Structures and Activities of Prions and Prion Proteins

National Institute Of Allergy And Infectious Diseases

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Abstract

CWD prion structure: In a given type of mammalian host, prion strains are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. We previously determined the near-atomic structures of 3 rodent prion strains. This fiscal year we determined the structure of the first naturally occurring prion, isolated from the brain of a chronic-wasting disease (CWD)-infected Montana deer. CWD is a widely distributed prion disease of cervids with implications for wildlife conservation and also for human and livestock health. We determined and published a 2.8 Å resolution cryogenic electron microscopy-based structure of CWD prion fibrils from a white-tailed deer expressing the most common wildtype PrP sequence. Like recently solved rodent-adapted scrapie prion fibrils, our atomic model of CWD fibrils contains single stacks of PrP molecules forming parallel in-register intermolecular β-sheets and intervening loops comprising major N- and C-terminal lobes within the fibril cross-section. However, CWD fibrils from a natural cervid host differ markedly from the rodent structures in many other features, including a ~180° twist in the relative orientation of the lobes. This CWD structure suggests mechanisms underlying the apparent CWD transmission barrier to humans and should facilitate more rational approaches to the development of CWD vaccines and therapeutics. Spontaneous prion disassembly? Infectious prion assemblies must fragment to replicate, spread, and trigger disease. However, the extent to which various types of amyloid fibrils fragment on their own versus being driven by other cellular processes is unclear. In the case of highly infectious, tissue-derived prion (PrPSc) preparations, over 40 years of previous studies have yielded starkly contradictory indications on this question. Many have reported high stability of PrPSc multimers in even strong detergents. However, others using non-disinfecting detergents and size exclusion chromatography combined with light scattering measurements, have described complete spontaneous disassembly into dimeric-tetrameric units. In attempting to replicate the latter experiments, we determined that PrPSc size exclusion elution behavior was dominated by binding to the column matrix, not particle size. The light scattering behavior of fractions containing PrPSc was dominated by the co-elution of detergent micelles similar in size to hypothetical PrPSc dimers-trimers. Furthermore, sedimentation velocity centrifugation and electron microscopy indicated that most detergent-treated PrPSc particles remained larger than 70-mers. When added to live cells that lacked PrPC and were therefore incapable of new PrPSc assembly, most PrPSc remained in the form of large multimers for ≥24 h, confirming substantial stability in a cellular model. Thus, we found no evidence that the much larger assemblies that predominate in brain homogenates or purified PrPSc preparations fragment spontaneously into small oligomers. Moreover, our identification of prion-associated size exclusion chromatography artifacts reconciles previously disparate reports about prion disassembly. Molecular dynamics of small prion oligomers: In many proteinopathies, the relative conformations of amyloid fibrils versus smaller oligomers remain unclear. Most tissue-derived isolates of infectious prion protein (PrP) prions are predominantly fibrillar. As noted above, a few studies from others have asserted that prion amyloid fibrils efficiently disassemble into dimeric to tetrameric “elemental bricks” under certain detergent or chaotropic conditions, but our work described in the section above provided strong evidence to the contrary. Given our difficulties in isolating detectable amounts of small oligomeric (2-4-mer) prions, we performed molecular dynamics simulations to test the abilities of small fragments (dimers to 25-mers) of cryo-EM-based infectious prion fibril core structures to retain their conformational integrity. We showed that dimers of the aRML prion structure lost most of their original secondary and tertiary structure within <<1 ms, while trimers maintained some intermolecular beta sheets. Further increases in fragment size helped preserve major structural motifs and the integrity of the templating surfaces responsible for self-propagation. In simulations of octamers and/or 25-mers, even at elevated temperatures, no fragmentation was observed for either the aRML, 22L, or 263K prion strains, although the terminal chains were substantially destabilized. Together, our results provided evidence that oligomeric fragments of prion fibril cores as small as tetramers retain substantial structural integrity. Our findings suggest that, as exemplified by PrP fibrils, short cores as small as tetramers may be stable enough to account for bioactive oligomeric species detected in brain extracts from proteinopathy patients. However, the lack of observed spontaneous core fragmentation suggests that prion oligomers might be rare in vivo and/or produced by non-autonomous physiological cleavage processes.