We started studying the structures of yeast prions in 1998, in collaboration with R Wickner (NIDDK), focussing initially on Ure2p, a negative regulator of nitrogen catabolism. We showed that its N-terminal domain is responsible for prionogenesis, while the C-terminal domain which performs its regulatory function remains folded in filaments but is inactivated by a steric mechanism. In our amyloid backbone concept, the prion domains form the filament backbone and are surrounded by the C-terminal domains. In 2005, we published the parallel superpleated beta-structure model for the amyloid backbone. It envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Topologically similar structures are good candidates for certain other amyloid fibrils, including amylin and growing support for models of this kind is appearing in the scientific literature. Ongoing work is aimed at testing and refining this model;exploring its range of applicability;and investigating fibril polymorphism. In FY09, we focussed on three areas: (1) Systematic analysis of beta-arcade motifs in amyloid fibril models and beta-solenoid protein structures. Amyloid fibrils accumulate in diseases, such as Alzheimers or type II diabetes. The amyloid-forming protein is disease-specific. Amyloids may also be formed in vitro from many other proteins. Unlike the diverse native folds of these proteins, their amyloids are fundamentally similar in being rigid, smooth-sided, and in having cross-β-structures. Despite the difficulties attendant upon obtaining high resolution experimentally determined fibril structures, increasingly credible models are being derived by integrating data from multiple techniques. Most current models of disease-related amyloids invoke β-arcades, columnar structures produced by in-register stacking of β-arches. A β-arch is a strand-turn-strand motif in which the two β-strands interact via their side-chains, not via the polypeptide backbone as in a conventional β-hairpin. Crystal structures of β-solenoids, a class of proteins with amyloid-like properties, offer insight into the β-arc turns found in arches. General thermodynamic considerations suggest that complexes of two or more β-arches may nucleate amyloid fibrillogenesis. (2) Formation of both infectious and non-infectious amyloid fibrils by the HET-s prion protein of the filamentous fungus, Podospora anserina. This prion differs from the yeast prions protein, Ure2p and Sup35p, in producing a gain-of-function prion, and in not having an abnormally high content of Asn/Gln residues. In 2007, we published a paper showing by electron diffraction that Het-s fibrils have a cross-beta structure (as anticipated), and by EM-based mass measurements that it has an axial packing density of 1 subunit per 0.94 nm - half the density of Ure2p and Sup35p fibrils, and in agreement with a published model, the stacked beta-solenoid. In another study, also completed in 2007, we compared the amyloids formed by the HET-s prion domain at different pH's. Fibrils formed at pH 7 from those formed at pH 2 on morphological grounds and in having higher specific infectivity. In FY09, we investigated the correlation between infectivity and fibril morphology further, by cryo-EM. Above pH 3, singlet fibrils are produced while, below pH 3, triplet fibrils are obtained. Singlets have an axial periodicity of 400 and a left-handed twist. Triplet fibrils have three supercoiled protofibrils whose diameter (50 ), and axial packing density (1 subunit per 9.4 ) resemble those of singlet fibrils but whose axial repeat and supercoiling differ. In triplet fibrils formed at higher pH, the interactions among protofibrils appear to loosen, eventually leading to their separation into individual protofibrils. By fitting a published atomic model derived from solid state NMR into tthe electron density of triplet fibrils, we investigated the inter-protofibril surface and suggest some key residues (E235, R238 and R274) that could be important in the interaction. (3) Fibrillation in situ and in vitro of the yeast prion protein Ure2p. For the most part, biochemical studies of amyloid formation are carried out in vitro with purified protein, often from exogenous recombinant sources. Genetic studies are carried out in situ. In this context, there has been long-standing uncertainty as to whether the same form of aggregate is being studied in both cases. With Ure2p, in vitro-formed fibrils have been electroporated into uninfected yeast cells and shown to elicit infection, albeit with low efficiency. However, it has not been possible to isolate fibrils from infected cells for structural analysis. In FY09, we have approached this problem by studying the Ure2p fibrils present in infected cells by electron tomography of freeze-substituted preparations. In this way, we have been able to estimate the total amount of fibrillar Urep present in these cells and found that it correlates with the amount per cells ascertained by biochemical quantitation. Thus all or almost all of the Ure2p protein in these cells is in fibrillar. The in situ fibrils appear to be slightly thicker than their in vitro-assembled counterparts. Our interpretation is that both kinds of fibrils have essentially the same structures and the in situ thickening probably represents a deposition of other cytoplasmic proteins onto the fibrils in response to specimen preparation for EM.
