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1 Astbury Centre for Structural Molecular Biology & Institute for Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
2 Department of Biochemistry, University of Sussex, John Maynard Smith Building, Falmer BN1 9QG, United Kingdom
3 Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette Cedex, France
(RECEIVED March 14, 2006; FINAL REVISION August 4, 2006; ACCEPTED August 12, 2006)
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Keywords: prion protein; yeast; structure; fibrils; electron microscopy; image analysis
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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The protein Ure2 (Ure2p) is at the origin of the [URE3] trait in S. cerevisiae (Wickner 1994). Native Ure2p is an active, dimeric, and soluble cytosolic protein that becomes inactive and insoluble in [URE3] yeast. Ure2p is a two-domain protein containing a short, poorly structured N-terminal region (1–93) and a globular, folded C-terminal domain (94–354) (Thual et al. 1999). The latter domain shares sequence homology with the glutathione S-transferase (GST) family (Coschigano and Magasanik 1991). The X-ray crystal structure confirmed that it shares a fold with GST (Bousset et al. 2001a; Umland et al. 2001) and that the monomer has dimensions 70 Å x 28 Å x 52 Å. In its native, soluble state Ure2p binds glutathione (Bousset et al. 2001b) and has glutathione peroxidase activity (Bai et al. 2004). It has been shown that fibrillar Ure2p retains these properties (Bousset et al. 2002; Bai et al. 2004). Recombinant Ure2p oligomerizes in vitro at neutral pH and under physiological salt concentrations into a globular, high molecular mass species. This globular species then further assembles into fibrillar structures that show some similarities to amyloid fibrils (Taylor et al. 1999; Thual et al. 1999; Schlumpberger et al. 2000). Atomic force microscopy (AFM) of assembled Ure2p fibrils showed relatively smooth fibers with some variation in height along their length (Bousset et al. 2002), with a periodicity of 
30 nm. AFM has also been used to follow the assembly process and reveals a series of Ure2p fibril types, including protofilaments, intermediate fibrils, and two distinct types of mature fibrils, with either a smooth or twisted appearance (Jiang et al. 2004). The fibrils show variation in height with a periodicity of about 40–70 nm and the twisted fibrils have a height of 8 nm. Twisted fibrils have also been observed by electron microscopy upon assembly of Ure2(1–80)-GFP, as well as some other Ure2(1–80) fusion proteins. In these cases, the repeat distance appeared to vary between different fibrils within the same sample (Baxa et al. 2002). Although twisted fibrils of Ure2p were first observed in 1999 (Thual et al. 1999), no detailed characterization of this morphology has yet been made. Protofilaments twisting together to form a number of different fibrillar morphologies have been previously observed for other fibril-forming proteins (Goldsbury et al. 1999; Jimenez et al. 2002; Kad et al. 2003; Khurana et al. 2003; Anderson et al. 2006).
Amyloid fibrils are insoluble ordered aggregates that can be formed from many diverse proteins in vivo and are associated with a group of diseases known as the amyloidoses (Pepys 2001). In vitro there is increasing evidence that amyloid-like fibrils can be formed from a very wide range of proteins (Dobson 2001). They share structural characteristics, including appearance by electron microscopy and a cross-
diffraction pattern by X-ray diffraction (Serpell et al. 1997). Amyloid fibrils are thought to be composed predominantly of a cross- core structure (Sunde et al. 1997). Ure2p fibrils however, have several characteristics that set them apart from conventional amyloid fibrils. Limited proteolysis patterns of the soluble and fibrillar forms of the protein are very similar (Thual et al. 1999). Fourier-transform infrared spectroscopy (FTIR) showed a very slight structural change between soluble and fibrillar Ure2p and showed that both contained a large proportion of 
-helical structure (Bousset et al. 2002). Furthermore, partially aligned fibrils assembled from full-length Ure2p give X-ray fiber diffraction patterns that are inconsistent with cross- structure. Such patterns consist of low-angle diffraction signals at 47 Å on the meridian and 25 Å and 52 Å on the equator (Bousset et al. 2003), consistent with the regular packing of Ure2p molecules. A cross- diffraction pattern with the characteristic signals at 4.7 Å and 10 Å can be obtained from heat-treated fibrils (65°C) (Bousset et al. 2003) or from dried fibrils (Baxa et al. 2005). A weak 4.7 Å ring is obtained from sucrose-embedded fibrils formed by full-length Ure2p and a very weak ring from Ure2p fibrils embedded in vitreous and cubic ice.
The N-terminal region of Ure2p is required for fibril formation and for prion-like activity (Masison and Wickner 1995; Thual et al. 2001; Jiang et al. 2004); however, recent evidence suggests that the C-terminal domain is also involved in the fibrillar scaffold (Bousset et al. 2004; Fay et al. 2005). An "amyloid-backbone" model has been suggested in which the unstructured N terminus of Ure2p associates to form a -sheet rich "spine" surrounded by globular C-terminal domains that retain their native structure (Baxa et al. 2003). More recently, a structural model in which 70 amino acid residues from the flexible N-termini of Ure2p form a parallel super-pleated -structure running along the fibrils was proposed to account for the assembly of Ure2p into protein fibrils (Kajava et al. 2004; Baxa et al. 2005).
In this study, we characterize regularly twisted fibrils of Ure2p assembled under native-like conditions for the first time. Using negative-stain and cryo-electron microscopy with image processing, we show that the fibrils are composed of a helical array of stacked Ure2p molecules associated laterally either into a sheet twisting around the axis of the fibril or into a cylindrical unit.
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Results |
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2 microglobulin amyloid formed in vitro (Kad et al. 2003). We chose to examine the morphology and structure of mature fibrils of Ure2p with a clear, regularly repeating helical twist, as this regular morphology allowed further structural analysis. The assembly reaction is a multistep process. Under normal native growth conditions, a series of fibrillar intermediates accumulate (data not shown) (Thual et al. 1999; Bousset et al. 2002; Jiang et al. 2004; Catharino et al. 2005). In order to assemble twisted fibrils preferentially, a number of conditions were explored. Reproducible results were obtained by assembling 50 µM Ure2p for 1 wk at 8°C at neutral pH (in buffer A) without shaking. The amount of twisted Ure2p fibrils in the sample was 80% as judged by observation using TEM (Fig. 1), and >95% by cryo-EM.
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200 Å across) were also observed around the periphery of the fibrils (data not shown).
Helical image analysis of twisted Ure2p fibrils
To investigate the helicity of the fibrils, Fourier transforms were calculated from selected areas of the fibril images. Several electron micrographs were examined and varying sizes of boxes were selected from the images. The fibril image was interpolated to vertical (Fig. 2a), and the Fourier transform (Fig. 2) was calculated and examined for high-intensity signals. The Fourier transform shown in Figure 2b shows a layer line corresponding to 690 Å, commensurate with the repeat distance along the helix. This indicates that the layer line arises from a one-start helix. The precise length of the helical repeat varied between different fibrils and in different regions of individual fibrils from 540 Å to 700 Å, averaging 660 Å (Fig. 2c). A maximum of three layer lines was observed from single fibril images, and this was insufficient for helical image reconstruction to be performed.
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Cryo-electron microscopy of twisted fibrils
Cryo-electron micrographs (cryo-EM) were taken of Ure2p fibrils embedded in unsupported vitreous ice. The resulting low-contrast images showed numerous fibrils, the vast majority of which (95%) displayed a pronounced, regular twist. Accurate diameter and repeat measurement directly from the micrographs was hampered by the inherently low contrast of raw cryo-EM images. However, the fibrils appeared to measure 220–260 Å across. Individual fibril images were examined and selected box Fourier transforms were calculated interactively (as described for negatively stained images). Many images of twisted fibrils were investigated, but in cryo-electron micrograph images only a single layer line was observed (corresponding to the helical repeat at 700 Å) (Fig. 3). The fact that the fibril images do not give more than one layer line may arise from variations in twist and their generally curved appearance in ice, which reduces long-range order.
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60–90 Å apart, and a punctate appearance is observed in some classes that may correspond to the stacking of subunits along the fibril axis. This punctate appearance became clearer upon further processing and averaging of more images (Fig. 5a).
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60 Å was also frequently observed, which may arise from the apparent punctate appearance. The improved signal-to-noise ratio of the class average views allows a more accurate determination of fibril diameter to be made at 240–250 Å. Little-g calculations (averaged, cylindrical cross-sections) calculated from the equatorial layer line of Fourier transforms were consistent with solid fibrils (data not shown). |
Discussion |
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700 Å. From these data we observed longitudinal striations twisting around the fibril, with a punctate appearance. Fourier transforms indicate a helical striation at 60 Å. Previous experimental results have contributed to an understanding of the composition of Ure2p fibrils, and two models have been suggested. The first proposes that assembly is driven by a significant conformational change of a portion of the N-terminal domain (40–70 of its 93 amino acid residues), leading to its organization into a cross--core running along the fibrils (Baxa et al. 2003; Kajava et al. 2004). The second model hypothesizes that the assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and intramolecular interactions between Ure2p molecules (Bousset et al. 2002; Fay et al. 2005). The N-terminal domain of Ure2p plays a critical role in its assembly into protein fibrils (Thual et al. 2001; Jiang et al. 2004). Indeed, it is widely accepted that the assembly of Ure2p into fibrils is driven by a conformational change occurring in the flexible N-terminal domain of the protein. There is, however, disagreement as to the extent of this conformational change.
In the cross- model, termed the "parallel super-pleated -structure model" (Baxa et al. 2003; Kajava et al. 2004), the globular C-terminal domains of Ure2p molecules protrude from the cross--core of the fibrils. The width of such putative fibrils is 245 Å and that of their cross--core is 28 Å, compatible with that of the fibrils shown here (250 Å). In this model, the N-terminal moieties of Ure2p molecules (residues 5–70) extend to form -strands that form the rigid core of the fibrils. The N-terminal 70 amino acid stretch is followed by a flexible linker region of 25 amino acid residues critical for the loose stacking of the globular C-terminal domains of the polypeptide chains within the fibrils. This linker region allows a considerable degree of freedom (Kajava et al. 2004). It is suggested that the C-terminal domains cluster to one side of the -core, and that one -spine layer from one Ure2p molecule twists axially by 0.7–3.4 degrees relative to the preceding layer (Kajava et al. 2004), giving rise to a helical pitch of 500 Å to 2500 Å. The parallel super-pleated -structure model was proposed to account for (1) the observation of reflections at 4.7 Å from X-ray fiber diffraction images of dried Ure2p fibrils and from electron diffraction of cryo-embedded fibrils of Ure2p10–39 (Baxa et al. 2005), (2) the longitudinal striations observed in cryo-electron micrograph images of Ure2p fibrils, and (3) the mass-per-unit length measurements from scanning transmission electron microscopy that gave one subunit rise per 4.7 Å (Kajava et al. 2005).
The second model for the Ure2p fibril (Bousset et al. 2002; Fay et al. 2005) is based on experiments that have shown an intimate involvement of the C-terminal domain within the fibrils, and that this domain retains its native structure and ability to bind GSH (Bousset et al. 2003). Previous X-ray diffraction from oriented native hydrated Ure2p fibrils gave a diffraction pattern that was not consistent with cross- structure (Bousset et al. 2003), but instead reflections were observed at 25 Å, 47 Å, and 52 Å (Bousset et al. 2003). These observations are consistent with size measurements from the crystal structure of the globular domain of Ure2p (Bousset et al. 2001a; Umland et al. 2001). It is only when Ure2p loses its native structure within the fibrils (for example, upon heating above 60°C, incubation at pH 
3.0 [Bousset et al. 2003], or extensive drying) that the fibrils acquire the amyloid characteristics: (1) reflections at 4.7 Å and (2) increased absorbance at 1620 cm–1 in FTIR spectroscopy (Bousset et al. 2003). In addition, native Ure2p fibrils promote catalytic assembly in vitro, whereas these "denatured" fibrils are unable to do so (Bousset et al. 2003). Under oxidizing conditions, an intramolecular disulfide bond is formed within fibrillar Ure2p C6C137 (where cysteine residues are located in the N- and C-terminal moieties of Ure2p) (Fay et al. 2005), whereas cysteine residues located within the N-terminal part of Ure2p at amino acid residue position 6 do not form disulfides. The parallel super-pleated -structure model suggests that the residues from adjacent molecules are in register within fibrillar Ure2p. This model therefore suggests that Cys 6 should form disulfides within the fibrils. This is not what is observed (Fay et al. 2005; Fayard et al. 2006), although it has been previously shown for A fibrils that disulfides can form within cross- structures (Shivaprasad and Wetzel 2004). Intermolecular disulfide bonds are formed under oxidative conditions between Ure2pC221 molecules within the fibrils (Fayard et al. 2006), which means that the globular moieties of Ure2p molecules are highly ordered within the polymers, unlike what is proposed in the parallel super-pleated -structure model. It is clear that there is a tight involvement of the C-terminal domain of Ure2p in the fibrillar scaffold following specific cleavage between the N- and C-terminal domains of fibrillar Ure2p (Bousset et al. 2004). The proteolytic patterns of soluble and fibrillar Ure2p are similar and there is an absence of a highly resistant polypeptide corresponding to the N-terminal domain of Ure2p following treatment of the fibrillar protein by a variety of proteases (Thual et al. 1999; Bousset et al. 2004). The punctate appearance of the fibrils shown here is not consistent with variably axially arranged layers, with the C-terminal domains flanking the serpentine core on one side as proposed in the parallel super-pleated -structure model. We show an average pitch for the fibrils of 700 Å, which is at the lowest limit of the 500–2500 Å pitch variation range predicted for the model (Kajava et al. 2005) and would require a regular axial twist of 2.4°.
The microscopy data described in this study indicates that Ure2p fibrils are likely to be composed of a highly repetitive structure made up of ordered arrangement of Ure2p molecules stacked in a helical array. This could also be described as several protofilaments composed of stacked Ure2p molecules. Striations observed in cryo-EM images as well as filtered FT images are suggestive of these protofilaments. This data is consistent with a model in which both N- and C-terminal domains are intimately involved with the fibrillar structure.
The work presented here does not allow for an assessment of the nature of the lateral interactions between Ure2p protofilaments in the fibrils. Further characterization of the surfaces exposed to the solvent in fibrillar Ure2p using, for example, hydrogen/deuterium exchange monitored by mass spectrometry, will allow modeling Ure2p in the fibril density, thus allowing generation of a more detailed three-dimensional model for Ure2p fibrils.
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Materials and methods |
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Assembly of Ure2p into fibrils
Twisted Ure2p fibrils were obtained upon assembly of soluble full-length wild-type Ure2p in buffer A at 8°C. The latter experimental conditions differ slightly from those used previously in that both the ionic strength and the temperature are higher. The assembly reactions were monitored by thioflavin T binding (McParland et al. 2000), using a Quantamaster QM 2000-4 spectrofluorimeter (Photon Technology International).
Negatively-stained and cryo-electron microscopy
Ure2p fibrils were examined following negative staining with 1%–3% uranyl acetate on carbon-coated grids (200 mesh) in a Philips EM 410 electron microscope operated at 80 kV and images were taken on film at magnifications of 24,000 to 40,000 times (Philips Inc.). Micrographs were scanned with a Zeiss SCAI scanner using a scanning step-size of 7.0 µm. The images were compressed by two and converted to MRC format using the Image2000 processing suite (tld2mrc) (Crowther et al. 1996). Data were also recorded on a Gatan CCD camera at a nominal magnification of 6000 and 10,000 using a Hitachi-7100 electron microscope operated at 100 kV.
Ure2p fibrils in vitreous ice were imaged using Quantifoil R2/1 holey carbon grids that were vitrified by standard methods (Dubochet et al. 1988). Vitreous specimens were imaged using an FEI TF20 field emission gun microscope operating at 200 kV and equipped with a Gatan 626 cryo-transfer holder. Images were recorded at a magnification of 36,613x on a Gatan US4000SP CCD camera with a pixel size of 15 µm, giving a final object sampling of 4.1 Å/pixel. Micrograph defocus was determined using the program CTFFIND3 (Mindell and Grigorieff 2003).
Fourier analysis of negatively-stained Ure2p fibrils
The digital micrographs were examined using Ximdisp (Smith 1999) and straight regions of twisted fibrils selected, boxed, and floated interactively. A Fourier transform was then calculated from the boxed floated image using the MRC Suite of image processing programs (Crowther et al. 1996). The positions of the layer lines were measured and noise masked out to give a filtered pattern. An inverse Fourier transform was then calculated from the filtered pattern to give an enhanced image of the fibril.
Single particle analysis of frozen-hydrated Ure2p fibrils
Fibril segments were selected with the "unbend helix" option in the program "boxer" (Ludtke et al. 1999), incorporating a 25% overlap between adjacent segments, and then excised into 672 x 672 pixel boxes. The resulting "particle" images were corrected for the effects of the microscope contrast transfer function (CTF) by multiplication of the individual image Fourier transforms with a binary CTF function for the originating micrograph. The resulting phase-flipped particle images were then further windowed into 448 x 448 pixel boxes, each of which encompassed approximately two copies of the major helical repeat. A total of 3400 fibril segments were selected. The entire data set was aligned using reference-free rotational alignment, and the resulting average was made vertical. All images were then realigned to the vertical average before being subjected to multivariate statistical analysis (MSA) and classification. Several representative class average views of helical segments were then selected and used as references for further rounds of multi-reference alignment, again followed by MSA and classification. All image processing steps were performed in SPIDER v11.12 compiled with FFTW libraries (Frank et al. 1996; Frigo and Johnson 1998), except for MSA classification, which was performed in IMAGIC (van Heel et al. 1996).
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Footnotes |
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Reprint requests to: Louise C. Serpell, Department of Biochemistry, University of Sussex, John Maynard Smith Building, Falmer BN1 9QG, UK; e-mail: L.C.Serpell@sussex.ac.uk; fax: 44-1273-678433; or Ronald Melki, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette Cedex, France; e-mail: melki{at}lebs.cnrs-gif.fr; fax: 33-169-823129.
Abbreviations: DTT, dithiothreitol; EM, electron microscopy; EGTA, ethylene glycol bis-(-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; Tris, tris(hydroxymethyl)aminomethane.
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062215206.
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References |
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