It is demonstrated that kinase-specific phosphorylation outside the amyloid core can be sufficient to bias tau toward a defined fibril structure, establishing a direct mechanistic link between kinase specificity, post-translational modification, and tau strain formation.
Abstract
Tauopathies are defined by the accumulation of filamentous tau assemblies that adopt disease-specific molecular conformations, or tau strains. Although tau is extensively hyperphosphorylated in Alzheimer’s disease, how phosphorylation patterns influence tau folding and strain selection remains unclear. Here, we show that site-specific phosphorylation of 0N3R tau by extracellular signal-regulated kinase 2 (ERK2), occurring predominantly within the proline-rich region and largely excluding the microtubule-binding domain, is sufficient to direct tau assembly into a structurally homogeneous fibril conformation. Residue-resolved NMR spectroscopy identifies a defined and quantifiable ERK2 phosphorylation pattern in the proline-rich region of tau. Cryo-electron microscopy at 3.0 Å resolution reveals that ERK2-phosphorylated 3R tau assembles into filaments with an ordered cross-β fold adopting an Alzheimer’s disease PHF fold, despite the absence of phospho-sites within the fibril core. These filaments exhibit robust seeding activity in tau biosensor cells. Together, these results demonstrate that kinase-specific phosphorylation outside the amyloid core can be sufficient to bias tau toward a defined fibril structure, establishing a direct mechanistic link between kinase specificity, post-translational modification, and tau strain formation.
Tau is an intrinsically disordered protein that binds and stabilizes the microtubules (MTs) of neurons. Abnormal phosphorylation of tau is hypothesized to cause its dissociation from MTs, leading to aggregation and fibril formation, hallmarks of neurodegenerative disorders. With 85 phosphorylation sites, it remains unclear which specific sites promotes and stabilizes fibril formation. Previous studies using techniques such as pseudo-phosphorylation and semi-synthetic strategies have yielded fibrils that often differ from structurally from those observed in patients. In this study, we used molecular dynamics simulations to investigate the effect of site-specific phosphorylation at S316, S320, S324, S341, S352, and S356 on all strands of tau paired helical filaments (PHFs), the predominant fibril form found in Alzheimer’s patients. We characterized the structural and dynamic properties of the fibrils using analyses such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent-accessible surface area (SASA), hydrogen bond analysis, MM/GBSA binding free energy calculations, and per-residue energy decomposition. Our analyses revealed that site-specific phosphorylation affects PHF tau fibril stability in a strongly site-dependent manner. Notably, phosphorylation at S356 and S324 preserved structural features and inter-chain interaction energies comparable to the WT fibril, whereas phosphorylation at S316, S320, S341 and S352 induced greater structural perturbations and reduced fibril stability. Compared with the corresponding all-chain phosphorylated system, single-chain phosphorylation largely preserved fibrillar architecture and favorable inter-chain interactions, suggesting that mixed phosphorylated and non-phosphorylated fibrillar assemblies may remain structurally compatible. These findings provide molecular-level insights into how the site and extent of phosphorylation modulate the structural stability of PHF tau fibrils and may influence their interactions within heterogeneous fibrillar assemblies.
Sathish Dasari, S. Kalyaanamoorthy· Journal of Computational Bio...· 0 citations
Phosphorylation of the microtubule-associated protein tau plays important roles in both normal physiology and disease. While tau phosphorylation levels are similarly elevated in processes such as fetal development and hibernation and in pathological states like Alzheimer’s disease, the pattern of phosphorylation differs between these contexts. Whether distinct phosphorylation patterns differentially impact tau’s function on microtubules and its propensity to form neurofibrillary tangles (NFTs) has remained unclear. Here we investigated how differential phosphorylation affects microtubule binding, cooperative tau envelope formation, and NFT-relevant filament assembly. Consistent with prior work, phosphorylation decreases tau’s overall microtubule affinity. However, we find that phosphorylation more dramatically impacts cooperativity between tau molecules during envelope formation. Strikingly, the specific pattern of phosphorylation, rather than overall level, strongly promotes tau filament assembly in vitro. These findings provide insights into how tau phosphorylation modulates its physiological microtubule interactions and its pathoconversion into NFTs.
Xiangyu Fan, Joey B. Truong, Kyoko Okada et al.· Communications Biology· 0 citations
Tau filaments are a hallmark of neurodegenerative tauopathies, such as Alzheimer’s disease (AD). Structural studies have revealed that patient-derived tau fibrils adopt distinct folds in different tauopathies; however, it is unclear what forces guide this process. To explore this question, we investigated the assembly of a tau fragment containing four disease-associated phospho-mimetics (termed Tau(297–407)-4D) in vitro. Under cofactor-free and quiescent conditions, Tau(297–407)-4D forms fibrils with a core structure that partially resembles the AD fold after about 7 days. Strikingly, we noticed that this filament behaves as a hydrogel and evolves into two new polymorphs as it ages over the next 35 days. Thus, tau fibrils formed under cofactor-free conditions are dynamic, exhibiting substantial nonequilibrium behavior. To probe what types of perturbations might stabilize these structures, we applied mechanical agitation, which drove the filaments toward thermodynamic equilibrium in a mechanism consistent with Ostwald ripening into solid-phase, micrometer-sized particles. Likewise, the addition of polyanionic cofactors to preformed Tau(297–407)-4D fibrils significantly stabilized them, as judged by solubility equilibria and chemical denaturation experiments. A subset of the polyanions also remodeled the fibril structure and tuned the extent of fibril–fibril interactions (i.e., “clumping”). We conclude that environmental factors, such as mechanical stress and/or polyanions, play an important role in promoting the thermodynamic stability of otherwise dynamic tau fibrils. We speculate that, in patients, such factors might contribute to the maturation of disease-specific conformers.
Wyatt C. Powell, Nicholas L. Yan, Eric Tse et al.· Journal of the American Chem...· 0 citations
Tau aggregation into amyloid fibrils is a central pathological feature of multiple neurodegenerative diseases, yet identical tau sequences can adopt structurally distinct fibril conformations associated with different disorders. How such disease-specific folds emerge from the same sequence remains poorly understood. Here, we use metadynamics and all-atom molecular dynamics simulations to elucidate the folding mechanisms of tau trimers comprising the R3-R4 region, the minimal aggregation nucleus of tau fibrils. By constructing the folding free-energy landscape within a pre-organized fibril-like scaffold, we identify a shared, partially folded intermediate from which two energetically comparable minimum free-energy pathways (MEPs) diverge, leading to either a compact AD-like or a more open CTE-like C-shaped conformation. Structural analysis reveals that the β4-β6 triangular region acts as the central determinant of this bifurcation. Hydrophobic-core rearrangements within this region provide the primary energetic driving force for compaction, whereas the conformational flexibility of the 332PGGG335 hinge modulates whether this tendency can be structurally realized. Additional simulations show that stable formation of the triangular region requires cooperative multichain interactions; reducing oligomer size destabilizes this scaffold and abolishes ordered folding. Together, our results establish a mechanistic framework in which identical tau sequences access alternative folding routes through a common intermediate, explaining the emergence of disease-specific fibril polymorphism at the oligomeric level and highlighting early folding intermediates as potential targets for therapeutic intervention.
Tong Zhang, Lingling Dai, Yanping Ma et al.· Biophysical Journal· 0 citations
The aggregation of the tau protein into intraneuronal fibrillar tangles is closely associated with the pathology of Alzheimer’s disease. The endogenous defense system against this process includes molecular chaperones, among which DNAJB6b has emerged as a key component. Using a tau model system comprising the tau fragment 304-380C322S, which spans the amyloidogenic core of ex vivo Alzheimer’s disease fibrils, we investigated the impact of DNAJB6b on tau fibril formation. Here, we show that DNAJB6b potently delays tau aggregation by co-assembling with small tau aggregates and by binding to mature fibrils, thereby reducing their ability to catalyze further fibril growth. This interplay between tau and the chaperone results in greatly reduced fibril formation rate and a lower final fibril mass, which we interpret as increased tau solubility. Moreover, solution-state NMR spectroscopy confirms that DNAJB6b does not interact with tau monomers. Human chaperone DNAJB6b has emerged as a key component involved in the endogeneous defense system against tau protein aggregation in the context of Alzheimer’s disease. Here, the authors study the impact of DNAJB6b on tau fibril formation, showing that it delays tau aggregation by co-assembling with small tau aggregates and by binding to mature fibrils, reducing their ability to catalyze further fibril growth.
Andreas Carlsson, Emil Axell, Johan Wallerstein et al.· Communications Chemistry· 0 citations
Fibrils formed by 40- and 42-residue amyloid-β peptides (Aβ40 and Aβ42) are polymorphic, containing molecular structures that vary with growth conditions in ways that are not fully understood. Here we use cryogenic electron microscopy to characterize the structure of rapidly twisting Aβ40 fibrils, for which the distance between apparent width minima in electron microscope images (“cross-over distances”) is approximately 25 nm. From samples grown under a single set of growth conditions, we obtain high-resolution structures for three different rapidly twisting polymorphs. Although their cross-over distances are similar, the three rapidly twisting polymorphs differ in twist handedness, symmetry, molecular conformations, and intermolecular contacts. Two of the rapidly twisting polymorphs resemble Aβ40 polymorphs with longer cross-over distances that have been described previously, including polymorphs extracted from brain tissue of Alzheimer’s disease patients or created by seeded growth from amyloid in brain tissue, but have shorter conformationally ordered segments and other specific conformational differences. These results contribute to our understanding of amyloid polymorphism, connections between morphology and molecular structure, and relationships between brain-derived and in vitro-grown fibrils.
M. G. Larimi, K. Thurber, R. Tycko· Biophysical Journal· 0 citations