin RG (1998) Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems. Mol Phys 95:1197–1207CrossRefGoogle Scholar
  54. 54.
    Fung BM, Khitrin AK, Ermolaev K (2000) An improved broadband decoupling sequence for liquid crystals and solids. J Magn Reson 142:97–101CrossRefGoogle Scholar
  55. 55.
    Harris RK, Becker ED, Cabral De Menezes SM, Granger P, Hoffman RE, Zilm KW et al (2008) Further conventions for NMR shielding and chemical shifts IUPAC recommendations 2008. Solid State Nucl Magn Reson 33:41–56CrossRefGoogle Scholar
  56. 56.
    Shaka AF, Frenkeil T, Freeman R (1983) NMR broadband decoupling with low radio-frequency power. J Magn Reson. 52:159–163Google Scholar
  57. 57.
    Baldus M, Geurts DG, Hediger S, Meier BH (1996) Efficient 15N-13C polarization transfer by adiabatic passage Hartmann-Hahn cross-polarization. J Magn Reson Ser A 118:140–144CrossRefGoogle Scholar
  58. 58.
    Andronesi OC, Becker S, Seidel K, Heise H, Young HS, Baldus M (2005) Determination of membrane protein structure and dynamics by magic-angle-spinning solid-state NMR spectroscopy. J Am Chem Soc 127:12965–12974CrossRefGoogle Scholar
  59. 59.
    Ader C, Frey S, Maas W, Schmidt HB, Gorlich D, Baldus M (2010) Amyloid-like interactions within nucleoporin FG hydrogels. Proc Natl Acad Sci U S A 107:6281–6285CrossRefGoogle Scholar
  60. 60.
    Szeverenyi NM, Sullivan MJ, Maciel GE (1982) Observation of spin exchange by two-dimensional Fourier-transform C-13 cross polarization-magic-angle spinning. J Magn Reson 47:462–475Google Scholar
  61. 61.
    Verel R, Ernst M, Meier BH (2001) Adiabatic dipolar recoupling in solid-state NMR: the DREAM scheme. J Magn Reson 150:81–99CrossRefGoogle Scholar
  62. 62.
    Westfeld T, Verel R, Ernst M, Bockmann A, Meier BH (2012) Properties of the DREAM scheme and its optimization for application to proteins. J Biomol NMR 53:103–112CrossRefGoogle Scholar
  63. 63.
    Scholz I, Hodgkinson P, Meier BH, Ernst M (2009) Understanding two-pulse phase-modulated decoupling in solid-state NMR. J Chem Phys 130:114510CrossRefGoogle Scholar
  64. 64.
    Luca S, Filippov DV, van Boom JH, Oschkinat H, de Groot HJ, Baldus M (2001) Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under magic angle spinning. J Biomol NMR 20:325–331CrossRefGoogle Scholar
  65. 65.
    Lewandowski JR, De Paepe G, Griffin RG (2007) Proton assisted insensitive nuclei cross polarization. J Am Chem Soc 129:728–729CrossRefGoogle Scholar
  66. 66.
    Bardiaux B, Malliavin T, Nilges M (2012) ARIA for solution and solid-state NMR. Methods Mol Biol 831:453–483CrossRefGoogle Scholar
  67. 67.
    Guerry P, Herrmann T (2012) Comprehensive automation for NMR structure determination of proteins. Methods Mol Biol 831:429–451CrossRefGoogle Scholar
  68. 68.
    Morag O, Sgourakis NG, Baker D, Goldbourt A (2015) The NMR-Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope. Proc Natl Acad Sci U S A 112:971–976CrossRefGoogle Scholar
  69. 69.
    Rieping W, Habeck M, Nilges M (2005) Inferential structure determination. Science 309:303–306CrossRefGoogle Scholar
  70. 70.
    Schraidt O, Marlovits TC (2011) Three-dimensional model of Salmonella’s needle complex at subnanometer resolution. Science 331:1192–1195CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Institute of Chemistry & Biology of Membranes & Nanoobjects (UMR5248 CBMN), CNRSUniversity of Bordeaux, Institut Européen de Chimie et BiologiePessacFrance

Personalised recommendations