Filaments of the protein tau are a characteristic occurrence in Alzheimer disease and many other neurodegenerative disorders and the distribution of tau filaments correlates well with the loss of neurons and cognitive functions in Alzheimer disease. Filament formation of tau filaments is based on structural transitions from random coil to b-structure, to give the paired helical filaments (PHFs) which share common characteristics of amyloid fibrils. Protease digestion and solvent-accessibility studies demonstrated that the “core” of PHFs is mainly built from repeat sequences in the C-terminal half of the tau protein. The PHF core is surrounded by a “fuzzy coat”, of more than 200 residues that come from the N-terminal half of the protein as well as the C-terminus (Figure 1a). Electron paramagnetic resonance and nuclear magnetic resonance (NMR) suggested that residues within the fuzzy coat are highly flexible. Biochemical studies have shown that the fuzzy coat is important for tau aggregation as well as neurotoxicity. Herein we characterized the dynamic structure of PHFs formed by 441-residue tau (htau40), the longest isoform of tau present in the human central nervous system (Figure 1a), at single-residue level using NMR spectroscopy. We aggregated N-labeled htau40 into insoluble filaments. NMR diffusion experiments demonstrated that the observed NMR signals arises from aggregated tau protein with a molecular mass of more than 1 MDa (Figure 1b). In a two-dimensional heteronuclear single quantum coherence (HSQC) spectrum employing high-resolution magic-angle spinning (HR-MAS) (see Figure S1 in the Supporting Information), we observed about 260 signals (Figure 1c and Figure S2 and S3 in the Supporting Information). Sequencespecific resonance assignment of 244 of these signals (BMRB accession number: 17920; see Figure S2 in the Supporting Information) identified most of the residues in the N-terminal domain up to Thr212 and at the C-terminus starting at Val399. No signals were detected for residues between Thr212 and Val399, suggesting that residues in the central domain are too immobile to be detected by liquid-state NMR spectroscopy in agreement with previous studies. Comparison with monomeric htau40 revealed that the NMR resonances of many residues were strongly reduced in filamentous htau40 (Figure 1d,e). Most strikingly, the sections His121– Lys130 and Met1–Gly37 that are separated from the fibril core by 170 residues or more, showed changes in position and intensity of NMR signals (Figure 1e and Figure S2c in the Supporting Information). The presence of chemical exchange in these regions was further supported by N spin relaxation measurements (Figure 1 f and Supporting Figure S3). In agreement with chemical exchange, additional peaks were observed in close proximity to several of these residues (Figure 1 f and Figure S4 in the Supporting Information). The additional signals could not be connected in triple-resonance experiments or using exchange spectroscopy because of low signal-to-noise and signal overlap. Therefore, we assigned the additional peaks to the residue for which the assigned crosspeak of the major peak set had the greatest similarity in chemical shifts and paramagnetic relaxation enhancement (see Figure S5 in the Supporting Information). This procedure indicates that the additional peaks arise from residues at the Nand C-terminus. No peak doubling was observed at the Nand C-terminus in monomeric tau (see Figure S4 in the Supporting Information), highlighting the specificity of the multiple conformations in PHF tau. We revealed the identity of the PHF-specific conformations through measurements of paramagnetic relaxation enhancements (PREs), in which nitroxide spin labels are attached to cysteine residues at various positions in the PHF tau. The resulting broadening of amide resonances caused by enhanced relaxations rate through the paramagnetic nitroxide label, is quantified through the intensity ratios in the paramagnetic and diamagnetic states (Figure 2). The PRE effect scales as the inverse sixth power of the distance between the unpaired electron of the nitroxide unit and the NMR spin, providing a powerful probe of distances. N spin relaxation times (Figure 1 f) indicate that the fuzzy coat of PHFs is highly dynamic on a broad scale suggesting that the correlation time of the electron—amide proton internuclear vector is comparable to that of small water soluble proteins. Initially, we measured PRE broadening for PHFs with a [*] S. Bibow, Dr. M. D. Mukrasch, Prof. Dr. C. Griesinger, Prof. Dr. M. Zweckstetter Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 Gcttingen (Germany) E-mail: mzwecks@gwdg.de
Search from 250 M+ research papersSearch from 250 M+ research papers
Jan 1, 2022
Felix A Roth + 3
Open Access