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

The intrinsically disordered human protein alpha-synuclein (alpha SN) can self-associate into oligomers and amyloid fibrils. Several lines of evidence suggest that oligomeric alpha SN is cyto-toxic, 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 alpha SN self-assembly, as it reduces the flexibility of the C-terminal region of alpha SN 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 alpha SN. We find that EGCG can bind to all parts of monomeric alpha SN but exhibits highest affinity for the N-terminal region. Monomeric alpha SN binds similar to 54 molecules of EGCG in total during oligomerization. Furthermore, kinetic data suggest that EGCG dimerization is coupled with the alpha SN association reaction. In contrast, preformed oligomers only bind similar to 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 alpha SN reduce oligomer toxicity by 50%. Our study thus demonstrates that alpha SN 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.

Automated summary

The study demonstrates that (-)-epigallocatechin gallate (EGCG) can reduce the toxicity of α-synuclein (α SN) oligomers by binding to both monomeric and oligomeric forms of α SN. EGCG binds to different regions of α SN with highest affinity for the monomeric form. Furthermore, kinetic data suggest a coupling between EGCG dimerization and α SN association.