Alpha-synuclein (αSyn) is a 14-kDa intrinsically disordered protein that aggregates into insoluble fibrils in synucleinopathies, including Lewy bodies, multiple system atrophy, and Parkinson's disease, contributing to neurotoxicity and disease progression. The ability of these fibrils to seed further aggregation of native protein is central to αSyn pathology. Here, we examined the broader non-amyloid component (NAC) domain, focusing on how residues flanking the hydrophobic 68-71 (GAVV) motif of αSyn (residues 8-11 in NAC35) modulate nucleation, stability, and pathological seeding. Using full-length NAC peptide and truncated variants, we show that the 68-71 (GAVV) stretch is critical for nucleation and aggregation into prion-like fibrils. Peptide inhibitors targeting this hydrophobic region block the formation of seed-competent fibrils. Molecular dynamics simulations showed that these inhibitors alter peptide-peptide interactions and contact key hydrophobic residues within the NAC domain. Further analysis indicates that residues beyond the 68-71 (GAVV) motif, such as 79-95, are critical for stabilizing fibrils and promoting seeding competency. Peptide B interactions with key hydrophobic motifs within the NAC domain were visualized in silico, offering mechanistic insights into how it disrupts aggregation.

Department of Biochemistry Faculty of Biological Sciences Tarbiat Modares University Tehran Iran

Department of Biophysics Faculty of Science Palacký University Olomouc Czech Republic

Department of Medical Biophysics Faculty of Medicine and Dentistry Palacký University Olomouc Czech Republic

Department of Nanobiotechnology and Biomimetics School of Life Science Engineering University of Tehran Iran

Department of Nanobiotechnology Faculty of Biological Sciences Tarbiat Modares University Tehran Iran

Faculty of Medicine and Dentistry Institute of Molecular and Translational Medicine Palacký University and University Hospital Olomouc Czech Republic

Institute of Molecular and Translational Medicine Czech Advanced Technologies and Research Institute Palacký University Olomouc Czech Republic

Nano Drug Delivery Research Centre Health Technology Institute Kermanshah University of Medical Sciences Iran

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