Impact of Tyr to Ala mutations on α-synuclein fibrillation and structural properties

Substantial evidence suggests that the fibrillation of α-synuclein is a critical step in the development of Parkinson's disease. In vitro, α-synuclein forms fibrils with morphologies and a staining characteristic similar to those extracted from disease-affected brain. Monomeric α-synuclein is an intrinsically disordered protein, with three Tyr residues in the C-terminal region, one in the N-terminus, and lacking Trp. It is thought that interactions between the C-terminus and the central portion of the molecule may prevent or minimize aggregation/fibrillation. To test this hypothesis we examined the importance of the Tyr residues on the propensity for α-synuclein to fibrillate in vitro. Fibril formation of α-synuclein was completely inhibited, in the timescale over which measurements were made, by replacing the three C-terminal Tyr residues with Ala. In addition, substitution of Tyr133 by Ala also resulted in the absence of fibrillation, whereas the individual Y125A and Y136A mutants showed limited inhibition. Replacement of Tyr39 by Ala also resulted in substantial inhibition of fibrillation. Structural analysis showed that the Y133A mutant had a substantially different conformation, rich in α-helical secondary structure, as compared with the wild-type and other mutants, although the formation of any tertiary structure has not been observed as can be judged from near-UV-CD spectra. These observations suggest that the long-range intramolecular interactions between the N- and C-termini of α-synuclein are likely to be crucial to the fibrillation process.