Cellular condensation of intrinsically disordered proteins (IDPs) through liquid–liquid phase separation (LLPS) allows dynamic compartmentalization and regulation of biological processes. The IDP ...tau, which promotes the assembly of microtubules and is hyperphosphorylated in Alzheimer's disease, undergoes LLPS in solution and on the surface of microtubules. Little is known, however, about the influence of tau phosphorylation on its ability to nucleate microtubule bundles in conditions of tau LLPS. Herein, we show that unmodified tau as well as tau phosphorylated at disease‐associated epitopes condense into liquid‐like droplets. Although tubulin partitioned into and reached high concentrations inside all tau droplets, it was unable to grow into microtubules form the inside of droplets formed by tau phosphorylated at the AT180 epitope (T231/S235). In contrast, neither phosphorylation of tau in the repeat domain nor at its tyrosine residues inhibited the assembly of tubulin from tau droplets. Because LLPS of IDPs has been shown to promote different types of cytoskeletal assembly, our study suggests that IDP phosphorylation might be a broadly used mechanism for the modulation of condensate‐mediated cytoskeletal assembly.
Active tau recruits tubulin into liquid‐like condensates and promotes microtubule assembly. Upon phosphorylation at the disease‐associated AT180‐epitope intramolecular salt bridges are formed and the microtubule‐assembly activity is lost.
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.
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.
Recent advances in the field of protein structure determination using liquid-state NMR enable the elucidation of multi-state protein conformations that can provide insight into correlated and ...non-correlated protein dynamics at atomic resolution. So far, NMR-derived multi-state structures were typically evaluated by means of visual inspection of structure superpositions, target function values that quantify the violation of experimented restraints and root-mean-square deviations that quantify similarity between conformers. As an alternative or complementary approach, we present here the use of a recently introduced structural correlation measure, PDBcor, that quantifies the clustering of protein states as an additional measure for multi-state protein structure analysis. It can be used for various assays including the validation of experimental distance restraints, optimization of the number of protein states, estimation of protein state populations, identification of key distance restraints, NOE network analysis and semiquantitative analysis of the protein correlation network. We present applications for the final quality analysis stages of typical multi-state protein structure calculations.
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.
Abstract Die zelluläre Kondensation von intrinsisch ungeordneten Proteinen (IDPs) durch Flüssig‐flüssig‐Phasentrennung (LLPS) ermöglicht die dynamische Kompartimentierung und Regulation biologischer ...Prozesse. Das IDP Tau, das den Aufbau von Mikrotubuli fördert und bei der Alzheimer‐Krankheit hyperphosphoryliert wird, kann in Lösung und auf der Oberfläche von Mikrotubuli LLPS‐Prozesse durchlaufen. Der Einfluss der Tau‐Phosphorylierung auf Tau‐LLPS‐vermittelte Tubulinpolymerisation ist jedoch weitestgehend unbekannt. Wir zeigen hier, dass unmodifiziertes Tau sowie an krankheitsassoziierten Epitopen phosphoryliertes Tau zu flüssigkeitsähnlichen Tröpfchen kondensieren. Obwohl sich Tubulin in allen Tau‐Tröpfchen in hohen Konzentrationen anreichert, ist es nicht in der Lage, aus dem Inneren von Tröpfchen, die durch am AT180‐Epitop phosphoryliertes Tau (T231/S235) gebildet wurden, zu Mikrotubuli zu wachsen. Im Gegensatz dazu hemmte weder die Phosphorylierung von Tyrosinresten in Tau noch die Phosphorylierung in der Repeatdomäne die Polymerisation von Tubulin aus Tau‐Tröpfchen. Da gezeigt wurde, dass LLPS von IDPs verschiedene Arten der Zytoskelettassemblierung fördert, legt unsere Studie nahe, dass die IDP‐Phosphorylierung ein weit verbreiteter Mechanismus für die Regulation der kondensatvermittelten Zytoskelettassemblierung sein könnte.
A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective ...recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease.
Governing function, half‐life and subcellular localization, the 3D structure and dynamics of proteins are in nature constantly changing in a tightly regulated manner to fulfill the physiological and ...adaptive requirements of the cells. To find evidence for this hypothesis, we applied in‐cell NMR to three folded model proteins and propose that the splitting of cross peaks constitutes an atomic fingerprint of distinct structural states that arise from multiple target binding co‐existing inside mammalian cells. These structural states change upon protein loss of function or subcellular localisation into distinct cell compartments. In addition to peak splitting, we observed NMR signal intensity attenuations indicative of transient interactions with other molecules and dynamics on the microsecond to millisecond time scale.
In buffer the NMR spectrum of a folded protein (A) shows no peak splitting effect due to the absence of multiple structural species. Inside cells (B) a subpopulation of the same protein binds different partners which results in structural changes leading to chemical shift perturbations of the cross peaks. The co‐existence of these structurally different variants in cells leads to the peak splitting effect and the appearance of two daughter peaks.
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.
Allostery and correlated motion are key elements linking protein dynamics with the mechanisms of action of proteins. Here, we present PDBCor, an automated and unbiased method for the detection and ...analysis of correlated motions from experimental multi-state protein structures. It uses torsion angle and distance statistics and does not require any structure superposition. Clustering of protein conformers allows us to extract correlations in the form of mutual information based on information theory. With PDBcor, we elucidated correlated motion in the WW domain of PIN1, the protein GB3, and the enzyme cyclophilin, in line with reported findings. Correlations extracted with PDBcor can be utilized in subsequent assays including nuclear magnetic resonance (NMR) multi-state structure optimization and validation. As a guide for the interpretation of PDBcor results, we provide a series of protein structure ensembles that exhibit different levels of correlation, including non-correlated, locally correlated, and globally correlated ensembles.
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•PDBcor algorithm extracts protein-correlated motion from protein ensembles•PDBcor is based on GMM clustering and information theory•High sensitivity to correlated motion comes from the use of protein distances•PDBcor is unbiased, as the structure superposition step is not required
Ashkinadze et al. present an unbiased algorithm, PDBcor, for the extraction of protein-correlated motion from protein structural ensembles. Using clustering and mutual information, this algorithm is based on the statistical analysis of protein interresidual distances. The authors validate it on three model proteins with known structural correlations
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