β-Sheet Core of Tau Paired Helical Filaments Revealed by Solid-State NMR

Abstract

One of the hallmarks of Alzheimer's disease is the self-assembly of the microtubule-associated protein tau into fibers termed "paired helical filaments" (PHFs). However, the structural basis of PHF assembly at atomic detail is largely unknown. Here, we applied solid-state nuclear magnetic resonance (ssNMR) spectroscopy to investigate in vitro assembled PHFs from a truncated three-repeat tau isoform (K19) that represents the core of PHFs. We found that the rigid core of the fibrils is formed by amino acids V306 to S324, only 18 out of 99 residues, and comprises three β-strands connected by two short kinks. The first β-strand is formed by the well-studied hexapeptide motif VQIVYK that is known to self-aggregate in a steric zipper arrangement. Results on mixed [(15)N:(13)C]-labeled K19 fibrils show that β-strands are stacked in a parallel, in-register manner. Disulfide bridges formed between C322 residues of different molecules lead to a disturbance of the β-sheet structure, and polymorphism in ssNMR spectra is observed. In particular, residues K321-S324 exhibit two sets of resonances. Experiments on K19 C322A PHFs further confirm the influence of disulfide bond formation on the core structure. Our structural data are supported by H/D exchange NMR measurements on K19 as well as a truncated four-repeat isoform of tau (K18). Site-directed mutagenesis studies show that single-point mutations within the three different β-strands result in a significant loss of PHF aggregation efficiency, highlighting the importance of the β-structure-rich regions for tau aggregation.

The fuzzy coat of pathological human Tau fibrils is a two-layered polyelectrolyte brush

“…The asymmetrical distribution of adhesion minima along twisted and straight hTau40 fibrils suggests that the NDs protrude sideways from hTau40 fibrils (i.e., parallel to the mica support), where they expose high structural flexibility. Such an arrangement of NDs in the fuzzy coat would be consistent with a parallel β-strand stacking of Tau molecules in the fibril backbone (12,23). In this case, all N-terminal ends protrude to the same side in straight fibrils and form a twist-dependent brush surrounding twisted fibrils.…”

“…S2C and S3). Because both hTau40 and TauRD fibrils are assumed to have a similar core of stacked repeat domains (RDs) (22,23) (Fig. 1A), we conclude that the deformation lanes running parallel to the hTau40 fibril cores resulted from the fuzzy coat.…”

Progress towards structural understanding of infectious sheep PrP-amyloid

“…21,[38][39][40][41] In contrast to rigid and highly flexible residues though, signals from semi-flexible regions, such as the fuzzy coat of amyloid fibrils, may be completely missing due to intermediate range molecular motion resulting in severe line broadening and/or disappearance of signals. 12,21,38,42 In order to compare the amounts of rigid and mobile protein segments, we recorded 1D-13 C-CP-and 1D- 13 C-INEPT-NMR spectra. CP-spectra, which selectively detect rigid protein segments, revealed identically strong and appropriately resolved resonances in samples after spontaneous conversion or PrP Sc -seeding of ovrecPrP .…”

Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein

“…8c. The TP4-bound ␤-sheet features intramolecular backbone hydrogen bonds between the ␤-strands, whereas the ␤-sheets of Tau filaments are intermolecular, with parallel, in-register orientation (20,42,46). However, due to the absence of NMR resonance signals the number, lengths, and relative orientations of ␤-strands of TP4-bound Tau could not be determined.…”

“…Two motifs with high ␤-sheet propensity in the repeats R3 and R2, called PHF6 ( 306 VQIVYK 311 ) and PHF6* ( 275 VQIINK 280 ), respectively, are involved in the initiation of the oligomerization process (14 -18). PHFs, the products of the aggregation reaction, are characterized by a rigid cross-␤-sheet core (19,20), built up by the imperfect repeats (21)(22)(23), and an outer fuzzy coat formed by the N-and C-terminal parts of the protein (24). Due to its importance for the aggregation process, the repeat domain constitutes an interesting target for interference with Tau assembly.…”