Solid-state NMR sequential assignments of the amyloid core of full-length Sup35p

Abstract

Sup35p is a yeast prion and is responsible for the [PSI +] trait in Saccharomyces cerevisiae. With 685 amino acids, full-length soluble and fibrillar Sup35p are challenging targets for structural biology as they cannot be investigated by X-ray crystallography or NMR in solution. We present solid-state NMR studies of fibrils formed by the full-length Sup35 protein. We detect an ordered and rigid core of the protein that gives rise to narrow and strong peaks, while large parts of the protein show either static disorder or dynamics on time scales which interfere with dipolar polarization transfer or shorten the coherence lifetime. Thus, only a small subset of resonances is observed in 3D spectra. Here we describe in detail the sequential assignments of the 22 residues for which resonances are observed in 3D spectra: their chemical shifts mostly corresponding to β-sheet secondary structure. We suspect that these residues form the amyloid core of the fibril.

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References

  1. Böckmann A, Meier BH (2010) Prions: en route from structural models to structures. Prion 4(2):72–79

    Article  Google Scholar 

  2. Böckmann A, Gardiennet C, Verel R, Hunkeler A, Loquet A, Pintacuda G, Emsley L, Meier BH, Lesage A (2009) Characterization of different water pools in solid-state NMR protein samples. J Biomol NMR 45(3):319–327

    Article  Google Scholar 

  3. Comellas G, Lemkau LR, Nieuwkoop AJ, Kloepper KD, Ladror DT, Ebisu R, Woods WS, Lipton AS, George JM, Rienstra CM (2011) Structured regions of α-synuclein fibrils include the early-onset parkinsons disease mutation sites. J Mol Biol 411:881–895

    Article  Google Scholar 

  4. Cox BS (1965) PSI, a cytoplasmic suppressor of super-suppressor in yeast. Heredity 20(4):505–521

    Article  Google Scholar 

  5. Debelouchina GT, Platt GW, Bayro MJ, Radford SE, Griffin RG (2010) Magic angle spinning NMR analysis of beta(2)-microglobulin amyloid fibrils in two distinct morphologies. J Am Chem Soc 132(30):10414–10423

    Article  Google Scholar 

  6. DePace AH, Santoso A, Hillner P, Weissman JS (1998) A critical role for amino-terminal glutamine/asparagine repeats in the formation and propagation of a yeast prion. Cell 93(7):1241–1252

    Article  Google Scholar 

  7. Fitzpatrick AWP, Debelouchina GT, Bayro MJ, Clare DK, Caporini MA, Bajaj VS, Jaroniec CP, Wang L, Ladizhansky V, Müller SA, MacPhee CE, Waudby CA, Mott HR, De Simone A, Knowles TPJ, Saibil HR, Vendruscolo M, Orlova EV, Griffin RG, Dobson CM (2013) Atomic structure and hierarchical assembly of a cross-β amyloid fibril. Proc Natl Acad Sci USA 110:5468–5473

    ADS  Article  Google Scholar 

  8. Fogh R, Ionides J, Ulrich E, Boucher W, Vranken W, Linge JP, Habeck M, Rieping W, Bhat TN, Westbrook J, Henrick K, Gilliland G, Berman H, Thornton J, Nilges M, Markley J, Laue E (2002) The CCPN project: an interim report on a data model for the NMR community. Nat Struct Biol 9(6):416–418

    Article  Google Scholar 

  9. Gath J, Habenstein B, Bousset L, Melki R, Meier BH, Böckmann A (2012) Solid-state NMR sequential assignments of α-synuclein. Biomol NMR Assign 6(1):51–55

    Article  Google Scholar 

  10. Habenstein B, Wasmer C, Bousset L, Sourigues Y, Schütz A, Loquet A, Meier BH, Melki R, Böckmann A (2011) Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion. J Biomol NMR 51(3):235–243

    Article  Google Scholar 

  11. Heise H, Celej MS, Becker S, Riede D, Pelah A, Kumar A, Jovin TM, Baldus M (2008) Solid-state NMR reveals structural differences between fibrils of wild-type and disease-related A53T mutant alpha-synuclein. J Mol Biol 380(3):444–450

    Article  Google Scholar 

  12. Krishnan R, Lindquist S (2005) Structural insights into a yeast prion illuminate nucleation and strain diversity. Nature 435(7043):765–772

    ADS  Article  Google Scholar 

  13. Krzewska J, Melki R (2006) Molecular chaperones and the assembly of the prion Sup35p, an in vitro study. EMBO J 25(4):822–833

    Article  Google Scholar 

  14. Krzewska J, Tanaka M, Burston SG, Melki R (2007) Biochemical and functional analysis of the assembly of full-length Sup35p and its prion-forming domain. J Biol Chem 282(3):1679–1686

    Article  Google Scholar 

  15. Lewandowski JR, van der Wel PCA, Rigney M, Grigorieff N, Griffin RG (2011) Structural complexity of a composite amyloid fibril. J Am Chem Soc 133(37):14686–14698

    Article  Google Scholar 

  16. Loquet A, Bousset L, Gardiennet C, Sourigues Y, Wasmer C, Habenstein B, Schütz A, Meier BH, Melki R, Böckmann A (2009) Prion fibrils of Ure2p assembled under physiological conditions contain highly ordered, natively folded modules. J Mol Biol 394(1):108–118

    Article  Google Scholar 

  17. Loquet A, Giller K, Becker S, Lange A (2010) Supramolecular interactions probed by 13C–13C solid-state NMR spectroscopy. J Am Chem Soc 132(43):15164–15166

    Article  Google Scholar 

  18. Luckgei N, Schütz AK, Bousset L, Habenstein B, Sourigues Y, Gardiennet C, Meier BH, Melki R, Böckmann A (2013a) The conformation of the prion domain of Sup35p in isolation and in the full-length protein is different (submitted)

  19. Luckgei N, Schütz AK, Habenstein B, Bousset L, Sourigues Y, Melki R, Meier BH, Böckmann A (2013b) Solid-state NMR sequential assignments of the amyloid core of Sup35pN. doi:10.1007/s12104-013-9518-y

  20. Nelson R, Sawaya M, Balbirnie M, Madsen A, Riekel C, Grothe R, Eisenberg D (2005) Structure of the cross-beta spine of amyloid-like fibrils. Nature 435(7043):773–778

    ADS  Article  Google Scholar 

  21. Paravastu AK, Leapman RD, Yau W-M, Tycko R (2008) Molecular structural basis for polymorphism in Alzheimer’s beta-amyloid fibrils. Proc Natl Acad Sci USA 105(47):18349–18354

    ADS  Article  Google Scholar 

  22. Sangill R, Rastrupandersen N, Bildsoe H, Jakobsen HJ, Nielsen NC (1994) Optimized spectral editing of 13C MAS NMR spectra of rigid solids using cross-polarization methods. J Magn Reson A 107(1):67–78

    ADS  Article  Google Scholar 

  23. Schuetz A, Wasmer C, Habenstein B, Verel R, Greenwald J, Riek R, Böckmann A, Meier BH (2010) Protocols for the sequential solid-state NMR spectroscopic assignment of a uniformly labeled 25 kDa protein: HET-s(1-227). Chembiochem 11(11):1543–1551

    Article  Google Scholar 

  24. Shewmaker F, Wickner RB, Tycko R (2006) Amyloid of the prion domain of Sup35p has an in-register parallel beta-sheet structure. Proc Natl Acad Sci USA 103(52):19754–19759

    ADS  Article  Google Scholar 

  25. Stansfield I, Jones K, Kushnirov V, Dagkesamanskaya A, Poznyakovski A, Paushkin S, Nierras C, Cox B, Teravanesyan M, Tuite M (1995) The products of the Sup45 (eRF1) and Sup35 genes interact to mediate translation termination in saccharomyces-cerevisiae. EMBO J 14(17):4365–4373

    Google Scholar 

  26. Stevens TJ, Fogh RH, Boucher W, Higman VA, Eisenmenger F, Bardiaux B, van Rossum B-J, Oschkinat H, Laue ED (2011) A software framework for analysing solid-state MAS NMR data. J Biomol NMR 51(4):437–447

    Article  Google Scholar 

  27. Toyama B, Kelly M, Gross J, Weissman J (2007) The structural basis of yeast prion strain variants. Nature 449(7159):233–237

    ADS  Article  Google Scholar 

  28. Tycko R (2006) Solid-state NMR as a probe of amyloid structure. Protein Pept Lett 13:229–234

    Article  Google Scholar 

  29. van der Wel PC, Hu K, Lewandowski JR, Griffin RG (2006) Dynamic nuclear polarization of amyloidogenic peptide nanocrystals: GNNQQNY, a core segment of the yeast prion protein Sup35p. J Am Chem Soc 128(33):10840–10846

    Article  Google Scholar 

  30. van der Wel PCA, Lewandowski JR, Griffin RG (2007) Solid-state NMR study of amyloid nanocrystals and fibrils formed by the peptide GNNQQNY from yeast prion protein Sup35p. J Am Chem Soc 129(16):5117–5130

    Article  Google Scholar 

  31. van der Wel PCA, Lewandowski JR, Griffin RG (2010) Structural characterization of GNNQQNY amyloid fibrils by magic angle spinning NMR. Biochemistry 49(44):9457–9469

    Article  Google Scholar 

  32. van Melckebeke H, Wasmer C, Lange A, Ab E, Loquet A, Böckmann A, Meier BH (2010) Atomic-resolution three-dimensional structure of HET-s(218–289) amyloid fibrils by solid-state NMR spectroscopy. J Am Chem Soc 132(39):13765–13775

    Article  Google Scholar 

  33. Vranken W, Boucher W, Stevens T, Fogh R, Pajon A, Llinas P, Ulrich E, Markley J, Ionides J, Laue E (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59(4):687–696

    Article  Google Scholar 

  34. Wang Y, Jardetzky O (2002) Probability-based protein secondary structure identification using combined NMR chemical-shift data. Protein Sci 11(4):852–861

    Article  Google Scholar 

  35. Wasmer C, Lange A, van Melckebeke H, Siemer AB, Riek R, Meier BH (2008) Amyloid fibrils of the HET-s(218–289) prion form a beta solenoid with a triangular hydrophobic core. Science 319(5869):1523–1526

    ADS  Article  Google Scholar 

  36. Wickner RB, Masison DC, Edskes HK (1995) [PSI] and [URE3] as yeast prions. Yeast 11(16):1671–1685

    Article  Google Scholar 

  37. Wishart DS, Sykes BD (1994) The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR 4(2):171–180

    Google Scholar 

  38. Wu X, Zilm K (1993) Complete spectral editing in CPMAS NMR. J Magn Reson A 102(2):205–213

    ADS  Article  Google Scholar 

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Acknowledgments

We thank Dr. Christian Wasmer for help with recording the NMR spectra. This work was supported by the Agence Nationale de la Recherche (ANR-12-BS08-0013-01), the ETH Zurich, the Swiss National Science Foundation (Grant 200020_124611) and the Centre National de la Recherche Scientifique. We also acknowledge support from the European Commission under the Seventh Framework Programme (FP7), contract Bio-NMR 261863.

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Correspondence to Ronald Melki or Anja Böckmann or Beat H. Meier.

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Anne K. Schütz, Nina Luckgei, Birgit Habenstein, and Luc Bousset have contributed equally to this work.

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Schütz, A.K., Habenstein, B., Luckgei, N. et al. Solid-state NMR sequential assignments of the amyloid core of full-length Sup35p. Biomol NMR Assign 8, 349–356 (2014). https://doi.org/10.1007/s12104-013-9515-1

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Keywords

  • Sup35p
  • Fibrils
  • Solid-state NMR
  • Assignments
  • Secondary structure