The protein Tau aggregates into tangles in the brain of patients with Alzheimer's disease. In solution, however, Tau is intrinsically disordered, highly soluble, and binds to microtubules. It is ...still unclear what initiates the conversion from an innocuous phase of high solubility and functionality to solid-like neurotoxic deposits. Here, we show that the microtubule-binding repeats of Tau, which are lysine-rich, undergo liquid-liquid phase separation in solution. Liquid-liquid demixing causes molecular crowding of amyloid-promoting elements of Tau and drives electrostatic coacervation. Furthermore, we demonstrate that three-repeat and four-repeat isoforms of Tau differ in their ability for demixing. Alternative splicing of Tau can thus regulate the formation of Tau-containing membrane-less compartments. In addition, phosphorylation of Tau repeats promotes liquid-liquid phase separation at cellular protein conditions. The combined data propose a mechanism in which liquid droplets formed by the positively charged microtubule-binding domain of Tau undergo coacervation with negatively charged molecules to promote amyloid formation.Tau forms aggregates in the brains of Alzheimer patients. Here, the authors identify conditions, where the microtubule-binding repeats of Tau undergo a phosphorylation-dependent liquid-liquid phase separation, leading to molecular crowding in the formed Tau liquid droplets and characterize them by NMR and other biophysical methods.
Significance Tau is an important microtubule-associated protein. Although the structureâfunction relationship of Tau has been intensively studied for many years primarily by molecular biology and ...biochemical approaches, little is still known about the molecular mechanisms by which Tau interacts with microtubules and promotes microtubule assembly. Here, we provide detailed insight into the Tauâmicrotubule association by using NMR spectroscopy and mass spectrometry. We show that Tau binds to microtubules by using small groups of residues, which are important for pathological aggregation of Tau. We further show that Tau stabilizes a straight protofilament conformation by binding to a hydrophobic pocket in between tubulin heterodimers. Our work is thus relevant to normal Tau action development and in Tau-related neurodegenerative diseases.
The structure, dynamic behavior, and spatial organization of microtubules are regulated by microtubule-associated proteins. An important microtubule-associated protein is the protein Tau, because its microtubule interaction is impaired in the course of Alzheimerâs disease and several other neurodegenerative diseases. Here, we show that Tau binds to microtubules by using small groups of evolutionary conserved residues. The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Tau residues in between the microtubule-binding sites remain flexible when Tau is bound to microtubules in agreement with a highly dynamic nature of the Tauâmicrotubule interaction. By binding at the interface between tubulin heterodimers, Tau uses a conserved mechanism of microtubule polymerization and, thus, regulation of axonal stability and cell morphology.
Cells form and use biomolecular condensates to execute biochemical reactions. The molecular properties of non-membrane-bound condensates are directly connected to the amino acid content of disordered ...protein regions. Lysine plays an important role in cellular function, but little is known about its role in biomolecular condensation. Here we show that protein disorder is abundant in protein/RNA granules and lysine is enriched in disordered regions of proteins in P-bodies compared to the entire human disordered proteome. Lysine-rich polypeptides phase separate into lysine/RNA-coacervates that are more dynamic and differ at the molecular level from arginine/RNA-coacervates. Consistent with the ability of lysine to drive phase separation, lysine-rich variants of the Alzheimer's disease-linked protein tau undergo coacervation with RNA in vitro and bind to stress granules in cells. Acetylation of lysine reverses liquid-liquid phase separation and reduces colocalization of tau with stress granules. Our study establishes lysine as an important regulator of cellular condensation.
Liquid-liquid phase separation (LLPS) of proteins enables the formation of non-membrane-bound organelles in cells and is associated with cancer and neurodegeneration. Little is known however about ...the structure and dynamics of proteins in LLPS conditions, because of the polymorphic nature of liquid-like protein droplets. Using carbon-detected NMR experiments we here show that the conversion of the aggregation-prone repeat region of the Alzheimer's-related protein tau from the dispersed monomeric state to phase-separated liquid-like droplets involves tau's aggregation-prone hexapeptides and regulatory KXGS motifs. Droplet dissolution in presence of 1,6-hexanediol revealed that chemical shift perturbations in the hexapeptide motifs are temperature driven, while those in KXGS motifs report on phase separation. Residue-specific secondary structure analysis further indicated that tau's repeat region exists in extended conformation in the dispersed state and attains transient β-hairpin propensity upon LLPS. Taken together our work shows that NMR spectroscopy can provide high-resolution insights into LLPS-induced changes in intrinsically disordered proteins.
Visualizing liquid-liquid phase separation by NMR spectroscopy: LLPS of the Alzheimer's-related protein tau involves aggregation-prone hexapeptides and activity regulating KXGS motifs.
Microtubule-associated proteins regulate microtubule dynamics, bundle actin filaments, and cross-link actin filaments with microtubules. In addition, aberrant interaction of the ...microtubule-associated protein Tau with filamentous actin is connected to synaptic impairment in Alzheimer's disease. Here we provide insight into the nature of interaction between Tau and actin filaments. We show that Tau uses several short helical segments to bind in a dynamic, multivalent process to the hydrophobic pocket between subdomains 1 and 3 of actin. Although a single Tau helix is sufficient to bind to filamentous actin, at least two, flexibly linked helices are required for actin bundling. In agreement with a structural model of Tau repeat sequences in complex with actin filaments, phosphorylation at serine 262 attenuates binding of Tau to filamentous actin. Taken together the data demonstrate that bundling of filamentous actin and cross-linking of the cellular cytoskeleton depend on the metamorphic and multivalent nature of microtubule-associated proteins.
The aggregation of the intrinsically disordered tau protein into highly ordered β-sheet-rich fibrils is implicated in the pathogenesis of a range of neurodegenerative disorders. The mechanism of tau ...fibrillogenesis remains unresolved, particularly early events that trigger the misfolding and assembly of the otherwise soluble and stable tau. We investigated the role the lipid membrane plays in modulating the aggregation of three tau variants, the largest isoform hTau40, the truncated construct K18, and a hyperphosphorylation-mimicking mutant hTau40/3Epi. Despite being charged and soluble, the tau proteins were also highly surface active and favorably interacted with anionic lipid monolayers at the air/water interface. Membrane binding of tau also led to the formation of a macroscopic, gelatinous layer at the air/water interface, possibly related to tau phase separation. At the molecular level, tau assembled into oligomers composed of ~ 40 proteins misfolded in a β-sheet conformation at the membrane surface, as detected by in situ synchrotron grazing-incidence X-ray diffraction. Concomitantly, membrane morphology and lipid packing became disrupted. Our findings support a general tau aggregation mechanism wherein tau's inherent surface activity and favorable interactions with anionic lipids drive tau-membrane association, inducing misfolding and self-assembly of the disordered tau into β-sheet-rich oligomers that subsequently seed fibrillation and deposition into diseased tissues.
Folding of the Tau Protein on Microtubules Kadavath, Harindranath; Jaremko, Mariusz; Jaremko, Łukasz ...
Angewandte Chemie (International ed.),
August 24, 2015, Letnik:
54, Številka:
35
Journal Article
Recenzirano
Microtubules are regulated by microtubule‐associated proteins. However, little is known about the structure of microtubule‐associated proteins in complex with microtubules. Herein we show that the ...microtubule‐associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a β‐sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau.
Tau the line: NMR spectroscopy shows that the protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and forms a β‐sheet structure in amyloid fibrils, the conserved hexapeptides at the beginning of the second and third repeats in Tau adopt a hairpin conformation when bound to microtubules. Thus, binding to microtubules stabilizes a unique conformation in Tau.
Blues brothers: Aggregation of the Tau protein is a key event in Alzheimer's disease and Tau aggregation inhibitors are important as potential drugs. Phenothiazines, such as methylene blue and its ...azure derivatives, have a unique mechanism, specifically modifying the Tau cysteine residues. The modification keeps Tau in a monomeric disordered conformation preventing the formation of filaments and their toxic precursors.
Phosphorylation of the microtubule-associated protein Tau influences the assembly and stabilization of microtubules and is deregulated in several neurodegenerative diseases. The high flexibility of ...Tau, however, has prevented an atomic-level description of its phosphorylation-induced structural changes. Employing an extensive set of distance and orientational restraints together with a novel ensemble calculation approach, we determined conformational ensembles of Tau fragments in the non-phosphorylated state and, when phosphorylated at T231/S235 or T231/S235/S237/S238, four important sites of phosphorylation in Alzheimer disease. Comparison of the molecular ensembles showed that phosphorylation of the regulatory T231 does not perturb the backbone conformation of the proximal microtubule-binding 225KVAVVR230 motif. Instead, phosphorylated T231 selectively engages in a salt bridge with R230 that can compete with the formation of intermolecular salt bridges to tubulin. Our study provides an ensemble description which will be useful for the analysis of conformational transitions in Tau and other intrinsically disordered proteins.
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•A novel ensemble calculation approach was developed•Molecular ensembles of phosphorylated Tau fragments were determined•Phosphorylated T231 selectively engages in a salt bridge with R230•Integration of NOE restraints with ensemble calculations are highly useful for IDPs
Alzheimer disease-related protein Tau is a phosphoprotein, and a number of different residues are subject to phosphorylation. Schwalbe et al. develop a molecular ensemble approach and use it to reveal an atomic-level description of the phosphorylation-induced structural changes in Tau phosphorylated at Thr231.
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