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. 2021 Jan-Jun:296:100788.
doi: 10.1016/j.jbc.2021.100788. Epub 2021 May 18.

How epigallocatechin gallate binds and assembles oligomeric forms of human alpha-synuclein

Camilla B Andersen  1 , Yuichi Yoshimura  2 , Janni Nielsen  1 , Daniel E Otzen  3 , Frans A A Mulder  4
Affiliations

How epigallocatechin gallate binds and assembles oligomeric forms of human alpha-synuclein

Camilla B Andersen et al. J Biol Chem. 2021 Jan-Jun.

Abstract

The intrinsically disordered human protein α-synuclein (αSN) can self-associate into oligomers and amyloid fibrils. Several lines of evidence suggest that oligomeric αSN is cytotoxic, making it important to devise strategies to either prevent oligomer formation and/or inhibit the ensuing toxicity. (-)-epigallocatechin gallate (EGCG) has emerged as a molecular modulator of αSN self-assembly, as it reduces the flexibility of the C-terminal region of αSN in the oligomer and inhibits the oligomer's ability to perturb phospholipid membranes and induce cell death. However, a detailed structural and kinetic characterization of this interaction is still lacking. Here, we use liquid-state NMR spectroscopy to investigate how EGCG interacts with monomeric and oligomeric forms of αSN. We find that EGCG can bind to all parts of monomeric αSN but exhibits highest affinity for the N-terminal region. Monomeric αSN binds ∼54 molecules of EGCG in total during oligomerization. Furthermore, kinetic data suggest that EGCG dimerization is coupled with the αSN association reaction. In contrast, preformed oligomers only bind ∼7 EGCG molecules per protomer, in agreement with the more compact nature of the oligomer compared with the natively unfolded monomer. In previously conducted cell assays, as little as 0.36 EGCG per αSN reduce oligomer toxicity by 50%. Our study thus demonstrates that αSN cytotoxicity can be inhibited by small molecules at concentrations at least an order of magnitude below full binding capacity. We speculate this is due to cooperative binding of protein-stabilized EGCG dimers, which in turn implies synergy between protein association and EGCG dimerization.

Keywords: NMR; alpha-synuclein (α-synuclein); kinetics; oligomer; oligomerization; protein aggregation; stoichiometry.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
EGCG induces αSN oligomerization.A and B, 1D 1H-NMR of αSN and EGCG at different stoichiometries. A, the methyl region from αSN (dotted box). Signals because of a glycerol impurity from protein concentration filter (3.6 ppm) and DSS (0.0, 0.625, and 2.9 ppm—but not the one at 1.75 ppm) have been removed to simplify the spectra (the unedited spectra can be seen in Fig. S2). B, the aromatic region (dominated by EGCG—H2”,6”, H2’,6’, H6,8, and H3 are assigned). C, integration of peak from EGCG H2’,6’ (at 6.6 ppm, see B) and integration of αSN methyl peaks (1.05–0.75 ppm). The dashed line and solid line in the figure are intended to guide the eye. αSN, α-synuclein; DSS, 2,2-dimethyl-2-silapentane-5-sulphonic acid; EGCG, (−)-epigallocatechin gallate.
Figure 2
Figure 2
Oligomerization of αSN in the presence of EGCG over time.A, EGCG-induced oligomerization of αSN, followed by integration of αSN peaks (2.5–0.6 ppm) in 1D 1H-NMR spectroscopy. Molar ratio [EGCG]:[αSN] and color code are provided alongside. B, growth rate from fit seen in A as a function of [EGCG]:[αSN] in a log–log plot. C, burst phase amplitude as a function of [EGCG]:[αSN] stoichiometry. Red line is a linear fit to the three data points at highest stoichiometry. The intercept at y = 1.0 occurs at a stoichiometry of ∼54. D, TEM images of αSN with EGCG taken after the NMR experiments. Red arrows are pointing at oligomers, and the black scale bar indicates 200 nm. αSN, α-synuclein; EGCG, (−)-epigallocatechin gallate.
Figure 3
Figure 3
Oligomerization of αSN in the presence of EGCG involves mainly the first 80 residues.A, 15N–1H HSQC of αSN upon incubation with EGCG ([αSN]:[EGCG] = 1:20) over time. B, relative intensity of peaks in 1H–15N HSQC spectra (A) of 0 min, 1 h, 2 h, and 4 days relative to αSN alone. C, 1D spectrum of 3 mM EGCG mixed with 150 μM αSN ([EGCG]:[αSN] = 20:1) immediately after mixing (black) and after 4 days of incubation (red). 28 mM EGCG was stepwise added to give first an [EGCG]:[αSN] of 50:1 (blue) and then 100:1 (magenta). The sharp peak at 7.1 ppm stems from an impurity in the EGCG sample. The assigned 15N–1H HSQC spectrum of αSN with high resolution is provided as Figure S4. αSN, α-synuclein; EGCG, (−)-epigallocatechin gallate; HSQC, heteronuclear single quantum coherence.
Figure 4
Figure 4
Binding of EGCG to preformed oligomer.A, titration of EGCG to αSN oligomer. At 1:10 = [αSN]:[EGCG], all EGCG peaks are broadened beyond observation. The peaks around 3.6 ppm are due to a small glycerol contamination in the oligomer sample. At excess EGCG (1:100 [αSN]:[EGCG]), peaks from EGCG reappear. B, integration of H2”,6” peak at different stoichiometries gives a binding stoichiometry of 7 ± 1 ([EGCG]:[αSN]) from the intercept at the x-axis. C, line width of 2”,6” (filled circles) and 2’,6’ peaks (open circles) of EGCG at the different stoichiometries. D, STD amplification factor. STD intensity as a function of saturation time at 1:100 ([αSN]:[EGCG]) (saturation field applied at −1 ppm) for 2”,6” (crosses), 2’,6’ (circles), 3 (squares), and 4Eq,4Ax (triangles). αSN, α-synuclein; EGCG, (−)-epigallocatechin gallate; STD, saturation transfer difference.
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