A structural basis for prion strain diversity

Abstract

Recent cryogenic electron microscopy (cryo-EM) studies of infectious, ex vivo, prion fibrils from hamster 263K and mouse RML prion strains revealed a similar, parallel in-register intermolecular β-sheet (PIRIBS) amyloid architecture. Rungs of the fibrils are composed of individual prion protein (PrP) monomers that fold to create distinct N-terminal and C-terminal lobes. However, disparity in the hamster/mouse PrP sequence precludes understanding of how divergent prion strains emerge from an identical PrP substrate. In this study, we determined the near-atomic resolution cryo-EM structure of infectious, ex vivo mouse prion fibrils from the ME7 prion strain and compared this with the RML fibril structure. This structural comparison of two biologically distinct mouse-adapted prion strains suggests defined folding subdomains of PrP rungs and the way in which they are interrelated, providing a structural definition of intra-species prion strain-specific conformations.

Fig. 1. ME7 fibril morphologies and the atomic structure of their constituent PrP subunit.

a, Representative cryo-EM image from a dataset comprising 6,370 multi-frame movies (300-kV FEI Krios G3i, K3 camera) showing examples of single ME7 protofilaments (s) alongside their paired assemblies (p) with their approximate diameters. b, Protein-only density of a single amyloid rung (pink) with the fitted atomic model of the mouse PrP chain shown with sticks colored by heteroatom: C, white; N, blue; O, red; S, yellow. The start (T94) and end (R229) residues of the fitted polypeptide and both N-glycosylation sites are indicated.

Fig. 2. Comparison of ME7 and RML protofibrils and assignment of folding subdomains in their core.

a, Rendered density views of the helix crossover or half-pitch (180° helical turn) distance, with annotated locations of PTA polyanions (semi-transparent white), N196 (yellow) and GPI anchor (blue). b, Density cross-sections with overlaid PrP backbone models colored by relative deviation based on global 3D alignment (UCSF Chimera; d), with annotations corresponding to a. C-α atoms of both N-glycosylation sites (N180 and N196) are marked with yellow circles. c, Diagrams of the PrP subunits with approximate dimensions of the inter-lobe grooves and the longest C-α distances in each model, measured between the indicated C-α atoms (dotted lines). Positions of amino acid side chains are shown with circles (positively charged, blue; negatively charged, red; neutral, green; hydrophobic, white; aromatic, gray) on either side of the backbone (black line). β-strands are indicated with thick black arrowheaded lines. d, Top, superposition of PrP backbones colored as in b and shaded according to the folding subdomain assignment. N-glycosylation sites are marked with yellow circles. PK-resistant core starts with residue 89. Bottom, mouse PrP sequence from the start of the amyloid core (T94) to the C-terminus (S230-GPI anchor), with color-coded folding subdomain assignment. Secondary structure annotation for PrP^C (gray), ME7 fibril (magenta) and RML fibril (green) is included below the sequence (α-helix, zig-zag; β-sheet, arrow; disordered, undulated dashed line). Start and end residues are numbered, and the β2-α2 region of PrP^C is indicated. N-glycosylation sites (N180 and N196) are marked with yellow circles.

Fig. 3. Hydrophobic and polar domains of mouse prion protofibrils and details of their PIRIBS arrangements.

Models of three consecutive PrP rungs shown as: a, solvent-excluded surface colored by hydrophobicity and by electrostatic charge distribution (ChimeraX), and hydrophobic clusters are labeled 1–5; b, ribbons with secondary structures and solvent-excluded surface models colored by chain.

Fig. 4. Lateral interactions stabilizing mouse prion fibrils.

Depiction of polar and non-polar lateral contacts. Protein backbone is shown with cartoon (licorice) representation, amino acid side chains as white sticks colored by heteroatom (O, red; N, blue; S, yellow) and selected hydrogen bonds as black lines. The hydrophobic patches are numbered 1–5.

Fig. 5. Summary of structural differences between ME7 and RML prion protofibril structures.

a, Licorice backbone models (ChimeraX) of ME7 and RML protofibrils colored according to PrP^Sc folding subdomains (blue, CC region; red, CV region; yellow, DS hairpin; white, intervening regions). Positive charge of the major basic patch (transparent surface) is indicated with + signs, and the sialoglycan occupancy corresponds to the intensity of red shading. The N-glycosylation sites (N180 and N196 side chains) and the disulphide bond are shown as sticks and colored by heteroatom (C, yellow; N, blue; O, red; S, yellow). b, Alignment of PrP sequences from different mammals focused on the PrP^Sc folding subdomains. Human residues 127 and 129 in the CV region (highlighted with dark red) are polymorphic (G/V and M/V, respectively).

Extended Data Fig. 1. Silver-stained SDS-PAGE (left) and western blot (right) of purified ME7 and RML rods.

Labelling shows the migration positions of di-, mono- and non-glycosylated PrP. The samples were prepared as described in Methods. Uncropped and unprocessed SDS-PAGE and western blot data are provided in a Source Data file. Findings are representative of ~30 purifications. Source data

Extended Data Fig. 2. Comparisons between cryo-EM maps and atomic models of mouse prion fibrils.

a Density cross-sections (pixel size: RML, 1.067 Å; ME7, 0.828 Å) showing the protein core and the non-protein extra densities in the final cryo-EM map. b Top views of rendered reconstructions with indicated common folding sub-domains. c backbone alignments of the common folding sub-domains and the root mean square deviations (RMSD) between all of their respective atom pairs, as calculated using UCSF Chimera. CC, conformationally conserved; CV, conformationally variable; DS, disulphide-stapled.

Extended Data Fig. 3. Comparison of rodent prion structures.

Backbones of the three structures are aligned on the conformationally conserved (CC) region in the N-terminal lobe (MERGE, dimmed beyond the CC region). The C-terminal lobe of mouse prion strains has divergent orientations in line with differences in the N-terminal lobe’s conformationally variable (CV) region that interfaces with the C-terminal lobe. The C-terminal lobe of the hamster strain diverges further from the mouse strains due to additional differences in its primary structure (PrP sequence). The 165-176 region, which corresponds to the β2-α2 loop in PrP^C, is part of the inter-lobe interface in all three rodent prion fibril structures.