Alzheimer's disease is the most common neurodegenerative disease, and there are no mechanism-based therapies. The disease is defined by the presence of abundant neurofibrillary lesions and neuritic ...plaques in the cerebral cortex. Neurofibrillary lesions comprise paired helical and straight tau filaments, whereas tau filaments with different morphologies characterize other neurodegenerative diseases. No high-resolution structures of tau filaments are available. Here we present cryo-electron microscopy (cryo-EM) maps at 3.4-3.5 Å resolution and corresponding atomic models of paired helical and straight filaments from the brain of an individual with Alzheimer's disease. Filament cores are made of two identical protofilaments comprising residues 306-378 of tau protein, which adopt a combined cross-β/β-helix structure and define the seed for tau aggregation. Paired helical and straight filaments differ in their inter-protofilament packing, showing that they are ultrastructural polymorphs. These findings demonstrate that cryo-EM allows atomic characterization of amyloid filaments from patient-derived material, and pave the way for investigation of a range of neurodegenerative diseases.
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•Cryo-EM enables high resolution structure determination of ex vivo amyloid fibres.•Patient derived tau fibrils adopt multiple folds and protofilament interactions.•β-helix fold has ...much more sequence specificity than cross-β.•Amyloidogenic species can form liquid phase compartments with physiological roles.•Liquid phase compartments can convert into fibrillar aggregates in cells.
Neurodegenerative and other protein misfolding diseases are associated with the aggregation of a protein, which may be mutated in genetic forms of disease, or the wild type form in late onset sporadic disease. A wide variety of proteins and peptides can be involved, with aggregation originating from a natively folded or a natively unstructured species. Large deposits of amyloid fibrils are typically associated with cell death in late stage pathology. In this review, we illustrate the contributions of cryo-EM and related methods to the structure determination of amyloid fibrils extracted post mortem from patient brains or formed in vitro. We also discuss cell models of protein aggregation and the contributions of electron tomography to understanding the cellular context of aggregation.
Tau aggregation into insoluble filaments is the defining pathological hallmark of tauopathies. However, it is not known what controls the formation and templated seeding of strain-specific structures ...associated with individual tauopathies. Here, we use cryo-electron microscopy (cryo-EM) to determine the structures of tau filaments from corticobasal degeneration (CBD) human brain tissue. Cryo-EM and mass spectrometry of tau filaments from CBD reveal that this conformer is heavily decorated with posttranslational modifications (PTMs), enabling us to map PTMs directly onto the structures. By comparing the structures and PTMs of tau filaments from CBD and Alzheimer’s disease, it is found that ubiquitination of tau can mediate inter-protofilament interfaces. We propose a structure-based model in which cross-talk between PTMs influences tau filament structure, contributing to the structural diversity of tauopathy strains. Our approach establishes a framework for further elucidating the relationship between the structures of polymorphic fibrils, including their PTMs, and neurodegenerative disease.
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•Cryo-EM structures of human brain-derived tau filaments from a four-repeat tauopathy•Cryo-EM and MS of tau filaments enable PTM mapping directly onto atomic models•Comparing tauopathies reveals that ubiquitination of tau can mediate fibril diversity•Integrated structural biology elucidates principles of PTM-mediated fibril assembly
Structural and mass spectrometry-based proteomics of tau filaments, including their posttranslational modifications, from corticobasal degeneration and Alzheimer’s disease point to how cross-talk between posttranslational modifications influences fibril structure, contributing to the structural diversity of tauopathy strains.
X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up ...endogenous structural proteomics approach whereby near-atomic-resolution cryo electron microscopy (cryoEM) maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria-causing parasite Plasmodium falciparum, an organism that has resisted conventional structural-biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.
The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable ...chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale—including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy—we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.
Amyloid is an important class of proteinaceous material because of its close association with protein misfolding disorders such as Alzheimer’s disease and type II diabetes. Although the degree of ...stiffness of amyloid is critical to the understanding of its pathological and biological functions, current estimates of the rigidity of these β-sheet–rich protein aggregates range from soft (10 ⁸ Pa) to hard (10 ¹⁰ Pa) depending on the method used. Here, we use time-resolved 4D EM to directly and noninvasively measure the oscillatory dynamics of freestanding, self-supporting amyloid beams and their rigidity. The dynamics of a single structure, not an ensemble, were visualized in space and time by imaging in the microscope an amyloid–dye cocrystal that, upon excitation, converts light into mechanical work. From the oscillatory motion, together with tomographic reconstructions of three studied amyloid beams, we determined the Young modulus of these highly ordered, hydrogen-bonded β-sheet structures. We find that amyloid materials are very stiff (10 ⁹ Pa). The potential biological relevance of the deposition of such a highly rigid biomaterial in vivo are discussed.
Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament ...structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or α-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.7 Å from postmortem human brain tissue afflicted with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP, n = 8), progressive supranuclear palsy (PSP, n = 2), or dementia with Lewy bodies (DLB, n = 1). The commonality of abundant amyloid fibrils composed of TMEM106B, a lysosomal/endosomal protein, to a broad range of debilitating human disorders indicates a shared fibrillization pathway that may initiate or accelerate neurodegeneration.
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•Cryo-EM structures of brain-derived TMEM106B fibrils from neurodegenerative diseases•Endolysosomal membrane protein TMEM106B C-terminal fragment forms amyloid fibrils•TMEM106B fibrillization is widespread among diverse neurodegenerative proteinopathies•Identification of fibrillization pathway potentially implicated in neurodegeneration
Cryo-EM and mass spectrometry-based proteomics of insoluble amyloid fibrils derived from postmortem human brains afflicted with diverse neurodegenerative diseases reveals widespread fibrillization of an endolysosomal membrane protein, TMEM106B, pointing toward a potentially pathogenic commonality between distinct proteinopathies.
α-Synuclein is a small protein strongly implicated in the pathogenesis of Parkinson's disease and related neurodegenerative disorders. We report here the use of in-cell NMR spectroscopy to observe ...directly the structure and dynamics of this protein within E. coli cells. To improve the accuracy in the measurement of backbone chemical shifts within crowded in-cell NMR spectra, we have developed a deconvolution method to reduce inhomogeneous line broadening within cellular samples. The resulting chemical shift values were then used to evaluate the distribution of secondary structure populations which, in the absence of stable tertiary contacts, are a most effective way to describe the conformational fluctuations of disordered proteins. The results indicate that, at least within the bacterial cytosol, α-synuclein populates a highly dynamic state that, despite the highly crowded environment, has the same characteristics as the disordered monomeric form observed in aqueous solution.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The amyloid state of polypeptides is a stable, highly organized structural form consisting of laterally associated β-sheet protofilaments that may be adopted as an alternative to the functional, ...native state. Identifying the balance of forces stabilizing amyloid is fundamental to understanding the wide accessibility of this state to peptides and proteins with unrelated primary sequences, various chain lengths, and widely differing native structures. Here, we use four-dimensional electron microscopy to demonstrate that the forces acting to stabilize amyloid at the atomic level are highly anisotropic, that an optimized interbackbone hydrogen-bonding network within β-sheets confers 20 times more rigidity on the structure than sequence-specific sidechain interactions between sheets, and that electrostatic attraction of protofilaments is only slightly stronger than these weak amphiphilic interactions. The potential biological relevance of the deposition of such a highly anisotropic biomaterial in vivo is discussed.
Significance The biomechanics of amyloid underlies its function in living organisms. We use four-dimensional electron microscopy to systematically dissect the nanoscale origins of amyloid elasticity by measuring the bond stiffnesses of the intermolecular forces stabilizing each of its three characteristic packing interfaces. We find amyloid to have a pronounced mechanical anisotropy with longitudinal, hydrogen bonding 20 times stiffer than transverse, amphiphilic, and electrostatic interactions. Such strongly anisotropic elastic properties are likely to give rise to length-dependent mechanical behavior with short fibrils possessing significantly different material properties than longer fibrils. This is of great importance in understanding fibril–membrane interactions and fragmentation mechanisms, both of which are thought to play a crucial role in the spread of amyloid diseases.
The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence ...factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism.
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