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bioRxiv · 10.64898/2026.05.19.726321

Phosphorylation of S396 and S400 Promotes Tau Self-Assembly and Favors the Chronic Traumatic Encephalopathy (CTE) Protofilament Fold

Powell, W.·Yan, N.·Tse, E.·Sin, N.·Melo, A.·Southworth, D. R.·Gestwicki, J. E.
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Abstract

Tau hyperphosphorylation is linked to tauopathy aggregates, but the effects of individual sites on tau assembly remain unclear. Here, we used protein semisynthesis to generate defined Tau(297-407) proteoforms containing specific combinations of phosphorylation within the PHF1 epitope. Spontaneous aggregation revealed that phosphorylation of S396, and S400 to a lesser extent, promoted nucleation, while phosphorylation of T403 and S404 suppressed assembly. A similar reactivity trend of pS396 > pS400 > WT > pT403 > pS404 was observed for seeded assembly, and all proteoforms, even anti-aggregation ones, were incorporated. The fibrils have similar thermodynamic stability, suggesting that phosphorylation selectively tunes reactivity and not thermodynamics. Cryo-EM revealed that pS400 produces a chronic traumatic encephalopathy (CTE) protofilament conformation. Strikingly, one of the structures observed in the pS400 sample appeared to capture a secondary nucleation step. Together, these studies reveal the importance of positional effects of phosphorylation on tau self-assembly.

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Powell, W., Yan, N., Tse, E., Sin, N., Melo, A., Southworth, D. R., Gestwicki, J. E.. 2026-05-21. Phosphorylation of S396 and S400 Promotes Tau Self-Assembly and Favors the Chronic Traumatic Encephalopathy (CTE) Protofilament Fold. https://doi.org/10.64898/2026.05.19.726321

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