Elongated Oligomers Assemble into Mammalian PrP Amyloid Fibrils

doi:10.1016/j.jmb.2006.01.052

J. Mol. Biol. (2006) 357, 975–985

Elongated Oligomers Assemble into Mammalian PrP Amyloid Fibrils M. Howard Tattum1, Sara Cohen-Krausz 2, Azadeh Khalili-Shirazi1 Graham S. Jackson1, Elena V. Orlova2, John Collinge1 Anthony R. Clarke1 and Helen R. Saibil2* 1

MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College London Queen Square, London WC1N 3BG, UK 2

School of Crystallography and Institute for Structural Molecular Biology, Birkbeck College, London WCIE 7HX UK

In prion diseases, the mammalian prion protein PrP is converted from a monomeric, mainly a-helical state into b-rich amyloid fibrils. To examine the structure of the misfolded state, amyloid fibrils were grown from a b form of recombinant mouse PrP (residues 91–231). The b-PrP precursors assembled slowly into amyloid fibrils with an overall helical twist. The fibrils exhibit immunological reactivity similar to that of ex vivo PrPSc. Using electron microscopy and image processing, we obtained threedimensional density maps of two forms of PrP fibrils with slightly different twists. They reveal two intertwined protofilaments with a subunit repeat of ˚ . The repeating unit along each protofilament can be accounted for w60 A by elongated oligomers of PrP, suggesting a hierarchical assembly mechanism for the fibrils. The structure reveals flexible crossbridges between the two protofilaments, and subunit contacts along the protofilaments that are likely to reflect specific features of the PrP sequence, in addition to the generic, cross-b amyloid fold. q 2006 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: prion protein; amyloid formation; electron microscopy; FTIR; single particle analysis

Introduction The prion diseases are a group of fatal, transmissible neurodegenerative disorders that include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE)1 in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. These conditions may be inherited, arise sporadically or be acquired by environmental exposure to infectious prions. According to the protein-only hypothesis,2 prions are composed principally or entirely of abnormal forms of host prion protein (PrP).1,3,4 In recent years, work on understanding the underlying biology of these diseases has intensified, due to the appearance of variant CreutzfeldtJakob disease and the experimental confirmation Present address: S. Cohen-Krausz, Department of Membrane and Ultrastructure Research, Hebrew University, Jerusalem, 91220, Israel. Abbreviations used: ATR, attenuated total reflection; FTIR, Fourier-transformed infrared spectroscopy; EM, electron microscopy. E-mail address of the corresponding author: [email protected]

that the aetiology of this new human disease is associated with exposure to bovine spongiform encephalopathy.5–7 The crucial step in the transmission of prion diseases, according to the protein-only hypothesis,2 is the conversion of the host’s normal, cellular form of the prion protein (PrPC), via a post-translational process to a protease-resistant, aggregated form (PrPSc).3 NMR solution structures of PrPC reveal a monomeric structure with three a-helices and a short, two-stranded b-sheet. These properties are conserved in a wide range of vertebrates,8–14 as well as both common polymorphic forms of human PrP, HuPrP(129M)15 and HuPrP(129V).16 PrPSc is found in brain tissue as extracellular deposits of varying size and morphology that can be labelled with antiPrP antibodies. These may form amyloid plaques with characteristic staining and optical properties. When isolated from tissue, this material is detergentinsoluble and, if isolated under non-denaturing conditions, retains its highly infectious properties; it appears fibrillar when observed by negative-stain electron microscopy (EM).17 It was shown by FTIR spectroscopy to contain a high content of b-structure and to be relatively resistant to proteolytic

0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.

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and subsequently refolded at low pH into a metastable, b-sheet-rich, conformation (b-PrP)31 that displays some of the hallmarks of PrPSc. Increasing the ionic strength of the b-PrP buffer induced the monomeric protein to form spherical aggregates within an hour. After 24 h, these associate to form fibrillar material that shows resistance to digestion by proteinase K but is too disordered for 3D analysis. Slow polymerisation of b-PrP over a period of several months at pH 3.0 produces long, ordered PrP fibrils suitable for structural analysis by EM. PrP conformation in the fibrils CD spectra of PrP fibrils purified by centrifugation indicate that the fibrils have a higher content of b-sheet than monomeric a-PrP (Figure 1), consistent with that expected in amyloid fibrils.32 The fibril spectrum is slightly different from that collected from b-PrP, the fibril precursor, but the fibril spectrum retains the characteristic b-sheet minimum at 216 nm. Figure 2 shows solution and attenuated total reflection (ATR)-Fourier-transformed infrared spectroscopy (FTIR) spectra of PrP in a, b and fibrillar states. The spectra cover the amide I absorption region, which is dominated by the carbonyl stretching vibrations of the backbone amides. The ATR data (Figure 2(a)) show that the fibril precursor state, soluble b-PrP, yielded a spectrum very similar to that of the fibrils. For each, there is a strong maximum at 1622 cmK1 that is considered to be characteristic of the amyloid b-sheet structure.33 For comparison, we show the ATR spectrum of a-PrP, which shows a maximum around 1652 cmK1 characteristic of helical structure in ATR spectra of hydrated films.34 The freesolution FTIR spectra (Figure 2(b)) of a-PrP and b-PrP in 2H2O are very similar to their ATR counterparts, showing that deposition of the

8 fibrils α β

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cleavage.18 These findings reveal a profound switch in properties between PrPC and PrPSc, demonstrating that the protein must undergo a significant conformational rearrangement during conversion. Indeed, hydrogen/deuterium exchange data suggest that the conversion of PrPC to PrPSc passes through an unfolded state before refolding into b-structure.19 As well as the prion diseases, both extracellular and intracellular deposits of fibrillar material are associated with a number of neurodegenerative diseases, including Alzheimer’s, Parkinson’s and Huntington’s diseases. More than 20 different proteins or peptides have been shown to be associated with amyloid formation in humans,20 and it has been proposed that amyloid fibrils formed by the polymerisation of these normally soluble proteins or peptides requires either a partial unfolding of natively folded proteins or a partial gain of structure in natively unfolded peptides.21,22 Despite the complete lack of primary sequence identity, structural or functional homology among the precursor proteins, amyloid fibrils display remarkable similarity in their gross morphology and secondary structure. Amyloid fibrils are characteristically long and unbranched, and are formed of two or more protofilaments.23 X-ray fibre diffraction studies have revealed the underlying cross-b core structure of amyloid fibrils,24,25 in which the polypeptide forms b-strands that are arranged perpendicular to ˚ the fibril axis in ribbon-like b-sheets. The 4.7 A cross-b repeat has been observed directly by cryoEM of amyloid fibrils.26,27 EM and 3D reconstruction have provided a view of the overall structure of amyloid fibrils, which display a range of assemblies. There is diversity in the number and packing arrangement of protofilaments, and the helical twist within and between fibrils.28–30 A combination of single-particle and helical reconstruction approaches has proved useful in determining the structure of amyloid fibrils.28 The 3D reconstructions of SH328 and insulin 30 fibrils revealed similar protofilament dimensions of ˚ !40 A ˚ , consistent with two b-sheet around 20 A layers in each protofilament. Given the burgeoning interest in the structure of amyloid fibrils in general and the demonstration that purified, infectious prion material is highly enriched in PrP amyloid fibrils, we have sought conditions for growing ordered PrP fibrils suitable for structural analysis by EM and image processing. Here, we present 3D density maps of PrP amyloid ˚ resolution. fibrils at 28 A

Structure of Mammalian PrP Amyloid Fibrils

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Results Growth of ordered fibrils Previous studies of alternative folding conformations of recombinant PrP have shown that a-PrP can be unfolded in reducing conditions

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Figure 1. Far-UV CD spectra of a-PrP, b-PrP and PrP fibrils. The CD spectra for 6 mM PrP are averages of 30 scans and were collected at 20 8C between 190 nm and 250 nm. PrP fibrils have a high content of b-secondary structure and no detectable a-structure.

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Structure of Mammalian PrP Amyloid Fibrils

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Figure 2. FTIR spectra of PrP samples. (a) ATR-FTIR spectra in the amide I region of a-PrP, b-PrP and PrP fibrils. (b) Solution FTIR spectra in the amide I region of a-PrP and soluble b-PrP. Regions of the spectrum characteristic of specific secondary structures are indicated by horizontal bars. The PrP fibril spectrum shows a peak at 1622 cmK1 that is characteristic of amyloid b-structure.

protein in a hydrated film did not perturb the secondary structure significantly. A notable finding is that the soluble b-PrP gives the same b-sheet signature as the fibrils. Natively folded b-sheet rich proteins typically have an amide I peak in the region 1630 cmK1–1643 cm K1.33 However, conversion of native proteins to the amyloid conformation shifts this amide I peak to the region 1611 cmK1–1630 cmK1. The similarity of the b-PrP and fibril spectra implies strongly that b-PrP is the fibril precursor and that the b-sheet interactions in soluble b-PrP are non-native and amyloid-like. The conformation of PrP in the fibrils was further probed by immunoprecipitation using monoclonal antibodies generated using either recombinant human a-PrP or b-PrP as the immunogen. The antibody ICSM 18, raised against a-PrP, has a higher affinity for native PrPC than PrPSc in immunoprecipitation reactions from tissue homogenates.35 In comparison, ICSM 35, produced from immunisation with b-PrP, is selective for PrPSc over PrPC. To determine if recombinant PrP within the fibrils adopts a conformation similar to that found in PrPSc, immunoprecipitations were performed of fibrils isolated by centrifugation and of a-PrP (Figure 3). ICSM 35 precipitated the fibrils preferentially, whereas ICSM 18 had a higher affinity for a-PrP. This result is in keeping with immunoprecipitations of normal and RMLinfected mouse brain homogenates with these antibodies, and suggests that recombinant PrP within the fibrils adopts a conformation that is consistent with that of PrPSc.35

Figure 3. Analysis of PrP conformation by reactivity to monoclonal antibodies. ICSM 35 shows a higher specificity for PrP fibrils, whereas ICSM 18 has a higher affinity for capturing a-PrP. The control antibody BRIC126 indicates that there is little background non-specific binding of PrP to the magnetic beads. This result suggests that the conformation of PrP in the fibrils is similar to that of ex vivo PrPSc.

Cysteine oxidation state in PrP fibrils The disulphide bond between helices 2 and 3 of PrP may play an important role in aggregation and fibrillisation. Our fibrils are formed from a reduced b-PrP precursor.31 However, full-length and truncated recombinant PrP with an intact disulphide bond have recently been shown to form amyloid fibrils in denaturing conditions and under agitation at neutral pH.36,37 An assembly mechanism for PrP fibrils via a dimer intermediate in which the dimer is stabilised by domain swapping of helix 3 and the formation of two inter-molecular disulphide bonds has been proposed on the basis of the crystal structure of an a-PrP dimer.38 Therefore, it is important to determine the oxidation state of the cysteine residues in the fibrils and to distinguish between inter- and intramolecular linkages. To address this question, samples of PrP fibrils were denatured in urea to ensure chemical exposure and reacted with iodoacetyl biotin, which labels free sulphydryl groups by alkylation. The material was then subjected to SDS-PAGE, blotted and visualized with a streptavidin-phosphatase system (Figure 4(a)). This treatment did not produce a significant signal from the fibril preparation, indicating that the sulphydryl groups were unavailable for alkylation and therefore involved in disulphide bridges. As a positive control for this experiment, another sample of fibrils was treated with urea and 20 mM dithiothreitol (Figure 4(a)) to reduce the bridges and then outtitrated with the iodoacetyl biotin compound. This pre-treatment gave rise to a strong streptavidinphosphatase signal, showing that the reduction process had created reactive sulphydryl groups. A control experiment on a-PrP also showed

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Structure of Mammalian PrP Amyloid Fibrils

Figure 4. Analysis of the sulphydryl state in PrP fibrils. (a) Iodoacetyl-biotin labelling of urea-denatured a-PrP (control) and PrP amyloid fibrils in the absence or in the presence of DTT. (b) Western blot analysis of a-PrP and PrP fibrillar species in the absence or presence of DTT.

protected sulphydryl groups in the oxidized (native) state and reactive cysteine residues post reduction. In the context of the fibril, the above experiment does not show whether the disulphide bonds are within or between PrP subunits. Figure 4(b) shows the result of an experiment in which fibrils were urea-denatured in the presence or in the absence of reducing agent. If the disulphide bonds were intermolecular then we would expect to see an increase in the intensity and or number of high molecular mass bands in the non-reduced sample by SDS-PAGE. Figure 4(b) shows this not to be the case. The band pattern and band intensities were little changed upon reduction. This Western-blot analysis also shows that very little proteolysis has occurred in the fibril samples, even those that are more than one year old (Figure 4(b)). Single-particle analysis of PrP fibril repeats Negative stain EM of recombinant PrP fibrils shows very long (5–10 mm) amyloid fibrils (Figure 5(a)). Most of the fibrils (O90%) have a twisted morphology (Figure 5(a) and (b)), with a minor fraction being untwisted (not shown). The twisted fibrils display a crossover repeat of ˚ in length and are w120 A ˚ approximately 1000 A in diameter at their widest cross-section. Figure 5(b) shows an enlarged view of two fibril crossover repeats. Examination of the raw data suggests that the fibrils consist of two protofilaments and appear to be formed of distinct subunits. A diffraction pattern calculated from a straightened fibril shows ˚ spacing, corresponding to layer-lines at w1000 A the crossover repeat unit of the fibrils (Figure 5(c)). The lack of layer-lines at higher resolution reflects the considerable variation in the crossover repeat ˚ ). (850–1200 A

To overcome this variability, individual repeats were excised from images of PrP fibrils and analysed as single particles. The extracted repeats were aligned in a horizontal orientation and centred. Multivariate statistical analysis was used to sort the repeats into five subgroups, which were then processed separately. The sorted, averaged images confirm the presence of two intertwining protofilaments and a subunit repeat ˚ (Figure 5(d)). of w60 A The 3D reconstruction of PrP fibrils Alignment and classification of images within the length subgroups produced class averages with better-resolved structural information, leading to consistent 3D reconstructions. The 3D analysis of a long and a short class (Figure 6(a) and (g)) is shown here. The 3D maps produced from these classes showed two protofilaments linked by crossbridges (Figure 6(c) and (i)). Reprojections of the maps (Figure 6(b) and (h)) compare well with the input class averages, supporting the consistency of the maps with the image data. The long class (Figure 6(a)–(f)) shows a crossover ˚ and an axial rise per subunit of repeat of 1100 A ˚ (Figure 6(d)). In the short class w65 A ˚ (Figure 6(g)–(l)), the crossover repeat of 900 A ˚ and subunit repeat of w55 A (Figure 6(j)) are consistent with a similar protofilament structure in a more tightly wound helix. Cross-sections of the maps show a dumbbell shape with two separate protofilaments (Figure 6(e) and (k)) or density bridges between the filaments (Figure 6(f) and (l)) depending upon the height of the section. The contour level (3.5s) was chosen to give a volume corresponding to a PrP dimer in each protofilament repeat. At this contour level, the ˚ !30–35 A ˚ in protofilaments are about 25–30 A

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Structure of Mammalian PrP Amyloid Fibrils

Figure 5. Negative stain transmission EM of PrP fibrils. (a) A field of fibrils; the scale bar represents 100 nm. The ˚ crossover repeat of the fibrils is indicated by the arrows. (b) An enlarged view ‘of part of a fibril; the scale bar w1000 A ˚ . Each repeat appears to consist of two protofilaments, and repeating substructure within each represents 225 A protofilament can be detected (white lines). (c) A calculated diffraction pattern of a computationally straightened fibril ˚ layer line corresponding to the crossover repeat distance. The diffraction pattern is calculated for a showing a w1000 A ˚ spacing. (d) Single-particle alignment and MSA rectangular image in order to resolve the layer-lines at 1000 A classification allowed repeats with variable helical repeat lengths to be separated into five separate subgroups varying ˚ to w1200 A ˚ in length, measured between the centres of the helical crossovers (white triangles). Subunit from w850 A repeats are clearly visible (parallel lines).

cross-section. The subunits are connected by bridges of density between the protofilaments, presumably formed by loops or terminal regions of PrP. The bridges vary in angle between the two structures, suggesting that they are connected to the main subunit density by a flexible hinge, so that the stagger between protofilaments is different in the two structures. Unlike the generic, cross-b core structure of the fibril, these connecting regions are likely to reflect sequencespecific aspects of the PrP fibril assembly.

Discussion The amyloid precursor state Previous studies of recombinant PrP have shown that a-PrP can be converted to a metastable b-rich form (b-PrP) that displays increased resistance to digestion by proteinase K as well as the ability to readily form fibrillar material and is recognised differentially by the host immune system.31,39 Conditions were identified for the growth of highly

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Structure of Mammalian PrP Amyloid Fibrils

Figure 6. A 3D reconstruction of PrP fibrils with long and short helical repeats. (a) Averaged image after alignment and ˚ crossover repeat. It shows two inter-twined protofilaments with a distinct subunit repeat, classification of the 1100 A offset between the two protofilaments. (b) A reprojected image from the 3D structure, showing comparable features. (c) A surface view of the 3D density map. (d) The calculated diffraction pattern of the class average of a single repeat in a ˚ subunit repeat. (e) A contour plot of crosssquare image, showing diffuse diffraction corresponding to the w65 A sections through the 3D density map at two different heights, showing the two protofilaments and (f) the bridge of ˚ ) and w55 A ˚ subunit density between them. (g)–(l) Equivalent views for a fibril class with a short crossover repeat (900 A ˚ in height and 30–35 A ˚ in width. The repeat. In both cases, the contour plots show that the protofilaments are 25–30 A short class has less offset between the subunits in the two protofilaments.

ordered amyloid fibrils from the reduced monomeric b form of recombinant mouse PrP91-231 protein. However, iodoacetate labelling analysis indicates that the reduced b-PrP precursor undergoes cysteine oxidation during or after fibril formation. Analysis by CD and IR spectroscopy shows that both the b-PrP starting material and the resulting fibrils are rich in b-conformation, and that there is significant ordering of these secondary structural elements. Moreover, the IR spectra of both precursor and product showed a dominant maximum at 1622 cmK1, a feature that is considered characteristic of b-amyloid structure.33,40 It is surprising that the 1622 cmK1 peak should be so pronounced in the soluble starting material, which clearly cannot contain the extensive b-strand ladders that exist in ordered fibrils. The 1622 cmK1 peak therefore suggests a non-standard b-strand packing, distinct from regular parallel and antiparallel b-sheets, in the precursor state. PrP conformation and subunit repeat in the fibrils Immunoprecipitation analysis indicates that the PrP conformation in the fibrils has some similarity

to that found in authentic PrPSc. The protofilament ˚ !35 A ˚ dimensions of approximately 30 A (Figure 6(e) and (k)) are consistent with at least two b-sheets in each and are similar to those reported for other amyloid fibril structures.28–30 ˚ subunit repeat is a remarkable feature of The 60 A the fibrils. It implies that the protofilaments do not contain a uniform, continuous ladder of cross-b structure, but that there is a distinct set of assembly contacts along the protofilament. In addition, the inter-protofilament density bridges indicate that their lateral assembly into fibrils is determined by ˚ repeat is regions outside the cross-b core. The 60 A rather long for a 17 kDa protein, and must contain at least a dimer of b-PrP molecules in each protofilament subunit, which is consistent with the volume enclosed in the surface-rendered views (Figures 6 and 7). However, it is not possible to rule out a higher-order oligomer forming the subunit repeat in the case that part of the subunit is disordered and does not contribute to the visible density. In Figure 7 we show enlarged views of the fibril structure along with a model of how elongated, head-to-head dimers of PrP could stack to form the cross-b backbone of each protofilament. In this context, it is interesting to note that a headto-head dimer interaction has been reported in

Structure of Mammalian PrP Amyloid Fibrils

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Figure 7. Enlarged view of the 3D map of the long form. (a) A surface view of the 3D density map of the ˚ crossover fibril with the 1100 A repeat. (b) A further enlarged view of several subunit repeats, with the fibril axis rotated to the horizontal direction. The subunit density appears bi-lobed (arrow), consistent with a dimeric subunit repeat along each protofilament. (c) A diagram showing the proposed packing of PrP subunits in the fibril. The blue stripes indicate that there is space for ˚ a ladder of 12 b-strands per 60 A repeat, i.e. six strands per subunit. The thickness of the protofilaments cross-section could accommodate two or three ribbon-like b-sheets.

fibres of the yeast prion sup35.41 The observation of an oligomeric assembly unit suggests a hierarchical assembly mechanism for the PrP fibrils in which dimers or higher oligomers first associate and then assemble into fibrils. A model has been proposed for formation of PrPSc through dimerisation and domain swapping.38 The crystal structure of the a-helical domain-swapped dimer of PrP fits roughly into our subunit density but this conformation is ruled out by the fibril CD and FTIR data, which are incompatible with any significant fraction of a-helical structure. A b-helix fold has been pro˚! posed as a model of prion amyloid.42 The 30 A ˚ 35 A cross-sections of the protofilaments and the cross-bridges in the fibril structure are not obviously compatible with the b-helix arrangement ˚ in diameter proposed with a circular profile w75 A on the basis of 2D arrays isolated from infectious prion material42. Some form of individual b-helix in the protofilament cannot be ruled out at the resolution of our maps, although it would have to ˚ axial repeat. account for the 60 A The ability of prion proteins to adopt an alternative conformation is the central tenet of the

“protein-only” hypothesis of prion propagation. However, little is known about this conversion process in vivo, or about the structure of the abnormal form of PrP that constitutes the infectious species. In vitro, it has been shown that the a-helical conformation of PrP can be converted to a b-sheet form that resembles the disease state, and that this form can oligomerise to form amyloid.31 Moreover, we have found that antibodies raised against b-PrP are highly effective in immunoprecipitating authentic, disease-related PrPSc from human and mouse prion-infected brain homogenates. Given that b-PrP is the building block of the fibrils we describe here, and that these fibrils bind preferentially the same antibodies that recognise PrPSc, we deduce that there are at least some structural features in common between the disease-related PrPSc form and the ordered material we have grown for these studies.35 Recent work by Prusiner and colleagues43 provides evidence that fibrillar forms of PrP may be infectious, although primary transmission of this material was reported only in transgenic mice with very high levels of overexpression of a truncated PrP, and not in wild-type animals. Transgenic mice

982 with overexpression of wild-type or mutant PrP may develop spontaneous disease.44,45 In this respect, understanding the structure and assembly of PrP monomers to form amyloid fibrils would identify key steps along the assembly process or reveal sites of intermolecular interaction in amyloid fibril formation. Several distinct biological strains of prions are recognised that can be propagated serially in hosts encoding the same PrP, and these are associated with biochemically distinct PrPSc types differing in PrP conformation and glycosylation.7,46–48 These differences in PrP conformation and glycosylation can be transmitted serially in suitable animals.7,48 The formation of multiple PrP fibril morphologies, which also involve differential recruitment of PrP glycoforms,35 may underlie the ability of prions to propagate different strain types. A high-resolution structure of b-PrP in combination with the prion fibril structure presented here would identify regions within the PrP molecule that are responsible for fibril assembly and for the conformation conversion reaction and the propagation of prion strains.

Materials and Methods PrP fibril preparation Recombinant mouse PrP 91-231 (Mo PrP91-231) was expressed, purified and converted from the a-helical to b-sheet conformation, as described.31 b-PrP at 1.2 mg/ml in 10 mM Tris, 10 mM sodium acetate buffer (pH 4), was adjusted to pH 3 by addition of 1 M HCl. For fibril assembly, samples were incubated at room temperature over a period ranging from four weeks to six months. PrP fibrils were concentrated by centrifugation at 13,000 rpm in an Eppendorf 5415D microfuge for 45 min and resuspension in 10 mM Tris, 10 mM sodium acetate (pH 3.0) immediately before use. Spectroscopic measurements Circular dichroism spectra were recorded on a Jasco J715 circular dichroism spectropolarimeter. All data were collected between 250 nm and 190 nm in a 1 mm pathlength cuvette at 25 8C and were accumulated from 30 spectra. Solution IR spectra of a-PrP and b-PrP after centrifugation at 15,000 rpm in a Beckman microfuge for 120 min, were recorded in 2H2O buffers containing 10 mM Tris–acetate (pH 8) and 10 mM Tris–acetate (pH 4), respectively, at a protein concentration of 0.5 mg/ml. The b-PrP used in this study was prepared as described.31,49 Spectra were recorded on a Bruker IFS66 spectrometer, using 256 scans at 2 cmK1 resolution. The apparatus incorporated an MCT liquid nitrogen-cooled detector and 100 ml samples were loaded into an in situ cuvette to minimise interference by water vapour. The cuvette had CaF2 windows with a 100 mm separation. Hydrated film samples were analysed on an ATR Nicolet Avatar 360 spectrometer with a ZnSe “multibounce” ATR accessory. Protein spectra were compiled from 1024 scans at 4 cmK1 resolution and were processed as a ratio of the reference spectra from the bare plate. Samples of a-PrP, b-PrP and fibrils were deposited on the spectrometer plate at concentrations of 0.1 mg/ml and

Structure of Mammalian PrP Amyloid Fibrils

0.01 mg/ml in 5 ml drops. The solutions were air-dried before data collection. Immunoprecipitation with conformation-specific antibodies Indirect immunoprecipitaion was applied using Protein G Dynabeads (Dynal Biotech), as described.35 In brief, after isolation by centrifugation, fibrils were resuspended in PBS containing 0.5% (v/v) Tween-20, at 0.25 mg/ml. Samples were made up to a final volume of 100 ml with PBS containing 0.5% Tween-20 and incubated for 12 h at room temperature, with 100 ml of antibodies ICSM 18, ICSM 35 and negative control antibody BRIC126, at a final concentration of 4 mg/ml. Magnetic Protein G Dynabeads were added and incubated at room temperature for 1 h. The beads were washed three times with 1 ml of PBS containing 2% Tween-20 and 2% (v/v) NP40, three times with 1 ml of PBS containing 2% Tween20 and twice with 1 ml of PBS. Beads were resuspended in a final volume of 20 ml of PBS and 20 ml of 2!SDS sample buffer was added and the samples heated for 10 min at 100 8C. After SDS-PAGE and Western blotting, blots were blocked in PBS containing 0.05% Tween-20 and 5% (w/v) non-fat milk powder and were probed with ICSM 35 (DGen Ltd, London, UK) and an alkaline phosphataseconjugated anti-mouse polyclonal antibody (Sigma, Poole, UK). Blots were developed with the chemiluminescent substrate CDP-Star (Tropix Inc) and exposed to Kodak Biomax films. Analysis of sulphydryl state by iodoacetate labelling Fibrils were separated from non-fibrillar material by centrifugation and resuspended in 8 M urea, 50 mM Tris (pH 8.0), 5 mM EDTA at a final protein concentration of 0.25 mg/ml, in the presence or absence of 20 mM dithiothreitol (DTT). As a control, a-PrP was diluted to 0.25 mg/ml in the same buffer. Samples were incubated at 37 8C for 30 min to allow full denaturation. Denaturation of fibrils was confirmed by treatment of fibril samples with increasing concentrations of urea. After application of samples to grids, the samples were washed three times with 20 ml of 10 mM Tris, 10 mM sodium acetate buffer and then negatively stained. Examination of fibrils in the absence of urea indicates that the fibrils remain on the grid after the washing process. At 2 M urea, most of the fibrils are disrupted. The few remaining fibrils are fragmented and display a morphology that suggests that they dissociate into individual protofilaments. No structures remained at 4 M urea. Therefore, after treatment with 8 M urea it is extremely unlikely that any fibrils remain in the preparation. For sulphydryl labelling, EZ-Link PEOIodoacetyl Biotin (Pierce, USA) was added at a final concentration of 80 mM. The reaction was allowed to proceed at room temperature for 2 min and then quenched by addition of 2-mercaptoethanol to 5% (v/ v). After SDS-PAGE and Western blotting, blots were blocked in PBS containing 0.05% Tween-20 and 5% nonfat milk powder, and were probed using a streptavidinalkaline phosphatase conjugate (Novagen). Unlabelled, control blots were probed using the biotinylated anti-PrP monoclonal antibody ICSM35 (D-Gen Ltd, London, UK) in conjunction with the streptavidin-alkaline phosphatase. All blots were developed with the chemiluminescent substrate CDP-Star (Tropix Inc).

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Structure of Mammalian PrP Amyloid Fibrils

Electron microscopy For negative stain EM, 3.5 ml of fibril preparation was applied to a carbon-coated, glow-discharged, 300-mesh copper grid and blotted after 2–3 min. The grids were stained with 3.5 ml of 2% (w/v) uranyl acetate, blotted after 3 min and allowed to air-dry. Images were recorded using minimal electron dose at a magnification of 27,000! in a Tecnai T10 microscope (FEI, Eindhoven, NL) with a tungsten filament operating at 100 kV. The defocus for these images ranged between 300 nm and 1000 nm, allowing the inclusion of data to a spatial ˚ K1) without CTF correction. Microfrequency of (20 A graphs were digitised on a Zeiss SCAI photoscanner (ZI imaging, Swindon, UK) using a step size of 7 mm and ˚ on the specimen level. Fibrils were averaged to 5.2 A scanned individually, aligned approximately parallel with the detector array of the scanner. Single-particle analysis Digitised fibre images were cut into individual repeats using SPIDER50 scripts in a semi-automated procedure. The centres of the repeats and their approximate orientations in the plane of the micrograph were calculated from manually selected crossover points. These coordinates were used to extract repeats into boxes of 350!350 pixels for single-particle analysis using IMAGIC-5.51 The images were band-pass filtered ˚ for the less defocused images and 173–20 A ˚ for (520–20 A the more defocused images), normalized and masked with a soft-edged rectangular mask. The final data set included 2510 repeats that were aligned initially to the sum of all repeats to centre the images with respect to the helical crossover points. The centred repeats were classified by multivariate statistical analysis. Class averages of repeats displaying clear features resembling the raw images were selected as references for subsequent rounds of multi-reference alignment. Repeats were sorted into five subclasses according to their length, and were aligned and classified separately to sort each length group into more homogeneous subsets. The 3D reconstruction Class averages with the clearest features and most prominent layer-lines in the calculated diffraction patterns were chosen for the 3D reconstruction using SPIDER.50 The crossover separation was first estimated by manual measurement on each class average image, and the subunit repeat was estimated from the position of the meridional diffraction peak. These estimated helical parameters were used to reconstruct a short segment of the fibril. The full helical structure was generated by helically symmetrizing the reconstructed segment. To refine the helical parameters, a set of maps was created with a range of different crossover and subunit repeat values. The resulting maps were evaluated by examining the standard deviation of the density and the similarity of reprojections to the input projections for each pair of input parameters. Alignment and reconstructions were further refined by using the class averages that produced the best maps as references for new rounds of multireference alignment and multivariate statistical analysis. Images were not realigned to reprojections of the maps, so ˚ subunit repeat was not imposed at any stage that the 60 A before 3D reconstruction. Final class averages used for 3D reconstructions of a long and a short helical repeat

contained 628 and 477 aligned images, respectively. The resolution of the class averages was determined by Fourier ring correlation and of the maps by Fourier shell correlation at 0.5 correlation, and was found to be ˚ in all cases. 26–28 A Electron density maps of the long and short crossover repeats have been deposited in the EM database with the accession codes EMD-1186 and EMD-1187 respectively.

Acknowledgements We thank Luchun Wang for EM support, Richard Westlake and David Houldershaw for computing support, Daniel Clare and Sarah Tilley for helpful advice and discussion, Mark Batchelor and Samantha Jones for help with protein production, and Ray Young for help with Figures. This work was funded by the UK Medical Research Council.

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Edited by P. Wright (Received 13 December 2005; received in revised form 12 January 2006; accepted 12 January 2006) Available online 31 January 2006 Note added in proof: PrP dimer formation has recently been proposed as a rate-limiting step in fibril formation. The fibrils reported also showed signs of a subunit repeat (Lu¨hrs et al. (2006). J. Mol. Biol.; doi:10.1016/ j.jmb.2006.01.016)