Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure

Abstract

We have searched for a minimal interaction motif in tau protein that supports the aggregation into Alzheimer-like paired helical filaments. Digestion of the repeat domain with different proteases yields a GluC-induced fragment comprising 43 residues (termed PHF43), which represents the third repeat of tau plus some flanking residues. This fragment self assembles readily into thin filaments without a paired helical appearance, but these filaments are highly competent to nucleate bona fide PHFs from full-length tau. Probing the interactions of PHF43 with overlapping peptides derived from the full tau sequence yields a minimal hexapeptide interaction motif of (306)VQIVYK(311) at the beginning of the third internal repeat. This motif coincides with the highest predicted beta-structure potential in tau. CD and Fourier transform infrared spectroscopy shows that PHF43 acquires pronounced beta structure in conditions of self assembly. Point mutations in the hexapeptide region by proline-scanning mutagenesis prevent the aggregation. The data indicate that PHF assembly is initiated by a short fragment containing the minimal interaction motif forming a local beta structure embedded in a largely random-coil protein.

Figures

Figure 1

Bar diagram of τ constructs and peptides. (A) hτ40, containing four repeats of ≈31 residues (labeled 1–4) and two inserts near the N terminus. Residue numbering follows that of hτ40 throughout. (B) hτ23, the smallest and fetal isoform. (C) K19 represents only the repeat domain of hτ23, i.e., R1, R3, R4. (D) PHF43, containing the end of R1, all of R3, and the beginning of R4. PHF47 was used for proline-scanning mutagenesis. (E) PHF8. (F) PHF6, highlighted also in the sequences of PHF43 and PHF8. (G) K19 and hτ23 mutants in the region 306-311. ΔVQI mutants lack 306VQI308, NAE mutants contain NAE instead of 306VQI308, and Y310E, Tyr-310 is exchanged to Glu.

Figure 2

Kinetics of PHF assembly. The assembly of τ or τ fragments was measured by the fluorescence of ThS (10). Protein concentrations were 20 μM in the presence of 5 μM heparin and 20 mM NH4Ac, at RT. PHF43 assembles rapidly and spontaneously (diamonds, top curve, t1/2 = 0.75 min), but hτ23 is very slow by comparison (circles, bottom curve, t1/2 = 180 min). K19 shows intermediate kinetics (triangles, t1/2 = 12 min). Hτ23 can be speeded up to a similar rate as K19 in the presence of seeds from sonicated fibers obtained after PHF43 assembly (squares, t1/2 = 14 min).

Figure 3

Electron micrographs of fibers obtained from the self assembly of τ or τ peptides. Assembly conditions were the same as in Fig. 2 (20 μM protein, 5 μM heparin), except E and F (660 μM peptide, 660 μM heparin). A–C show mostly twisted filaments (width ≈10–20 nm) resembling Alzheimer PHFs polymerized from, (A) hτ23, (B) hτ23 plus seeds made from PHF43, and (C) PHF43. D shows the seeds obtained by sonication of PHF43 fibers. E and F show filamentous aggregates with variable diameters obtained from the short peptides PHF8 and PHF6.

Figure 4

Peptide spot membrane interactions between PHF43 and peptides derived from the repeat region of τ. The region of τ from L253 in repeat 1 to D348 in repeat 4 (encompassing the sequence of PHF43) was subdivided into consecutive 15-mer peptides staggered by three residues, synthesized and covalently attached to a cellulose membrane (22), and incubated with iodinated PHF43. Bars represent the bound radioactivity determined by autoradiography. The strongest interaction occurs around the spots that contain the sequence of PHF6 (306VQIVYK311, highlighted) at the beginning of R3. Another cluster of strong interaction occurs around the spots that harbor the analogous sequence in R2 (275VQIINK280, highlighted).

Figure 5

CD spectroscopy of τ constructs and τ polymers. CD spectra were obtained at 50 μM protein concentrations in 10 mM NH4 acetate at RT, in the absence or presence of 50 μM heparin and/or 50% TFE. (A) hτ23 monomers (circles), dimers (triangles), and assembled PHFs (solid line) show similar CD curves with or without heparin (filled or open symbols). (B) K19 monomers (without heparin, open circles; with heparin, filled circles) and dimers (without heparin, open triangles; with heparin, filled triangles; after aggregation, solid line). (C) PHF43 monomers (without heparin, open circles; with heparin, filled circles) and dimers without heparin (open triangles) and after aggregation (solid line). (D) PHF8 (without heparin, open circles; with heparin, filled circles) and PHF6 (without heparin, open triangles; with heparin, filled triangles). (E) Three peptides derived from PHF43, but not including 306-311 (313VDLSKVTSK321; without heparin, open circles; with heparin, filled circles; 318VTSKCGSLGNIHHKPGGG335 without heparin, open triangles; with heparin, filled triangles; 335GQVEVKSE342, without heparin, open squares; with heparin, filled squares). (F) K19 (open circles) and PHF43 dimers (open triangles) and PHFs (K19, filled circles; PHF43, filled triangles) in the presence of 50% TFE.

Figure 6

FTIR of τ constructs and τ-derived peptides. (A) Infrared absorbance spectrum of the peptide PHF43 in the absence (solid line) and presence (dotted line) of heparin. Spectral contributions of heparin and the solvent D2O have been subtracted. (B) Aggregation-induced difference spectra of K19 and PHF43.

Figure 7

Analysis of the ability of τ mutants to form filaments. (A) A series of substitution mutations to proline was analyzed by ThS fluorescence in comparison to wild-type τ after 24-h incubation under PHF-assembly conditions. (B) PHF assembly of K19 wild type and the mutants K19ΔVQI, K19NAE, and K19Y310E was performed for 24 h, and the supernatants and pellets after ultracentrifugation of the reaction mixtures were applied to a 15% SDS/PAGE and stained by Coomassie blue. (C) PHF assembly of hτ23 wild type and the mutants hτ23ΔVQI and hτ23NAE was performed for 3 days, and the supernatants and pellets after ultracentrifugation of the reaction mixtures were applied to a 10% SDS/PAGE and stained by Coomassie blue.

Figure 8

Model of structural changes during PHF assembly. PHF43 dimer and PHF43 monomer (in blue) are able to bind polyanions (red), but only the dimer shows a transition from random coil (wiggle) to β sheet (straight) during aggregation.