β-Turn exchanges in the α-synuclein segment 44-TKEG-47 reveal high sequence fidelity requirements of amyloid fibril elongation

The folding of turns and β-hairpins has been implicated in amyloid formation, with diverse potential consequences such as promotion or inhibition of fibril nucleation, fibril elongation, or off-pathway oligomer formation. In the Parkinson's disease-associated protein α-synuclein (αS), a β-hairpin comprised of residues 36-56 was detected in complex with an engineered binding protein, with a turn formed by the αS sequence segment 44-TKEG-47. Molecular dynamics simulations revealed extensive populations of transient β-hairpin conformations in this region in free, monomeric αS. Here, we investigated potential effects of turn formation on αS fibril formation by studying the aggregation kinetics of an extensive set of αS variants with between two and four amino acid exchanges in the 44-TKEG-47 segment. The exchanges were chosen to specifically promote formation of β1-, β1'-, or β2'-turns. All variants assembled into amyloid fibrils, with increased β1'- or β2'-turn propensity associated with faster aggregation and increased β1-turn propensity with slower aggregation compared to wild-type (WT) αS. Atomic force microscopy demonstrated that β-turn exchanges altered fibril morphology. In cross-elongation experiments, the turn variants showed a low ability to elongate WT fibril seeds, and, vice versa, WT monomer did not efficiently elongate turn variant fibril seeds. This demonstrates that sequence identity in the turn region is crucial for efficient αS fibril elongation. Elongation experiments of WT fibril seeds in the presence of both WT and turn variant monomers suggest that the turn variants can bind and block WT fibril ends to different degrees, but cannot efficiently convert into the WT fibril structure. Our results indicate that modifications in the 44-TKEG-47 segment strongly affect amyloid assembly by driving αS into alternative fibril morphologies, whose elongation requires high sequence fidelity.