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. 2018 Aug 3;430(16):2360-2371.
doi: 10.1016/j.jmb.2018.05.024. Epub 2018 May 18.

Multi-Pronged Interactions Underlie Inhibition of α-Synuclein Aggregation by β-Synuclein

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Free PMC article

Multi-Pronged Interactions Underlie Inhibition of α-Synuclein Aggregation by β-Synuclein

Jonathan K Williams et al. J Mol Biol. .
Free PMC article

Abstract

The intrinsically disordered protein β-synuclein is known to inhibit the aggregation of its intrinsically disordered homolog, α-synuclein, which is implicated in Parkinson's disease. While β-synuclein itself does not form fibrils at the cytoplasmic pH 7.4, alteration of pH and other environmental perturbations are known to induce its fibrilization. However, the sequence and structural determinants of β-synuclein inhibition and self-aggregation are not well understood. We have utilized a series of domain-swapped chimeras of α-synuclein and β-synuclein to probe the relative contributions of the N-terminal, C-terminal, and the central non-amyloid-β component domains to the inhibition of α-synuclein aggregation. Changes in the rates of α-synuclein fibril formation in the presence of the chimeras indicate that the non-amyloid-β component domain is the primary determinant of self-association leading to fibril formation, while the N- and C-terminal domains play critical roles in the fibril inhibition process. Our data provide evidence that all three domains of β-synuclein together contribute to providing effective inhibition, and support a model of transient, multi-pronged interactions between IDP chains in both processes. Inclusion of such multi-site inhibitory interactions spread over the length of synuclein chains may be critical for the development of therapeutics that are designed to mimic the inhibitory effects of β-synuclein.

Keywords: Parkinson's disease; chimeras; fluorescence; intrinsically disordered proteins; protein–protein interactions.

Conflict of interest statement

Declarations of Interest: none

Figures

Figure 1.
Figure 1.
(a) Comparison of the primary sequences of αS and βS, broken down by the N-terminal, non-amyloid β component (NAC) and C-terminal domains. The sequences were aligned using EMBOSS Stretcher [78]. Residues that are different between the two aligned sequences are indicated in bold font. (b) 1H-15N HSQC spectra of the synuclein chimeras at pH 6 (20 mM MES, 100 mM NaCl), at 288 K. The small chemical shift dispersion and narrow linewidths indicate that all of the chimeras are intrinsically disordered.
Figure 2.
Figure 2.
Normalized ThT fluorescence of self-incubated (black) or co-incubated (red) fibril formation of (a) XBX chimeras and (b) XAX chimeras at pH 7.3. Each fluorescence trace is an average of at least three measurements, and the standard deviation is reported. For the sake of clarity, traces that showed no increase in ThT fluorescence from the baseline were normalized and shifted to appear near zero by subtracting 1 from the normalized intensities (5ABA, 5ABB, 5BBB). The stoichiometry for each assay is indicated in parentheses for co-incubated data sets, with a 1 indicating a protein concentration of 1 mg/mL, and a 5 indicating a 5 mg/mL concentration. In the case of self-incubated data, the stoichiometry is indicated before the chimera name (e.g. 5XXX, 5 mg/mL protein concentration).
Figure 3.
Figure 3.
Comparison of the time it takes the fluorescence intensity to reach 10% of its normalized maximum (ie. lag time, tlag) for the chimeras at (a) pH 7.3 and (b) pH 5.8. The lag times of the control AAA are subtracted from the co-incubated (middle, red) lag times to give the lag time differences (Δtlag) shown in blue at the bottom. In each panel, the lag time is presented as a bar with text label, and the error bars shown represent the range of times at which the standard deviation reaches 10% normalized fluorescence intensity.
Figure 4.
Figure 4.
Normalized ThT fluorescence of βS with the E61A mutation (green), showing (a) self-incubated or (b) co-incubated fibril formation at pH 7.3. AAA (black), BBB (red), and BAB (blue) are shown for comparison. The stoichiometry for each assay is indicated in parentheses for co-incubated data sets, with a 1 indicating a protein concentration of 1 mg/mL, and a 5 indicating a 5 mg/mL concentration. In the case of self-incubated data, the stoichiometry is indicated before the chimera name (e.g. 5XXX, 5 mg/mL protein concentration).
Figure 5.
Figure 5.
Schematic representation of synuclein aggregation pathways (a), in the cases of self-incubation (top) and co-incubation (bottom). Synuclein exists as an intrinsically disordered monomer in solution. In the case of self-incubation, monomers can interact in a predominately head-to-head configuration, which can lead to fibril formation. The propensity of the different chimeras to form fibrils falls along a continuum which can be generally separated by the NAC domain: XAX chimeras tend to quickly form fibrils at neutral pH, while XBX chimeras do not form fibrils (or are very resistant). When co-incubated together at neutral pH, the αS and chimera monomers can transiently interact in either a head-to-head orientation, which leads to on-pathway fibril formation, or in an off-pathway, head-to-tail orientation, which acts to kinetically trap the αS monomers and increase the time it takes to form fibrils. The propensity to inhibit, or slow down (i.e. increase Δtlag), fibril formation again falls along a continuum: inhibition is very high for BXB chimeras (large Δtlag), and lower for all others (small Δtlag). The relative proportions of αS (blue) and chimera (red) found in mature, co-incubated fibrils at pH 7.3 are shown as pie charts, and are summarized in Table 1. The BB*B chimera in this figure refers to the βS E61A chimera, indicating the fact that this mutant has exactly the same domains as the BBB chimera except for the single mutation in the NAC region. (b) Schematic of single-domain swaps and their effects on inhibition propensity, relative to A-NAC (left) or B-NAC (right) domains.

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