Prion strains viewed through the lens of cryo-EM

Abstract

Mammalian prions are lethal transmissible pathogens that cause fatal neurodegenerative diseases in humans and animals. They consist of fibrils of misfolded, host-encoded prion protein (PrP) which propagate through templated protein polymerisation. Prion strains produce distinct clinicopathological phenotypes in the same host and appear to be encoded by distinct misfolded PrP conformations and assembly states. Despite fundamental advances in our understanding of prion biology, key knowledge gaps remain. These include precise delineation of prion replication mechanisms, detailed explanation of the molecular basis of prion strains and inter-species transmission barriers, and the structural definition of neurotoxic PrP species. Central to addressing these questions is the determination of prion structure. While high-resolution definition of ex vivo prion fibrils once seemed unlikely, recent advances in cryo-electron microscopy (cryo-EM) and computational methods for 3D reconstruction of amyloids have now made this possible. Recently, near-atomic resolution structures of highly infectious, ex vivo prion fibrils from hamster 263K and mouse RML prion strains were reported. The fibrils have a comparable parallel in-register intermolecular β-sheet (PIRIBS) architecture that now provides a structural foundation for understanding prion strain diversity in mammals. Here, we review these new findings and discuss directions for future research.

Keywords: Cryo-EM; Prion; Prion disease; Prion strains; Prion structure.

Fig. 1

Prion protein conversion into infectious prion fibrils. a, b Atomistic model of mature mouse PrP^C (residues 23–230), including post-translational modifications (carbohydrate groups, pink; sialic acid groups, red), anchored in the phospholipid bilayer (coloured by heteroatom: C, white; P, orange; N, blue; O, red). Proteinase K (PK)-sensitive region (residues 23–89, light grey) refers to PK-sensitivity after conversion to prion fibrils (see panel d). The model was built in CHARMM-GUI (https://www.charmm-gui.org/) (Jo et al. 2008) and UCSF Chimera (Pettersen et al. 2004), using a solution NMR structure of the GPI-anchor from human complement regulatory protein CD59 (pdb ID: 1CDR) (Fletcher et al. 1994), an X-ray structure of the mouse PrP (pdb ID: 4H88) (Sonati et al. 2013), and X-ray structures of tri-antennary N-linked sialylated glycans from human prostate specific antigen glycoprotein (pdb ID: 3QUM) (Stura et al. 2011). The close-up view (b) shows the portion of the PrP^C chain (ribbon representation) that contributes specific sub-domains in PrP^Sc, as indicated with distinct colours. Selected secondary structures are labelled. c Mouse PrP sequence with colour-coded prion fibril PrP^Sc sub-domain ranges. PK-resistant RML fibril core (panel d, top; residues 89–230) includes amyloid core (residues 94–225; grey highlight). N180 and N196 glycosylation sites are numbered in red. d RML (pdb ID: 7QIG) (Manka et al. 2022b) and 263K (pdb ID: 7LNA) (Kraus et al. 2021) PrP^Sc fibril structures (3 subunits, ribbon representation) coloured as in (a–b). N- and C-terminal flexible tails (residues 89/90–93/94 and 226/227–230/231, mouse/hamster numbering) have been added to the models, together with post-translational modifications. The 263K model has additional residues 94–96 modelled at the tips of the C-terminal lobe hairpins, due to their absence in the original structure. Major internal hydrophobic clusters that contribute to fold stability (1–6) are shown with surface representation. Mouse-to-hamster substitutions in PrP sequence are indicated in the 263K structure (hamster numbering). S–S, disulphide bond

Fig. 2

PrP chain conformations in recombinant and ex vivo PrP fibrils a–c PrP folds and protofilament pairing interfaces in fibrils grown in vitro from recombinantly expressed PrP substrates (human sequence): a 106–145 9.7 kDa fragment (M129 variant) (Glynn et al. 2020); b 23–231 (full-length) PrP (Wang et al. 2020) with schematically indicated theoretical sialoglycan occupancy (red cones), as modelled by Artikis et al. (2020); c full-length PrP with familial prion disease-related mutation E196K (Wang et al. 2021). d RML protofibril (Manka et al. 2022b). e 263K protofibril (Kraus et al. 2021). Structures in all panels (a–e) represent fibril cores with species-specific numbering of start and end residues. PrP subunits are shown predominantly as backbones coloured according to ex vivo fibril sub-domain assignment defined in Fig. 1. Residues involved in PrP protofibril pairing (contributing to experimentally confirmed inter-protofilament interfaces) are shown with side chains coloured by heteroatom (N, blue; O, red; S, yellow; C, as backbone) and salt bridges or hydrogen bonds stabilising the two-protofilament architecture are indicated with black lines. Selected interfacing and glycosylated residues are labelled. S–S, disulphide bond