Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrP C ) to the scrapie form (PrP Sc ) is still unknown. Here we discovered a chemical chaperone to stabilize the PrP C conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a ''pocket'' created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states (PrP*) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8, efficiently reduced PrP Sc . Subsequently, administration of GN8 was found to prolong the survival of TSE-infected mice. Heteronuclear NMR and computer simulation showed that the specific binding sites are the A-S2 loop (N159) and the region from helix B (V189, T192, and K194) to B-C loop (E196), indicating that the intercalation of these distant regions (hot spots) hampers the pathogenic conversion process. Dynamics-based drug discovery strategy, demonstrated here focusing on the hot spots of PrP C , will open the way to the development of novel anti-prion drugs.anti-prion compound ͉ binding sites ͉ chemical chaperone ͉ dynamicsbased drug discovery ͉ transmissible spongiform encephalopathy

A crucial step for transformation of the normal cellular isoform of the prion protein (PrP(C)) to the infectious prion protein (PrP(Sc)) is thought to entail a previously uncharacterized intermediate conformer, PrP*, which interacts with a template PrP(Sc) molecule in the conversion process. By carrying out (15)N-(1)H two-dimensional NMR measurements under variable pressure on Syrian hamster prion protein rPrP(90-231), we found a metastable conformer of PrP(C) coexisting at a population of approximately 1% at pH 5.2 and 30 degrees C, in which helices B and C are preferentially disordered. While the identity is still unproven, this observed metastable conformer is most logically PrP* or a closely related precursor. The structural characteristics of this metastable conformer are consistent with available immunological and pathological information about the prion protein.

PrP106 -126, a peptide corresponding to residues 107-127 of the human prion protein, induces neuronal cell death by apoptosis and causes proliferation and hypertrophy of glia, reproducing the main neuropathological features of prion-related transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Although PrP106 -126 has been shown to form amyloid-like fibrils in vitro, their structural properties have not been elucidated. Here, we investigate the conformational characteristics of a fibril-forming fragment of the mouse prion protein, MoPrP106 -126, by using electron microscopy, CD spectroscopy, NMR-detected hydrogen-deuterium exchange measurements, and molecular dynamics simulations. The fibrils contain Ϸ50% ␤-sheet structure, and strong amide exchange protection is limited to the central portion of the peptide spanning the palindromic sequence VAGAAAAGAV. Molecular dynamics simulations indicate that MoPrP106 -126 in water assumes a stable structure consisting of two four-stranded parallel ␤-sheets that are tightly packed against each other by methyl-methyl interactions. Fibril formation involving polyalanine stacking is consistent with the experimental observations. P rions are infectious particles that cause transmissible spongiform encephalopathies in animals and humans. Prions are composed of PrP Sc , a conformationally altered form of a hostencoded glycoprotein, PrP C (1). Although the two isoforms are chemically identical, they possess very different physicochemical properties. In particular, PrP C is mostly helical, whereas the scrapie form PrP Sc contains Ϸ40% ␤-sheet (2). A synthetic peptide, PrP106-126, was shown to aggregate into proteaseresistant amyloid fibrils and induce neuronal cell death by apoptosis, causing proliferation and hypertrophy of cultured glia (3, 4). This segment corresponds to an unstructured region just outside of the globular C-terminal domain of PrP C (5, 6). Analysis of deletion mutants of human prion protein (PrP) showed that a large N-terminal fragment (residues 23-88) and a segment within the structured domain of PrP C (residues 141-176) could be deleted without affecting its conversion into the protease-resistant PrP Sc , whereas deletion of segments 95-107, 108-121, or 122-140 abolished the conformational transition (7). PrP106-126 is located within this critical region (residues 95-140), has been shown to adopt different secondary structures under different solution conditions (8, 9), and is thus a relevant model for investigating the mechanism of fibril formation and PrP Sc -mediated cell death. Recent solid-state NMR results showed that fibrils of the mouse prion peptide 89-143 are composed predominantly of ␤-structure, and they suggested that its pathogenicity is related to the specific ␤-sheet conformation (10, 11). However, little is known presently about the detailed structure of PrP Sc or any fibril-forming fragments of the PrP. In fact, there are very few direct experimental observations available for any...

Using heteronuclear NMR spectroscopy, we studied the solution structure and dynamics of bovine b-lactoglobulin A at pH 2.0 and 45 8C, where the protein exists as a monomeric native state. The monomeric NMR structure, comprising an eight-stranded continuous antiparallel b-barrel and one major a-helix, is similar to the X-ray dimeric structure obtained at pH 6.2, including b Istrand that forms the dimer interface and loop EF that serves as a lid of the interior hydrophobic hole. $ 1 H%-15 N NOE revealed that b F , b G , and b H strands buried under the major a-helix are rigid on a pico-to nanosecond time scale and also emphasized rapid fluctuations of loops and the N-and C-terminal regions.

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We investigated the dissociation process of tri-N-acetyl-d-glucosamine from hen egg white lysozyme using parallel cascade selection molecular dynamics (PaCS-MD), which comprises cycles of multiple unbiased MD simulations using a selection of MD snapshots as the initial structures for the next cycle. Dissociation was significantly accelerated by PaCS-MD, in which the probability of rare event occurrence toward dissociation was enhanced by the selection and rerandomization of the initial velocities. Although this complex was stable during 1 μs of conventional MD, PaCS-MD easily induced dissociation within 10-10 ns. We found that velocity rerandomization enhances the dissociation of triNAG from the bound state, whereas diffusion plays a more important role in the unbound state. We calculated the dissociation free energy by analyzing all PaCS-MD trajectories using the Markov state model (MSM), compared the results to those obtained by combinations of PaCS-MD and umbrella sampling (US), steered MD (SMD) and US, and SMD and the Jarzynski equality, and experimentally determined binding free energy. PaCS-MD/MSM yielded results most comparable to the experimentally determined binding free energy, independent of simulation parameter variations, and also gave the lowest standard errors.

We have defined the structural and dynamic properties of an early folding intermediate of beta-lactoglobulin known to contain non-native alpha-helical structure. The folding of beta-lactoglobulin was monitored over the 100 micros--10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 micros is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta-strands F, G and H, and the main alpha-helix, which was thus identified as the folding core of beta-lactoglobulin. At the same time, weak protection (up to approximately 10-fold) of amide protons in a segment spanning residues 12--21 is consistent with formation of marginally stable non-native alpha-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain.

We performed fragment molecular orbital (FMO) calculations to examine the molecular interactions between the prion protein (PrP) and GN8, which is a potential curative agent for prion diseases. This study has the following novel aspects: we introduced the counterpoise method into the FMO scheme to eliminate the basis set superposition error and examined the influence of geometrical fluctuation on the interaction energies, thereby enabling rigorous analysis of the molecular interaction between PrP and GN8. This analysis could provide information on key amino acid residues of PrP as well as key units of GN8 involved in the molecular interaction between the two molecules. The present FMO calculations were performed using an original program developed in our laboratory, called "Parallelized ab initio calculation system based on FMO (PAICS)".