Molecular Mechanism for the (−)-Epigallocatechin Gallate-Induced Toxic to Nontoxic Remodeling of Aβ Oligomers Rashik Ahmed, † Bryan VanSchouwen, ‡ Naeimeh Jafari, ‡ Xiaodan Ni, † Joaquin Ortega, † and Giuseppe Melacini* ,†,‡ † Department of Biochemistry and Biomedical Sciences and ‡ Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada *S Supporting Information ABSTRACT: (−)-Epigallocatechin gallate (EGCG) effectively reduces the cytotoxicity of the Alzheimer’s disease β-amyloid peptide (Aβ) by remodeling seeding-competent Aβ oligomers into off- pathway seeding-incompetent Aβ assemblies. However, the mech- anism of EGCG-induced remodeling is not fully understood. Here we combine 15 N and 1 H dark-state exchange saturation transfer (DEST), relaxation, and chemical shift projection NMR analyses with fluorescence, dynamic light scattering, and electron microscopy to elucidate how EGCG remodels Aβ oligomers. We show that the remodeling adheres to a Hill−Scatchard model whereby the Aβ(1− 40) self-association occurs cooperatively and generates Aβ(1−40) oligomers with multiple independent binding sites for EGCG with a K d ∼10-fold lower than that for the Aβ(1−40) monomers. Upon binding to EGCG, the Aβ(1−40) oligomers become less solvent exposed, and the β-regions, which are involved in direct monomer−protofibril contacts in the absence of EGCG, undergo a direct-to-tethered contact shift. This switch toward less engaged monomer−protofibril contacts explains the seeding incompetency observed upon EGCG remodeling and suggests that EGCG interferes with secondary nucleation events known to generate toxic Aβ assemblies. Unexpectedly, the N-terminal residues experience an opposite EGCG-induced shift from tethered to direct contacts, explaining why EGCG remodeling occurs without release of Aβ(1−40) monomers. We also show that upon binding Aβ(1−40) oligomers the relative positions of the EGCG B and D rings change with respect to that of ring A. These distinct structural changes occurring in both Aβ(1−40) oligomers and EGCG during remodeling offer a foundation for understanding the molecular mechanism of EGCG as a neurotoxicity inhibitor. Furthermore, the results reported here illustrate the effectiveness of DEST-based NMR approaches in investigating the mechanism of low-molecular-weight amyloid inhibitors. ■ INTRODUCTION Protein misfolding and the consequent self-association into toxic oligomers and amyloid deposits are central elements of the etiology of a wide range of disorders, from type II diabetes to neurodegenerative conditions such as Alzheimer’s disease. 1−7 Understanding how small molecules modulate the self- association pathways of amyloidogenic polypeptides is there- fore of pathological and pharmacological relevance. 8−21 Among the small molecules with the most promising therapeutic potential is the polyphenol (−)-epigallocatechin gallate (EGCG) extracted from green tea. 8,9,12,16,17 EGCG inhibits the formation of toxic, seeding-competent, on-pathway intermediates arising from the self-association of amyloidogenic peptides and stabilizes nontoxic, seeding-incompetent, off- pathway oligomers. 9−11,13−15,18 This reduction in cellular toxicity is observed also in the presence of preformed, mature amyloid fibrils and oligomers, which are bound by EGCG and are effectively remodeled into smaller and less toxic off-pathway assemblies. 8 The ability of EGCG to bind and redirect amyloidogenic peptides in either the early or late stages of self-association into non-neurotoxic assemblies (Figure 1) has prompted intense scrutiny of the complexes formed by EGCG and amyloidogenic peptides. 8,9,12 Solid-state NMR (ssNMR) has provided an important initial picture of how EGCG remodels the oligomers formed by the prototypical amyloidogenic peptide Aβ(1−40) linked to Alzheimer’s disease, 12 denoted here as Aβ40. On the basis of the ssNMR data, it is clear that the EGCG-induced Aβ40 oligomers preserve a β-sheet structure for residues 29−36 and an intact salt bridge between D23 and K28. The latter stabilizes a hairpin spanning residues ∼23−28, 12 similar to what has been observed for mature cross-β fibrils and toxic Aβ oligomers in the absence of EGCG. 22,23 These observations indicate that the effect of EGCG on the structure of the C- terminal Aβ40 residues alone is insufficient to fully explain how EGCG remodels Aβ40 into nontoxic assemblies; i.e., the toxicity reduction elicited by EGCG arises from perturbations of other aspects of Aβ40 oligomers. For example, EGCG may perturb the dynamic contacts of Aβ40 monomers with the surface of Aβ40 protofibrils and/or affect regions that are invisible to ssNMR. These include the N-terminal Aβ40 region Received: May 19, 2017 Article pubs.acs.org/JACS © XXXX American Chemical Society A DOI: 10.1021/jacs.7b05012 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX