Disordered regions of inhibitor-bound α-synuclein suppress seed-induced fibril nucleation in cells

Abstract

Inhibitors of amyloid fibril formation can act in diverse ways and aid in elucidating the mechanisms of protein aggregation. The engineered binding protein β-wrapin AS69 binds monomers of Parkinson-disease-associated α-synuclein (αS), yet achieves inhibition at substoichiometric concentration. The substoichiometric activity was not attributed to the binding protein per se, but to its 1:1 complex with αS, in which AS69 sequesters αS residues 30–60 into a globular protein fold, whereas other αS parts remain intrinsically disordered regions (IDRs). Here, we investigate AS69-αS fusion constructs that form the AS69:αS complex by intramolecular folding and expose different IDRs. We find that not only the globular part of the complex but also αS IDRs are critical for substoichiometric inhibition, which is achieved by interference with primary and secondary fibril nucleation. The effects in vitro are reproduced in cellular seeding assays, indicating that secondary nucleation drives seeding in aggregate biosensing.