Structure of an infectious mammalian prion

ABSTRACTClassical mammalian prions are assemblies of prion protein molecules that are extraordinarily transmissible, with a microgram of protein containing up to 108 lethal doses of infectivity1,2. Unlike most other pathologic and amyloidogenic proteins, prions typically contain glycolipid anchors 3 and abundant asparagine‐linked glycans4‐6. The infectious nature, complexity, and biophysical properties of prions have complicated structural analyses and stymied any prior elucidation of 3D conformation at the polypeptide backbone level7. Here we have determined the structure of the core of a fully infectious, brain‐derived prion by cryo‐electron microscopy with ∼3.1 Å resolution. The purified prions are amyloid fibrils comprised of monomers assembled with parallel in‐register intermolecular beta sheets and connecting chains. Residues ∼95‐227 of each monomer provide one rung of the ordered fibril core, with the glycans and glycolipid anchor projecting from the lateral surfaces of the fibril. The fibril ends, where prion growth occurs, are formed by single monomers in an extended serpentine combination of β‐ arches, a Greek key, and loops that presumably template the refolding of incoming monomers. Our results describe an atomic model to underpin detailed molecular hypotheses of how pathologic prion proteins can propagate as infectious agents, and how such propagation and associated pathogenesis might be impeded.

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Prion protein dynamics before aggregation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1620400114 ◽

2017 ◽

Vol 114 (14) ◽

pp. 3572-3577 ◽

Cited By ~ 13

Author(s):

Kinshuk Raj Srivastava ◽

Lisa J. Lapidus

Keyword(s):

Protein Dynamics ◽

Neurodegenerative Disorders ◽

Syrian Hamster ◽

Time Scale ◽

Molecular Basis ◽

Prion Proteins ◽

Prion Diseases ◽

Hydrophobic Residues ◽

Protein Deposits ◽

Polypeptide Backbone

Prion diseases, like Alzheimer’s disease and Parkinson disease, are rapidly progressive neurodegenerative disorders caused by misfolding followed by aggregation and accumulation of protein deposits in neuronal cells. Here we measure intramolecular polypeptide backbone reconfiguration as a way to understand the molecular basis of prion aggregation. Our hypothesis is that when reconfiguration is either much faster or much slower than bimolecular diffusion, biomolecular association is not stable, but as the reconfiguration rate becomes similar to the rate of biomolecular diffusion, the association is more stable and subsequent aggregation is faster. Using the technique of Trp–Cys contact quenching, we investigate the effects of various conditions on reconfiguration dynamics of the Syrian hamster and rabbit prion proteins. This protein exhibits behavior in all three reconfiguration regimes. We conclude that the hamster prion is prone to aggregation at pH 4.4 because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs, whereas the rabbit sequence avoids aggregation by reconfiguring 10 times faster than the hamster sequence.

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Examining the structure of the mature amyloid fibril

Biochemical Society Transactions ◽

10.1042/bst0300521 ◽

2002 ◽

Vol 30 (4) ◽

pp. 521-525 ◽

Cited By ~ 43

Author(s):

O. S. Makin ◽

L. C. Serpell

Keyword(s):

Self Assembly ◽

Rational Design ◽

Amyloid Fibrils ◽

Structural Information ◽

X Ray ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Fibre Diffraction ◽

Complementary Techniques ◽

Mature Fibril

The pathogenesis of the group of diseases known collectively as the amyloidoses is characterized by the deposition of insoluble amyloid fibrils. These are straight, unbranching structures about 70–120 å (1 å = 0.1 nm) in diameter and of indeterminate length formed by the self-assembly of a diverse group of normally soluble proteins. Knowledge of the structure of these fibrils is necessary for the understanding of their abnormal assembly and deposition, possibly leading to the rational design of therapeutic agents for their prevention or disaggregation. Structural elucidation is impeded by fibril insolubility and inability to crystallize, thus preventing the use of X-ray crystallography and solution NMR. CD, Fourier-transform infrared spectroscopy and light scattering have been used in the study of the mechanism of fibril formation. This review concentrates on the structural information about the final, mature fibril and in particular the complementary techniques of cryo-electron microscopy, solid-state NMR and X-ray fibre diffraction.

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Parkinson’s disease-related phosphorylation at Tyr39 rearranges α-synuclein amyloid fibril structure revealed by cryo-EM

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922741117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 20305-20315 ◽

Cited By ~ 3

Author(s):

Kun Zhao ◽

Yeh-Jun Lim ◽

Zhenying Liu ◽

Houfang Long ◽

Yunpeng Sun ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Posttranslational Modifications ◽

Cortical Neurons ◽

Amyloid Fibrils ◽

Lewy Bodies ◽

Interaction Network ◽

Lewy Neurites ◽

Cryo Electron Microscopy ◽

Fibril Structure

Posttranslational modifications (PTMs) of α-synuclein (α-syn), e.g., phosphorylation, play an important role in modulating α-syn pathology in Parkinson’s disease (PD) and α-synucleinopathies. Accumulation of phosphorylated α-syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to α-syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous α-syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-electron microscopy (cryo-EM) structure of the pY39 α-syn fibril, which reveals a fold of α-syn with pY39 in the center of the fibril core forming an electrostatic interaction network with eight charged residues in the N-terminal region of α-syn. This structure composed of residues 1 to 100 represents the largest α-syn fibril core determined so far. This work provides structural understanding on the pathology of the pY39 α-syn fibril and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.

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Cryo-EM structure and polymorphism of Aβ amyloid fibrils purified from Alzheimer’s brain tissue

Nature Communications ◽

10.1038/s41467-019-12683-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 85

Author(s):

Marius Kollmer ◽

William Close ◽

Leonie Funk ◽

Jay Rasmussen ◽

Aref Bsoul ◽

...

Keyword(s):

Electron Microscopy ◽

Brain Tissue ◽

Amyloid Fibrils ◽

Structural Basis ◽

Amyloid Angiopathy ◽

Cryo Electron Microscopy ◽

Aβ Amyloid ◽

Cerebral Amyloid ◽

Aβ Fibrils

Abstract The formation of Aβ amyloid fibrils is a neuropathological hallmark of Alzheimer’s disease and cerebral amyloid angiopathy. However, the structure of Aβ amyloid fibrils from brain tissue is poorly understood. Here we report the purification of Aβ amyloid fibrils from meningeal Alzheimer’s brain tissue and their structural analysis with cryo-electron microscopy. We show that these fibrils are polymorphic but consist of similarly structured protofilaments. Brain derived Aβ amyloid fibrils are right-hand twisted and their peptide fold differs sharply from previously analyzed Aβ fibrils that were formed in vitro. These data underscore the importance to use patient-derived amyloid fibrils when investigating the structural basis of the disease.

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Molecular insights into amyloid regulation by membrane cholesterol and sphingolipids: common mechanisms in neurodegenerative diseases

Expert Reviews in Molecular Medicine ◽

10.1017/s1462399410001602 ◽

2010 ◽

Vol 12 ◽

Cited By ~ 112

Author(s):

Jacques Fantini ◽

Nouara Yahi

Keyword(s):

Neurodegenerative Diseases ◽

Conformational Changes ◽

Amyloid Fibrils ◽

Fine Tuning ◽

Neural Cells ◽

Amyloidogenic Proteins ◽

Innovative Therapies ◽

Conformational Plasticity ◽

Direct Binding ◽

Α Helix

Alzheimer, Parkinson and other neurodegenerative diseases involve a series of brain proteins, referred to as ‘amyloidogenic proteins’, with exceptional conformational plasticity and a high propensity for self-aggregation. Although the mechanisms by which amyloidogenic proteins kill neural cells are not fully understood, a common feature is the concentration of unstructured amyloidogenic monomers on bidimensional membrane lattices. Membrane-bound monomers undergo a series of lipid-dependent conformational changes, leading to the formation of oligomers of varying toxicity rich in β-sheet structures (annular pores, amyloid fibrils) or in α-helix structures (transmembrane channels). Condensed membrane nano- or microdomains formed by sphingolipids and cholesterol are privileged sites for the binding and oligomerisation of amyloidogenic proteins. By controlling the balance between unstructured monomers and α or β conformers (the chaperone effect), sphingolipids can either inhibit or stimulate the oligomerisation of amyloidogenic proteins. Cholesterol has a dual role: regulation of protein–sphingolipid interactions through a fine tuning of sphingolipid conformation (indirect effect), and facilitation of pore (or channel) formation through direct binding to amyloidogenic proteins. Deciphering this complex network of molecular interactions in the context of age- and disease-related evolution of brain lipid expression will help understanding of how amyloidogenic proteins induce neural toxicity and will stimulate the development of innovative therapies for neurodegenerative diseases.

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Exploring the structure and formation mechanism of amyloid fibrils by Raman spectroscopy: a review

The Analyst ◽

10.1039/c5an00342c ◽

2015 ◽

Vol 140 (15) ◽

pp. 4967-4980 ◽

Cited By ~ 96

Author(s):

Dmitry Kurouski ◽

Richard P. Van Duyne ◽

Igor K. Lednev

Keyword(s):

Raman Spectroscopy ◽

Formation Mechanism ◽

Structural Characterization ◽

Amyloid Fibrils ◽

Label Free ◽

Amyloidogenic Proteins ◽

Non Destructive

Applications of Raman spectroscopy, a label-free non-destructive technique, for the structural characterization of amyloidogenic proteins, prefibrilar oligomers, and mature fibrils.

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Structural identification of individual helical amyloid filaments by integration of cryo-electron microscopy-derived maps in comparative morphometric atomic force microscopy image analysis

10.1101/2021.10.19.464873 ◽

2021 ◽

Author(s):

Liisa Lutter ◽

Youssra Al-Hilaly ◽

Christopher J. Serpell ◽

Mick F. Tuite ◽

Claude M. Wischik ◽

...

Keyword(s):

Electron Microscopy ◽

Atomic Force Microscopy ◽

Image Analysis ◽

Structural Analysis ◽

Amyloid Fibrils ◽

Structural Identification ◽

Force Microscopy ◽

Atomic Force ◽

Cryo Electron Microscopy

The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations. Here, we report a method for structural identification of individual fibril structures imaged by atomic force microscopy (AFM) by integration of high-resolution maps of amyloid fibrils determined by cryo-EM in comparative AFM image analysis. This approach was demonstrated using the hitherto structurally unresolved amyloid fibrils formed in vitro from a fragment of tau (297-391), termed 'dGAE'. Our approach established unequivocally that dGAE amyloid fibrils bear no structural relationship to heparin-induced tau fibrils formed in vitro. Furthermore, our comparative analysis resulted in the prediction that dGAE fibrils are closely related structurally to the paired helical filaments (PHFs) isolated from Alzheimer's disease (AD) brain tissue characterised by cryo-EM. These results show the utility of individual particle structural analysis using AFM, provide a workflow of how cryo-EM data can be incorporated into AFM image analysis and facilitate an integrated structural analysis of amyloid polymorphism.

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Molecular modelling indicates that the pathological conformations of prion proteins might be β-helical

Biochemical Journal ◽

10.1042/bj3430453 ◽

1999 ◽

Vol 343 (2) ◽

pp. 453-460 ◽

Cited By ~ 13

Author(s):

Donald T. DOWNING ◽

N. D. LAZO

Keyword(s):

Molecular Modelling ◽

Amyloid Fibrils ◽

Prion Proteins ◽

Prion Diseases ◽

Spectroscopic Studies ◽

Cellular Protein ◽

Precursor Protein ◽

Random Coil ◽

Helical Conformation ◽

Spongiform Encephalopathy

Creutzfeldt-Jakob disease, kuru, scrapie and bovine spongiform encephalopathy are diseases of the mammalian central nervous system that involve the conversion of a cellular protein into an insoluble extracellular isoform. Spectroscopic studies have shown that the precursor protein contains mainly α-helical and random-coil conformations, whereas the prion isoform is largely in the β conformation. The pathogenic prion is resistant to denaturation and protease digestion and can promote the conversion of the precursor protein to the pathogenic form. These properties have yet to be explained in terms of the structural conformations of the proteins. In the present study, molecular modelling showed that prion proteins could adopt the β-helical conformation, which has been established for a number of fibrous proteins and has been suggested previously as the basis of amyloid fibrils. The β-helical conformation provides explanations for the biophysical and biochemical stability of prions, their ability to form templates for the transmission of pathological conformation, and the existence of phenotypical strains of the prion diseases.

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Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.23.10962 ◽

1993 ◽

Vol 90 (23) ◽

pp. 10962-10966 ◽

Cited By ~ 1556

Author(s):

K M Pan ◽

M Baldwin ◽

J Nguyen ◽

M Gasset ◽

A Serban ◽

...

Keyword(s):

Chemical Modification ◽

Prion Protein ◽

Conformational Transition ◽

Prion Proteins ◽

Limited Proteolysis ◽

Alpha Helix ◽

Beta Sheets ◽

Cellular Prion Protein ◽

Beta Sheet ◽

Alpha Helices

Prions are composed largely, if not entirely, of prion protein (PrPSc in the case of scrapie). Although the formation of PrPSc from the cellular prion protein (PrPC) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPSc synthesis. To assess this possibility, we purified both PrPC and PrPSc by using nondenaturing procedures and determined the secondary structure of each. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrPSc was 43% and the alpha-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrPSc derived by limited proteolysis, designated PrP 27-30, has an even higher beta-sheet content (54%) and a lower alpha-helix content (21%). Neither PrPC nor PrPSc formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing findings argue that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrPSc initiates this process. Since PrPSc seems to be the only component of the "infectious" prion particle, it is likely that this conformational transition is a fundamental event in the propagation of prions.

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Insights into the Structure of Amyloid Fibrils

The Open Biology Journal ◽

10.2174/1874196700902010185 ◽

2009 ◽

Vol 2 (1) ◽

pp. 185-192

Author(s):

Karen E. Marshall ◽

Louise C. Serpell

Keyword(s):

Amyloid Fibrils ◽

Amyloid Formation ◽

X Ray Diffraction ◽

Generic Structure ◽

Structural Characterisation ◽

Cryo Electron Microscopy ◽

Atomic Interactions ◽

Heterogeneous Nature ◽

Structural Insights ◽

Proteins And Peptides

Various proteins and peptides are able to self assemble into amyloid fibrils that are associated with disease. Structural characterisation of these fibres is limited by their insoluble and heterogeneous nature. However, advances in various techniques including X-ray diffraction, cryo-electron microscopy and solid state NMR have provided detailed information on various amyloid fibrils, from the long range order and macromolecular structure to the atomic interactions that promote assembly and stabilise the amyloid core. The cross-β model has been widely accepted as a generic structure for most amyloid fibrils and is discussed in detail. It is clear, however, that polymorphisms are present, even in fibrils formed from the same precursor protein, and that these may represent differences in packing at a molecular level. To fully understand the roles of particular residues in amyloid formation and structure, short peptides can be used in conjunction with mutagenesis studies to assess their effects. The structural insights gained using a combination of techniques to study both full-length, disease related peptides and short fragments are essential if progress is to be made towards understanding why these fibres form and how to prevent their formation.

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