Hydrodynamic Radius Determination of Tau and AT8 Phosphorylated Tau Mutants: A Combined Simulation and Experimental Study

The Intrinsically Disordered Protein (IDP) Tau is, under a hyperphosphorylated form, one of the molecular hallmarks of Alzheimers disease. This abnormal phosphorylation induces both a functional loss to regulate microtubule assembly and the formation of intraneuronal fibrillar inclusions. Notably, phosphorylation sites recognized by the AT8 antibody are particularly relevant in the development of Alzheimers disease. The main objective of this work is to understand how AT8 phosphorylation site affects the conformation and dynamics of Tau protein as early steps in Tau pathological transformation. We engineered an alanine Tau mutant to target specific phosphorylation sites restricted to the AT8 epitope, for which we generated two distinct phosphorylation states differing by the number of phosphosites. The combination of Dynamic Light Scattering (DLS), Analytical UltraCentrifugation (AUC) and pCALVADOS (a forcefield based on CALVADOS 2 with phosphorylation-specific parameters) allowed us to determine that both alanine mutation and phosphorylation on the AT8 epitope do not significantly change the hydrodynamic radius of the conformational ensemble. Our calculations of Local Curvatures and Local Flexibilities corroborated by cw EPR experimental data indicated that the mutations for selective phosphorylation did not modify Taus global dynamics. Nevertheless, simulations revealed that local stiffening and extension at the AT8 epitope scale with the extent of phosphorylation. Interestingly, phosphorylation does not just affect the immediate AT8 area but leads to distant contact losses, directed towards the N-terminus, potentially influencing Taus function. The study paves the way for future research on Taus structure-function-dynamics relationship and the role of phosphorylation in neurodegenerative diseases. Statement of significanceWe combined experimental and simulation approaches to investigate the role of Tau site-specific protein phosphorylation at the AT8 epitope, which is implicated in Tau transformation associated with Alzheimers disease. We demonstrated that while phosphorylation does not significantly alter the overall size of Tau, it induces local structural changes at the AT8 epitope and long-range contact losses. These findings suggest a potential mechanism in which specific phosphorylation could modulate Taus interactions and function, contributing to neurodegeneration. Combining experimental studies with the pCALVADOS model, we provided a robust approach for future studies on Taus structure-function-dynamics and more generally for studying intrinsically disordered proteins and the impact of post-translational modifications on their conformational ensembles.