Differences in prion strain conformations result from non-native interactions in a nucleus

Journal name:
Nature Chemical Biology
Year published:
Published online


Aggregation-prone proteins often misfold into multiple distinct amyloid conformations that dictate different physiological impacts. Although amyloid formation is triggered by a transient nucleus, the mechanism by which an initial nucleus is formed and allows the protein to form a specific amyloid conformation has been unclear. Here we show that, before fiber formation, the prion domain (Sup35NM, consisting of residues 1–254) of yeast prion Sup35, the [PSI+] protein determinant, forms oligomers in a temperature-dependent, reversible manner. Mutational and biophysical analyses revealed that 'non-native' aromatic interactions outside the amyloid core drive oligomer formation by bringing together different Sup35NM monomers, which specifically leads to the formation of highly infectious strain conformations with more limited amyloid cores. Thus, transient non-native interactions in the initial nucleus are pivotal in determining the diversity of amyloid conformations and resulting prion strain phenotypes.

At a glance


  1. Sup35NM forms oligomers in a temperature-dependent, reversible manner.
    Figure 1: Sup35NM forms oligomers in a temperature-dependent, reversible manner.

    (a) Sedimentation coefficient distributions (c(S)) of Sup35NM at 4 °C (blue) and 37 °C (red). c(S) and S denote sedimentation coefficient distrubution and sedimentation coefficient, respectively. (b) SAXS profile of Sup35NM protein. SAXS profile was first measured at 4 °C (blue solid line) and the temperature was increased to 37 °C (red solid line), returned to 4 °C (blue broken line) and finally shifted again to 37 °C (red broken line). See Supplementary Methods for definition of I(S). (c) Concentration-dependent changes in scattering intensity (I(0)) of Sup35NM protein at 4 °C. Maximum scattering intensities of Sup35NM protein at 4 °C (blue) and 37 °C (red) are plotted against the concentration of Sup35NM. (d) Kratky plot of the SAXS profile shown in b. The peak at 4 Å−1 for Sup35NM at 4 °C indicates the presence of globular structures20. (e) HSQC NMR spectra of Sup35NM protein. Spectra were first measured at 25 °C (left), and then the temperature was decreased to 10 °C (center) and finally returned to 25 °C (right). The boxed area shows signals from glycines in Sup35NM. (f) Images of Sup35NM oligomer by transmission electron microscopy (left). A zoomed view of Sup35NM oligomer (right). Scale bar, 50 nm.

  2. Low-temperature Sup35NM oligomers are on the pathway to formation of Sc4 amyloid fibers.
    Figure 2: Low-temperature Sup35NM oligomers are on the pathway to formation of Sc4 amyloid fibers.

    (a) Temperature-dependent changes in the amounts of Sup35NM oligomer. Rg values determined by the Guinier approximation are plotted against temperature. (b) Relationships between levels of Sup35NM oligomer and prion strain phenotypes. Sup35NM amyloids formed at various temperatures were introduced into [psi] yeast, and the resulting degree of prion infectivity as well as the fraction of strong (white bar) and weak (pink bar) [PSI+] phenotypes were determined. The infection experiments were performed at least twice, and fractions (%) of the strain phenotypes were calculated using more than 100 colonies in each experiment. (c) Schematic representation of on-pathway or off-pathway Sup35NM oligomer in the fiber formation. (d) Effects of changes in concentration of Sup35NM protein on the ratio of strong to weak [PSI+] strain phenotypes. Asterisk denotes P < 0.05. All error bars represent s.d.

  3. Distinct amino acid regions in the prion domain of Sup35NM are involved in nucleation and amyloid growth.
    Figure 3: Distinct amino acid regions in the prion domain of Sup35NM are involved in nucleation and amyloid growth.

    (a) Effects of proline- or leucine-encoding mutations on oligomer formation. Maximum SAXS intensities of Sup35NM proline mutants (4 °C, blue; 37 °C, red) and leucine mutants (4 °C, cyan; 37 °C, orange) are plotted against the amino acid positions of the mutations. The dotted lines denote the intensity of wild-type Sup35NM at 4 °C (upper, blue) and 37 °C (lower, red). tyrosine residues are highlighted by open symbols. (b) Effects of tryptophan-encoding mutations on oligomer formation. Maximum SAXS intensities of Sup35NM tryptophan mutants (4 °C, blue; 37 °C, red) are plotted against the amino acid positions of the mutations. The dotted lines indicate intensities of wild-type Sup35NM at 4 °C (upper, blue) and 37 °C (lower, red). (c,d) Effects of proline (c) or tryptophan (d) mutants on prion strain phenotypes. Fractions of strong (white bar) and weak (pink bar) [PSI+] phenotypes were determined. Fibers of Sup35NM proline and tryptophan mutants were formed at 8 °C and 11 °C, respectively, and used for amyloid infection.

  4. Non-native interactions outside of the Sc4 amyloid core drive oligomer formation of Sup35NM.
    Figure 4: Non-native interactions outside of the Sc4 amyloid core drive oligomer formation of Sup35NM.

    (a,b) Normalized excimer fluorescence spectra of pyrene-labeled Sup35NM mutants at 4 °C (a) or 37 °C (b). Shown are fluorescence spectra of Cys16 (red), Cys89 (blue), Cys108 (black) and Cys238 (pink) mutants. (c) The ratio of excimer fluorescence intensities (470 nm) to non-excimer fluorescence intensities (385 nm). Normalized intensities of excimer fluorescence of the pyrene-labeled Sup35NM mutants at 4 °C (blue) or 37 °C (red) are plotted against amino acid position. The error bars represent s.d. (d) Normalized intensities of excimer fluorescence are plotted against temperature for pyrene-labeled Sup35NM Cys16 (red), Cys26 (orange), Cys46 (green), Cys89 (blue) and Cys108 (black) mutants.

  5. Global relationships between Sup35NM monomer, initial nucleus, amyloid conformation and prion strain phenotype.
    Figure 5: Global relationships between Sup35NM monomer, initial nucleus, amyloid conformation and prion strain phenotype.

    Non-native interactions (green lines) outside of the Sc4 amyloid core drive oligomer formation. This interaction brings Sup35NM monomers into close proximity while leaving the amyloid core regions free to interact, which eventually leads to highly infectious Sc4 strain conformations that have more limited amyloid cores. The nucleus created during fibrillation at 37 °C may be larger than a monomer36 but is still much smaller than the oligomer formed at 4 °C.


  1. Chien, P., Weissman, J.S. & DePace, A.H. Emerging principles of conformation-based prion inheritance. Annu. Rev. Biochem. 73, 617656 (2004).
  2. Chiti, F. & Dobson, C.M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333366 (2006).
  3. Kodali, R. & Wetzel, R. Polymorphism in the intermediates and products of amyloid assembly. Curr. Opin. Struct. Biol. 17, 4857 (2007).
  4. Collinge, J. Prion diseases of humans and animals: their causes and molecular basis. Annu. Rev. Neurosci. 24, 519550 (2001).
  5. Wickner, R.B. [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae . Science 264, 566569 (1994).
  6. Tuite, M.F. & Koloteva-Levin, N. Propagating prions in fungi and mammals. Mol. Cell 14, 541552 (2004).
  7. Tessier, P.M. & Lindquist, S. Unraveling infectious structures, strain variants and species barriers for the yeast prion [PSI +]. Nat. Struct. Mol. Biol. 16, 598605 (2009).
  8. Derkatch, I.L., Chernoff, Y.O., Kushnirov, V.V., Inge-Vechtomov, S.G. & Liebman, S.W. Genesis and variability of [PSI] prion factors in Saccharomyces cerevisiae . Genetics 144, 13751386 (1996).
  9. Kochneva-Pervukhova, N.V. et al. [PSI +] prion generation in yeast: characterization of the 'strain' difference. Yeast 18, 489497 (2001).
  10. Tanaka, M., Chien, P., Naber, N., Cooke, R. & Weissman, J.S. Conformational variations in an infectious protein determine prion strain differences. Nature 428, 323328 (2004).
  11. Caughey, B. & Lansbury, P.T. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu. Rev. Neurosci. 26, 267298 (2003).
  12. Johnson, S.M. et al. Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: a focus on the transthyretin amyloidoses. Acc. Chem. Res. 38, 911921 (2005).
  13. Haass, C. & Selkoe, D.J. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat. Rev. Mol. Cell Biol. 8, 101112 (2007).
  14. Balch, W.E., Morimoto, R.I., Dillin, A. & Kelly, J.W. Adapting proteostasis for disease intervention. Science 319, 916919 (2009).
  15. Serio, T.R. et al. Nucleated conformational conversion and the replication of conformational information by a prion determinant. Science 289, 13171321 (2000).
  16. King, C.Y. & Diaz-Avalos, R. Protein-only transmission of three yeast prion strains. Nature 428, 319323 (2004).
  17. Scheibel, T. & Lindquist, S.L. The role of conformational flexibility in prion propagation and maintenance for Sup35p. Nat. Struct. Biol. 11, 958962 (2001).
  18. Collins, S.R., Douglass, A., Vale, R.D. & Weissman, J.S. Mechanism of prion propagation: amyloid growth occurs by monomer addition. PLoS Biol. 2, e321 (2004).
  19. Tanaka, M. et al. Expansion of polyglutamine induces the formation of quasi-aggregate in the early stage of protein fibrillization. J. Biol. Chem. 278, 3471734724 (2003).
  20. Glatter, O. & Kratky, O. Small Angle X-ray Scattering (Academic Press, New York, 1982).
  21. Walsh, D.M. et al. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535539 (2002).
  22. Kayed, R. et al. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486489 (2003).
  23. Lee, S. & Eisenberg, D. Seeded conversion of recombinant prion protein to a disulfide-bonded oligomer by a reduction-oxidation process. Nat. Struct. Biol. 10, 725730 (2003).
  24. Plakoutsi, G. et al. Evidence for a mechanism of amyloid formation involving molecular reorganisation within native-like precursor aggregates. J. Mol. Biol. 351, 910922 (2005).
  25. Eakin, C.M., Berman, A.J. & Miranker, A.D. A native to amyloidogenic transition regulated by a backbone trigger. Nat. Struct. Mol. Biol. 13, 202208 (2006).
  26. Jahn, T.R., Parker, M.J., Homans, S.W. & Radford, S.E. Amyloid formation under physiological conditions proceeds via a native-like folding intermediate. Nat. Struct. Mol. Biol. 13, 195201 (2006).
  27. Toyama, B.H., Kelly, M.J., Gross, J.D. & Weissman, J.S. The structural basis of yeast prion strain variants. Nature 449, 233237 (2007).
  28. Liu, J.J. & Lindquist, S. Oligopeptide-repeat expansions modulate 'protein-only' inheritance in yeast. Nature 400, 573576 (1999).
  29. Krishnan, R. & Lindquist, S.L. Structural insights into a yeast prion illuminate nucleation and strain diversity. Nature 435, 765772 (2005).
  30. Tessier, P.M. & Lindquist, S. Prion recognition elements govern nucleation, strain specificity and species barriers. Nature 447, 556561 (2007).
  31. Glover, J.R. et al. Self-seeded fibers formed by Sup35, the protein determinant of [PSI +], a heritable prion-like factor of S. cerevisiae . Cell 89, 811819 (1997).
  32. Tanaka, M., Chien, P., Yonekura, K. & Weissman, J.S. Mechanism of cross-species prion transmission: an infectious conformation compatible with two highly divergent yeast prion proteins. Cell 121, 4962 (2005).
  33. Nelson, R. et al. Structure of the cross-β spine of amyloid-like fibrils. Nature 435, 773778 (2005).
  34. Shewmaker, F., Wickner, R.B. & Tycko, R. Amyloid of the prion domain of Sup35p has an in-register parallel beta-sheet structure. Proc. Natl. Acad. Sci. USA 103, 1975419759 (2006).
  35. Chen, S., Ferrone, F.A. & Wetzel, R. Huntington's disease age-of-onset linked to polyglutamine aggregation nucleation. Proc. Natl. Acad. Sci. USA 99, 1188411889 (2002).
  36. Xue, W.F., Homans, S.W. & Radford, S.E. Systematic analysis of nucleation-dependent polymerization reveals new insights into the mechanism of amyloid self-assembly. Proc. Natl. Acad. Sci. USA 105, 89268931 (2008).
  37. Shorter, J. & Lindquist, S. Hsp104 catalyzes formation and elimination of self-replicating Sup35 prion conformers. Science 304, 17931797 (2004).
  38. Inoue, Y., Taguchi, H., Kishimoto, A. & Yoshida, M. Hsp104 binds to yeast Sup35 prion fiber but needs other factor(s) to sever it. J. Biol. Chem. 279, 5231952323 (2004).
  39. Tipton, K.A., Verges, K.J. & Weissman, J.S. In vivo monitoring of the prion replication cycle reveals a critical role for Sis1 in delivering substrates to Hsp104. Mol. Cell 32, 584591 (2008).
  40. Tanaka, M., Collins, S.R., Toyama, B.H. & Weissman, J.S. The physical basis of how prion conformations determine strain phenotypes. Nature 442, 585589 (2006).
  41. Duennwald, M.L., Jagadish, S., Muchowski, P.J. & Lindquist, S. Flanking sequences profoundly alter polyglutamine toxicity in yeast. Proc. Natl. Acad. Sci. USA 103, 1104511050 (2006).
  42. Thakur, A.K. et al. Polyglutamine disruption of the huntingtin exon 1 N terminus triggers a complex aggregation mechanism. Nat. Struct. Mol. Biol. 16, 380389 (2009).
  43. Fujisawa, T. et al. Small-angle X-ray scattering station at the SPring-8 RIKEN beamline. J. Appl. Cryst. 33, 797800 (2000).
  44. Ameyama, Y. et al. Large-aperture TV detector with a beryllium-windowed image intensifier for x-ray diffraction. Rev. Sci. Instrum. 66, 22902294 (1995).
  45. Guinier, A. & Fournet, G. Small-Angle Scattering of X-rays (John Wiley & Sons, New York, 1955).
  46. Svergun, D.I., Semenyuk, A.V. & Feigin, L.A. Small-angle-scattering-data treatment by the regularization method. Acta Crystallogr. 44, 244250 (1988).
  47. Lehrer, S.S. Intramolecular pyrene excimer fluorescence: a probe of proximity and protein conformational change. Methods Enzymol. 278, 286295 (1997).

Download references

Author information


  1. Tanaka Research Unit, RIKEN Brain Science Institute, Wako, Saitama, Japan.

    • Yumiko Ohhashi &
    • Motomasa Tanaka
  2. RIKEN Spring-8 Center, Sayo, Hyogo, Japan.

    • Kazuki Ito
  3. Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.

    • Brandon H Toyama &
    • Jonathan S Weissman
  4. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Saitama, Japan. .

    • Motomasa Tanaka


M.T. and Y.O. designed the experiments. Y.O. performed most experiments, K.I. contributed to setup of the SAXS experiments and B.H.T. helped the NMR experiments. B.H.T. and J.S.W. provided new reagents. Y.O., J.S.W. and M.T. analyzed and discussed the data and wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (00000)

    Supplementary Methods and Supplementary Figures 1–9

Additional data