Abstract

The conformational ensembles of intrinsically disordered proteins (IDPs) contain substantial fractions of conformers that exhibit long-range intramolecular interactions. We have investigated beta-hairpin motifs of islet amyloid polypeptide (IAPP), alpha-synuclein, and amyloid-beta peptide (Abeta). Applying NMR spectroscopy, we have identified a beta-hairpin conformation of IAPP in complex with an engineered binding protein, the beta-wrapin HI18 . The beta-strands of the IAPP beta-hairpin correspond to two amyloidogenic sequence motifs which are connected by a turn established around Ser-20. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations, indicating that the IAPP beta-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation. In a similar vein, the related beta-wrapin AS10 inhibits amyloid formation, exhibiting sub-micromolar affinity for each of the three amyloidogenic IDPs IAPP , alpha-synuclein and Abeta, demonstrating that multi-specific monomer-binding agents can be generated. In the case of alpha-synuclein, we have identified a beta-hairpin formed in the sequence region 35-59 which contains the beta-strand segments b1 and b2 of amyloid fibril models and most disease-related mutations. We show by disulfide engineering, biophysical techniques, and cell viability assays, that intramolecular tertiary interactions between the b1 and b2 segments of alpha-synuclein interfere with its aggregation, and moreover inhibit aggregation of IAPP and amyloid-beta peptide. Our results reveal a common preference of different amyloidogenic proteins for formation of beta-hairpin motifs and demonstrate a critical role of hairpin conformers in the control of amyloid formation.