Abstract The structure of the infectious prion protein (PrPSc), 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. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc 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 Å3, 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.

Author Summary The structure of the infectious prion (PrPSc), 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 PrPSc and allow the formulation of a molecular mechanism for the replication of prions.

Citation: Vázquez-Fernández E, Vos MR, Afanasyev P, Cebey L, Sevillano AM, Vidal E, et al. (2016) The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy. PLoS Pathog 12(9): e1005835. https://doi.org/10.1371/journal.ppat.1005835 Editor: Surachai Supattapone, Dartmouth Medical School, UNITED STATES Received: March 18, 2016; Accepted: August 1, 2016; Published: September 8, 2016 Copyright: © 2016 Vázquez-Fernández et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Most relevant data are contained within the paper and its Supporting Information files. Additional raw data (electron micrographs) and a 3D reconstruction were deposited in figshare and are freely available under: https://dx.doi.org/10.6084/m9.figshare.c.3292481. Funding: This project has been supported by grants from the Alberta Prion Research Institute / Alberta Innovates Bio Solutions (201100010; 201100011; 201300012; 201300024), the Alberta Livestock & Meat Agency (2012A001R), the Canada Foundation for Innovation (NIF 21633 and IOF 21633 awards to D. Westaway), the European Commission grant FP7 222887 "Priority", a Spanish Ministry of Education grant (BFU2006-04588/BMC), and Spanish Ministry of Economy and Competitiveness grants (BFU2013-48436-C2-1-P & TIN2012-37483-C03-02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: MRV is an employee of FEI Company (Eindhoven, The Netherlands), this does not alter our adherence to all PLoS Pathogens policies on sharing data and materials.

Introduction Little is known about the structure of the infectious prion protein, the infectious agent causing prion diseases such as sheep and goat scrapie, bovine spongiform encephalopathy or “mad cow disease”, chronic wasting disease in cervids (deer, elk, moose, and reindeer), and Creutzfeldt-Jakob disease in humans. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. The non-infectious, cellular prion protein (PrPC), which has its highest expression levels in neurons, is misfolded through a posttranslational process into an altered, infectious conformer termed PrPSc or prion [1]. The structure of recombinant PrP, which approximates the structure of PrPC, has been solved repeatedly by NMR spectroscopy [2] and X-ray crystallography [3]. PrPC consists of an unfolded N-terminal domain and a largely α-helical C-terminal domain, which contains three α-helices and a short, two-stranded ß-sheet [2,3]. In contrast, PrPSc has been found by a variety of methods to contain predominantly ß-sheet structure [4]. PrPSc and its N-terminally truncated variant, PrP 27–30, are generally insoluble and prone to aggregation into an assortment of quaternary structures, such as amorphous aggregates, 2D crystals, and amyloid fibrils. None of these aggregation products are amenable to conventional structural analysis techniques such as X-ray crystallography or solution NMR spectroscopy. To overcome the paucity of experimental data on the structure of the infectious prion, molecular modeling has been used by a variety of researchers to predict its structure. Little agreement exists among the published molecular models regarding the nature of the infectious conformer and a large range of different folds have been put forward [5], with the most recent entry proposing a parallel in-register ß-sheeted structure [6]. While none of the published models satisfy all experimental restraints [5], a ß-helical architecture has been suggested as a likely candidate for the structure of the infectious prion [7,8]. To investigate the structure of the infectious prion, we used electron cryomicroscopy (cryo-EM) to record and analyze the structure of brain-derived, murine prion protein amyloid. Brain-derived PrPSc has a high level of molecular heterogeneity due to its GPI-anchor and N-linked carbohydrates, which increases the difficulty to analyze its structure considerably. On the other hand, brain-derived PrPSc, as the disease-relevant conformer, can provide insights into the infectious state, which the more well-behaved, recombinantly-derived PrP amyloid, cannot [9]. By using transgenic mice expressing a GPI-anchorless form of the prion protein, which is also substantially underglycosylated [10], a more homogeneous version of the prion protein could be analyzed. GPI-anchorless PrPSc is deposited predominantly as large fibrillar amyloid plaques, which allows for a milder purification procedure compared to traditional purification approaches [11]. In prion-infected mice expressing GPI-anchorless PrP, PrPSc retains its full infectivity, while the neuropathology is similar to a cerebral amyloid angiopathy (CAA) as seen in Alzheimer’s disease [12] and hereditary prion protein amyloidosis [13,14]. This difference in apparent neuropathology is not surprising given that infectivity and toxicity of PrPSc are not unequivocally linked [15]. After all, GPI-anchorless PrPSc cannot attach to the cell membrane, and therefore exhibits a different cellular and tissue distribution. Nonetheless, the homogeneous preparation of GPI-anchorless, infectious prions facilitated novel insights into the structure of PrPSc by cryo-EM.

Discussion Our cryo-EM images of GPI-anchorless PrPSc fibrils, and their subsequent analysis, show that they consist of two intertwined protofilaments, in agreement with a recent study of negatively stained PrPSc fibrils [35]. Each protofilament exhibits an approximately ellipsoidal cross-section with a linear volume of ~9,900 Å3/nm. Given the fact that cryo-EM preserves the native structure of specimens, this information sets a structural restraint for the conformation of GPI-anchorless PrPSc. One important implication is that PrPSc subunits can only fit into protofilaments with the observed dimensions (Table 1), if they are folded up onto themselves. Based on the routine observation of regular 4.8 Å cross-ß signals in individual GPI-anchorless PrP 27–30 fibrils (Fig 3), a ß-solenoid arrangement is the easiest way to accommodate the peptide into the available protofilament volume. This arrangement would result in 4 x 4.8 Å (~19 Å) repeats along the protofilament axis, which is exactly what our two independent single particle analyses revealed (Figs 5 and S5), together with an additional ~40 Å signal that likely corresponds to a vertical pairing of two PrPSc subunits, as seen in HET-s prion fibrils [30]. Therefore, our cryo-EM data revealed a ß-solenoid architecture as the basic element for the structure of the mammalian prion GPI-anchorless PrPSc (Fig 8), which is in agreement with previous results obtained by X-ray fiber diffraction for other prions such as RML and Sc237 PrP 27–30 [25]. As an important corollary, our cryo-EM data are incompatible with models based on alternative architectures, such as the parallel in-register ß-sheet fold, which is based on a single, superpleated protofilament with a molecular height of only 4.8 Å [6]. It is noteworthy that a β-solenoidal architecture has been demonstrated, as already mentioned, for the HET-s prion [30,36], and has been proposed for insulin [31] and SH3 amyloid [37] fibrils based on cryo-EM data. While important structural elements still need to be defined, such as which residues participate in the ß-strands that form each solenoid rung, and which ones are located in turns and connecting loops, what we have learned about the structure of GPI-anchorless PrP 27–30 and its four-rung ß-solenoid architecture, allows us to extrapolate about possible templating mechanisms that control the replication of infectious prions in vivo. In-phase stacking of identical residues along the peptide chain, as was proposed by the parallel in-register ß-sheet model [6], can be ruled out, as mentioned above, due to the experimentally determined height constraints. Therefore an alternative templating mechanism must explain the replication of prions in general, and the fidelity required for transmitting distinct prion strains in particular [19]. Templating based on a four-rung ß-solenoid architecture must involve the upper- and lowermost ß-solenoid rungs. These edge strands are inherently aggregation-prone, as they are predestined to propagate their hydrogen-bonding pattern into any amyloidogenic peptide they encounter [38]. In fact, the ß-strands of native proteins that contain a ß-solenoid are capped by loops and other structures to block unregulated propagation of ß-sheets. Furthermore, the elimination of the capping structures results in edge-to-edge-driven oligomerization of the "de-capped" ß-solenoids [39]. Thus, it is easy to conceive that these upper and lower ß-solenoid rungs can template an incoming unfolded PrP molecule to create additional ß-solenoid rungs. It is noteworthy that the molecular forces responsible for the templating − hydrogen-bonding, charge and hydrophobic interactions, aromatic stacking, and steric constraints − are fundamentally similar to those operating during DNA replication. Obviously, the exquisite specificity of the A:T and G:C pairings is lacking and instead, a much more complex array of forces controls the pairing of the pre-existing and nascent ß-rungs. Once an additional ß-rung has formed, it creates a fresh "sticky" edge ready to continue templating until the incoming unfolded PrP molecule has been converted into another copy of the infectious conformer. Furthermore, the stacking of GPI-anchorless PrP 27–30 molecules into amyloid fibrils, or, in other words, the way in which templating occurs, is either based on a) a head-to-tail orientation resulting in amyloid fibrils with intrinsic polarity (Fig 8), or b) a head-to-head and tail-to-tail orientation, which would result in generally apolar amyloid fibrils. In the former case, templating of ß-sheets would involve direct contact between different parts of the molecule, i.e. heterotypic templating. In the latter case, the same protein stretches will come into contact principally, but homotypic templating would result in two PrPSc molecules with opposite handedness. Alternatively, a head-to-head arrangement could also rely on heterotypic templating, if different parts of the molecule interact with each other. Experimentally, we observed a ~40 Å signal in Fourier-transform analyses of the fibril segments (Figs 5, S4 and S5) that may originate from a dimeric arrangement. The presence of a distinct dimer signal supports a head-to-head and tail-to-tail arrangement, but, ultimately, higher resolution data are needed to distinguish between these dimer options. In summary, we present data based on cryo-EM analysis that strongly support the notion that GPI-anchorless PrPSc fibrils consist of stacks of four-rung ß-solenoids. Two of such protofilaments intertwine to form double fibrils, in agreement with a recent report based on tomography of negatively stained PrPSc samples [35]. The four-rung ß-solenoid architecture of GPI-anchorless PrPSc provides unique and novel insights into the molecular mechanism by which mammalian prions replicate.

Materials and Methods Animals Heterozygous GPI-anchorless PrP transgenic mice (tg44+/-) were developed by Dr. Bruce Chesebro (NIH Rocky Mountain Laboratories, Hamilton, MT, USA) [10]. The mice were crossed to generate homozygous GPI-anchorless PrP animals (tg44+/+) [16] and genotyped by tail DNA analysis using the PCR protocol described in Chesebro et al. [10]. Prion infection of mice Homozygous GPI-anchorless PrP transgenic mice (tg44+/+) [10,16] were inoculated intra-cerebrally (IC) in the right temporal lobe, at 6 weeks of age, with 20 μl of a 2% RML-infected mouse brain homogenate (BH), prepared in 2X (w/v) PBS. After 365 days post inoculation, the asymptomatic mice were euthanized, the brains were harvested, rinsed in PBS, and stored at -80°C until use. For the bioassays two groups of six wild-type C57BL/6 mice were inoculated IC with either 20 μl of a 2% GPI-anchorless RML prion BH or with an equivalent amount of purified GPI-anchorless PrP 27–30 resuspended in 5% (w/v) glucose in 2X PBS. Also a new cohort of six wild-type mice was inoculated IC with 20 μl 2X PBS as negative control. Mice were monitored until the appearance of clinical signs, at which time they were euthanized and the brains were removed. Ethics statement Animal experiments were carried out in accordance with the European Union Council Directive 86/609/EEC, and were approved by the University of Santiago de Compostela Ethics Committee (protocol 15005AE/12/FUN 01/PAT 05/JRR2). Statistical analysis The Kaplan-Meier plot survival analyses of the wild-type mouse groups inoculated with GPI-anchorless RML prion BH and purified GPI-anchorless PrP 27–30 were compared using the Gehan-Breslow-Wilcoxon test. Immunohistochemistry Immediately after extraction, the brain was fixed in formalin and then sliced into four transversal sections by cutting the brain caudally and rostrally to the midbrain and at the level of the basal nuclei. The sections were dehydrated by immersion in solutions of progressively higher ethanol concentration and, finally, with xylene before being embedded in paraffin. Haematoxylin-eosin was used to stain 4 μm thick sections. Additional sections were mounted on 3-triethoxysilyl-propylamine-coated glass slides for immunohistochemical (IHC) studies. IHC for the detection of PrPSc was performed as follows. Deparaffinized sections were subjected to epitope unmasking treatments: Immersed in formic acid and boiled at low pH (6.15) in a pressure cooker and pre-treated with proteinase K. Endogenous peroxidases were blocked by immersion in a 3% H 2 O 2 in methanol. Then, the sections were incubated overnight with anti-PrP mAb 2G11 primary antibody (1:100, kindly supplied by Dr. Eoin Monks, University College Dublin, Ireland) and subsequently visualized using the Dako EnVision system K400111/0 (Dako, Glostrup, Denmark) and 3,3’diaminobenzidine as the chromogenic substrate. Additional sections were incubated with a rabbit polyclonal antibody against glial fibrillary acidic protein Z0334, 1:600 (Dako, Glostrup, Denmark) to visualize astrocytic activation. For the glial fibrillary acidic protein detection epitope unmasking treatments were omitted, but the same visualization system was used. As a background control, the incubation with the primary antibodies was omitted. Protein purification GPI-anchorless PrP 27–30 was isolated using a slightly modified version of the method of Baron et al. [11]. During the purification, total PrPSc was treated with 10 μg/ml of proteinase K (PK) at 37°C for 1 h. PK treatment yielded a shortened, protease-resistant form termed PrP 27–30, due to its similarity to GPI-anchored PrP 27–30, which has an apparent molecular mass of 27–30 kDa [40]. The final GPI-anchorless PrP 27–30 pellet was resuspended in 100 μl of deionized water and treated with lipase (porcine pancreas lipase, Sigma No. 62300) at 1 μg/ml for 2 h at 37°C. To trap the fatty acids released by the lipase, bovine serum albumin (BSA) was added to a final concentration of 10 mg/ml. The sample was centrifuged at 22,000 g for 20 min, twice, and the pellet containing the PrP 27–30 fibrils was resuspended in 100 μl of deionized water. Finally, fibrils were sonicated with three pulses at a 50% amplitude with a probe ultrasonic homogenizer (Cole Parmer Instrument CO., Chicago, IL, USA), and the purified protein was stored at 4°C or -80°C. The sample purity was assessed by SYPRO Ruby Protein Gel Stain. The yield of GPI-anchorless PrP 27–30 was ~35 μg per mouse brain (BCA protein assay [41]). Immunoblotting and SYPRO Ruby protein gel stain SDS-PAGE was performed in 15% polyacrylamide gels. For Sypro Ruby staining, the gel was washed with ultrapure water, fixed for 1 hour with 10% methanol and 7% acetic acid, followed by overnight incubation with Sypro Ruby solution (Lonza, Rockland, ME, USA) at room temperature with gentle agitation and protection from light. For immunoblotting, the gel was transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA, USA) and probed with the 3F10 antibody (recognizing residues 137–151) at a 1:5,000 dilution. Peroxidase-labeled anti-mouse antibody was used as the secondary antibody at a 1:5,000 dilution. Electron cryomicroscopy Samples were prepared by pipetting a 3 μl sample, mixed with a gold fiducial solution (15 nm gold particle size), onto a freshly glow-discharged Lacey carbon grids (Ted Pella Inc., Redding, CA, USA). The grids were plunge-frozen in liquid ethane in a Vitrobot Mark IV (FEI, Eindhoven, The Netherlands). Cryo-EM data were collected using a Titan Krios microscope equipped with Falcon II direct electron detector (FEI, Eindhoven, The Netherlands), operated at 300 kV. Low-dose imaging conditions with 20 electrons per Å2 were applied. Images were collected at 1–3 μm underfocus. All micrographs were recorded at a pixel size of 1.34 Å per pixel. Tilt series data were obtained on a Titan Krios (FEI, Eindhoven, The Netherlands), operated at 300 kV on a Falcon II direct electron detector (FEI, Eindhoven, The Netherlands), under low-dose conditions with 50 electrons per Å2. Tomograms of selected areas were obtained from -70 to +70 degrees with a 1.5° tilt increment. The defocus range of the data set was 5–8 μm, with a pixel size of 6 Å. The tilted images were aligned and reconstructed with both the FEI Inspect3D software (FEI, Eindhoven, The Netherlands) and with Tomo3D [42] and the SIRT reconstruction method (30 iterations). Measurement of the fibril width 261 fibril widths were obtained from 9 different reconstructed tomograms. Measurements were determined using ImageJ software, following the “Measuring distances between points” manual. Image processing and data analysis Single particle analysis: Only cryo-EM images presenting clear Thon rings were used for processing. The CTF was determined by CTFFIND3 [43] and phases were corrected with Bsoft [44]. Straight fibril segments were extracted manually from 400 micrographs using EMAN’s boxer program [45]. A total of 1305 non-overlapping segments were picked with a box size of 200 x 200 pixels (26.8 x 26.8 nm). In order to align the segments along the y-axis, a symmetrized average from an iterative alignment using the straightest particle in the set as a reference, was used as a starting template. Subsequently, particles were classified by multivariate statistical analysis implemented in IMAGIC [46]. Also, the aligned particles were cut into single protofilaments, realigned and reclassified, resulting in a new data set of 2610 particles. Finally, the summed amplitude spectra of all class averages was calculated and further analyzed. Reference-free single particle analysis: Image processing was performed with the IMAGIC-4D software [47]. The same 400 selected micrographs (4096 x 4096 pixels) were normalized by a posteriori camera correction to remove camera artifacts [48] (S6 Fig). Amplitude spectra were used to determine the CTF parameters and perform CTF-correction by phase flipping (using CTF2D-FIND and CTF2D-FLIP programs). The particles were picked from the best patches of normalized and band-pass filtered micrographs by the PICK-M-ALL program, using a single featureless rectangular reference created by smearing a filament along its length. From the selected patches containing clear fibrils, the best 2725 particles were selected based on standard statistical analyses (averages, standard deviations, cross correlation coefficients). Multivariate eigenvector data compression was then applied, followed by automatic unsupervised classification, to create 20 classes. No alignments were applied to avoid reference bias. 3D fibril reconstruction: Suitable fibril images were selected from a set of 1284 cryo-EM images. An individual fibril, presenting at least half a helical turn (180°), was segmented along the helical axis using EMAN’s boxer program [45] into overlapping boxes of 300 x 300 pixels (40.2 x 40.2 nm). The boxes were centered and spaced 1–5 pixels apart along the helical axis. By measuring the repeat distance of the helical fibril, we could estimate the angular orientation of each box in the set. The underlying assumption was that each box represented a different view of identically the same fibril, where each view is rotated and translated according to the helical twist. By assigning the angles to each box in the set, a preliminary 3D reconstruction was generated by back projection. Two-fold symmetry was then imposed. The preliminary 3D reconstruction was refined against other 3D reconstructions generated from the same fibril using different step sizes [29]. SPIDER software was used for the reconstruction [49]. In order to remove the rippling artifact created by the overlapping segments, the procedure continued with a second stage, using IMAGIC software [46]. The refined 3D reconstruction was rotated 90° around its x-axis and sliced along the yz plane. The new set of cross-sectional projections was aligned, centered, and averaged. The averaged density was symmetrized, replicated along the fibril length, and in-plane rotation angles were assigned based on the helical twist. Finally, the collection of 2D projections was used to assemble the 3D volume. Reconstructions were visualized in UCSF Chimera [50]. The thresholds for the cryo-EM maps were fixed based on the fibril widths measured in the raw images. The monomer height calculation was accomplished by determining the cross-section area (Å2) of each protofilament in Chimera [50] and then calculating the corresponding volume using an experimentally determined molecular mass for the GPI-anchorless PrP 27–30 of 17,148 Da [18], and a mean protein density of 0.903 Da/Å3; calculated based on a HET-s monomer stacked in a HET-s prion fibril ([30] PDB: EMD-2946).

Acknowledgments The authors would like to thank Dr. Bruce Chesebro (NIH, Rocky Mountain Laboratory, Hamilton, MT, USA) for providing the GPI-anchorless PrP transgenic mice; Dr. Juan María Torres (CISA, INIA, Madrid, Spain) for providing RML prion inoculum, Dr. Leonardo Cortez (CPPFD, University of Alberta, Edmonton, Canada) for advice on statistical analyses, and Dr. David Westaway (CPPFD, University of Alberta, Edmonton, Canada) for his critical reading of the manuscript.

Author Contributions Conceptualization: MRV HSY JRR HW. Data curation: EVF LC LR. Formal analysis: EVF HSY JRR HW. Funding acquisition: JJF JRR HW. Investigation: EVF MRV AMS EV IR AR JRR HW. Methodology: MvH HSY. Project administration: JRR HW. Resources: PJP MvH JRR. Software: PA JJF MvH HSY. Supervision: HSY JRR HW. Visualization: EVF PA JJF. Writing – original draft: EVF JRR HW.