Mechanisms of Protein Oligomerization: Inhibitor of Functional Amyloids Templates α-Synuclein Fibrillation

Small organic molecules that inhibit functional bacterial amyloid fibers, curli, are promising new antibiotics. Here we investigated the mechanism by which the ring-fused 2-pyridone FN075 inhibits fibrillation of the curli protein CsgA. Using a variety of biophysical techniques, we found that FN075 promotes CsgA to form off-pathway, non-amyloidogenic oligomeric species. In light of the generic properties of amyloids, we tested whether FN075 would also affect the fibrillation reaction of human alpha-synuclein, an amyloid-forming protein involved in Parkinson's disease. Surprisingly, FN075 stimulates alpha-synuclein amyloid fiber formation as measured by thioflavin T emission, electron microscopy (EM), and atomic force microscopy (AFM). NMR data on N-15-labeled alpha-synuclein show that upon FN075 addition, alpha-synuclein oligomers with 7 nm radius form in which the C-terminal 40 residues remain disordered and solvent exposed. The polypeptides in these oligomers contain beta-like secondary structure, and the oligomers are detectable by AFM, EM, and size-exclusion chromatography (SEC). Taken together, FN075 triggers oligomer formation of both proteins: in the case of CsgA, the oligomers do not proceed to fibers, whereas for alpha-synudein, the oligomers are poised to rapidly form fibers. We conclude that there is a fine balance between small-molecule inhibition and templation that depends on protein chemistry.