Structures of AT8 and PHF1 phosphomimetic tau: Insights into the posttranslational modification code of tau aggregation

Abstract

The microtubule-associated protein tau aggregates into amyloid fibrils in Alzheimer's disease and other neurodegenerative diseases. In these tauopathies, tau is hyperphosphorylated, suggesting that this posttranslational modification (PTM) may induce tau aggregation. Tau is also phosphorylated in normal developing brains. To investigate how tau phosphorylation induces amyloid fibrils, here we report the atomic structures of two phosphomimetic full-length tau fibrils assembled without anionic cofactors. We mutated key Ser and Thr residues to Glu in two regions of the protein. One construct contains three Glu mutations at the epitope of the anti-phospho-tau antibody AT8 (AT8-3E tau), whereas the other construct contains four Glu mutations at the epitope of the antibody PHF1 (PHF1-4E tau). Solid-state NMR data show that both phosphomimetic tau mutants form homogeneous fibrils with a single set of chemical shifts. The AT8-3E tau rigid core extends from the R3 repeat to the C terminus, whereas the PHF1-4E tau rigid core spans R2, R3, and R4 repeats. Cryoelectron microscopy data show that AT8-3E tau forms a triangular multi-layered core, whereas PHF1-4E tau forms a triple-stranded core. Interestingly, a construct combining all seven Glu mutations exhibits the same conformation as PHF1-4E tau. Scalar-coupled NMR data additionally reveal the dynamics and shape of the fuzzy coat surrounding the rigid cores. These results demonstrate that specific PTMs induce structurally specific tau aggregates, and the phosphorylation code of tau contains redundancy.

Keywords: Alzheimer’s disease; helical reconstruction; magic-angle spinning; phosphorylation; solid-state NMR.

Fig. 1.

Amino acid sequences, fibril morphologies, and ssNMR spectra of full-length 0N4R tau fibrils containing phosphomimetic mutations. (A) Amino acid sequence domains of 0N4R tau. The estimated charge at pH 7.0 for each domain is indicated. The positions of the AT8 epitope and PHF1 epitope are indicated. The full amino acid sequence shows the AT8-3E mutations (S202E, T205E, and S208E) and PHF1-4E mutations (S396E, S400E, T403E, and S404E). (B) Negative stain TEM images of full-length AT8 and PHF1 mutant tau fibrils formed in the absence of cofactors. These fibrils were used for ssNMR and cryo-EM characterization. (C) 2D NCACB spectra of AT8 and PHF1 mutant tau. Assignments are obtained from 3D correlation spectra. ¹⁵N-¹³Cα cross-peaks have positive intensities, whereas ¹⁵N-¹³Cβ cross-peaks have negative intensities due to the double-quantum Cα-Cβ magnetization transfer. A small number of negative ¹⁵N-¹³Cγ cross-peaks are observed due to direct N-Cβ polarization transfer followed by Cβ-Cγ transfer. (D) Secondary structure–dependent chemical shifts of the rigid cores of AT8 and PHF1 mutant tau fibrils. The difference between the secondary chemical shifts of Cβ and Cα (δCβ–δCα) is plotted. Positive differences indicate β-strands, while negative differences indicate coil or helical conformations.

Fig. 2.

Cryo-EM data of two phosphomimetic full-length tau fibrils. (A–C) AT8-3E tau fibril data. (A) Representative micrograph. (B) Helical reconstruction of AT8-3E tau fibrils with a cross-over length of 123 nm. (C) Projected slice of the central 4.8 Å of the final 3D reconstructed map of AT8-3E tau. (D–F) PHF1-4E tau fibril data. (D) Representative micrograph. (E) Helical reconstruction of PHF1-4E tau fibrils with a cross-over length of 79 nm. (F) Projected slice of the central 4.8 Å of the final 3D reconstructed map.

Fig. 3.

High-resolution structures of the rigid cores of phosphomimetic 0N4R tau fibrils. (A) The AT8-3E tau rigid core adopts a complex triangular structure that spans the R3 to the CT, except for the last 7 residues of R3 and the first half of the R4, which are disordered. (B) The PHF1-4E tau rigid core spans R2, R3, and R4, which form antiparallel stacked β-strands that are separated by PGGG motifs.

Fig. 4.

The fuzzy coat of the three phosphomimetic tau fibrils has different dynamics. (A) Glu region of the 2D ¹³C-¹³C TOCSY spectra of AT8, PHF1, and AT8/PHF1 mutant tau fibrils. For AT8-3E tau, the Pro-preceding Glu (E(P)) peak is more intense than the Glu (E) peak. But for the other two fibrils, the E(P) peak is weaker than the E peak. (B) Diagnostic regions of the 2D ¹⁵N-¹H INEPT spectra of the three tau fibrils, showing intensity variations of the N-terminal residues E3, T17, and G43 among the three samples. These intensity variations indicate that AT8/PHF1-7E tau has a highly dynamic NT1, whereas AT8 and PHF1 mutant tau fibrils have partially immobilized NT1. (C) Distribution of Glu (blue lines) and Glu(Pro) (pink lines) residues in the amino acid sequence and summary of the mobilities of the three phosphomimetic tau fibrils based on the NMR spectra. (D) Models of fuzzy coat arrangement around the rigid core of the three tau mutants, based on the TOCSY and INEPT spectral intensities, and accounting for the NT/CT interactions found from previous FRET and solution NMR data (40, 41). Light blue: the most mobile segments; light green: the semimobile segments; light orange: the semirigid domains.

Fig. 5.

Proposed aggregation pathways of AT8 and PHF1 phosphorylated tau. (A) Schematic of the residual conformation of soluble tau bearing Glu mutations, obtained from previous FRET data (40). (B) Schematic of AT8-3E tau and PHF1-4E tau structures and their fuzzy coat arrangement determined in this work. The color scheme of the dynamic domains of the protein is the same as in Fig. 4D: light blue: the most mobile segments; light green: semi-mobile segments; light orange: semi-rigid domains. The + and − signs approximately indicate the number of charges of each domain at pH 7. (C) Three-dimensional folds of three ex vivo brain tau fibrils (13). The pink shade indicates the ³²²CGS³²⁴ motif, the green shade indicates the R4-R′ U-turn, the gray shade indicates the R2-R3 U-turn, and the blue circle indicates the unassigned cofactor density.