Site-specific phosphorylation of Ser352 drives aggregation of Tau R4 under acidosis conditions

In Alzheimer's disease, neurons undergo acidosis as their pH drops from ∼7.1 to ∼6.5. Here we elucidate the molecular mechanism of specific Tau aggregation only at this lower pH. We show that a specific phosphorylation event in the Tau R4 domain is coupled to this pH drop to drive a defined phase transition from condensates to fibrils. Using a combination of experimental and computational studies, our results demonstrate that site-specific phosphorylation of Ser352 is the molecular switch that induces aggregation of Tau only at the more acidic, disease-related pH. We designed and synthesized a phosphopeptide library covering all phosphorylation patterns of the Tau-R4 domain and tested its response to controlled acidification using an array of complementary biophysical methods. This revealed that only a single phosphorylation of Ser352 led to acid-driven aggregation of Tau-R4. At neutral pH, Tau-R4 with a phosphorylation on Ser352 formed liquid condensates. These condensates converted irreversibly into amyloid filaments as the pH gradually decreased towards the level associated with pathological acidosis. Using a combination of ³¹P- and ¹H-NMR, MD simulations and microscopy studies, we show that the mechanism by which the phosphorylation of Ser352 Tau R4 exerts its effects is based on the unique position of this residue within the protein structure. pSer352 is located at the inside of the tip of a β-hairpin, pointing into the hydrophobic core of the amyloid fold. This buried position increases its effective pK _a, resulting in compacting of the hairpin following pSer352 protonation upon acidification. This shortens the inter-sheet distance at this position and tightens the filament. We conclude that this single protonation event of the pSer352 phosphate is responsible for the macroscopic phase transition from condensates to aggregates. Our results provide the molecular explanation for the specific aggregation of Tau under the pathologically relevant acidic pH, which is substantially different from its behavior at neutral pH.