Abstract

The atomic structure of the infectious, protease-resistant, β-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-ångström-resolution structure of a protofibril formed by a wild-type segment from the β2–α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we have named ‘polar clasps’.

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Acknowledgements

We thank C. Ophus (Molecular Foundry-NCEM), J. Miao (UCLA), and H. Nelson (UCLA) for helpful discussions. This work is supported by DOE Grant DE-FC02-02ER63421, the EICN in the CNSI at UCLA, the Janelia Research Visitor Program, and the NE-CAT beamline 24-ID-E, funded by NIH-NIGMS P41 GM103403. This work was also supported by STROBE: a National Science Foundation Science and Technology Center under Grant No. DMR 1548924. G.F.H. is supported by the BEC.AR program, Fundación Bunge y Born, Fundación Williams, and Fundación René Barón, and is a member of CONICET. M.G.J. is supported by a QCB Collaboratory Postdoctoral Fellowship. C.G. is supported by the Cellular and Molecular Biology Training Program. J.A.R. is supported by the Searle Scholar Program and the Beckman Young Investigator Program. T.G. and D.S.E. are supported by the Howard Hughes Medical Institute (HHMI), and J.E.E. and I.V.N. are supported by the DOE-BER Molecules to Mesoscale Bioimaging Project FWP #66382.

Author information

Author notes

  1. Marcus Gallagher-Jones and Calina Glynn contributed equally to this work.

Affiliations

  1. Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, University of California Los Angeles, Los Angeles, CA, USA

    • Marcus Gallagher-Jones
    • , Calina Glynn
    • , Evelyn Hernandez
    • , Jennifer Miao
    • , Chih-Te Zee
    •  & Jose A. Rodriguez
  2. Department of Biological Chemistry and Department of Chemistry and Biochemistry, University of California Los Angeles, Howard Hughes Medical Institute, UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, CA, USA

    • David R. Boyer
    • , Lukasz Goldschmidt
    • , Heather T. McFarlane
    • , Michael R. Sawaya
    • , Duilio Cascio
    •  & David S. Eisenberg
  3. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    • Michael W. Martynowycz
    •  & Tamir Gonen
  4. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA

    • Irina V. Novikova
    •  & James E. Evans
  5. Laboratory of Pharmaceutical Biotechnology, Institute of Biology and Experimental Medicine, Buenos Aires, Argentina

    • Gustavo F. Helguera

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Contributions

J.A.R. directed the work. J.A.R., J.M., E.H., M.W.M., and C.G. grew, evaluated, and optimized crystals. J.A.R., D.R.B., C.G., M.R.S., H.T.M., M.G.-J., C.-T.Z., I.V.N., J.E.E., and D.C. collected data. J.A.R., C.G., J.M., M.G.-J., M.R.S., D.C., M.W.M., G.F.H., E.H., L.G., D.S.E., and T.G. analyzed the data. C.G., M.G.-J., and J.A.R. wrote the article, with input from all authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Jose A. Rodriguez.

Integrated supplementary information

  1. Supplementary Figure 1 Sequence analysis of the common core of prionprotofilaments.

    a, Tightly packed prion core as defined by NMR (Zahn, R. et al. NMR solution structure of the human prion protein. Proc. Natl. Acad. Sci. 97, 145–150 (2000)), Proteinase K resistance (Bocharova, O. V., Breydo, L., Salnikov, V. V., Gill, A. C. & Baskakov, I. V. Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPsc from sporadic Creutzfeldt-Jakob Disease. Protein Sci. Publ. Protein Soc. 14, 1222–1232 (2005); Bocharova, O. V. et al. Annealing Prion Protein Amyloid Fibrils at High Temperature Results in Extension of a Proteinase K-resistant Core. J. Biol. Chem. 281, 2373–2379 (2006)), Hydrogen-Deuterium Exchange (Dutta, A., Chen, S. & Surewicz, W. K. The effect of β2-α2 loop mutation on amyloidogenic properties of the prion protein. FEBS Lett. 587, 2918 (2013); Smirnovas, V. et al. Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange. J. Biol. Chem. 284, 24233–24241 (2009); Lu, X., Wintrode, P. L. & Surewicz, W. K. β-Sheet core of human prion protein amyloid fibrils as determined by hydrogen/deuterium exchange. Proc. Natl. Acad. Sci. 104, 1510–1515 (2007); Cobb, N. J., Apostol, M. I., Chen, S., Smirnovas, V. & Surewicz, W. K. Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region. J. Biol. Chem. 289, 2643–2650 (2014).), and EPR (Cobb, N. J., Sönnichsen, F. D., Mchaourab, H. & Surewicz, W. K. Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure. Proc. Natl. Acad. Sci. U. S. A. 104, 18946–18951 (2007)). b, sequence alignment of bank vole PrP 27-30 with the 19 most similar sequences containing the proto-PrPScsequence (top, blue) and commonly studied PrP sequences (bottom, green) where only deviations from the bank vole sequence are shown.

  2. Supplementary Figure 2 Characterization of proto-PrPSc aggregates.

    a, MALDI-TOF/TOF mass spectrum of proto-PrPSc (Exact Mass of 1140.5 Da). b, Detection of Proto-PrPSc aggregates at an optical wavelength of 350 nm. Colors indicate solutions with increasing peptide concentrations with optical density plotted over the course of several hours. Values represent the mean OD 350 values and error bars represent ± 1 standard deviation (n = 3, technical replicates). c, Light microscope images of peptide solutions in a 96-well plate and electron micrographs of samples from those wells measured after 3 and 6 hours of incubation. d, Fiber diffraction with 10.2Å, 4.9Å, and 2.5Å rings labeled. Inset shows optical microscope image of the fibril from which diffraction was obtained.

  3. Supplementary Figure 3 Physical and chemical denaturation of proto-PrPSc aggregates.

    Visible light spectra (250-700nm) show absorbance from proto-PrPSc aggregates exposed to: a, a range of pH b, concentrations of guanidinium-HCl and c, concentrations of urea. Insets show each sample in a glass capillary above its corresponding figure legend. A 0.01% (w/v) solution of one µm latex spheres (grey lines) was used as a baseline against which to gauge particle density in various samples. The mean value of three replicates is shown (solid lines) with the shadowed range around each line representing ± 1 standard deviation (n = 3, technical replicates).

  4. Supplementary Figure 4 Microfocal x-ray structure determination of proto-PrPSc and comparison to MicroED structure of proto-PrPSc.

    a, Diffraction image collected by microfocal x-ray diffraction. Lower right inset shows hanging drop with a cluster of microcrystals (white arrow). Blue box in upper right shows a magnified view of high-resolution reflections near 1.1Å (black arrows) from the lower quadrant. b, Structure of proto-PrPSc determined by microfocal x-ray crystallography shown as a stick model. Waters are shown as cross marks, 2Fo-Fc density is shown at 1σ (blue mesh) and Fo-Fc at 2.8σ (green isosurface). c, Alignment of microfocal x-ray (blue) and MicroED (purple) structures. The peptide backbone is represented as an idealized beta strand and side chains as stick models. Residues are labeled according to their numbering in the sequence of bank vole prion.

  5. Supplementary Figure 5 Ab initio structure determination of proto-PrPSc.

    CFOM distribution obtained after 50,000 idenpendent trials of the SHELXD program. The ab initio model (top inset) results from the best CFOM in this distribution; the final solution is shown below. The blue box (CFOM > 80) defines regions where correct solutions are expected. The lower insets demonstrate the need to modify hydrogen positions from their idealized placement (magenta arrow) to better match the residual density (green arrow). Hydrogen bonding positions for the final model are shown as dashed lines.

  6. Supplementary Figure 6 B factors in proto-PrPSc.

    a, Side and b, top view of an isosurface model of proto-PrPSc analogous to those in Figure 1, but now colored by B-factor, where blue indicates low B-factors while red indicates high B-factors. A stick model is shown overlaid onto the isosurface model as a guide. The B-factor scale ranges from 0 to 18.0Å2.

  7. Supplementary Figure 7 Hydrogen bond networks formed by polar side chains in β-sheets.

    Five examples of hydrogen bonding networks are shown. From left to right, they are the proton wire (Nagle, J. F. & Morowitz, H. J. Molecular mechanisms for proton transport in membranes. Proc. Natl. Acad. Sci. U. S. A. 75, 298–302 (1978).), polar zipper (Perutz, M. F., Staden, R., Moens, L. & De Baere, I. Polar zippers. Curr. Biol. CB 3, 249–253 (1993).), glutamine ladder (Perutz, M. F., Johnson, T., Suzuki, M. & Finch, J. T. Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proc. Natl. Acad. Sci. U. S. A. 91, 5355–5358 (1994).), asparagine ladder (Yoder, M. D., Lietzke, S. E. & Jurnak, F. Unusual structural features in the parallel β-helix in pectate lyases. Structure 1, 241–251 (1993).), and the polar clasp (blue outline). For each, three layers are shown, each representing three beta strands stacked along a sheet. Hydrogen bonds are shown as dashed lines.

  8. Supplementary Figure 8 Refinement of proto-PrPSc as a function of resolution.

    High resolution refinement of proto-PrPSc allows visualization of density for hydrogen atoms in density maps (orange arrows) at 0.7σ; the same atoms cannot be seen at lower resolution. Bridging density for the hydrogen bond between Q172 and N174 (blue arrows) can similarly only be seen at sub-ångstrom resolution.

  9. Supplementary Figure 9 Comparison of ultrahigh-resolution features in maps solved by MicroED maps and X-ray crystallography.

    Comparison of ultrahigh resolution features in MicroED maps of proto-PrPSc to those of 0.48Å and 0.8Å x-ray crystallographic maps of the hiPiP protein (PDB IDs: 1IUA and 5D8V) (Hirano, Y., Takeda, K. & Miki, K. Charge-density analysis of an iron sulfur protein at an ultra-high resolution of 0.48 Å. Nature 534, 281–284 (2016); Liu, L., Nogi, T., Kobayashi, M., Nozawa, T. & Miki, K. Ultrahigh-resolution structure of high-potential iron sulfur protein from Thermochromatium tepidum. Acta Crystallogr. D Biol. Crystallogr. 58, 1085–1091 (2002).). 2mFoDFc maps are rendered as a blue mesh (0.8σ) and mFo-DFc maps are rendered as a solid green isosurface (2.8σ). Density for hydrogen atoms are indicated by red arrows and residual density in the difference map is indicated by blue arrows. The sidechain panel also includes contour maps for 2Fo-Fc and Fo-Fc density.

  10. Supplementary Figure 10 Ab initio structure determination of carbamazepine.

    CFOM distribution obtained after 100,000 independent trials of the SHELXD program. The ab initio model (top inset) results from the best CFOM in this distribution; the final solution is shown below. 2Fo-Fc maps are shown in blue, Fo-Fc maps in green inform the locations of hydrogens in the model. The blue box (CFOM > 80) defines the region where correct solutions are expected.

  11. Supplementary Figure 11 Comparison of ultrahigh-resolution features in MicroED maps of proto-PrPSc refined in different software packages.

    MicroED maps of proto-PrPSc were refined by either the Phenix (a and c) or Refmac (b and d) software packages. 2mFo-DFc maps are rendered as a blue mesh (0.8σ) and mFo-DFc maps are rendered as a solid green isosurface (2.8σ). Arrows indicate bridging hydrogen bonds. b, Fourier shell correlation between the maps produced by each refinement package, with a dashed black line indicating FSC cutoffs of 0.5 and 0.143.

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https://doi.org/10.1038/s41594-017-0018-0