Effects of charge residues on α-synuclein fibril formation.

Abstract

α-Synuclein (α-syn) amyloid fibrils are associated with Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. α-Syn fibrils derived from ex vivo patient materials as well as those formed in vitro adopt an array of structures or polymorphs. It is important to delineate the structure-function relationship of fibril polymorphs and the mechanisms that give rise to these molecular differences. In particular, salt bridges between Lys and Glu residues are widely observed in all α-syn fibril structures to date. On the one hand, this is not surprising given the preponderance of Lys (14) and Glu (17) residues in its 140 amino acid long sequence; but, because of their large numbers, it is cumbersome and challenging to identify key residues that modulate fibril formation. Here, we performed studies using a smaller PD-related N-terminally truncated α-syn variant, composed of residues 66‒140 (ΔN1-65), that forms amyloid fibrils. Within the proteolytic-resistant core, the number of charged residues reduces to 4 Lys, 4 Glu, and 1 Asp, which is tractable for alanine substitutions to determine their effects. Using atomic force microscopy (AFM) and transmission electron microscopy, we show that fibril structure is strongly impacted by charge neutralization. For example, while ΔN1-65 formed predominantly paired twisted protofilaments with uniform helical periodicity, E83A formed only paired straight fibrils, whereas K80A displayed both twisted and straight fibrils. Interestingly, different heights were seen by AFM for K80A. Of note, structural complexity within a single fibril was revealed in K96A/K97A, where varying helical periodicities were measured along the fibril axis. In conclusion, these results on ΔN1-65 offer residue-specific insights and bolster the importance of electrostatic interactions in fibril formation, which is also involved in the full-length protein.