In silico peptide self-assembly reveals the importance of N-terminal motifs and the inhibition mechanism of the mutation L38M in α-synuclein fibrillation.

Alpha-synuclein (αSyn) is a presynaptic protein associated with several neurodegenerative diseases. While the non-amyloid component (NAC) region of the αSyn sequence (residues 65-90) forms the core of all αSyn fibrils, recent findings suggest that the flanking regions play a key role in initiating or preventing amyloid formation. Two motifs in the N-terminal region, named P1 (αSyn [36-42]) and P2 (αSyn [45-57]), have been shown to be key modulators of fibril formation, with deletion of these regions or single-point mutations in the P1 region inhibiting amyloid formation of full-length αSyn. In this study, we use the coarse-grained molecular dynamics package DMD/PRIME20 to simulate the self-assembly of the P1 and P2 regions, alongside longer segments P3 (αSyn [36-57]) and P3Next (αSyn [27-57]), and single-point mutations: focusing primarily on L38M, L38A, and V40A, and additionally examining Y39A and S42A as secondary variants, all of which have different effects on fibril formation of the full-length protein in vitro. The results show that P1, P2 and P3 have a high propensity to form parallel β-sheets while P3Next tends to form β-hairpins within fibrillar structures. The L38M substitution reduces the formation of both parallel β-sheets and β-hairpins, consistent with the inability of full-length αSyn containing L38M to form amyloid fibrils in vitro at neutral pH and to aggregate in vivo in Caenorhabditis elegans. In contrast, simulations of L38A and V40A show no such effect, consistent with their minimal impact on full-length αSyn fibrillation. The simulation results suggest that the presence of P1/P2 hairpins are required to unleash the amyloid potential of αSyn and offer a structural explanation of how L38M mutation in this region protects the protein from amyloid formation.