Mutant protein A30P α-synuclein adopts wild-type fibril structure, despite slower fibrillation kinetics

Abstract

α-Synuclein (AS) is associated with both sporadic and familial forms of Parkinson disease (PD). In sporadic disease, wild-type AS fibrillates and accumulates as Lewy bodies within dopaminergic neurons of the substantia nigra. The accumulation of misfolded AS is associated with the death of these neurons, which underlies many of the clinical features of PD. In addition, a rare missense mutation in AS, A30P, is associated with highly penetrant, autosomal dominant PD, although the pathogenic mechanism is unclear. A30P AS fibrillates more slowly than the wild-type (WT) protein in vitro and has been reported to preferentially adopt a soluble, protofibrillar conformation. This has led to speculation that A30P forms aggregates that are distinct in structure compared with wild-type AS. Here, we perform a detailed comparison of the chemical shifts and secondary structures of these fibrillar species, based upon our recent characterization of full-length WT fibrils. We have assigned A30P AS fibril chemical shifts de novo and used them to determine its secondary structure empirically. Our results illustrate that although A30P forms fibrils more slowly than WT in vitro, the chemical shifts and secondary structure of the resultant fibrils are in high agreement, demonstrating a conserved β-sheet core.

FIGURE 1.

In vitro fibrillation conditions provide microscopically well ordered A30P AS fibrils that form slower than WT. a, average fibril formation assay of (red triangles) and A30P AS fibrils (blue circles) monitored by Thioflavin T fluorescence. Error bars were determined from seven replicates for each. b, comparison of the electron micrographs of WT (left) and A30P AS (right) fibrils. ¹³C′ (c), ¹³CA (d), and ¹³CB (e) chemical shift plots between two individual batches of A30P AS fibrils. f, ¹³C-¹³C two-dimensional with 50-ms DARR mixing of A30P AS fibrils. Overlaid expansions of WT (red) on to A30P (blue) AS fibrils for the Thr/Ser (CB-C′) (g) and Thr/Ser (CB-CA) (h) regions.

FIGURE 2.

Multidimensional spectra with high sensitivity and resolution allowed for the chemical shift assignments of A30P AS fibrils. Left, backbone walk schematic. Right, illustration of backbone connectivity through the NCACX (red), NCOCX (blue), and CAN(CO)CX (purple) spectra of residues Val⁷¹-Asn⁶⁵ for A30P AS fibrils. All spectra were acquired with 50-ms DARR mixing and processed with 0.5 ppm of line broadening in each dimension.

FIGURE 3.

Comparison of the chemical shift assignments of A30P and WT AS fibrils (22) demonstrates that the fibril is mostly unchanged upon A30P mutation. ¹⁵N (a), ¹³CA (b), ¹³C′ (c) and ¹³CB (d) chemical shift plots of WT versus A30P AS fibrils are shown. Residues that differ by more than 0.5 ppm are labeled (open circles).

FIGURE 4.

Comparison of the secondary structures between WT (22) and A30P AS fibrils demonstrates that the fibril core is mostly unchanged upon A30P mutation. a, representation of the secondary structure of WT and A30P AS fibrils based on TALOS+ analysis (β-strands, arrows; turn or loop, curved lines; not predicted, dashed line). TALOS+ predicted backbone dihedral angles φ (black squares) and ψ (gray circles), with error bars based on the 10 best TALOS+ data base matches (b) and the normalized peak heights from CANCO as a function of residue number for A30P (c).