Department of Biochemistry and Biomedical Sciences and ‡Department of Chemistry and Chemical Biology, McMaster University , 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
J Am Chem Soc. 2017 Oct 4;139(39):13720-13734. doi: 10.1021/jacs.7b05012. Epub 2017 Aug 25.
(-)-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 mechanism of EGCG-induced remodeling is not fully understood. Here we combine N and 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 ∼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.
（-）-表没食子儿茶素没食子酸酯（EGCG）通过重塑具有成核能力的 Aβ 寡聚物为无成核能力的 Aβ 组装体，有效降低了阿尔茨海默病β-淀粉样肽（Aβ）的细胞毒性。然而，EGCG 诱导重塑的机制尚不完全清楚。在这里，我们结合 N 和 H 暗态交换饱和转移（DEST）、弛豫和化学位移投影 NMR 分析以及荧光、动态光散射和电子显微镜来阐明 EGCG 如何重塑 Aβ 寡聚物。我们表明，这种重塑符合 Hill-Scatchard 模型，其中 Aβ（1-40）自组装协同发生，并产生具有多个独立的 EGCG 结合位点的 Aβ（1-40）寡聚物，其 K 值比 Aβ（1-40）单体低约 10 倍。与 EGCG 结合后，Aβ（1-40）寡聚物变得不易溶剂暴露，并且β-区域，其在不存在 EGCG 的情况下涉及直接单体-原纤维接触，经历直接到连接接触的转变。这种向较少参与的单体-原纤维接触的转变解释了在 EGCG 重塑后观察到的成核能力丧失，并表明 EGCG 干扰了已知产生毒性 Aβ 组装体的次级成核事件。出乎意料的是，N 端残基经历了从连接到直接接触的相反的 EGCG 诱导转变，解释了为什么 EGCG 重塑不会释放 Aβ（1-40）单体。我们还表明，在结合 Aβ（1-40）寡聚物后，EGCG B 和 D 环的相对位置相对于 A 环发生变化。在重塑过程中，Aβ（1-40）寡聚物和 EGCG 中均发生这些独特的结构变化为理解 EGCG 作为神经毒性抑制剂的分子机制提供了基础。此外，这里报道的结果说明了基于 DEST 的 NMR 方法在研究低分子量淀粉样蛋白抑制剂的机制方面的有效性。
