Complexation of NAC-Derived Peptide Ligands with the C-Terminus of α-Synuclein Accelerates Its Aggregation

Aggregation of alpha-synuclein (alpha-Syn) into neurotoxic oligomers and amyloid fibrils is suggested to be the pathogenic mechanism for Parkinson's disease (PD). Recent studies'have indicated that oligomeric species of alpha-Syn are more cytotoxic than their mature fibrillar counterparts, which are responsible for dopaminergic neuronal cell death in PD. Therefore, the effective therapeutic strategies for tackling aggregation-associated diseases would be either to prevent aggregation or to modulate the aggregation process to minimize the formation of toxic oligomers during aggregation. In this work, we showed that arginine-substituted alpha-Syn ligands, based on the most aggregation-prone sequence of alpha-Syn, accelerate the protein aggregation in a concentration-dependent manner. To elucidate the mechanism by which Arg-substituted peptides could modulate alpha-Syn aggregation kinetics, we performed surface plasmon resonance (SPR) spectroscopy, nuclear magnetic resonance (NMR) studies, and all-atom molecular dynamics (MD) simulation. The SPR analysis showed a high binding potency of these peptides with alpha-Syn but one that was nonspecific in nature. The two-dimensional NMR studies suggest that a large stretch within the C-terminus of alpha-Syn displays a chemical shift perturbation upon interacting with Arg-substituted peptides, indicating C-terminal residues of alpha-Syn might be responsible for this class of peptide binding. This is further supported by MD simulation studies in which the Arg-substituted peptide showed the strongest interaction with the C-terminus of alpha-Syn. Overall, our results suggest that the binding of Arg-substituted ligands to the highly acidic C-terminus of alpha-Syn leads to reduced charge density and flexibility, resulting in accelerated aggregation kinetics. This may be a potentially useful strategy while designing peptides, which act as alpha-Syn aggregation modulators.