Targeting Hydrophobic Residues in the Alpha-Synuclein NAC Domain Disrupts Aggregation and Seed-Competent Fibril Formation

Alpha-synuclein (αSyn) is a 14 kDa soluble and intrinsically disordered protein ubiquitously expressed in neurons. It plays a crucial role in synucleinopathies, where insoluble fibrils contribute to neurotoxicity and disease progression. The formation of these fibrils and their ability to seed further aggregation are central to αSyn pathology. Our study focused on the hydrophobic non-amyloid component (NAC) region of αSyn. Using full-length NAC peptide and its truncated variants, inhibitory peptides, and a combination of experimental and computational approaches, we demonstrate that the 68GAVV71 region and residues adjacent to it, such as T72, T75, and A76, are essential for αSyn aggregation and its prion-like behavior. Inhibitor peptides that target the hydrophobic region effectively block αSyn dimerization and aggregation into seed-competent fibrils. Molecular dynamics simulations revealed that the peptide inhibitor disrupted hydrophobic interactions within the NAC domain, preventing stable β-sheet structure formation. These results show that the 68GAVV71 stretch in the NAC domain is critical for αSyn aggregation into prion-like fibrils and could be a potential molecular target for treating synucleinopathies.