Studies using low-resolution fiber diffraction, electron microscopy, and atomic force microscopy on various amyloid fibrils indicate that the misfolded conformers must be modular, compact, and adopt a cross-β structure. In an earlier study, we used electron crystallography to delineate molecular models of the N-terminally truncated, disease-causing isoform (PrP ^Sc ) of the prion protein, designated PrP 27–30, which polymerizes into amyloid fibrils, but we were unable to choose between a trimeric or hexameric arrangement of right- or left-handed β-helical models. From a study of 119 all-β folds observed in globular proteins, we have now determined that, if PrP ^Sc follows a known protein fold, it adopts either a β-sandwich or parallel β-helical architecture. With increasing evidence arguing for a parallel β-sheet organization in amyloids, we contend that the sequence of PrP is compatible with a parallel left-handed β-helical fold. Left-handed β-helices readily form trimers, providing a natural template for a trimeric model of PrP ^Sc . This trimeric model accommodates the PrP sequence from residues 89–175 in a β-helical conformation with the C terminus (residues 176–227), retaining the disulfide-linked α-helical conformation observed in the normal cellular isoform. In addition, the proposed model matches the structural constraints of the PrP 27–30 crystals, positioning residues 141–176 and the N-linked sugars appropriately. Our parallel left-handed β-helical model provides a coherent framework that is consistent with many structural, biochemical, immunological, and propagation features of prions. Moreover, the parallel left-handed β-helical model for PrP ^Sc may provide important clues to the structure of filaments found in some other neurodegenerative diseases.

Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrP ^Sc ) interacts with the substrate cellular PrP (PrP ^C ) during conversion into nascent PrP ^Sc . While PrP ^Sc appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrP ^C is converted into PrP ^Sc . We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cells contain PrP ^C and PrP ^Sc . After lysis of ScN2a cells in ice-cold Triton X-100, both PrP isoforms and an N-terminally truncated form of PrP ^C (PrP ^C -II) were found concentrated in detergent-insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H-ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo- N -hydroxysuccinimide-biotin, both PrP ^C and PrP ^Sc were found biotinylated in CLD fractions. Similar results on the colocalization of PrP ^C and PrP ^Sc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrP ^C and PrP ^Sc are present in CLDs and, thus, support the hypothesis that the PrP ^Sc formation occurs within this subcellular compartment.

Recent evidence from several laboratories shows that the paired helical filaments of Alzheimer's disease brains consist mainly of the protein tau in an abnormally phosphorylated form, but the mode of assembly is not understood. Here we use EM to study several constructs derived from human brain tau and expressed in Escherichia coli. All constructs or tau isoforms are rodlike molecules with a high tendency to dimerize in an antiparallel fashion, as shown by antibody labeling and chemical crosslinking. The length of the rods is largely determined by the region of internal repeats that is also responsible for microtubule binding. One unit length of the repeat domain (three or four repeats) is around 22-25 nm, comparable to the cross-section of Alzheimer PHF cores. Constructs corresponding roughly to the repeat region of tau can form synthetic paired helical filaments resembling those from Alzheimer brain tissue. A similar self-assembly occurs with the chemically cross-linked dimers. In both cases there is no need for phosphorylation of the protein.

Because the insolubility of the scrapie prion protein (PrP ^Sc ) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP ^Sc (PrP 27-30) and a miniprion (PrP ^Sc 106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 Å resolution. By comparing projection maps of PrP 27-30 and PrP ^Sc 106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP ^Sc . Only models featuring parallel β-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.