Inhibitor and substrate cooperate to inhibit amyloid fibril elongation of α-synuclein

In amyloid fibril elongation, soluble growth substrate binds to the fibril-end and converts into the fibril conformation. This process is targeted by inhibitors that block fibril-ends. Here, we investigated how the elongation of α-synuclein (αS) fibrils, which are associated with Parkinson's disease and other synucleinopathies, is inhibited by αS variants with a preformed hairpin in the critical N-terminal region comprising residues 36–57. The inhibitory efficiency is strongly dependent on the specific position of the hairpin. We find that the inhibitor and substrate concentration dependencies can be analyzed with models of competitive enzyme inhibition. Remarkably, the growth substrate, i.e., wild-type αS, supports inhibition by stabilizing the elongation-incompetent blocked state. This observation allowed us to create inhibitor–substrate fusions that achieved inhibition at low nanomolar concentration. We conclude that inhibitor–substrate cooperativity can be exploited for the design of fibril growth inhibitors.