Fig. 1: Sequential hyperphosphorylation of tau in vitro generates AD-specific epitopes.

a A pictorial representation of the experimental design: WT tau was sequentially hyperphosphorylated, first by PKA and then by either GSK-3β or SAPK4 kinase. Hyperphosphorylation is shown to be able to cause an opening of such transient paperclip conformation⁵⁰ and simultaneously stabilizes the α-helical structures, which is associated to the aggregation process⁵¹ Recent cryo-EM structures and computational study reveals heparin can stabilize the interaction between R2 and R3 (repeat domain 2 and 3) and hyperphosphorylation lowers the free-energy landscape of R3 and R4 for forming aggregation^15,55. b Representative SDS-PAGE (4–12%, stained by Coomassie blue) across three independent experiments showed an upward shift of electrophoretic mobility for both g-tau and s-tau species, indicating successful phosphorylation reaction. c LC-MS/MS analysis revealed that both g-tau and s-tau tau were hyperphosphorylated at AD-specific epitopes, such as targeting sites of AT8, AT100, AT180, and PHF-1, highlighted along with the pictorial representation of WT tau. d High-resolution native mass spectrometry results indicated on average there were 7 phosphate groups per PKA-tau molecule, 11 phosphate groups per g-tau molecule and over 19 phosphate groups per s-tau molecule. Error bars represent ±s.d. from MCMC analysis.