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

This study investigates the effect of multiple phosphorylation of the Intrinsically Disordered Protein (IDP) Tau on its hydrodynamic radius (Rh). Hyperphosphorylated Tau is notably implicated in Alzheimer’s disease The kinases responsible for adding phosphate groups are highly involved in the regulation of biological processes such as cell division, cell signaling, and apoptosis and, particularly, in the development of Alzheimer’s disease, especially those recognized by the AT8 antibody. The main objective of this work is to understand how phosphorylation at the AT8 site affects the conformation and dynamics of the Tau protein through the study of its hydrodynamic radius. To achieve this, we used a Tau protein mutant with all phosphorylation sites mutated to alanine except those recognized by the AT8 antibody to achieve a specific phosphorylation pattern. The phosphorylation of this mutant was performed, firstly, by incubation with CDK2/ cyclin A kinase to obtain a first phosphorylated state, termed p-state 1 (serine at positions 202 and threonines at position 205 and 212 are phosphorylated), and secondly by incubation with CDK2/cyclin A kinase followed by GSK3β kinase to obtain a phosphorylated state termed call p-state 2 (serines 198 and 208 are phosphorylated in addition to the three previously described). The Rh of each sample was measured experimentally by dynamic light scattering (DLS) and analytical ultracentrifugation (AUC), and simulated using a new simulation model, pCALVADOS, combining the CALVADOS 2 model for IDPs with parameters for phosphorylated residues. We find that both wild-type Tau and AT8-phosphorylated Tau mutants exhibit a relatively monodisperse distribution in solution suggesting that phosphorylation do not significantly alter the overall size of the conformational ensemble of Tau. Our calculations of Local Curvatures and Local Flexibilities confirm that the mutations made on the Tau sequence to perform selective phosphorylation do not significantly impact local dynamics, which is corroborated by several experimental results. We also identify a local stiffening and extension of the AT8 epitope that is proportional to the amount of phosphorylation. Interestingly, phosphorylation does not just affect the immediate AT8 area. We also report contact losses at a distance, with a noticeable directionality pointing towards the N-terminal end of the monomer, which could have functional implications for the phosphorylated Tau protein. The study paves the way for future research on the structure-function relationship of Tau and the role of phosphorylation in neurodegenerative diseases.