Amyloid fibrils formed from different proteins, each associated with a particular disease, contain a common cross-beta spine. The atomic architecture of a spine, from the fibril-forming segment GNNQQNY of the yeast prion protein Sup35, was recently revealed by X-ray microcrystallography. It is a pair of beta-sheets, with the facing side chains of the two sheets interdigitated in a dry 'steric zipper'. Here we report some 30 other segments from fibril-forming proteins that form amyloid-like fibrils, microcrystals, or usually both. These include segments from the Alzheimer's amyloid-beta and tau proteins, the PrP prion protein, insulin, islet amyloid polypeptide (IAPP), lysozyme, myoglobin, alpha-synuclein and beta(2)-microglobulin, suggesting that common structural features are shared by amyloid diseases at the molecular level. Structures of 13 of these microcrystals all reveal steric zippers, but with variations that expand the range of atomic architectures for amyloid-like fibrils and offer an atomic-level hypothesis for the basis of prion strains.

Numerous soluble proteins convert to insoluble amyloid-like fibrils having common properties. Amyloid fibrils are associated with fatal diseases such as Alzheimer's, and amyloid-like fibrils can be formed in vitro. For the yeast protein Sup35, conversion to amyloid-like fibrils is associated with a transmissible infection akin to that caused by mammalian prions. A seven-residue peptide segment from Sup35 forms amyloid-like fibrils and closely related microcrystals, which here reveal the atomic structure of the cross-β spine. It is a double β-sheet, with each sheet formed from parallel segments stacked in-register. Sidechains protruding from the two sheets form a dry, tightly self-complementing steric zipper, bonding the sheets. Within each sheet, every segment is bound to its two neighbouring segments via stacks of both backbone and sidechain hydrogen bonds. The structure illuminates the stability of amyloid fibrils, their self-seeding characteristic, and their tendency to form polymorphic structures.Four decades of research have established that amyloid-like fibrils of different proteins have a common structural 'cross-β' spine 1 . In 1959 Cohen and Calkins 2 observed elongated, unbranched fibrils in electron micrographs of diseased tissues, and in 1968 Glenner and Eanes 3 discovered that the fibrils exhibit an X-ray diffraction signature known as the cross-β pattern. This pattern shows 4 that the strongest repeating feature of the fibril is a set of β-sheets that are parallel to the fibril axis with their strands perpendicular to this axis. The hypothesis of a common molecular organization was supported by the finding 5 that amyloid fibrils from 6 different proteins, each associated with its own clinical syndrome, showed similar cross-β diffraction patterns. The degree of similarity pointed to 'a common core molecular structure.'Revealing the atomic details of this cross-β spine has been impeded by the limited order of fibrils isolated from diseased tissues, infected cells, and in vitro conversions of proteins to fibrils. There is also evidence for a diversity of crystalline and fibril structures 6-8 . Nevertheless, an arsenal of biophysical tools has defined important features. These tools include solid-state NMR 9-11 , model-building constrained by X-ray fiber and powder diffraction 6,7,12,13 , site-directed spin labeling 14,15 , cryo-electron microscopy 16,17 , and proline-scanning mutagenesis 18 . Despite numerous models suggested by these studies, until now no refined, fully objective atomic model has been available for the common spine structure.Correspondence and requests for materials should be addressed to D.E. (david@mbi.ucla.edu). The structures of GNNQQNY and NNQQNY have been deposited in the Protein Data Bank with accession codes 1yjp and 1yjo, respectively.. Competing Interests StatementThe authors declare that they have no competing financial interests.Supplementary Information accompanies the paper on www.nature.com/nature. We selected the yeast protein Sup35 for X-ray diffraction analy...

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Strains are phenotypic variants, encoded by nucleic acid sequences in chromosomal inheritance and by protein “conformations” in prion inheritance and transmission. But how is a protein “conformation” stable enough to endure transmission between cells or organisms? Here new polymorphic crystal structures of segments of prion and other amyloid proteins offer structural mechanisms for prion strains. In packing polymorphism, prion strains are encoded by alternative packings (polymorphs) of β-sheets formed by the same segment of a protein; in a second mechanism, segmental polymorphism, prion strains are encoded by distinct β-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring “conformations,” capable of encoding strains. These molecular mechanisms for transfer of information into prion strains share features with the familiar mechanism for transfer of information by nucleic acid inheritance, including sequence specificity and recognition by non-covalent bonds.

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In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer’s disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross-sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using di-glycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. Here, we determine the corkscrew-like structure of a cytotoxic segment of superoxide dismutase 1 (SOD1) in its oligomeric state. Mutations that prevent formation of this structure eliminate cytotoxicity of the segment in isolation as well as cytotoxicity of the ALS-linked mutants of SOD1 in primary motor neurons and in a Danio rerio (zebrafish) model of ALS. Cytotoxicity assays suggest that toxicity is a property of soluble oligomers, and not large insoluble aggregates. Our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register β-sheet structures and identifies a target for structurebased therapeutics in ALS.oligomer | SOD1 | ALS

Amyloid fibrils are found in association with at least two dozen fatal diseases. The tendency of numerous proteins to convert into amyloid-like fibrils poses fundamental questions for structural biology and for protein science in general. Among these are the following: What is the structure of the cross-beta spine, common to amyloid-like fibrils? Is there a sequence signature for proteins that form amyloid-like fibrils? What is the nature of the structural conversion from native to amyloid states, and do fibril-forming proteins have two distinct stable states, the native state and the amyloid state? What is the basis of protein complementarity, in which a protein chain can bind to itself? We offer tentative answers here, based on our own recent structural studies.