The Conformation and the Aggregation Kinetics of α-Synuclein Depend on the Proline Residues in Its C-Terminal Region

The neuronal protein alpha-synuclein (alpha-syn) plays a central role in Parkinson's disease (PD). The pathological features of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of Lewy bodies. The C-terminal domain of alpha-syn is characterized by the presence of 15 acidic amino acids and all five proline residues of the protein (P108, P117, P120, P128, and P138). The aggregation of this natively unfolded protein is accelerated in vitro by FK506 binding proteins (FKBPs) showing peptidyl-prolyl cis-trans isomerase activity. These proteins catalyze the cis-trans conformational change of the X Pro peptide bond, often a rate-limiting step in protein folding. The acceleration of the folding of alpha-syn by FKBPs may accelerate disease-associated aggregation. To further elucidate the role of the proline residues in the conformation and aggregation of alpha-syn, we constructed several mutants of alpha-syn in which one or more proline residues are mutated to alanine via site-directed mutagenesis. For this purpose, we produced and purified His-WT alpha-syn, a recombinant alpha-syn with a polyhistidine tag (six His residues) and a linker, and a number of Pro-to-Ala mutants. The aggregation kinetics of these mutants and His-WT alpha-syn were studied by turbidity, thioflavin T fluorescence, and CD measurements. We can conclude that mutation of the proline residues to alanine accelerates the aggregation kinetics of alpha-syn while all proline mutants formed fibrils similar to His-WT alpha-syn, as visualized via transmission electron microscopy. We also demonstrate that the accelerating effect of hFKBP12 is abolished via removal of the proline residues from the C-terminus. Finally, we show that the mutant of His alpha-syn with all five proline residues mutated to alanine is more structured (more alpha-helix) than His-WT alpha-syn, indicating the role of the Pro residues as potential helix breakers in the inhibitory conformation of the C-terminus.