A Rationally Designed Six-Residue Swap Generates Comparability in the Aggregation Behavior of α-Synuclein and β-Synuclein

The aggregation process of a-synuclein, a protein closely associated with Parkinson's disease, is highly sensitive to sequence variations. It is therefore of great importance to understand the factors that define the aggregation propensity of specific mutational variants as well as their toxic behavior in the cellular environment. In this context, we investigated the extent to which the aggregation behavior of alpha-synuclein can be altered to resemble that of beta-synuclein, an aggregation-resistant homologue of alpha-synuclein not associated with disease, by swapping residues between the two proteins. Because of the vast number of possible swaps, we have applied a rational design procedure to single out a mutational variant, called alpha 2 beta, in which two short stretches of the sequence in the NAC region have been replaced in a-synuclein from beta-synuclein. We find not only that the aggregation rate of alpha 2 beta is close to that of beta-synuclein, being much lower than that of alpha-synuclein, but also that alpha 2 beta effectively changes the cellular toxicity of alpha-synuclein to a value similar to that of beta-synuclein upon exposure of SH-SYSY cells to preformed oligomers. Remarkably, control experiments on the corresponding mutational variant of beta-synuclein, called beta 2 alpha, confirmed that the mutations that we have identified also shift the aggregation behavior of this protein toward that of alpha-synuclein. These results demonstrate that it is becoming possible to control in quantitative detail the sequence code that defines the aggregation behavior and toxicity of alpha-synuclein.