Saturation mutagenesis of α-synuclein reveals monomer fold that modulates aggregation

α-Synuclein (aSyn) aggregation underlies neurodegenerative synucleinopathies. aSyn seeds are proposed to replicate and propagate neuronal pathology like prions. Seeding of aSyn can be recapitulated in cellular systems of aSyn aggregation; however, the mechanism of aSyn seeding and its regulation are not well understood. We developed an mEos-based aSyn seeding assay and performed saturation mutagenesis to identify with single-residue resolution positive and negative regulators of aSyn aggregation. We not only found the core regions that govern aSyn aggregation but also identified mutants outside of the core that enhance aggregation. We identified local structure within the N terminus of aSyn that hinders the fibrillization propensity of its aggregation-prone core. Based on the screen, we designed a minimal aSyn fragment that shows a ~4-fold enhancement in seeding activity and enabled discrimination of synucleinopathies. Our study expands the basic knowledge of aSyn aggregation and advances the design of cellular systems of aSyn aggregation to diagnose synucleinopathies based on protein conformation.