Twelve phosphomimetic mutations induce the assembly of recombinant full-length human tau into paired helical filaments
- MRC Laboratory of Molecular Biology, United Kingdom
Figures
Figure 1 with 5 supplements
Electron cryo-microscopy (cryo-EM) analysis of PAD12 tau filaments.
(A) Schematic of PAD12 tau. The amino-terminal inserts N1 (residues 44–73) and N2 (74–102) are shown in grey, the proline-rich region (151–243) is in light grey, the microtubule-binding repeats are in purple (R1, 244–274), blue (R2, 275–305), green (R3, 306–336), and yellow (R4, 337–368), and the carboxy-terminal domain (369–441) is shown in orange. The 12 phosphomimetic mutations of PAD12 are indicated with vertical lines. (B) Cryo-EM micrograph of paired helical filaments (PHFs) composed of a 1:1 mixture of 0N3R and 0N4R PAD12 tau. Arrows show PHFs (blue) and single protofilaments with the Alzheimer tau fold (light blue). (C) Cross-sections of cryo-EM reconstructions perpendicular to the helical axis, with a thickness of approximately 4.7 Å for assembly reactions with 0N3R PAD12 tau, 0N4R PAD12 tau, and a 1:1 mixture of 0N3R and 0N4R PAD12 tau. Inserts show pie charts with the particle distribution per filament types (PHFs in blue; single protofilaments with the Alzheimer fold in light blue; single protofilaments with the chronic traumatic encephalopathy [CTE] fold in yellow; discarded solved particles [coloured] and discarded filaments in grey). (D) Cryo-EM density map of the 1:1 0N3R:0N4R PAD12 tau mixture in transparent grey, superimposed with its refined atomic model. (E) Main chain trace of the atomic model shown in D (blue), overlaid with the model of PHFs from Alzheimer’s disease (AD) brain (PDB-ID: 6hre) (grey).
Figure 1—figure supplement 1
Purification of 0N3R and 0N4R PAD12 tau.
(A) Schematic of protein purification and assembly procedure. (B) Coomassie-stained SDS-PAGE (4–20%) after cation exchange chromatography of 0N3R PAD12 tau. (C) SDS-PAGE after size exclusion chromatography (SEC) of 0N3R PAD12 tau. (D) SEC profile of 0N3R PAD12 tau. (E-G) As B–D, but for 0N4R PAD12 tau.
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Figure 1—figure supplement 1—source data 1
Original gels annotated from Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/104778/elife-104778-fig1-figsupp1-data1-v1.pdf
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Figure 1—figure supplement 1—source data 2
Original gels from Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/104778/elife-104778-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
Fourier shell correlation curves.
(A) Fourier shell correlation curves between two independently refined half-maps (black); between the atomic model fitted in half-map 1 and half-map 1 (blue); between the atomic model fitted in half-map 1 and half-map 2 (yellow); and between the atomic model fitted in the sum of the two half-maps and the sum of the two half-maps (red), for the PAD12 paired helical filament (PHF) structure assembled from a 1:1 mixture of PAD12 0N3R and 0N4R tau (left) and from PAD12 0N3R tau, seeded with filaments that were extracted from the brain of an individual with Alzheimer’s disease (AD) (middle) and 297–441 Δ392–395 (right).
Figure 1—figure supplement 3
Electron cryo-microscopy (cryo-EM) analysis of tau297–391 and PAD12 tau paired helical filaments (PHFs).
(A) Cross-sections of cryo-EM reconstructions perpendicular to the helical axis, with an approximate thickness of 4.7 Å and pie charts showing the distribution of filament types (blue: PHFs; light blue: single protofilaments with the Alzheimer fold [not shown]); for freeze-thawed filaments (flash-frozen in liquid nitrogen, thawed at room temperature), for filaments assembled in an Eppendorf tube, for filaments assembled from PAD12 tau0–391, for filaments assembled from PAD12 tau151–391, and for filaments assembled from 0N3R PAD12 with glutamate phosphomimetics instead of aspartate. (B–E) Cryo-EM overviews of PHFs composed of residues tau297–391 (B), a 1:1 mixture 0N3R and 0N4R PAD12 tau (C), tau 0–391 (D), and 151–391 PAD12 3R tau (E). From left to right: grid square overview, foilhole overview, and acquisition image. Arrows indicate clumped aggregates of filaments (pink) and ice particles (blue).
Figure 1—figure supplement 4
Quantification of PAD12 tau filament types with time.
Hierarchical classification of filament segments (left) based on their assigned 2D class averages (vertical axis) and corresponding filament IDs (horizontal axis). The pie charts on the top right show the relative amounts of different filament types. Pink represents paired helical filaments (PHFs) and ochre shows singlets. 2D class averages on the bottom right show examples of the boxed-out filament segments from the dendrogram, with PHFs in pink boxes and singlets in ochre boxes. 2D class averages in grey boxes are examples that were not included in the quantification of filament polymorphs. (A) After 4 days of shaking. (B) After 1 week of shaking. (C) After 1 week of shaking followed by 2 weeks of quiescent incubation.
Figure 1—figure supplement 5
Reproducibility of paired helical filaments (PHF) formation.
The percentage of PHFs formed in seven replicates of the assembly reaction with 0N3R PAD12 tau, with 1 week of shaking, followed by 2 weeks of quiescent incubation. Five replicates yielded 100% PHFs; one outlier contained 75% PHFs and 25% singlets. The mean percentage of PHFs is 95.3%, with a standard deviation of 9.4%.
Figure 2
EM analysis of 0N3R PAD12+4, PAD12–4, and PAD12±4 tau constructs.
(A) Schematic of tau sequence as in Figure 1A, with the four extra mutations of PAD12+4 in blue and the four mutations that were removed from PAD12–4 in red. (B) Negative-stain EM of filaments formed with PAD12+4 (left), PAD12–4 (middle), and PAD12±4 (right) 0N3R tau. (C) Electron cryo-microscopy (cryo-EM) reference-free 2D classes of filaments assembled from 0N3R PAD12–4. (D) Reference-free 2D classes of filaments assembled from 0N3R PAD12-4.
Figure 3
Labelling of pre-assembled PAD12 tau filaments.
(A) Cartoon showing that filaments can be labelled via NHS-ester chemistry, which specifically targets primary amines in lysine residues. (B) Immuno-EM showing biotinylated tau is labelled with streptavidin-coated 10 nm gold particles. (C) Electron cryo-microscopy (cryo-EM) reconstructions of PAD12 3R tau (top) labelled with different fluorophores. The circular inset shows a cross-section of the corresponding cryo-EM reconstruction, and the pie charts show the distributions of the different filament types (paired helical filaments [PHFs] in solid colours; singlets in lighter colours). The coloured pellets (bottom) indicate successful labelling of the filaments.
Figure 4
In vitro seeded assembly with PAD12 tau.
(A) Schematic of experimental approach for the seeded assembly of PAD12 0N3R. Small amounts of brain material are used to seed the assembly of PAD12 0N3R (round 1). The filaments formed from round one are used as seeds for a second round. (B) Thioflavin T (ThT) fluorescence profile of the Alzheimer’s disease (AD)-seeded (purple), second-round seeding (pink), and non-seeded control (yellow) N=9. The circles are individual measurements (normalised for each reaction). (C) Cross-sections of electron cryo-microscopy (cryo-EM) reconstructions perpendicular to the helical axis, with a thickness of approximately 4.7 Å and pie charts showing the distribution of filament types for the first (left) and second (right) round of seeding (pink/purple paired helical filaments [PHFs], yellow single protofilaments with the Alzheimer fold (not shown); grey discarded filaments). (D) Cryo-EM density map of AD-seeded 0N3R PAD12 tau (transparent grey) with the superimposed fitted atomic model. (E) Main chain trace of in vitro seeded PHF (purple) overlaid with AD PHF (grey; PDB-ID: 6hre).
Figure 5 with 1 supplement
Cellular seeding with recombinant tau filaments.
(A) Box plot showing the number of detected seeds, which were normalised to the number of cells and compared to mock-treated control cells (n≥10,000 cells/condition analysed). Graph represents mean values; error bars represent standard deviation. (B) Images from control (-seed) and cells seeded with 0.25 µg of assembled tau297–391 paired helical filaments (PHFs), PAD12 0N3R PHFs and PAD12 0N3R:0N4R PHFs. Fixed cells were stained against hemagglutinin (HA) for labelling over-expressed tau297–391 (green) and Hoechst (blue) for labelling of the nucleus. Scale bar, 50 µm. (C) Images from control cells without the addition of seeds (-seed) and cells seeded with 0.25 µg of PAD12 0N3R PHFs that were pre-labelled with DyLight-488. Fixed cells were stained against HA for labelling over-expressed tau (red) and Hoechst (blue) for labelling nuclei. Scale bar, 50 µm.
Figure 5—figure supplement 1
Cellular seeding with Alzheimer’s disease (AD) brain-derived tau and with PAD12 0N3R tau filaments labelled with DyLight-488.
(A, B) Box plots showing the number of detected seeds which were normalised to the number of cells and compared to mock-treated control cells (n≥10,000 cells/condition analysed), for cells seeded with AD-brain-derived seeds (A) and PAD12 0N3R DyLight-488-labelled filaments (B). Graphs represent mean values; error bars represent standard deviations. (C) Images from control cells without the addition of seeds (-seed) and cells seeded with insoluble tau from 200 µg of sAD brain tissue or 0.25 µg PAD12 0N3R tau filaments that were pre-labelled with DyLight-488. Fixed cells were stained against hemagglutinin (HA) for labelling over-expressed tau (green) and Hoechst dye (blue) for labelling nuclei. Scale bar, 50 µm.
Figure 6 with 6 supplements
Nuclear magnetic resonance (NMR) and electron cryo-microscopy (cryo-EM) of C-terminal tau297–441, tau297–441 PAD12, and tau297–441 Δ392–395.
(A) Peak height differences for selected residues in heteronuclear single quantum coherence (HSQC) spectra as shown by normalised peak intensity. Values for tau297–391 are shown in gold, tau297–441 in black, PAD12 tau297–441 in lilac, and tau297–441 Δ392–395 in blue. The grey dashed line box indicates the first intermediate amyloid (FIA) region (residues 301–316). Lilac dashed lines are PAD12 mutations and blue dashed lines are the residues deleted in the Δ392–395 tau construct. Horizontal dotted lines highlight the differences in peak intensity between tau297–441 (black, does not form paired helical filaments [PHFs]) and PAD12 tau297–441 (purple, does form PHFs) in the FIA region and around the PAD12 mutation site. Both tau297–391 (gold) and tau297–441 Δ392–395 (blue) have peak intensity values like those of PAD12 tau297–441. Full residue information is shown in Figure 6—figure supplement 5C. (B) The cryo-EM structure of tau297–441 Δ392–395. Cryo-EM density map in transparent grey with the superimposed fitted atomic model in blue. All filaments were of the same type.
Figure 6—figure supplement 1
Nuclear magnetic resonance (NMR) spectroscopy of wild-type and PAD12 tau151–391.
(A) Chemical shift perturbation (CSP) map of the peak location differences between wild-type and PAD12 tau151–391, with mutated residues shown in lilac. (B) Secondary chemical shift analysis of the backbone Cα and Cβ resonances shows similar torsion angle preferences for both constructs. Residues with a preference for helical torsion angles have positive values, and those with an extended backbone preference have negative values. Secondary chemical shifts for tau297–391 are shown in gold, wild-type tau151–391 in black, and PAD12 tau151-391 in lilac. (C) Heteronuclear NOE (HetNOE) values for wild-type and PAD12 tau151–391 are shown in black and lilac, respectively. HetNOE values are sensitive to backbone motion on the picosecond timescale. Residues within a permanent secondary structure element will have HetNOE values of ~0.8; lower or negative values indicate increased backbone flexibility. Relative increases in rigidity are seen for residues that are mutated in PAD12 tau297–441. (D) Normalised peak intensity differences in heteronuclear single quantum coherence (HSQC) spectra are shown for values of tau151–391 in black and PAD12 tau151–391 in lilac, with differences in the proline-rich N-terminus, indicating that PAD12 tau151–391 has reduced conformational sampling. The dashed box highlights residues that form the FIA and dashed lilac lines indicate mutated residues. Residues that we were unable to assign in the tau151–391 or the PAD12 tau151–391 constructs are highlighted with a purple or black asterisk, respectively.
Figure 6—figure supplement 2
Heteronuclear single quantum coherence (HSQC) peak assignment of wild-type and PAD12 tau151–391.
Assigned 700 MHz 15N-1H HSQC spectrum of left, wild-type tau151–391 (black), and right PAD12 tau151–391 (lilac).
Figure 6—figure supplement 3
Nuclear magnetic resonance (NMR) spectroscopy of wild-type and PAD12 tau297–441.
(A) Secondary shift analysis of the backbone Cα and Cβ chemical shifts. Residues that have a preference for helical torsion angles have positive values, and those with an extended backbone preference have negative values. Secondary chemical shifts for tau297–391 are shown in gold, tau297–441 in black, and PAD12 tau297–441 in lilac. Residues 393–404 show an increased preference for extended structure in PAD12 tau297–441 compared to wild-type tau297–441, as well as a preference for helical structure in residues 405–407. (B) Chemical shift perturbation (CSP) map of the peak location differences between wild-type and PAD12 tau297–441 highlight that the effects of the PAD12 mutations extend beyond the mutation sites. (C) Heteronuclear NOE (HetNOE) values for wild-type and PAD12 tau297–441 are shown in black and lilac, respectively. HetNOE values are sensitive to backbone motion on the picosecond timescale. Residues within a permanent secondary structure element will have HetNOE values of ~0.8; lower or negative values indicate increased backbone flexibility. Residues around the first intermediate amyloid (FIA) region and the PAD12 mutation sites appear more rigid in the PAD12 tau297–441 construct, compared to the wild-type tau297–441 construct. (D) Normalised peak intensity differences in HSQC spectra are shown for values of tau297–391 in gold, tau297–441 in black, and PAD12 tau297–441 in lilac. While tau297–391 (gold) has peak intensity values similar to PAD12 tau297–441 (lilac), wild-type tau297–441 (black) has reduced peak intensities in both the FIA region and residues 392–404. The dashed box highlights residues that form the ordered core of the FIA, and dashed lilac lines indicate mutated residues.
Figure 6—figure supplement 4
Heteronuclear single quantum coherence (HSQC) peak assignment of wild-type and PAD12 tau297–441.
Assigned 700 MHz 15N-1H HSQC spectrum of, from left to right, tau297–441 (black), overlaid and PAD12 tau297–441 (lilac). Residues from the PHF6 region (306–311, VQIVYK) and the C-terminal IVYK motif (392–395) are indicated in the central overlaid panel.
Figure 6—figure supplement 5
Nuclear magnetic resonance (NMR) spectroscopy of tau297–441 Δ392–395.
(A) Secondary chemical shift analysis of the backbone Cα and Cβ resonances. Residues that have a preference for helical torsion angles have positive values, and those with an extended backbone preference have negative values. Secondary chemical shifts for wild-type tau297–441 in black, PAD12 tau297–441 in lilac, and tau297–441 Δ392–395 in blue. The deletion of residues 392–395 has no major effect on the secondary structure preference of the remaining residues. (B) Heteronuclear NOE (HetNOE) values for wild-type 297–441, PAD12 tau297–441, and tau297–441 Δ392–395 are shown in black, lilac, and blue, respectively. HetNOE values are sensitive to motion on the picosecond timescale. Residues within a permanent secondary structure element will have HetNOE values of ~0.8; lower or negative values indicate increased backbone flexibility. The motions of tau297–441 Δ392–395 are like those of PAD12 tau297–441. (C) Normalised peak intensity differences in HSQC spectra are shown for values of tau297–391 in gold, tau297–441 in black, PAD12 tau297–441 in lilac, and tau297–441 Δ392–395 in blue. Both tau297–391 (gold) and tau297–441 Δ392–395 (blue) have peak intensities like those of PAD12 tau297–441. In contrast, wild-type tau297–441 (black) has lower peak intensities in the first intermediate amyloid (FIA) region and residues 392–404. The dashed box highlights residues that form the FIA, the dashed lilac lines indicate mutated residues, and the dashed blue lines indicate the truncated residues.
Figure 6—figure supplement 6
Heteronuclear single quantum coherence (HSQC) peak assignment of tau297–441 Δ392–395.
Assigned 700 MHz 15N-1H HSQC spectrum of tau297–441 Δ392–395 (blue), overlaid with tau297–441 (black). Residues 392–395 of tau297–441 are indicated in red.
Figure 7
Hairpin model of phosphorylated tau filament assembly.
In unmodified tau monomer, residues 392IVYK395 (orange) in the carboxy-terminal domain interact with residues 306VQIVYK311 (purple) in the core-forming region of tau. A similar interaction between the amino-terminal domain of tau (green) may also exist, but the residues involved in this interaction are unknown. Upon phosphorylation (red) of residues in the amino-terminal and the carboxy-terminal domains of tau, these interactions are disrupted, which then leads to filament nucleation through inter-molecular interactions of residues 306VQIVYK311 in the first intermediate amyloid (FIA).
Tables
Table 1
Filament assembly conditions.
| Construct | Concentration (μM) | Buffer | Shaking | Time (days) | Fold |
|---|---|---|---|---|---|
| 0N3R PAD12 | 100 | 100 mM KPhos*, 10 mM TCEP | 500 rpm, 1 min on:1 min off | 7 | AD |
| 0N4R PAD12 | 100 | 100 mM KPhos, 4 mM TCEP | 500 rpm, 1 min on:1 min off | 7 | CTE- singlet |
| 0N3R:0N4R | 50:50 | 100 mM KPhos, 10 mM TCEP | 500 rpm, 1 min on:1 min off | 7 | AD |
| tau0–391 | 100 | 100 mM KPhos, 10 mM TCEP | 500 rpm, 1 min on:1 min off | 7 | AD |
| tau151–391 | 100 | 100 mM KPhos, 10 mM TCEP | 500 rpm, 1 min on:1 min off | 7 | AD |
| 0N3R PAD12 (freeze-thaw) | 100 | 100 mM KPhos, 10 mM TCEP | 500 rpm, 1 min on:1 min off | 7 | AD |
| 0N3R PAD12 (tube) | 100 | 100 mM KPhos, 10 mM TCEP | 500 rpm | 7 | AD |
| 0N3R PAD12 (+seeds from 0.8 μg AD brain) | 40 | 20 mM HEPES at pH 7.3 and 4 mM KPhos at pH 7.2, plus 300 mM sodium citrate and 4 mM TCEP | 500 rpm, 1 min on:1 min off | 2 | AD |
| 0N3R PAD12 | 50 | 100 mM KPhos, 10 mM TCEP | 500 rpm, 1 min on:1 min off | 7 shaking; 14 quiescent | AD |
| 0N3R PAD12 | 50 | 20 mM HEPES at pH 7.3 and 4 mM KPhos at pH 7.2, plus 300 mM sodium citrate and 4 mM TCEP | 500 rpm, 5 s on:5 s off | 7 shaking; 14 quiescent | AD |
| tau297–441 Δ392–395 | 50 | 100 mM KPhos, 4 mM TCEP | 500 rpm, 1 min on:1 min off | 3.17 | New |
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*
KPhos is potassium phosphate.
Table 2
Electron cryo-microscopy (cryo-EM) statistics.
| LMB Krios G4 | PAD12 0N3R:0 N4R (EMDB-51884) (PDB-9H5G) | PAD12 0N3R AD-seeded (EMDB-51886) (PDB-9H5J) | 297–441 Δ392–395 (EMDB 54485) (PDB-9S2B) |
|---|---|---|---|
| Data acquisition | |||
| Electron gun | CFEG | CFEG | FEG |
| Detector | Falcon 4i | Falcon 4i | Falcon 4i |
| Energy filter slit (eV) | 10 | 10 | na |
| Magnification | 165,000 | 165,000 | 96,000 |
| Voltage (kV) | 300 | 300 | 300 |
| Electron dose (e-/Å2) | 40 | 40 | 35 |
| Defocus range (μM) | 0.5–2.5 | 0.5–2.5 | 0.5–2.5 |
| Pixel size (Å) | 0.744 | 0.744 | 0.824 |
| Data processing | |||
| Initial particle images (no.) | 664,772 (Autopick) | 163,335 (manual) | 643,712 (Autopick) |
| Final particle images (no.) | 56,351 | 125,334 | 213,971 |
| Helical twist (°) | 179.47 | 179.39 | –2.9 |
| Helical rise (Å) | 2.43 | 2.42 | 4.85 |
| Symmetry imposed | C1 | C1 | C1 |
| Map resolution FSC 0.143 (Å) | 2.48 | 2.72 | 2.9 |
| Refinement | |||
| Initial model used (PDB code) | 6hre | 6hre | ModelAngelo |
| Model resolution FSC 0.5 (Å) | 2.2 | 2.6 | 3.5 |
| Map sharpening B factor (Å2) | –43 | –71 | –97.8 |
| Model composition | |||
| Non-hydrogen atoms | 3300 | 3300 | 978 |
| Protein residues | 432 | 432 | 132 |
| B factors (Å2) | |||
| Protein | 48.4 | 48.4 | 96 |
| R.m.s. deviations | |||
| Bond lengths (Å) | 0.011 | 0.011 | 0.011 |
| Bond angles (°) | 2.009 | 1.961 | 1.935 |
| Validation | |||
| MolProbity score | 0.9 | 0.98 | 0.5 |
| Clashscore | 0 | 0 | 0 |
| Poor rotamers (%) | 0 | 0 | 0 |
| Ramachandran plot | |||
| Favoured (%) | 94.05 | 92.14 | 100 |
| Allowed (%) | 5.95 | 6.19 | 0 |
| Disallowed (%) | 0 | 1.67 | 0 |
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Twelve phosphomimetic mutations induce the assembly of recombinant full-length human tau into paired helical filaments
eLife 14:RP104778.
https://doi.org/10.7554/eLife.104778.4
