Gradual N-terminal truncation of tau dGAE (297–391) reveals the importance of 321–325 sequence for early steps of fibril formation

Abstract

The pathological conversion of intrinsically disordered proteins into β-sheet–rich amyloid filaments is a hallmark of numerous neurodegenerative disorders. In the case of tau protein, misfolding into aggregation-prone species can be promoted by truncation, motivating the present study. We employed an integrative approach combining established experimental techniques, including AFM and ThT fluorescence assay, with MD simulations to study amyloidogenic propensities of five tau truncation variants in the presence of aggregation inducers in vitro. The experimental observations were further validated by CD and NMR spectroscopy. Our experiments demonstrated that tau variants exhibit distinct amyloidogenic propensities. Tau variant spanning residues 321–391 (numbered according to the longest CNS tau isoform 1–441) represents the minimal E391-truncated construct capable of amyloid aggregation in vitro, whereas the shorter 326–391 variant failed to form fibrillar assemblies, even in the presence of aggregation inducers. All-atom (AA) MD simulations highlighted the importance of hairpin-like structural motifs during the early stages of aggregation, which appear to template subsequent fibril growth and are preferentially adopted by the Tau321–391 variant. In contrast, coarse-grained (CG) simulations revealed a pronounced α-helical propensity in Tau321–391, particularly at the N-terminal region. This elevated N-terminal helicity constitutes the most prominent structural distinction between the aggregation-competent and aggregation-incompetent variants. In both AA and CG MD simulations, the structural transitions of tau were driven by amyloid-nucleating sequence motifs, including the G-motif, PHF6**, and PAM4. Despite sharing these amyloidogenic regions, the Tau326–391 variant lacks the intrinsic amyloid propensity required to undergo productive self-assembly into ordered amyloid fibrils.