Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton ON L8S4M1, Canada.
Department of Chemistry and Chemical Biology, McMaster University, Hamilton ON L8S4M1, Canada.
J Am Chem Soc. 2021 Mar 31;143(12):4668-4679. doi: 10.1021/jacs.0c13289. Epub 2021 Mar 18.
Soluble oligomers formed by amyloidogenic intrinsically disordered proteins are some of the most cytotoxic species linked to neurodegeneration. Due to the transient and heterogeneous nature of such oligomeric intermediates, the underlying self-association events often remain elusive. NMR relaxation measurements sensitive to zero-frequency spectral densities (J(0)), such as the N - R rates, are ideally suited to map sites of self-association at atomic resolution without the need of exogenous labels. Such experiments exploit the dynamic exchange between NMR visible monomers and slowly tumbling oligomers. However,N - R rates are also sensitive to intrinsic monomer dynamics, and it is often difficult to discern these contributions from those arising from exchange with oligomers. Another challenge pertains to defining a hierarchy of self-association. Here, using the archetypical amyloidogenic protein alpha synuclein (αS), we show that the temperature-dependence of N - R effectively identifies self-association sites with reduced bias from internal dynamics. The key signature of the residues involved in self-association is a nonlinear temperature-dependence of N - R with a positive ΔR/ΔT slope. These two hallmarks are systematically probed through a thermal R correlation matrix, from which the network of residues involved in self-association as well as the hierarchy of αS self-association sites is extracted through agglomerative clustering. We find that aggregation is initiated by residues within the NAC region that is solvent inaccessible in αS fibrils and eventually extends to the N-terminal segment harboring familial PD mutations. These hierarchical self-association maps help dissect the essential drivers of oligomerization and reveal how amyloid inhibitors affect oligomer formation.
淀粉样蛋白构象紊乱的可溶性低聚物是与神经退行性变相关的最具细胞毒性的物质之一。由于这些低聚物中间体具有瞬态和异质的性质，因此潜在的自缔合事件往往难以捉摸。对零频谱密度（J(0)）敏感的 NMR 弛豫测量（如 N - R 速率）非常适合在无需外源性标记的情况下以原子分辨率绘制自缔合位点。这些实验利用 NMR 可见单体和缓慢翻滚低聚物之间的动态交换。然而，N - R 速率也对固有单体动力学敏感，并且通常很难将这些贡献与来自与低聚物交换的贡献区分开来。另一个挑战涉及定义自缔合的层次结构。在这里，我们使用典型的淀粉样蛋白构象紊乱蛋白α-突触核蛋白（αS）表明，N - R 的温度依赖性可有效地识别自缔合位点，从而减少了来自内部动力学的偏差。参与自缔合的残基的关键特征是 N - R 的非线性温度依赖性，具有正的 ΔR/ΔT 斜率。通过热 R 相关矩阵系统地探测这两个特征，从该矩阵中提取出涉及自缔合的残基网络以及αS 自缔合位点的层次结构，通过凝聚聚类进行提取。我们发现，聚合由 NAC 区域内的残基引发，这些残基在αS 原纤维中溶剂不可及，最终扩展到含有家族 PD 突变的 N 端片段。这些分层自缔合图谱有助于剖析寡聚化的基本驱动因素，并揭示淀粉样抑制剂如何影响寡聚体形成。
