The structure of the infectious prion protein (PrP^Sc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrP^Sc replicates by converting the non-infectious, cellular prion protein (PrP^C) into the misfolded, infectious conformer through an unknown mechanism. PrP^Sc and its N-terminally truncated variant, PrP 27–30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27–30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 ų, predicted the height of each PrP 27–30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding. The structure of the infectious prion (PrP^Sc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its propensity to aggregate. Here, we use the repetitive organization inherent in amyloid fibrils to analyze the structure of GPI-anchorless PrP 27–30 via electron cryomicroscopy. Fourier-transform analysis of averaged fibril segments indicates a repeating unit of 19.1 Å. In agreement with this observation, 3D reconstructions reveal that each fibril contains two distinct protofilaments and that the height of each PrP 27–30 molecule in these fibrils is ~17.7 Å. Together the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. The data conflict with all previous models for the structure of PrP^Sc and allow the formulation of a molecular mechanism for the replication of prions.

Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrP^Sc) of the cellular prion protein (PrP^C), caused epidemic and epizootic episodes [1]. Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-β-Sheet (PIRIBS) [2] or β-solenoid conformations [3]. Similar folding models have also been proposed for PrP^Sc, although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiber-diffraction studies provided evidence that PrP^Sc is structured as a 4-rung β-solenoid (4RβS) [4, 5]. Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrP^Sc, based on the 4RβS architecture. The stability of this new PrP^Sc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring β-solenoid. Importantly, the 4RβS arrangement allowed the first simulation of the sequence of events underlying PrP^C conversion into PrP^Sc. This study provides the most updated, experimentally-driven and physically-coherent model of PrP^Sc, together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions. Prions are unusual infectious pathogens that do not contain any nucleic acid. They consist of assemblies of misfolded proteins. The scrapie isoform of the mammalian prion protein, PrP^Sc, is the most notorious prion, and is responsible for deadly neurodegenerative diseases affecting humans, like Creutzfeldt-Jakob disease, and animals, such as bovine spongiform encephalopathy (“mad cow disease”) and chronic wasting disease affecting elk and deer in North America and, more recently, Europe). Understanding the structure (“shape”) of the PrP^Sc prion is critical to understand how it propagates. We have created a very detailed model of PrP^Sc, which includes all its atoms, using computational techniques. The model resembles a 4-rung cork-screw. The model is stable and agrees with all available experimental data on PrP^Sc. This structure allowed us to model for the first time the process of prion propagation at high resolution. These data inspire several new hypotheses to elucidate prion biology and design therapeutics for prion diseases

PrP^Sc (scrapie isoform of the prion protein) prions are the infectious agent behind diseases such as Creutzfeldt–Jakob disease in humans, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids (deer, elk, moose, and reindeer), as well as goat and sheep scrapie. PrP^Sc is an alternatively folded variant of the cellular prion protein, PrP^C, which is a regular, GPI-anchored protein that is present on the cell surface of neurons and other cell types. While the structure of PrP^C is well studied, the structure of PrP^Sc resisted high-resolution determination due to its general insolubility and propensity to aggregate. Cryo-electron microscopy, X-ray fiber diffraction, and a variety of other approaches defined the structure of PrP^Sc as a four-rung β-solenoid. A high-resolution structure of PrP^Sc still remains to be solved, but the four-rung β-solenoid architecture provides a molecular framework for the autocatalytic propagation mechanism that gives rise to the alternative conformation of PrP^Sc. Here, we summarize the current knowledge regarding the structure of PrP^Sc and speculate about the molecular conversion mechanisms that leads from PrP^C to PrP^Sc.

Elucidation of the structure of PrP^Sc continues to be one major challenge in prion research. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. Given that high resolution techniques such as NMR or X-ray crystallography cannot be used, a number of lower resolution analytical approaches have been attempted. Thus, limited proteolysis has been successfully used to pinpoint flexible regions within prion multimers (PrP^Sc). However, the presence of covalently attached sugar antennae and glycosylphosphatidylinositol (GPI) moieties makes mass spectrometry-based analysis impractical. In order to surmount these difficulties we analyzed PrP^Sc from transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. Such animals produce prions that are devoid of the GPI anchor and sugar antennae, and, thereby, permit the detection and location of flexible, proteinase K (PK) susceptible regions by Western blot and mass spectrometry-based analysis. GPI-less PrP^Sc samples were digested with PK. PK-resistant peptides were identified, and found to correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. The first 10 peptides (to position 153), match very well with PK cleavage sites we previously identified in wild type PrP^Sc. These results reinforce the hypothesis that the structure of PrP^Sc consists of a series of highly PK-resistant β-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrP^Sc is highly resistant to PK and therefore perhaps also contains β-sheet secondary structure.

Understanding the structure of PrP^Sc is without doubt a sine qua non to understand not only PrP^Sc propagation, but also critical features of that process such as the strain phenomenon and transmission barriers. While elucidation of the PrP^Sc structure has been full of difficulties, we now have a large amount of structural information that allows us to begin to understand it. This commentary article summarizes a round table that took place within the Prion 2018 meeting held in Santiago de Compostela to discuss the state of the art in this matter. Two alternative models of PrP^Sc exist: the PIRIBS and the 4-rung β-solenoid models. Both of them have relevant features. The 4-rung β-solenoid model agrees with experimental constraints of brain derived PrP^Sc obtained from cryo-EM and X-ray fiber diffraction studies. Furthermore, it allows facile accommodation of the bulky glycans that decorate brain-derived PrP^Sc. On the other hand, the infectious PrP23-144 amyloid exhibits a PIRIBS architecture. Perhaps, both types of structure co-exist.