Abstract
<jats:title>Abstract</jats:title> <jats:p> Tau assembles into fibrillar aggregates that are pathological hallmarks of a group of neurodegenerative diseases collectively called tauopathies. Templated aggregation of naïve 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 <jats:sup>1</jats:sup> H- <jats:sup>15</jats:sup> N 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. </jats:p> <jats:sec> <jats:title>Significance Statement</jats:title> <jats:p>Tau proteins must align and stack precisely with existing fibril ends to propagate pathology, yet the molecular signature that initiates and ensures this in-register alignment has been unclear. This study shows recruitment begins at a single, structurally well-defined contact site on the fibril surface that is enriched in release-prone hydration water. These findings reveal that water-release-prone hotspots, rather than the well-known amyloid-core regions alone, govern the initial steps in templating seeding and can hence be blocked, opening a new path for identifying therapeutic targets that could slow the progression of tau-related diseases.</jats:p> </jats:sec>