A water-pinning hotspot drives templated tau aggregation
Chung-Ta HaniD, Karen TsayiD, Samuel LoboiD, Saeed NajafiiD, Kieran Monahan, Erica HongiD, M. Scott ShelliD, Joan‐Emma SheaiD, Songi HaniD
Abstract
Tau assembles into fibrillar aggregates that are pathological hallmarks of a group of neurodegenerative diseases collectively called tauopathies. Templated aggregation of naive tau to seeding-competent fibrils that proceed from cell to cell is a key driver of prion-like progression of tauopathies. This study tests the hypothesis that tau, an intrinsically disordered protein (IDP), achieves in-register stacking to form seed-competent fibrils by a pinning action of tau to each other and/or the seed surface via a single dominant hotspot to avoid mismatch in tau stacking to fibrils. Structured solvation water has been proposed to be a signature of such hotspots at both the tau fibril-end surface and soluble tau monomers. Although jR2R3-P301L tau exhibits a heterogeneous hydration landscape in its intrinsically disordered state, with enhanced water structuring near the P301L mutation site, it is unclear whether a localized hotspot exists at the fibril end surface and surface water facilitates the initial contacts in templated aggregation. Using rapid 1H-15N SOFAST-HMQC NMR to track seed-induced aggregation of jR2R3-P301L in real time, complemented by molecular dynamics simulation of fibril surface hydration, we identify a residue-specific pinning hotspot that is prone to dewetting followed by sequential folding and incorporation of the remaining segment in a two-step dock-and-lock process. Site-specific spin labeling further demonstrates that blocking this pinning hotspot disrupts templated aggregation, leading to shorter fibrils. The identification of a dominant pinning site will facilitate the rational design of binders to effectively disrupt fibril extension or serve as diagnostic or therapeutic strategies.
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In-depth summary
Claude Opus 5.5 · xhigh effort · 7 Nov 2026 · from the full text of version 3 (25 Sept 2026) · CC BY 4.0
In a minimal tau fibril model, residue 300/301 binds incoming peptide first and carries the most release-prone hydration water. Spin-labelling site 300 impairs locking and suppresses seeded growth.
The dock-lock model of templated seeding leaves open how a disordered tau chain finds the right docking site on a fibril end. The paper tests whether one hotspot, marked by release-prone hydration water, initiates contact and in-register stacking.
Key results
ShownTime-resolved NMR of a seeded reaction shows residues immobilised in a fixed order. V300 goes first, and the segment 308-313 goes last. This points to a single dominant pinning site at V300.
Fast decay rate alpha was 5.74 ± 0.78 at V300, 4.44 ± 0.68 at V306, 3.74 ± 0.68 at L301 and 2.67 ± 0.61 at I297. Sites I308, V309 and V313 were below 0.55. Seven isotope-labelled residues were tracked. · Interaction hotspots in templated seeding of…
SuggestedBefore seeds are added, the soluble peptide is a mixture of monomers and small oligomers. Within 25 min of seeding the diffusion profile moves toward monomer-like. The authors read this as oligomers being incorporated into fibrils, so the NMR decay reports recruitment onto fibril ends.
The DOSY decay before seeding followed a simulated 8 kDa species. At 25 min it shifted toward the simulated 2 kDa monomer curve. · jR2R3-P301L oligomers are present before fibril…; Discussion
ShownIn MD of a single fibril-end layer, site 300 shows the largest loss of hydration waters relative to the disordered peptide. Water-structure analysis marks sites 300/301 as the most release-prone: site 300 has the highest vapor-like (PC2) signal and site 301 the highest side-chain tetrahedral (PC1) signal.
Per-residue comparison across D295 to V314 of fibril (PDB 8V1N) vs disordered peptide. Three-body water-angle PCA was run separately on backbone and side chain. Simulation was 5 ns with a99SB-disp. · Enhanced differential hydration around site 300…; Enhanced release-prone hydration water around…
ShownODNP relaxometry on spin-labelled fibril ends finds the most slow, structured water at site 300, in contact region 1 (CR1). It supports the simulation result.
klow was 1477 ± 70 s⁻¹M⁻¹ at site 300, 1041 ± 189 at site 306 and 957 ± 98 at site 314. Only the CR1 vs CR3 difference is clear; CR2 is intermediate. · Enhanced release-prone hydration water around…
ShownSpin labels at site 300 suppress templated seeding more than labels at 306 or 314. Initial docking still occurs but locking and elongation are impeded. Docking occurs on a faster timescale than the ThT-reported locking.
Site-300-labelled peptide gave the lowest ThT plateau, and negative-stain TEM showed fibrils close to the original seeds. cw-EPR showed the fastest docking component at 300 and the slowest locking component at 300 vs 314. The seeded ThT trace gave a locking rate near 0.8 hr⁻¹. · Seed-induced aggregation of jR2R3-P301L tau; Blocking the interaction hotspots on tau fibril…; Discussion
Methods, data and reproducibility
Time-resolved 1H-15N SOFAST-HMQC NMR and DOSY followed seeding of a 19-residue tau peptide (jR2R3-P301L) onto preformed fibril seeds. Atomistic MD, ODNP relaxometry, and ThT, TEM and cw-EPR of spin-labelled variants tested the role of hydration at the hotspot.
- Synthetic 19-residue tau jR2R3-P301L peptide (residues 295-313), seeded with preformed fibrils; 13C/15N labelling on seven V/L/I residues for NMR · n = 250 µM peptide with 250 µM seed for NMR; ThT in triplicate
- MD of one strand of the jR2R3-P301L cryo-EM fibril structure with residues 312-314 appended · n = one 5 ns NPT simulation · PDB 8V1N
- Spin-labelled peptide variants (sites 300, 306, 314) for ODNP, ThT, TEM and cw-EPR · n = triplicates; seed ratios 0X, 0.5X, 1X, 2X for ODNP
- Methods & tools
- SOFAST-HMQC NMR, DOSY, ODNP relaxometry, cw-EPR, Thioflavin T assay, negative-stain TEM, MD with a99SB-disp force field, PyMOL, MTSSL spin label, MestReNova, pyB12MPS, INDUS
- Reagents & environment
- Force field a99SB-disp; the pyB12MPS Python package was used for ODNP acquisition; no versions or containers are stated.
- Experimental conditions
- Each 2D HMQC spectrum took 16 min on a 600 MHz Bruker system; MD ran for 5 ns; no hardware is stated.
- Reproducibility gaps
- Supplementary files (bulk_water_triplets.csv, principalComps.csv, jR2R3_P301L_appended.pdb) are named but no URL is given; no code repository is stated.
Limitations (4)
- The mechanism of the slow NMR decay phase (alpha2) is not assigned. It mixes locking with continued fibril growth, and the authors say the main conclusion does not depend on it. · stated by the authors
- The hydrophobicity analysis (INDUS) places CR1 among three hydrophobic patches and cannot by itself single out one hotspot. Hydration of the soluble oligomeric states is left for future work. · stated by the authors
- Spin labelling works only at sites that tolerate a cysteine substitution, so only sites 300, 306 and 314 were probed for ODNP and blocking. CR2 is only marginally distinguishable from CR1 by ODNP. · stated by the authors
- All evidence comes from one 19-residue peptide with the P301L mutation and its fibril, with a single 5 ns MD simulation of one strand. The generality to full-length tau or other tauopathy folds is claimed but not tested. · noted in this summary
Who should read:Read this if you study tau seeding or amyloid elongation and want residue-resolved NMR, EPR and hydration-water evidence for a single docking hotspot.
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