The ₃₂₉ HHK ₃₃₁ motif is essential for Alzheimer’s disease tau filament fold

Cryo-electron microscopy (cryo-EM) has revealed disease-specific tau filament folds, yet the local sequence elements that determine them remain poorly understood. Here we focused on the ₃₂₉ HHK ₃₃₁ motif near an inter-protofilament interface in Alzheimer’s disease (AD)-type tau filaments, and analyzed recombinant dGAE filaments of wild-type (WT) and mutants in this motif. All mutants formed amyloid-like filaments in vitro , but their morphologies differed. In cultured cells, WT filaments efficiently seeded WT tau aggregation. Filaments with three-residue changes (deletion or Ala substitution) showed almost no seeding activity, whereas two-residue deletions retained partial activity. Cryo-EM showed that WT dGAE filaments form a quadruple helical filament of two protofilament dimers. Each dimer comprises two C-shaped protofilaments, centered on a ₃₃₃ GGG ₃₃₅ -mediated inter-protofilament interaction and supported by flanking ₃₂₉ HHK ₃₃₁ - ₃₃₆ QVE ₃₃₈ contacts. Three-residue alterations abolished interactions with the QVE motif at the protofilament interface, thereby displacing ₃₃₃ GGG ₃₃₅ and forming non-C-shaped protofilament structures that are intrinsically poor templates for tau seeding. By contrast, two-residue deletions maintained the C-shaped protofilament structure because the remaining residue formed alternative inter-protofilament interactions. These findings suggest that the ₃₂₉ HHK ₃₃₁ region is a key determinant of the AD-like C-shaped protofilament fold and link this motif to tau seeding, providing insight into disease-specific tau filament formation.