Parallel in-register intermolecular ß-sheet architectures for prion-seeded prion protein (PrP) amyloids.

Structures of the infectious form of prion protein (e.g. PrP(Sc) or PrP-Scrapie) remain poorly defined. The prevalent structural models of PrP(Sc) retain most of the native α-helices of the normal, noninfectious prion protein, cellular prion protein (PrP(C)), but evidence is accumulating that these helices are absent in PrP(Sc) amyloid. Moreover, recombinant PrP(C) can form amyloid fibrils in vitro that have parallel in-register intermolecular ß-sheet architectures in the domains originally occupied by helices 2 and 3. Here, we provide solid-state NMR evidence that the latter is also true of initially prion-seeded recombinant PrP amyloids formed in the absence of denaturants. These results, in the context of a primarily ß-sheet structure, led us to build detailed models of PrP amyloid based on parallel in-register architectures, fibrillar shapes and dimensions, and other available experimentally derived conformational constraints. Molecular dynamics simulations of PrP(90-231) octameric segments suggested that such linear fibrils, which are consistent with many features of PrP(Sc) fibrils, can have stable parallel in-register ß-sheet cores. These simulations revealed that the C-terminal residues ∼124-227 more readily adopt stable tightly packed structures than the N-terminal residues ∼90-123 in the absence of cofactors. Variations in the placement of turns and loops that link the ß-sheets could give rise to distinct prion strains capable of faithful template-driven propagation. Moreover, our modeling suggests that single PrP monomers can comprise the entire cross-section of fibrils that have previously been assumed to be pairs of laterally associated protofilaments. Together, these insights provide a new basis for deciphering mammalian prion structures.

Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.

A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP(C) to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD(50)). Brain tissue from 263K scrapie-affected hamsters gave SD(50) values of 10(11)-10(12)/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD(50) values of 10(3.5)-10(5.7)/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD(50) values of 10(2.0)-10(2.9)/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.

Structure of the flexible amino-terminal domain of prion protein bound to a sulfated glycan.

The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence, PHGGGWGQ, that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear ((1)H, (13)C, (15)N) NMR (nuclear magnetic resonance), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23-106 (PrP 23-106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23-106 remain disordered upon PPS binding, the octarepeat region adopts a repeating loop-turn structure that we have determined by NMR. The beta-like turns within the repeats are corroborated by CD data demonstrating that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan side chains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23-106 when bound to PPS, consistent with the alignment of tryptophan side chains. Chemical-shift mapping identified binding sites on PrP 23-106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations.

Prion protein misfolding and disease.

Transmissible spongiform encephalopathies (TSEs or prion diseases) are a rare group of invariably fatal neurodegenerative disorders that affect humans and other mammals. TSEs are protein misfolding diseases that involve the accumulation of an abnormally aggregated form of the normal host prion protein (PrP). They are unique among protein misfolding disorders in that they are transmissible and have different strains of infectious agents that are associated with unique phenotypes in vivo. A wealth of biological and biophysical evidence now suggests that the molecular basis for prion diseases may be encoded by protein conformation. The purpose of this review is to provide an overview of the existing structural information for PrP within the context of what is known about the biology of prion disease.

Structural studies of Apo NosL, an accessory protein of the nitrous oxide reductase system: insights from structural homology with MerB, a mercury resistance protein.

The formation of the unique catalytic tetranuclear copper cluster (Cu(Z)) of nitrous oxide reductase, N(2)OR, requires the coexpression of a multiprotein assembly apparatus encoded by the nosDFYL operon. NosL, one of the proteins encoded by this transcript, is a 20 kDa lipoprotein of the periplasm that has been shown to bind copper(I), although its function has yet to be detemined. Cu(I) EXAFS data collected on the holo protein demonstrated that features of the copper binding site are consistent with a role for this protein as a metallochaperone, a class of metal ion transporters involved in metal resistance, homeostasis, and metallocluster biosynthesis. To test this hypothesis and to gain insight into other potential functional roles for this protein in the N(2)OR system, the three-dimensional solution structure of apo NosL has been solved by solution NMR methods. The structure of apo NosL consists of two relatively independent homologous domains that adopt an unusual betabetaalphabeta topology. The fold of apo NosL displays structural homology to only one other protein, MerB, an organomercury lyase involved in bacterial mercury resistance (Di Lello et al. (2004) Biochemistry 43, 8322-32). The structural similarity between apo NosL and MerB, together with the absolute conservation of Met109 in all NosL sequences, indicates that this residue may be involved in copper ligation, and that the metal binding site is likely to be solvent-accessible and contiguous with a large binding cleft. The structural observations suggest that NosL is exceptionally adapted for a role in copper and/or sulfur delivery and possibly for metallochaperone function.