Effect of Site-Specific Phosphorylation of Serine on the Stability of PHF Tau Fibrils: A Molecular Dynamics Simulation Study

Abstract

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.