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 (3-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 alpha-synuclein (alpha S), a beta-hairpin comprised of residues 36-56 was detected in complex with an engineered binding protein, with a turn formed by the aS sequence segment 44-TKEG-47. Molecular dynamics simulations revealed extensive populations of transient beta-hairpin conformations in this region in free, monomeric alpha S. Here, we investigated potential effects of turn formation on alpha S fibril formation by studying the aggregation kinetics of an extensive set of alpha S variants with between two and four amino acid exchanges in the 44-TKEG-47 segment. The exchanges were chosen to specifically promote formation of beta 1-, beta 1'-, or beta 2'-turns. All variants assembled into amyloid fibrils, with increased beta 1'- or beta 2'-turn propensity associated with faster aggregation and increased beta 1-turn propensity with slower aggregation compared to wild-type (WT) aS. Atomic force microscopy demonstrated that beta-turn exchanges altered fibril morphology. In crosselongation 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 aS 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 aS into alternative fibril morphologies, whose elongation requires high sequence fidelity.

Automated summary

The study shows that the formation of turns in the αS protein significantly affects its fibril formation kinetics. Variants with faster beta 1'- or beta 2'-turn formation tend to aggregate more rapidly, while variants with beta 1-turn formation show slower aggregation compared to wild-type. Furthermore, turn exchanges alter fibril morphology and affect the efficiency of fibril elongation.