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.

Human islet amyloid polypeptide (IAPP or amylin) is a 37-residue hormone found as fibrillar deposits in pancreatic extracts of nearly all type II diabetics. Although the cellular toxicity of IAPP has been established, the structure of the fibrillar form found in these deposits is unknown. Here we have crystallized two segments from IAPP, which themselves form amyloid-like fibrils. The atomic structures of these two segments, NNFGAIL and SSTNVG, were determined, and form the basis of a model for the most commonly observed, full-length IAPP polymorph.

Based on the crystal structure of the cross-␤ spine formed by the peptide NNQQNY, we have developed a computational approach for identifying those segments of amyloidogenic proteins that themselves can form amyloid-like fibrils. The approach builds on experiments showing that hexapeptides are sufficient for forming amyloid-like fibrils. Each six-residue peptide of a protein of interest is mapped onto an ensemble of templates, or 3D profile, generated from the crystal structure of the peptide NNQQNY by small displacements of one of the two intermeshed ␤-sheets relative to the other. The energy of each mapping of a sequence to the profile is evaluated by using ROSETTADESIGN, and the lowest energy match for a given peptide to the template library is taken as the putative prediction. If the energy of the putative prediction is lower than a threshold value, a prediction of fibril formation is made. This method can reach an accuracy of Ϸ80% with a P value of Ϸ10 ؊12 when a conservative energy threshold is used to separate peptides that form fibrils from those that do not. We see enrichment for positive predictions in a set of fibril-forming segments of amyloid proteins, and we illustrate the method with applications to proteins of interest in amyloid research.amyloid ͉ prediction ͉ ROSETTADESIGN ͉ lysozyme ͉ myoglobin A myloid-like fibrils of protein are common to deposition diseases such as Alzheimer's, the spongiform encephalopathies including Creutzfeldt-Jakob disease and bovine spongiform encephalopathy, and the protein-based heredity of [PSI ϩ ] and other prions in yeast. Thus understanding the range of protein sequences that can undergo fibrillization and the basis for stability of fibrils could have wide significance. We address these problems with a computational method for predicting which segments of a given protein might form the cross-␤ spine in the fibrillar form.The ability to form amyloid fibers is not restricted to those proteins associated with amyloid or prion disease. Otherwise innocuous proteins can be fibrillized by altering the pH, temperature, or composition of their native solvent (1-3). In addition, numerous short peptides (e.g., four to seven residues) are found to form amyloid-like fibrils in isolation from the rest of the protein (4-11). De novo-designed synthetic peptides have also been shown to form fibers (12)(13)(14).The question of how both full proteins and short peptides can form fibrils was illuminated by the crystal structures (11) of NNQQNY and GNNQQNY, which showed that the fundamental structure of the protofibril is a pair of ␤-sheets, which mate at a dry interface where their side chains tightly interdigitate in a ''steric zipper.'' To form this steric zipper, the strands in the sheets need be only four to six residues in length. Therefore, we would expect that short peptides with a tendency to fibrillize can do so, either when cleaved from the rest of the protein chain, as for the ␤-amyloid (Abeta) peptide of Alzheimer's disease, or when they are unmasked from an inaccessible ...

Many globular and natively disordered proteins can convert into amyloid fibers. These fibers are associated with numerous pathologies1 as well as with normal cellular functions2,3, and frequently form during protein denaturation4,5. Inhibitors of pathological amyloid fibers could serve as leads for therapeutics, provided the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibers as templates, we have designed and characterized an all D-amino acid inhibitor of fibrillation of the tau protein found in Alzheimer’s disease, and a non-natural L-amino acid inhibitor of an amyloid fiber that enhances sexual transmission of HIV. Our results indicate that peptides from structure-based designs can disrupt the fibrillation of full-length proteins, including those like tau that lack fully ordered native structures.

In the rare medical condition termed injection amyloidosis, extracellular fibrils of insulin are observed. We found that the segment of the insulin B-chain with sequence LVEALYL is the smallest segment that both nucleates and inhibits the fibrillation of fulllength insulin in a molar ratio-dependent manner, suggesting that this segment is central to the cross-␤ spine of the insulin fibril. In isolation from the rest of the protein, LVEALYL forms microcrystalline aggregates with fibrillar morphology, the structure of which we determined to 1 Å resolution. The LVEALYL segments are stacked into pairs of tightly interdigitated ␤-sheets, each pair displaying the dry steric zipper interface typical of amyloid-like fibrils. This structure leads to a model for fibrils of human insulin consistent with electron microscopic, x-ray fiber diffraction, and biochemical studies.amyloid ͉ fibril structure