Strain-specific morphologies of yeast prion amyloid fibrils

We performed mass-per-length (MPL) measurements and electron cryomicroscopy (cryo-EM) with 3D reconstruction on an A␤(1-42) amyloid fibril morphology formed under physiological pH conditions. The data show that the examined A␤(1-42) fibril morphology has only one protofilament, although two protofilaments were observed with a previously studied A␤(1-40) fibril. The latter fibril was resolved at 8 Å resolution showing pairs of ␤-sheets at the cores of the two protofilaments making up a fibril. Detailed comparison of the A␤(1-42) and A␤(1-40) fibril structures reveals that they share an axial twofold symmetry and a similar protofilament structure. Furthermore, the MPL data indicate that the protofilaments of the examined A␤(1-40) and A␤(1-42) fibrils have the same number of A␤ molecules per cross-␤ repeat. Based on this data and the previously studied A␤(1-40) fibril structure, we describe a model for the arrangement of peptides within the A␤(1-42) fibril.Alzheimer's disease ͉ electron microscopy ͉ prion ͉ protein folding A myloid fibrils are fibrillar polypeptide aggregates that consist of a cross-␤ structure (1, 2). They accumulate inside the human body in the course of aging and are associated with several debilitating conditions such as Alzheimer disease (AD) (1, 2). AD amyloid fibrils are formed from A␤ peptide, which occurs in isoforms of different length. The 40-residue peptide A␤(1-40) represents the most abundant A␤ isoform in the brain (3), while the 42-residue A␤(1-42) shows a significant increase with certain forms of AD (4). A␤ amyloid fibrils have been analyzed with various biophysical and biochemical techniques, such as solid-state NMR (NMR) spectroscopy, solution state NMR spectroscopy coupled with hydrogen/deuterium exchange, or mutagenesis (5-8). These analyses have provided a wealth of information about specific structural details of the peptide in the fibril, for example dihedral torsional angles, protection factors or distances between specific atoms. Atomic models for A␤ peptides and their assembly in different amyloid fibrils have been constructed based on such data (6, 8-10) but these models have not been confirmed by more direct 3D imaging methods.A hallmark of A␤ amyloid fibrils is their substantial polymorphism (11)(12)(13)(14). We have recently shown by transmission electron cryomicroscopy (cryo-EM) and 3D reconstruction that A␤(1-40) fibrils form a range of morphologies with almost continuously altering structural properties (13). Despite their different morphologies, the cross-sectional areas of the reconstructed fibrils were similar. The study suggested that the observed polymorphism may be the result of different packing of protofilaments that have the same basic structure. To obtain an image of a fibril at higher resolution, we established growth conditions that promote a specific A␤(1-40) fibril morphology (15) and selected fibrils with this morphology from electron micrographs for further processing. Using newly developed image processing tools (16), we obtained a 3D image at 8 Å...

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“…Non-integer numbers of peptides per cross-␤ repeat were also obtained for other amyloid fibrils (25). This lends support to our MPL measurements.…”

Section: Discussionsupporting

confidence: 74%

“…The MPL measurements were calibrated with Tobacco Mosaic Virus (TMV) for each individual image as described in ref. 25, and a cross-␤ repeat of 4.7 Å for all fibril morphologies was assumed.…”

Section: Scanning Transmission Electron Microscopy (Stem)mentioning

confidence: 99%

Comparison of Alzheimer Aβ(1–40) and Aβ(1–42) amyloid fibrils reveals similar protofilament structures

Schmidt

Sachse

Richter

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

258

283

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“…Measurements of the mass per unit length of Sup35 1-65 fibrils showed approximately one monomer per 0.47 nm in each of three variants (52). This result is inconsistent with a single-helix ␤-helical model but is consistent with an in-register parallel ␤-sheet structure with purely intermolecular backbone hydrogen bonding.…”

Section: Discussioncontrasting

confidence: 31%

Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Shewmaker¹,

Wickner²,

Tycko

2006

Proc. Natl. Acad. Sci. U.S.A.

272

302

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The [PSI ؉ ] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor. Using solid-state NMR we have examined the structure of amyloid fibrils formed in vitro from purified recombinant Sup35 1-253 , consisting of the glutamine-and asparagine-rich Nterminal 123-residue prion domain (N) and the adjacent 130-residue highly charged M domain. Measurements of magnetic dipole-dipole couplings among 13 C nuclei in a series of Sup35NM fibril samples, 13 C-labeled at backbone carbonyl sites of Tyr, Leu, or Phe residues or at side-chain methyl sites of Ala residues, indicate intermolecular 13 C-13 C distances of Ϸ0.5 nm for nearly all sites in the N domain. Certain sites in the M domain also exhibit intermolecular distances of Ϸ0.5 nm. These results indicate that an in-register parallel ␤-sheet structure underlies the [PSI ؉ ] prion phenomenon. The Sup35p prion domain (Sup35N, residues 1-123) is asparagine-and glutamine-rich, is poor in charged residues, and has five imperfect nine-residue repeats with consensus YQQYN-PQGG. Sequence shuffling shows that the repeats are not necessary for prion generation or propagation and that amino acid content of the prion domain (not the sequence) determines whether a protein can form a prion (13). Certain point mutations in the prion domain can block propagation of [PSI ϩ ] introduced with the wild sequence (14, 15), although the mutant sequence may itself form a prion (16). Thus, propagation of an existing prion is very sequence-specific, as in the species barriers of mammalian prion diseases (reviewed in ref. 17).Amyloid fibrils are filamentous protein aggregates exhibiting ''cross-␤'' x-ray fiber diffraction patterns, indicating the presence of ␤-sheets formed by ␤-strands that are oriented approximately perpendicular to the fiber axis, with interstrand hydrogen bonds approximately parallel to the fiber axis (reviewed in ref. 18). The fact that the prion domains of Ure2p (another yeast prion protein with an N-terminal prion domain rich in asparagine and glutamine) and Sup35p can be shuffled and yet still form prions and amyloid (13,19) suggests that the amyloid on which these prions are based has an in-register parallel ␤-sheet structure (20, 21). A prion amyloid structure based on antiparallel ␤-sheets or ␤-helices would necessarily be stabilized by interactions among specific sets of unlike residues. These interactions would likely be destroyed by shuffling the sequence. In contrast, an in-register parallel ␤-sheet structure can be stabilized by intermolecular hydrophobic interactions (22,23) or polar side chain interactions [e.g., the ''polar zipper'' interactions suggested by Perutz et al. (24)] among like residues. Shuffling the sequence would still allow like residues to align and interact in such a structure. Thus, shuffleability of a prion domain suggests an in-register parallel ␤-sheet structure.The molecular structures of amyloid fibrils, particularly those formed by bona fide proteins, are difficult t...

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“…The efficiency of their cellular propagation is kinetically determined where the fastest multiplicating, truebreeding structures dominate. We previously suggested that the cellular frangibility of prion fibers played an important role in determining the strength of a prion strain (14). This notion was born out by a beautiful full treatment in Tanaka (Fig.…”

Section: Resultsmentioning

confidence: 99%

Strain-specific sequences required for yeast [ PSI ⁺ ] prion propagation

Chang

Lin

Lee

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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[PSI] strains ͉ amyloid ͉ Sup35 A myloid is a generic class of ordered protein aggregates self-assembled by many unrelated proteins (1). X-ray diffraction studies reveal that amyloid fibers are rich in ␤-strands, which run perpendicular to the fiber axis, forming the characteristic cross-␤ pattern (2). One of the most intriguing features of the amyloid is structural polymorphism, whereby the same protein polypeptide can adopt distinct chain-folding patterns to give rise to a variety of cross-␤ structures (3). Structural plasticity of the amyloid underlies the prion strain phenomenon. Selfnucleating amyloid conformers faithfully maintain their distinct folds in the host but sometimes can adjust their structures when encountering different sequences (such as during interspecies transmission), thus giving rise to novel prion strains (4, 5). It is not well understood how distinct cross-␤ folding patterns are determined and what is the mechanism for their interconversion.The yeast prion [PSI] is a self-propagating amyloid aggregate of Sup35, the yeast translation-termination factor whose aggregation in the [PSI ϩ ] state results in enhanced read-through of nonsense mutations (6, 7). Structural polymorphism of Sup35 amyloids gives rise to [PSI] strains that exhibit distinctive nonsense-suppression efficiencies and differential compatibility with Sup35 mutations (8-11). With the ease of biochemical and genetic manipulation, [PSI] strains provide excellent experimental systems to study amyloid polymorphism. However, because in vitro assembled amyloid samples often contain multiple fiber morphologies, great care must be taken to ensure correct interpretation of experimental results. Attributing genetic observations to pure structural causes, however, is complicated by the interplay between amyloid structures and cellular machineries. Here, we design a synthesized approach to avoid these difficulties, combining biochemistry and yeast genetics to examine the amyloid structures of three [PSI] strains: [VH], [VK], and [VL] (11). By individually substituting residues 5-55 of Sup35 with proline and inserting glycine in front of every other residue in this segment, we identify strain-specific Sup35 sequences in vivo. The necessity of these sequences for encoding infectious prion structures is then investigated by infecting yeast with amyloid fibers of Sup35 fragments assembled in vitro. Our goal is to define the minimally required Sup35 sequences that form core structures of the three [PSI] strains to understand how the same polypeptides are recruited in different ways to assemble polymorphic amyloid structures. ResultsProline Substitutions. We employ proline substitutions to map possible ␤-structures along the polypeptide chain. Proline residues with the unique cyclic structure generate bulges in ␤-strands and destabilize periodic amyloid structures. A panel of 51 [psi Ϫ ] haploid mutants, each having a single proline substitution in the amino acid residues 5-55 of the SUP35 allele, was created by homologous recombination. ...

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Strain-specific morphologies of yeast prion amyloid fibrils

Cited by 131 publications

References 36 publications

Comparison of Alzheimer Aβ(1–40) and Aβ(1–42) amyloid fibrils reveals similar protofilament structures

Comparison of Alzheimer Aβ(1–40) and Aβ(1–42) amyloid fibrils reveals similar protofilament structures

Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Strain-specific sequences required for yeast [ PSI ⁺ ] prion propagation

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Strain-specific morphologies of yeast prion amyloid fibrils

Cited by 131 publications

References 36 publications

Comparison of Alzheimer Aβ(1–40) and Aβ(1–42) amyloid fibrils reveals similar protofilament structures

Comparison of Alzheimer Aβ(1–40) and Aβ(1–42) amyloid fibrils reveals similar protofilament structures

Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Strain-specific sequences required for yeast [ PSI + ] prion propagation

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Strain-specific sequences required for yeast [ PSI ⁺ ] prion propagation