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Cryo-EM of prion strains from the same genotype of host identifies conformational determinants [1]

['Forrest Hoyt', 'Research Technologies Branch', 'Rocky Mountain Laboratories', 'National Institute Of Allergy', 'Infectious Diseases', 'National Institutes Of Health', 'Hamilton', 'Montana', 'United States Of America', 'Parvez Alam']

Date: 2022-12 

Prion strains in a given type of mammalian host are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. Near-atomic structures of prions from two host species with different PrP sequences have been determined but comparisons of distinct prion strains of the same amino acid sequence are needed to identify purely conformational determinants of prion strain characteristics. Here we report a 3.2 Å resolution cryogenic electron microscopy-based structure of the 22L prion strain purified from the brains of mice engineered to express only PrP lacking glycophosphatidylinositol anchors [anchorless (a) 22L]. Comparison of this near-atomic structure to our recently determined structure of the aRML strain propagated in the same inbred mouse reveals that these two mouse prion strains have distinct conformational templates for growth via incorporation of PrP molecules of the same sequence. Both a22L and aRML are assembled as stacks of PrP molecules forming parallel in-register intermolecular β-sheets and intervening loops, with single monomers spanning the ordered fibril core. Each monomer shares an N-terminal steric zipper, three major arches, and an overall V-shape, but the details of these and other conformational features differ markedly. Thus, variations in shared conformational motifs within a parallel in-register β-stack fibril architecture provide a structural basis for prion strain differentiation within a single host genotype.

Prions are protein-based pathogens that can spread within and between hosts without carrying a pathogen-specific nucleic acid genome. Given this protein-based propagation mechanism, a long-standing mystery in the prion disease field has been the molecular basis of distinct, faithfully propagating strains in a single type of mammalian host. Here we provide a direct, high-resolution cryo-EM-based comparison of the structures of two highly infectious prion strains isolated from the brains of mice of a single genotype. We show in detail how these two prion strains are protein filaments of mouse PrP molecules that display distinct conformational templates for growth on their tips. Our results identify purely conformational, rather than sequence-based, underpinnings of infectious and deadly prion strains.

Funding: This work was supported by Intramural Research Program, NIAID, NIH [Project ZIAAI000580-22 (BC) and core funding for the Research Technologies Branch]. All authors, except A.K., received salary from the NIAID. A.K. was supported by Case Western Reserve University, the Britton Fund, and the Clifford V. Harding and Mina K. Chung Professorship in Pathology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Introduction

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal infectious neurodegenerative diseases including CJD (Creutzfeldt-Jakob disease) in humans, bovine spongiform encephalopathy in cattle, scrapie in sheep, and CWD (chronic wasting disease) in cervids [1,2]. Neuropathological hallmarks of prion diseases include spongiform change, neuronal loss, astrocytosis, and accumulation of pathologic forms of the hosts’ prion protein (PrP) that, when infectious, have generically been termed PrPSc for PrP-scrapie [3]. The normal cellular form of PrP (PrPC) typically exists as a glycosylated, glycophosphatidylinositol (GPI)- linked, membrane bound monomer with a largely α-helical C- terminal domain and disordered N- terminal domain [4]. In contrast, PrPSc is multimeric and when purified, at least, usually takes the form of amyloid fibrils with ordered cores that are high in intermolecular β-sheets and loop structures [5–7]. This aggregated form of PrP is able to grow by refolding PrPC and incorporating it into an ordered, transmissible assembly.

Given the protein-based mechanism of prion propagation, and the fact that prions do not carry their own mutable nucleic acid genome from host-to-host, one of the most intriguing and long-standing questions in prion biology has been the molecular basis of prion strains. Prion strains are infectious isolates that when transmitted to a given type of host exhibit characteristic clinical phenotypes, neuropathological lesions, survival times, and PrP deposition patterns [8]. Many low-resolution biochemical and spectroscopic comparisons of preparations of different PrPSc strains have provided evidence of distinct conformations even when strains are formed from the same polypeptide sequence (e.g. [2,9–17]). Moreover, PrPSc strains have been shown to impose their general conformational attributes onto newly recruited PrP molecules in cell-free conversion and amplification reactions [9,18]. Accordingly, consistent prion strain propagation has been postulated to involve conformational templating by PrPSc [9,11].

In accordance with this concept are comparisons of the first high-resolution cryo-electron microscopy (cryo-EM) structures of fully infectious brain-derived PrPSc prions, namely the hamster 263K [6], and mouse wildtype (wt) RML [7] and GPI-anchorless (a) RML [5] prions. Each is an amyloid fibril with a parallel in-register intermolecular β-sheet (PIRIBS)-based core containing amino-proximal (N), middle, and disulfide arches, as well as a steric zipper that holds the extreme N-terminal residues of the core against the head of the middle arch. (Note: we have taken to using the term arch, instead of β-arch, because some cases do not fully meet the β-arch (or arc) criterion of having β-strands on both sides of the arch that interact via their sidechains). However, while wtRML and aRML prion structures are quite similar to one another, they differ markedly from the 263K structure in their conformational details [5–7]. Importantly, the RML and 263K structures also differ in amino acid sequences at 8 residues within their ordered amyloid cores, raising questions about the contributions of sequence differences to their respective templating activities and species tropisms.

To identify purely conformational determinants of prion strains, high-resolution comparisons of strains propagated in hosts of the same genotype are needed. Here we address this central question in prion biology by providing a high-resolution structure of the a22L prion strain isolated from a GPI-anchorless PrP-expressing transgenic mouse strain and compare it to our previously determined structure of the aRML strain propagated in the same genotype of mouse [5]. Previous studies have already suggested conformational differences between these two prion strains. For example, Sim et al reported the differences in ultrastructure of a22L and aRML using TEM and AFM [19]. Comparison of HDX- M/S data for a22L and aRML by Smirnovas et al showed localized differences in protection against deuterium exchange within their amyloid cores. a22L and aRML also differ in their infrared spectra, most notably in the β-sheet region of the amide I region [15]. Also suggestive of distinct conformations was a report from Bett et al that polythiophene acetic acid (PTAA) emission spectra from a22L were more red-shifted than those of aRML [20]. They also found that a22L is more resistant to proteinase K (PK) digestion than aRML. In terms of neuropathology, wildtype 22L PrPSc deposition occurs mainly in astroglia in several brain regions during early stages of infection while RML PrPSc associates with astroglia in the thalamus, cortex, as well as in neurons and neuropil of the substantia nigra and hypothalamus [21]. Detailed knowledge of the conformations of these prion strains should provide a foundation for understanding their respective pathophysiological mechanisms. Our current study specifies, with near atomic resolution, how the a22L strain conformation differs from that of the aRML and wtRML strains of the same murine PrP sequence. These findings reveal purely conformational bases for prion strain differentiation.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010947

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