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Cryo-EM structure of a human prion fibril with a hydrophobic, protease-resistant core

Nature Structural & Molecular Biology volume 27, pages 417–423 (2020)Cite this article

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Abstract

Self-templating assemblies of the human prion protein are clinically associated with transmissible spongiform encephalopathies. Here we present the cryo-EM structure of a denaturant- and protease-resistant fibril formed in vitro spontaneously by a 9.7-kDa unglycosylated fragment of the human prion protein. This human prion fibril contains two protofilaments intertwined with screw symmetry and linked by a tightly packed hydrophobic interface. Each protofilament consists of an extended beta arch formed by residues 106 to 145 of the prion protein, a hydrophobic and highly fibrillogenic disease-associated segment. Such structures of prion polymorphs serve as blueprints on which to evaluate the potential impact of sequence variants on prion disease.

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Fig. 1: PrP domain architecture and structure of the rPrPRes fibril, the protease-resistant core of rPrP 94–178.
Fig. 2: Secondary structure of rPrPRes in contrast to that of PrPC and topology of the rPrPRes protofilament.
Fig. 3: Stability of the core of rPrPRes.
Fig. 4: Compatibility of sequence variants at key residues in rPrPRes.

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Data availability

EM maps and atomic coordinates for rPrP 106–145 have been deposited in the EMDB and wwPDB as EMD-20900 and PDB 6UUR, respectively; atomic coordinates and structure factors have been deposited in the wwPDB for PrP 113–118 (PDB 6PQ5) and PrP119–124 (PDB 6PQA).

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Acknowledgements

We thank D. Cascio (UCLA), H. McFarlane (UCLA) and C. Sigurdson (UCSD). This work is supported by National Science Foundation (NSF) Grants DMR-1548924 and DBI-1338135, DOE Grant DE-FC02-02ER63421 and National Institutes of Health (NIH) grants R35 GM128867, AG054022 and 1U24GM116792, as well as NIH instrumentation grants 1S10OD016387-01, 1S10RR23057 and 1S10OD018111, which support our use of instruments at the Electron Imaging Center for NanoMachines and CNSI at UCLA. C.G. was funded by the Ruth L. Kirschstein NRSA GM007185 (NIH T32 Cellular and Molecular Biology Training Grant, UCLA) and is now funded by the Ruth L. Kirschstein Predoctoral Individual NRSA, 1F31 AI143368. J.A.R. is supported as a Searle Scholar, a Pew Scholar and a Beckman Young Investigator. D.S.E is supported by the Howard Hughes Medical Institute.

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  1. Marcin Apostol

    Present address: ADRx, Thousand Oaks, CA, USA

Authors and Affiliations

  1. Department of Chemistry and Biochemistry; UCLA-DOE Institute for Genomics and Proteomics; STROBE, NSF Science and Technology Center, University of California, Los Angeles, Los Angeles, CA, USA

    Calina Glynn, Marcus Gallagher-Jones, Connor W. Short, Ronquiajah Bowman & Jose A. Rodriguez

  2. Department of Biological Chemistry and Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, CA, USA

    Michael R. Sawaya, Marcin Apostol & David S. Eisenberg

  3. California NanoSystems Institute, University of California Los Angeles, Los Angeles, CA, USA

    Peng Ge & Z. Hong Zhou

  4. Department of Microbiology Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, USA

    Z. Hong Zhou

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Contributions

C.G. and R.B. produced, evaluated and optimized recombinant PrP fibril preparations. C.G. and P.G. performed electron microscopy. M.A. and M.R.S. performed X-ray structure determination of PrP segments. C.G. and C.W.S. selected particles for analysis. C.G., P.G. and M.G.J. performed fibril reconstruction. C.G. and M.R.S. built the fibril model. C.G., P.G., M.R.S., M.G.J., C.W.S., R.B., M.A., Z.H.Z., D.S.E. and J.A.R. critically analyzed and provided feedback on data. C.G. and J.A.R. wrote the manuscript, with input from all authors.

Corresponding author

Correspondence to Jose A. Rodriguez.

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D.S.E. is SAB chair and an equity holder in ADRx.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Summary sentence: The cryo-EM structure of a denaturant- and protease-resistant human prion fibril shows a parallel, in-register assembly with a hydrophobic core.

Extended data

Extended Data Fig. 1 Production, isolation and characterization of rPrP94–178 fibrils.

a, Purification of untagged human PrP94–178. Samples collected during size exclusion chromatography of PrP94–178 were run on a 4–12 % SDS-PAGE gel corresponding to labeled peaks b, Gel lanes left to right: 1. Pre-stained protein ladder (Thermo), 2. Post-induction fraction, 3. Solubilized material from inclusion bodies, 4. Void fraction (A) from (a), 5. Peak (B) from (a), 6. Peak (C) from (a) containing 9.7 kDa monomeric rPrP94–178, 7–9. Peaks D-F from (a) containing excess guanidine or other UV active small eluates. c, Mass spectrum showing most abundant peaks correspond to ions with an extracted molecular weight that matches rPrP94–178. d, Representative micrographs of a heterogeneous mix of untreated fibrils (left) in Growth Buffer; promising filaments (black arrows) and disordered, clumped, or amorphous material (blue arrows). Adjacent images are of filaments treated with 1:10 molar ratio proteinase K:rPrP94–178 monomer and bath sonicated for 10 minutes. Proteinase K-treated and sonicated filaments exchanged into water in a frozen-hydrated state (third column, top) or 2 % SDS (third column, bottom). Representative image of Proteinase K, sonication, and SDS treated filaments used for high-resolution imaging and reconstruction (right). Scale bars, 200 nm. Scale bar for high-resolution image (right) 50 nm.

Extended Data Fig. 2 Partial protease digestion of rPrP94–178 fibrils.

a, Plots of incubation time versus nephelometry units as a measure of insoluble character in fibril suspensions treated with proteinase K compared to proteinase K only and buffer controls. b, Representative electron micrographs of each sample in panel (a) before proteinase K digestion at the start of the incubation period. This image of fibrils is the same as that shown in Extended Data Fig. 1d. c, Representative micrographs of each sample in Panel (a) after the 24-hour incubation period. All scale bars are 200 nm.

Extended Data Fig. 3 Classification of major species of protease-resistant rPrP94–178 fibrils.

a, Final 2D class averages from the major species comprising 71 % of defined segments using a 535 Å box with a 530 Å mask (left) or 428 Å (400 pixel) box with a 400 Å mask (right). b, A composite image (left) formed by stitching of 2D class averages with the small box shown in (a), agrees with a composite image (right) formed by class averages obtained using a box size encompassing a full crossover distance in (a). The crossover distance and full pitch are both marked. c, Comparison between a 2D class (enlarged view of boxed class in (a)), the map backprojection, and model backprojection with accompanying Fourier transforms below. d, Slice through the 3D density with dimensions of the ordered region and surrounding diffuse density noted.

Extended Data Fig. 4 2D classification of minor morphological populations formed by rPrP94–178.

a, Stain-embedded, Proteinase K and sonication treated fibrils show several minor populations of ribbon-like polymorphs (white arrows), including wide filaments with regions that abruptly stop (red arrows). These morphologies remain a minor species after vitrification in 2 % SDS alongside the major twisted species (single example black arrow) (b) A 2D classification round selecting for minor species results in 2D classes that make up 29 % (75,860 segments) of total particles sorted into defined classes (c) with 2.1% of the total segments across major and minor populations (~6000 particles) being sorted into classes that resemble thick ribbons with columns of alternating electron dense and poor material (stars). Additional 2D classification of segments that did not sort into thick ribbon classes (d) revealed several classes containing high-resolution information, in some classes even revealing 4.8 Å strand separation. The 16 best looking classes are shown and make up 62 % (47,479 segments) of the particles shown in (c). 3D reconstruction of these filaments was unsuccessful. A magnified view of select classes from (c) and (d) show the range of fibril widths observed (e) Scalebar in (a) 200 nm, in (b) 50 nm.

Extended Data Fig. 5 Agreement of rPrP106–145 model with core density in rPrPRes.

a, Fourier Shell Correlation (FSC) between half-maps (blue) and the map and model (green) with resolutions at FSC = 0.5 (black dotted line) and 0.143 (gray dotted line) noted. b, Agreement of turn region with density looking down the fibril axis and (c) a side view of the same region shows clear strand separation. d, Overall fit of model into density. e, Magnified view of protofilament interface showing spacing between backbones at the center of the interface is at the most tightly spaced region between G114 of one chain and G119 of its mate. f, Magnified view of linchpin region with inferred salt bridge between residues H111 and D144 that seal off the interior of each chain and a side view (g) of the same region showing 4.8 Å separation between strands.

Extended Data Fig. 6 Structural agreement of hexapeptide prion zipper structures to rPrPRes.

a, ZipperDB26 profile of the segment encoding for rPrPRes. Bars extending below the line represent hexapeptides predicted to form steric zippers. Peptides with crystal structures aligned to the fibril model are boxed. Two of these were previously published27,28. Alignment of crystal structures 119GAVVGG124 (b) 113AGAAAA118 (c) 127GYMLGS132 (e) and 138IIHFGS143 (f) with rPrPRes (d) Symmetry mates in plane, all atom, and backbone (parentheses) RMSD values against rPrPRes are shown (g) Sequence alignment for species of common interest to prion disease. Darker blues correlates with more variability, loosely defined as more residue mismatches among the species compared, and mapping of these residues onto rPrPRes h, Hexapeptide structure for muPrP137–142 (mouse numbering) is compatible with rPrPRes (i) while shPrP138–143 is not (j).

Extended Data Fig. 7 Molecular contacts at the core of rPrPRes.

a, Cartoon representation of one protofilament with three stacked chains highlighting outer (yellow) and inner (blue) sheets. Stick representation of stacked inner (b) and outer (c) sheets with inferred hydrogen bonding networks based on refinement giving weight to standard beta sheet geometry, favored Ramachandran angles, and map to model fit. Residues where geometry deviated during refinement to break backbone hydrogen bonds are noted in blue text. d, Side view highlighting a favorable salt bridge between H111 and D144 and the same region rotated to show the interaction looking down the fiber axis (e).

Extended Data Fig. 8 Alignment of fast-relaxed mutant sequences to rPrPRes.

a, Left: Alignment of eight chains of the model (gray) to the same number of chains of a fast-relaxed model produced in PyRosetta (green) along with magnified views with sidechains in the turn region (center) and the protofilament interface (right). b, Eight chains of fast relaxed G114V mutant (left) along with magnified view of the protofilament interface near the mutation site (red arrow). The same set of three views are also shown for A117V (c) G127V (d) and M129V (e) mutants or polymorphisms.

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Glynn, C., Sawaya, M.R., Ge, P. et al. Cryo-EM structure of a human prion fibril with a hydrophobic, protease-resistant core. Nat Struct Mol Biol 27, 417–423 (2020). https://doi.org/10.1038/s41594-020-0403-y

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