Amyloid of the prion domain of Sup35p has an in-register parallel beta-sheet structure.
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ABSTRACT:
SUBMITTER: Shewmaker F
PROVIDER: S-EPMC1750918 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The [PSI+] prion is a self-propagating amyloid of the translation termination factor, Sup35p, of Saccharomyces cerevisiae. The N-terminal 253 residues (NM) of this 685-residue protein normally function in regulating mRNA turnover but spontaneously form infectious amyloid in vitro. We converted the three Ile residues in Sup35NM to Leu and then replaced 16 single residues with Ile, one by one, and prepared Ile-1-(13)C amyloid of each mutant, seeding with amyloid formed by the reference sequence Sup35NM. Using solid-state NMR, we showed that 10 of the residues examined, including six between residues 30 and 90, showed the ∼0.5-nm distance between labels diagnostic of the in-register parallel amyloid architecture. The five scattered N domain residues with wider spacing may be in turns or loops; one is a control at the C terminus of M. All mutants, except Q56I, showed little or no [PSI+] transmission barrier from the reference sequence, suggesting that they could assume a similar amyloid architecture in vitro when seeded with filaments of reference sequence Sup35NM. Infection of yeast cells expressing the reference SUP35 gene sequence with amyloid of several mutants produced [PSI+] transfectants with similar efficiency as did reference sequence Sup35NM amyloid. Our work provides a stringent demonstration that the Sup35 prion domain has the folded in-register parallel β-sheet architecture and suggests common locations of the folds. This architecture naturally suggests a mechanism of inheritance of conformation, the central mystery of prions.
| S-EPMC4217437 | biostudies-literature
Project description:The [PSI(+)] prion is a self-propagating amyloid of the Sup35 protein, normally a subunit of the translation termination factor, but impaired in this vital function when in the amyloid form. The Sup35 N, M, and C domains are the amino-terminal prion domain, a connecting polar domain, and the essential C-terminal domain resembling eukaryotic elongation factor 1alpha respectively. Different [PSI(+)] isolates (prion variants) may have distinct biological properties, associated with different amyloid structures. Here we use solid state NMR to examine the structure of infectious Sup35NM amyloid fibrils of two prion variants. We find that both variants have an in-register parallel beta-sheet structure, both in the fully hydrated form and in the lyophilized form. Moreover, we confirm that some leucine residues in the M domain participate in the in-register parallel beta-sheet structure. Transmission of the [PSI(+)] prion by amyloid fibrils of Sup35NM and transmission of the [URE3] prion by amyloid fibrils of recombinant full-length Ure2p are similar whether they have been lyophilized or not (wet or dry).
| S-EPMC2744896 | biostudies-literature
Project description:The [PIN(+)] prion, a self-propagating amyloid form of Rnq1p, increases the frequency with which the [PSI(+)] or [URE3] prions arise de novo. Like the prion domains of Sup35p and Ure2p, Rnq1p is rich in N and Q residues, but rnq1Delta strains have no known phenotype except for inability to propagate the [PIN(+)] prion. We used solid-state NMR methods to examine amyloid formed in vitro from recombinant Rnq1 prion domain (residues 153-405) labeled with Tyr-1-(13)C (14 residues), Leu-1-(13)C (7 residues), or Ala-3-(13)C (13 residues). The carbonyl chemical shifts indicate that most Tyr and Leu residues are in beta-sheet conformation. Experiments designed to measure the distance from each labeled residue to the next nearest labeled carbonyl showed that almost all Tyr and Leu carbonyl carbon atoms were approximately 0.5 nm from the next nearest Tyr and Leu residues, respectively. This result indicates that the Rnq1 prion domain forms amyloid consisting of parallel beta-strands that are either in register or are at most one amino acid out of register. Similar experiments with Ala-3-(13)C indicate that the beta-strands are indeed in-register. The parallel in-register structure, now demonstrated for each of the yeast prions, explains the faithful templating of prion strains, and suggests as well a mechanism for the rare hetero-priming that is [PIN(+)]'s defining characteristic.
| S-EPMC2268149 | biostudies-literature
Project description:Ure2p of Candida albicans (Ure2(albicans) or CaUre2p) can be a prion in Saccharomyces cerevisiae, but Ure2p of Candida glabrata (Ure2(glabrata)) cannot, even though the Ure2(glabrata) N-terminal domain is more similar to that of the S. cerevisiae Ure2p (Ure2(cerevisiae)) than Ure2(albicans) is. We show that the N-terminal N/Q-rich prion domain of Ure2(albicans) forms amyloid that is infectious, transmitting [URE3alb] to S. cerevisiae cells expressing only C. albicans Ure2p. Using solid-state nuclear magnetic resonance of selectively labeled C. albicans Ure2p(1-90), we show that this infectious amyloid has an in-register parallel ?-sheet structure, like that of the S. cerevisiae Ure2p prion domain and other S. cerevisiae prion amyloids. In contrast, the N/Q-rich N-terminal domain of Ure2(glabrata) does not readily form amyloid, and that formed upon prolonged incubation is not infectious.
| S-EPMC3144561 | biostudies-literature
Project description:The extracellular curli proteins of Enterobacteriaceae form fibrous structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-beta structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli structure is not based on an in-register parallel beta-sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a beta-helix-like structure but are not sufficient to establish such a structure definitively.
| S-EPMC2757210 | biostudies-literature
Project description:Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and alpha-helical prion protein, PrP(C), to the beta-sheet-rich PrP(Sc). This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrP(C) monomer, structures of the pathogenic PrP(Sc) or synthetic PrP(Sc)-like aggregates remain elusive. Here we have used site-directed spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrP(C). Our data show that, in contrast to earlier, largely theoretical models, the con formational conversion of PrP(C) involves major refolding of the C-terminal alpha-helical region. The core of the amyloid maps to C-terminal residues from approximately 160-220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of beta-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to beta-structure.
| S-EPMC2141888 | biostudies-other
Project description:The pathognomonic plaques of Alzheimer's disease are composed primarily of the 39- to 43-aa beta-amyloid (Abeta) peptide. Crosslinking of Abeta peptides by tissue transglutaminase (tTg) indicates that Gln15 of one peptide is proximate to Lys16 of another in aggregated Abeta. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Abeta peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Abeta. Peptides containing a single carbonyl 13C label at Gln15, Lys16, Leu17, or Val18 were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 A. Analysis of these data establish that this central core of Abeta consists of a parallel beta-sheet structure in which identical residues on adjacent chains are aligned directly, i. e., in register. Our data, in conjunction with existing structural data, establish that the Abeta fibril is a hydrogen-bonded, parallel beta-sheet defining the long axis of the Abeta fibril propagation.
| S-EPMC24832 | biostudies-literature
Project description: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 ?-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 ?-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 ?-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 ?-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 ?-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.
| S-EPMC4148845 | biostudies-literature
Project description:X-ray diffraction and other biophysical tools reveal features of the atomic structure of an amyloid-like crystal. Sup35, a prion-like protein in yeast, forms fibrillar amyloid assemblies intrinsic to its prion function. We have identified a polar peptide from the N-terminal prion-determining domain of Sup35 that exhibits the amyloid properties of full-length Sup35, including cooperative kinetics of aggregation, fibril formation, binding of the dye Congo red, and the characteristic cross-beta x-ray diffraction pattern. Microcrystals of this peptide also share the principal properties of the fibrillar amyloid, including a highly stable, beta-sheet-rich structure and the binding of Congo red. The x-ray powder pattern of the microcrystals, extending to 0.9-A resolution, yields the unit cell dimensions of the well-ordered structure. These dimensions restrict possible atomic models of this amyloid-like structure and demonstrate that it forms packed, parallel-stranded beta-sheets. The unusually high density of the crystals shows that the packed beta-sheets are dehydrated, despite the polar character of the side chains. These results suggest that amyloid is a highly intermolecularly bonded, dehydrated array of densely packed beta-sheets. This dry beta-sheet could form as Sup35 partially unfolds to expose the peptide, permitting it to hydrogen-bond to the same peptide of other Sup35 molecules. The implication is that amyloid-forming units may be short segments of proteins, exposed for interactions by partial unfolding.
| S-EPMC30146 | biostudies-literature
Project description:The mechanism of prion strain diversity remains unsolved. Investigation of inheritance and diversification of protein-based pathogenic information demands the identification of the detailed structures of abnormal isoforms of the prion protein (PrPSc); however, achieving purification is difficult without affecting infectivity. Similar prion-like properties are recognized also in other disease-associated in-register parallel ?-sheet amyloids including Tau and ?-synuclein (?Syn) amyloids. Investigations into structures of those amyloids via solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy recently made remarkable advances due to their relatively small sizes and lack of post-translational modifications. Herein, we review advances regarding pathogenic amyloids, particularly Tau and ?Syn, and discuss implications about strain diversity mechanisms of prion/PrPSc from the perspective that PrPSc is an in-register parallel ?-sheet amyloid. Additionally, we present our recent data of molecular dynamics simulations of ?Syn amyloid, which suggest significance of compatibility between ?-sheet propensities of the substrate and local structures of the template for stability of amyloid structures. Detailed structures of ?Syn and Tau amyloids are excellent models of pathogenic amyloids, including PrPSc, to elucidate strain diversity and pathogenic mechanisms.
| S-EPMC6410106 | biostudies-literature