Portal de Periódicos da CAPES

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

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100788

1083-351X

Camilla Andersen, Yuichi Yoshimura, Janni Nielsen, Daniel E. Otzen, Frans A. A. Mulder,

Ginkgo biloba and Cashew Applications

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. 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. The intrinsically disordered human protein α-synuclein (αSN) accumulates in the brains of patients with Parkinson's disease as intracellular deposits called Lewy bodies (1Spillantini M.G. Schmidt M.L. Lee V.M.Y. Trojanowski J.Q. Jakes R. Goedert M. α-Synuclein in Lewy bodies.Nature. 1997; 388: 839Crossref PubMed Scopus (5551) Google Scholar, 2Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2189) Google Scholar). Self-assembly of αSN also occurs in vitro, where the protein is found to form oligomeric species and amyloid fibrils (3Uversky V.N. Li J. Fink A.L. Evidence for a partially folded intermediate in alpha-synuclein fibril formation.J. Biol. Chem. 2001; 276: 10737-10744Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar). The soluble oligomeric form that is formed by self-assembly is cytotoxic, in the sense that it binds and permeates cell membranes (4Winner B. Jappelli R. Maji S.K. Desplats P.A. Boyer L. Aigner S. Hetzer C. Loher T. Vilar M. Campioni S. Tzitzilonis C. Soragni A. Jessberger S. Mira H. Consiglio A. et al.In vivo demonstration that alpha-synuclein oligomers are toxic.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 4194-4199Crossref PubMed Scopus (915) Google Scholar, 5Lorenzen N. Lemminger L. Pedersen J.N. Nielsen S.B. Otzen D.E. The N-terminus of alpha-synuclein is essential for both monomeric and oligomeric interactions with membranes.FEBS Lett. 2014; 588: 497-502Crossref PubMed Scopus (84) Google Scholar, 6Danzer K.M. Haasen D. Karow A.R. Moussaud S. Habeck M. Giese A. Kretzschmar H. Hengerer B. Kostka M. Different species of alpha-synuclein oligomers induce calcium influx and seeding.J. Neurosci. 2007; 27: 9220-9232Crossref PubMed Scopus (552) Google Scholar, 7Lashuel H.A. Hartley D. Petre B.M. Walz T. Lansbury P.T. Neurodegenerative disease - amyloid pores from pathogenic mutations.Nature. 2002; 418: 291Crossref PubMed Scopus (1077) Google Scholar, 8Fusco G. Chen S.W. Williamson P.T.F. Cascella R. Perni M. Jarvis J.A. Cecchi C. Vendruscolo M. Chiti F. Cremades N. Ying L.M. Dobson C.M. De Simone A. Structural basis of membrane disruption and cellular toxicity by alpha-synuclein oligomers.Science. 2017; 358: 1440-1443Crossref PubMed Scopus (255) Google Scholar, 9Volles M.J. Lee S.J. Rochet J.C. Shtilerman M.D. Ding T.T. Kessler J.C. Lansbury P.T. Vesicle permeabilization by protofibrillar a-synuclein: Implications for the pathogenesis and treatment of Parkinson's disease.Biochemistry. 2001; 40: 7812-7819Crossref PubMed Scopus (592) Google Scholar). Recently, it was shown that monomeric αSN and iron-induced oligomeric forms of αSN interact in different ways with lipid membranes: whereas monomers cause membrane thinning and oligomers interact with and lead to changes within lipid raft–like domains (10Perissinotto F. Stani C. De Cecco E. Vaccari L. Rondelli V. Posocco P. Parisse P. Scaini D. Legname G. Casalis L. Iron-mediated interaction of alpha synuclein with lipid raft model membranes.Nanoscale. 2020; 12: 7631-7640Crossref PubMed Google Scholar). Different types of oligomers have been identified; some are thought to be obligate intermediates in fibril formation, whereas others are off pathway, forming independently and even inhibiting fibrillation (11Lorenzen N. Nielsen S.B. Buell A.K. Kaspersen J.D. Arosio P. Vad B.S. Paslawski W. Christiansen G. Valnickova-Hansen Z. Andreasen M. Enghild J.J. Pedersen J.S. Dobson C.M. Knowles T.P.J. Otzen D.E. The role of stable alpha-synuclein oligomers in the molecular events underlying amyloid formation.J. Am. Chem. Soc. 2014; 136: 3859-3868Crossref PubMed Scopus (149) Google Scholar, 12Paslawski W. Andreasen M. Nielsen S.B. Lorenzen N. Thomsen K. Kaspersen J.D. Pedersen J.S. Otzen D.E. High stability and cooperative unfolding of cytotoxic α-synuclein oligomers.Biochemistry. 2014; 53: 6252-6263Crossref PubMed Scopus (50) Google Scholar, 13Paslawski W. Mysling S. Thomsen K. Jørgensen T.J.D. Otzen D.E. Co-existence of two different α-synuclein oligomers with different core structures determined by hydrogen/deuterium exchange mass spectrometry.Angew. Chem. Int. Ed. Engl. 2014; 53: 7560-7563Crossref PubMed Scopus (79) Google Scholar). For example, an off-pathway oligomer can readily be formed by shaking αSN solutions at 37 °C. This oligomer consists of 30 protomers that form a compact β-sheet core surrounded by a diffuse corona (14Zijlstra N. Blum C. Segers-Nolten I.M.J. Claessens M.M.A.E. Subramaniam V. Molecular composition of sub-stoichiometrically labeled a-synuclein oligomers determined by single-molecule photobleaching.Angew. Chem. Int. Ed. Engl. 2012; 51: 8821-8824Crossref PubMed Scopus (57) Google Scholar, 15Giehm L. Svergun D.I. Otzen D.E. Vestergaard B. Low resolution structure of a vesicle disrupting α-synuclein oligomer that accumulates during fibrillation.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 3246-3251Crossref PubMed Scopus (180) Google Scholar). Given the demonstrated toxicity of oligomeric αSN, there is great interest in compounds that prevent their formation, or, once formed, reduce their toxicity. One such compound is (−)-epigallocatechin gallate (EGCG). Although EGCG typically shows low affinity for folded proteins (16Ehrnhoefer D.E. Bieschke J. Boeddrich A. Herbst M. Masino L. Lurz R. Engemann S. Pastore A. Wanker E.E. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers.Nat. Struct. Mol. Biol. 2008; 15: 558-566Crossref PubMed Scopus (1031) Google Scholar), it binds numerous flexible proteins with no obvious sequence discrimination (17Fusco G. Sanz-Hernandez M. Ruggeri F.S. Vendruscolo M. Dobson C.M. De Simone A. Molecular determinants of the interaction of EGCG with ordered and disordered proteins.Biopolymers. 2018; 109e23117Crossref PubMed Scopus (18) Google Scholar). Thus, EGCG binds monomeric αSN, affecting the entire amino acid sequence (16Ehrnhoefer D.E. Bieschke J. Boeddrich A. Herbst M. Masino L. Lurz R. Engemann S. Pastore A. Wanker E.E. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers.Nat. Struct. Mol. Biol. 2008; 15: 558-566Crossref PubMed Scopus (1031) Google Scholar, 18Kurnik M. Sahin C. Andersen C.B. Lorenzen N. Giehm L. Mohammad-Beigi H. Jessen C.M. Pedersen J.S. Mente S. Christiansen G. Pedersen S.V. Staal R. Krishnamurthy G. Pitts K. Reinhart P.H. et al.Novel α-synuclein aggregation inhibitors, identified by HTS, mainly target the monomeric state.Cell Chem. Biol. 2018; 25: 1389-1402.e9Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and also inhibits the fibrillation of several other proteins (19Ehrnhoefer D.E. Duennwald M. Markovic P. Wacker J.L. Engemann S. Roark M. Legleiter J. Marsh J.L. Thompson L.M. Lindquist S. Muchowski P.J. Wanker E.E. Green tea (-)-epigallocatechin-gallate modulates early events in huntingtin misfolding and reduces toxicity in Huntington's disease models.Hum. Mol. Genet. 2006; 15: 2743-2751Crossref PubMed Scopus (316) Google Scholar, 20Meng F.L. Abedini A. Plesner A. Verchere C.B. Raleigh D.P. The flavanol (-)-epigallocatechin 3-gallate inhibits amyloid formation by islet amyloid polypeptide, disaggregates amyloid fibrils, and protects cultured cells against IAPP-induced toxicity.Biochemistry. 2010; 49: 8127-8133Crossref PubMed Scopus (206) Google Scholar, 21Hudson S.A. Ecroyd H. Dehle F.C. Musgrave I.F. Carver J.A. (-)-Epigallocatechin-3-gallate (EGCG) maintains kappa-casein in its pre-fibrillar state without redirecting its aggregation pathway.J. Mol. Biol. 2009; 392: 689-700Crossref PubMed Scopus (118) Google Scholar, 22Bieschke J. Russ J. Friedrich R.P. Ehrnhoefer D.E. Wobst H. Neugebauer K. Wanker E.E. EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 7710-7715Crossref PubMed Scopus (689) Google Scholar, 23Lee Y.H. Lin Y. Cox S.J. Kinoshita M. Sahoo B.R. Ivanova M. Ramamoorthy A. Zinc boosts EGCG's hIAPP amyloid Inhibition both in solution and membrane.Biochim. Biophys. Acta Proteins Proteom. 2019; 1867: 529-536Crossref PubMed Scopus (21) Google Scholar, 24Hauber I. Hohenberg H. Holstermann B. Hunstein W. Hauber J. The main green tea polyphenol epigallocatechin-3-gallate counteracts semen-mediated enhancement of HIV infection.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 9033-9038Crossref PubMed Scopus (122) Google Scholar). EGCG reduces the toxicity of αSN aggregates by remodeling amyloids into smaller nontoxic oligomers (22Bieschke J. Russ J. Friedrich R.P. Ehrnhoefer D.E. Wobst H. Neugebauer K. Wanker E.E. EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 7710-7715Crossref PubMed Scopus (689) Google Scholar), redirecting the aggregation of monomeric αSN into nontoxic oligomers (8Fusco G. Chen S.W. Williamson P.T.F. Cascella R. Perni M. Jarvis J.A. Cecchi C. Vendruscolo M. Chiti F. Cremades N. Ying L.M. Dobson C.M. De Simone A. Structural basis of membrane disruption and cellular toxicity by alpha-synuclein oligomers.Science. 2017; 358: 1440-1443Crossref PubMed Scopus (255) Google Scholar, 16Ehrnhoefer D.E. Bieschke J. Boeddrich A. Herbst M. Masino L. Lurz R. Engemann S. Pastore A. Wanker E.E. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers.Nat. Struct. Mol. Biol. 2008; 15: 558-566Crossref PubMed Scopus (1031) Google Scholar, 18Kurnik M. Sahin C. Andersen C.B. Lorenzen N. Giehm L. Mohammad-Beigi H. Jessen C.M. Pedersen J.S. Mente S. Christiansen G. Pedersen S.V. Staal R. Krishnamurthy G. Pitts K. Reinhart P.H. et al.Novel α-synuclein aggregation inhibitors, identified by HTS, mainly target the monomeric state.Cell Chem. Biol. 2018; 25: 1389-1402.e9Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and inhibiting the toxicity of preformed toxic αSN and amyloid-β oligomers in vitro (25Lorenzen N. Nielsen S.B. Yoshimura Y. Vad B.S. Andersen C.B. Betzer C. Kaspersen J.D. Christiansen G. Pedersen J.S. Jensen P.H. Mulder F.A.A. Otzen D.E. How epigallocatechin gallate can inhibit alpha-synuclein oligomer toxicity in vitro.J. Mol. Biol. 2014; 289: 21299-21310Scopus (122) Google Scholar, 26Hyung S.J. DeToma A.S. Brender J.R. Lee S. Vivekanandan S. Kochi A. Choi J.S. Ramamoorthy A. Ruotolo B.T. Lim M.H. Insights into antiamyloidogenic properties of the green tea extract (-)-epigallocatechin-3-gallate toward metal-associated amyloid-β species.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 3743-3748Crossref PubMed Scopus (168) Google Scholar). Both solid-state (8Fusco G. Chen S.W. Williamson P.T.F. Cascella R. Perni M. Jarvis J.A. Cecchi C. Vendruscolo M. Chiti F. Cremades N. Ying L.M. Dobson C.M. De Simone A. Structural basis of membrane disruption and cellular toxicity by alpha-synuclein oligomers.Science. 2017; 358: 1440-1443Crossref PubMed Scopus (255) Google Scholar) and liquid-state (25Lorenzen N. Nielsen S.B. Yoshimura Y. Vad B.S. Andersen C.B. Betzer C. Kaspersen J.D. Christiansen G. Pedersen J.S. Jensen P.H. Mulder F.A.A. Otzen D.E. How epigallocatechin gallate can inhibit alpha-synuclein oligomer toxicity in vitro.J. Mol. Biol. 2014; 289: 21299-21310Scopus (122) Google Scholar) NMR spectroscopy have demonstrated that EGCG-induced oligomers have altered structures compared with "naked" oligomers. Whereas naked oligomers insert into and disrupt the membrane (possibly in association with interaction between protein and lipids and conformational changes to the oligomer (27Sciacca M.F. Lolicato F. Tempra C. Scollo F. Sahoo B.R. Watson M.D. García-Viñuales S. Milardi D. Raudino A. Lee J.C. Ramamoorthy A. La Rosa C. Lipid-chaperone hypothesis: A common molecular mechanism of membrane disruption by intrinsically disordered proteins.ACS Chem. Neurosci. 2020; 11: 4336-4350Crossref PubMed Scopus (24) Google Scholar, 28Korshavn K.J. Satriano C. Lin Y. Zhang R. Dulchavsky M. Bhunia A. Ivanova M.I. Lee Y.H. La Rosa C. Lim M.H. Ramamoorthy A. Reduced lipid bilayer thickness regulates the aggregation and cytotoxicity of amyloid-β.J. Biol. Chem. 2017; 292: 4638-4650Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar)), EGCG-induced structural changes lead to reduced affinity for and an inability to perturb membranes (8Fusco G. Chen S.W. Williamson P.T.F. Cascella R. Perni M. Jarvis J.A. Cecchi C. Vendruscolo M. Chiti F. Cremades N. Ying L.M. Dobson C.M. De Simone A. Structural basis of membrane disruption and cellular toxicity by alpha-synuclein oligomers.Science. 2017; 358: 1440-1443Crossref PubMed Scopus (255) Google Scholar). To complicate matters, αSN can form multiple different species of coexisting oligomers (13Paslawski W. Mysling S. Thomsen K. Jørgensen T.J.D. Otzen D.E. Co-existence of two different α-synuclein oligomers with different core structures determined by hydrogen/deuterium exchange mass spectrometry.Angew. Chem. Int. Ed. Engl. 2014; 53: 7560-7563Crossref PubMed Scopus (79) Google Scholar, 29Kaylor J. Bodner N. Edridge S. Yamin G. Hong D.-P. Fink A.L. Characterization of oligomeric intermediates in α-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F α-synuclein.J. Mol. Biol. 2005; 353: 357-372Crossref PubMed Scopus (137) Google Scholar). This makes it difficult to study the effect of EGCG on the interaction between oligomeric and monomeric αSN. As a result, we still lack a detailed structural and kinetic characterization of the interaction between EGCG and αSN in the process of oligomer formation. Obtained under in vitro conditions to obtain maximal structural insight, such advances provide a foundation to better understand how these oligomers subsequently may interact with membranes and other components in the cell. Here, we use liquid-state NMR spectroscopy (30Ahmed R. Melacini G. A solution NMR toolset to probe the molecular mechanisms of amyloid inhibitors.Chem. Commun. 2018; 54: 4644-4652Crossref PubMed Google Scholar, 31Cawood E.E. Karamanos T.K. Wilson A.J. Radford S.E. Visualizing and trapping transient oligomers in amyloid assembly pathways.Biophys. Chem. 2021; 268: 106505Crossref PubMed Scopus (25) Google Scholar) to show that EGCG binds to monomeric and oligomeric αSN. We find that monomeric and oligomeric αSN bind 54 and 7 EGCG molecules, respectively, and that EGCG slowly (over a 4-day period) binds to and immobilizes all protein residues. We suggest that these binding properties may explain the broad spectrum of activity by EGCG toward cytotoxic protein oligomers, and that related polyphenols act to bind together disordered proteins in a comparable way. Determination of the stoichiometry of EGCG binding to αSN monomer by conventional titration is complicated by the fact that αSN oligomerizes in a time-dependent manner when EGCG is present (8Fusco G. Chen S.W. Williamson P.T.F. Cascella R. Perni M. Jarvis J.A. Cecchi C. Vendruscolo M. Chiti F. Cremades N. Ying L.M. Dobson C.M. De Simone A. Structural basis of membrane disruption and cellular toxicity by alpha-synuclein oligomers.Science. 2017; 358: 1440-1443Crossref PubMed Scopus (255) Google Scholar, 16Ehrnhoefer D.E. Bieschke J. Boeddrich A. Herbst M. Masino L. Lurz R. Engemann S. Pastore A. Wanker E.E. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers.Nat. Struct. Mol. Biol. 2008; 15: 558-566Crossref PubMed Scopus (1031) Google Scholar). Therefore, separate samples of αSN monomer were incubated with different amounts of EGCG, making sure to have identical incubation time (10 min) before recording 1D 1H-NMR spectra. Changes in the NMR spectra of both EGCG and αSN are expected upon interaction (for EGCG NMR assignments, see Fig. S1). These changes should manifest themselves as line broadening or chemical shift perturbation, with the exact outcome depending on binding kinetics (30Ahmed R. Melacini G. A solution NMR toolset to probe the molecular mechanisms of amyloid inhibitors.Chem. Commun. 2018; 54: 4644-4652Crossref PubMed Google Scholar). We hypothesized that our observations could be captured by a minimal model that considers (1) free and bound states for the protein as well as for EGCG and (2) that broadening of the NMR signals is caused by the modulation of the chemical (and magnetic) environment of the nuclei in the limiting states (free and bound), although more complex models can be envisaged (e.g., intermediate exchange line broadening intrinsic to the bound form of EGCG and influence of EGCG on the self-association state of αSN). In addition, once oligomers of high molecular weight have formed, even a small population of the bound form in a weak-affinity interaction can bring about severe line broadening (32Fawzi N.L. Ying J. Ghirlando R. Torchia D.A. Clore G.M. Atomic-resolution dynamics on the surface of amyloid-β protofibrils probed by solution NMR.Nature. 2011; 480: 268-272Crossref PubMed Scopus (289) Google Scholar). Figure 1A displays the aliphatic region of 1D 1H-NMR spectra at different EGCG:αSN ratios. The region (2.5–0.75 ppm) shows signals from aliphatic side chains (mostly methyl groups) of αSN (EGCG does not show any resonances here, see Fig. S1) and is used to gauge the fate of the protein in solution. In contrast, the aromatic region (7.5–5.5 ppm) is dominated by EGCG (in red boxes in Fig. 1B), although there are weak signals from αSN's four Tyr and two Phe residues around 7.4 to 6.8 ppm (see lowest trace in Fig. 1B). Strikingly, using 10-min incubation, all protein signals fully disappeared when the [EGCG]:[αSN] ratio was 60:1 (Fig. 1, A–C), whereas only a very modest decrease was observed for αSN signals when titrating in the first 20 equivalents of EGCG. At the same time, EGCG NMR signals initially increased linearly with concentration up to 20 EGCG per αSN, in proportion to the amount of EGCG added, but upon an increase from 20:1 to 60:1, also all the EGCG signals vanished (Fig. 1, A–C). Since we did not see any precipitation, this implies that at ligand-to-protein ratios in the range 20 to 60, all EGCGs become sequestered in large, soluble, or dispersed assemblies that simultaneously cause both αSN and EGCG signals to become NMR invisible. This is the kind of behavior expected for molecules in a slow tumbling complex. Negative-stain EM demonstrated that loss of the NMR spectrum coincided with the formation of oligomeric species of ∼20 nm diameter (16Ehrnhoefer D.E. Bieschke J. Boeddrich A. Herbst M. Masino L. Lurz R. Engemann S. Pastore A. Wanker E.E. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers.Nat. Struct. Mol. Biol. 2008; 15: 558-566Crossref PubMed Scopus (1031) Google Scholar). Upon further addition of EGCG, αSN continues to remain NMR invisible, whereas a re-emergence of visible EGCG NMR signals is seen (Fig. 1C). The EGCG signals scale with the amount added, indicating accumulation of unbound EGCG. Consistent with this explanation, the line width of the EGCG peak for H2',6' is very little affected up to [EGCG]:[αSN] = 20:1. This indicates that, over the course of a 10-min incubation, most EGCG molecules exist in solution, and are only little affected by the presence of αSN, until a critical ratio is reached beyond 20 EGCGs per αSN. Once the NMR-invisible species have been fully formed, the signals of free ligand once more increase in intensity with further EGCG addition, but the peaks are very wide (Fig. 1, B and D). Broad peaks observed for small molecules typically point to the existence of a dynamic equilibrium between a rapidly tumbling free state and a slowly tumbling bound state (30Ahmed R. Melacini G. A solution NMR toolset to probe the molecular mechanisms of amyloid inhibitors.Chem. Commun. 2018; 54: 4644-4652Crossref PubMed Google Scholar). As 1H-NMR spectra of free EGCG consistently show sharp signals over the concentration range (0.15–3 mM) used in the αSN titration study (Fig. S3), we can rule out that broad signals result from self-association of EGCG (33Wróblewski K. Muhandiram R. Chakrabartty A. Bennick A. The molecular interaction of human salivary histatins with polyphenolic compounds.Eur. J. Biochem. 2001; 268: 4384-4397Crossref PubMed Scopus (103) Google Scholar, 34Eaton J.D. Williamson M.P. Multi-site binding of epigallocatechin gallate to human serum albumin measured by NMR and isothermal titration calorimetry.Biosci. Rep. 2017; 37BSR20170209Crossref PubMed Scopus (14) Google Scholar). This is in keeping with a reported EGCG self-association constant of 0.014 mM−1 (i.e., 50% associated at 7 mM) at pH 6.0 (35Charlton A.J. Baxter N.J. Khan M.L. Moir A.J. Haslam E. Davies A.P. Williamson M.P. Polyphenol/peptide binding and precipitation.J. Agric. Food Chem. 2002; 50: 1593-1601Crossref PubMed Scopus (528) Google Scholar).We conclude that a stoichiometrically well-defined EGCG:αSN complex is formed in the presence of a critical number of EGCG molecules. To obtain mechanistic insight into the self-assembly process, we next investigated the time-dependent structural changes that take place during coincubation. To address this, EGCG and αSN were mixed at EGCG:αSN molar ratios ranging from 8.7 to 51.9, and a series of NMR spectra were recorded over time. A marked decline was observed for all 1D 1H-NMR signals (Fig. 2A), whereas no such decline in signals was seen in a control experiment (i.e., in the absence of EGCG; data not shown). Using the integrated intensity of αSN protein NMR signals (2.5–0.6 ppm), the signal loss could be fitted using an exponential decay function (assuming that all signal is eventually lost), giving a rate constant k, which increased in a power-law fashion with EGCG:αSN stoichiometry up to 34.6 (Fig. 2B) (at higher stoichiometries, the signal change was too small to provide a reliable estimate of the rate constant). The order of a chemical reaction with regard to a given reactant can formally be determined by plotting the logarithm of the rate v (estimated from the initial slope in Fig. 2A) versus the logarithm of the concentration of that reactant. Whether using k or v, we obtain a slope of ∼2, which indicates that two EGCG molecules associate per reaction step, suggesting an EGCG dimerization associated with the mechanism of interaction with αSN. We note that at neutral pH, EGCG dimerizes (on the minute–hour scale) because of oxidation (36Sang S. Yang I. Buckley B. Ho C.-T. Yang C.S. Autoxidative quinone formation in vitro and metabolite formation in vivo from tea polyphenol (-)-epigallocatechin-3-gallate: Studied by real-time mass spectrometry combined with tandem mass ion mapping.Free Radic. Biol. Med. 2007; 43: 362-371Crossref PubMed Scopus (118) Google Scholar, 37Sang S. Lee M.J. Hou Z. Ho C.T. Yang C.S. Stability of tea polyphenol (-)-epigallocatechin-3-gallate and formation of dimers and epimers under common experimental conditions.J. Agric. Food Chem. 2005; 53: 9478-9484Crossref PubMed Scopus (255) Google Scholar), and dimeric EGCG has been shown to possess a more potent disaggregating effect than monomeric EGCG (38Nie R.Z. Zhu W. Peng J.M. Ge Z.Z. Li C.M. Comparison of disaggregative effect of A-type EGCG dimer and EGCG monomer on the preformed bovine insulin amyloid fibrils.Biophys. Chem. 2017; 230: 1-9Crossref PubMed Scopus (16) Google Scholar). Dimerization is possible, but not conclusively demonstrated by these data, which provide a lower limit for cooperativity of the EGCG–αSN interaction. Interestingly, this slow signal decay was preceded by a very rapid loss of signal within the dead time of measurement (6–18 min) corresponding to a burst phase. The magnitude of this burst phase increased linearly with EGCG stoichiometry from 25.9 to 51.9 EGCG, and this linear phase extrapolated to the upper limit of 1.0 (i.e., complete disappearance of the αSN methyl signal) around 54 [EGCG]:[αSN] (Fig. 2C). Assuming that rapid binding of EGCG to αSN within the dead time is responsible for this signal disappearance, this suggests that αSN has a capacity to bind up to 54 EGCGs per αSN protomer in the formation of EGCG-induced oligomer. The appearance of EGCG-induced aggregates of αSN was monitored by transmission electron microscopy (TEM) (Fig. 2D), which revealed small round oligomers that resembled those observed in other studies (25Lorenzen N. Nielsen S.B. Yoshimura Y. Vad B.S. Andersen C.B. Betzer C. Kaspersen J.D. Christiansen G. Pedersen J.S. Jensen P.H. Mulder F.A.A. Otzen D.E. How epigallocatechin gallate can inhibit alpha-synuclein oligomer toxicity in vitro.J. Mol. Biol. 2014; 289: 21299-21310Scopus (122) Google Scholar, 39Pieri L. Madiona K. Melki R. Structural and functional properties of prefibrillar α-synuclein oligomers.Sci. Rep. 2016; 6: 24526Crossref PubMed Scopus (81) Google Scholar), either isolated or connected into chains. We have previously observed small amounts of oligomeric concatamers in the absence of EGCG, but not to the same extent as with EGCG, indicating that EGCG promotes formation of these high molecular weight assemblies. To investigate the oligomerization process with higher structural resolution, time-dependent signal loss was monitored by 2D 15N–1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy for a 20:1 ligand-to-protein ratio. We observe severe signal loss for all protein residues already after 20 min (Fig. 3A). Based on the observations made at higher EGCG ratio, this observation is consistent with a scenario in which most αSN is sequestered into NMR-invisible oligomeric species, removing all free EGCGs from solution in a cooperative process. As there is insufficient EGCG to aggregate all protein within the first rapid phase (<10% of the signal disappears in the burst phase at 20:1 EGCG:αSN, cf. Fig. 2C; this fraction is likely higher in the HSQC experiment where we have a fivefold increase in αSN and EGCG concentration), the remaining αSN will be subject to slower self-association processes. Plotting signal intensity versus residue number reveals a second and slower loss of the NMR-visible pool that is not evenly distributed over the sequence (Fig. 3B). Resonances belonging to the N-terminal portion of the protein (residues 1–70) are completely lost after 2 h, whereas signals from the C-terminal domain (residues 95–140) still remain detectable after multiple days. Previous 2D 15N–1H HSQC NMR studies have shown that the C-terminal ∼40 residues remain highly mobile (and thus visible) in αSN oligomers