NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
J Am Chem Soc. 2012 Aug 29;134(34):13982-9. doi: 10.1021/ja305470p. Epub 2012 Aug 15.
One of the hallmarks of Alzheimer's disease is the self-assembly of the microtubule-associated protein tau into fibers termed "paired helical filaments" (PHFs). However, the structural basis of PHF assembly at atomic detail is largely unknown. Here, we applied solid-state nuclear magnetic resonance (ssNMR) spectroscopy to investigate in vitro assembled PHFs from a truncated three-repeat tau isoform (K19) that represents the core of PHFs. We found that the rigid core of the fibrils is formed by amino acids V306 to S324, only 18 out of 99 residues, and comprises three β-strands connected by two short kinks. The first β-strand is formed by the well-studied hexapeptide motif VQIVYK that is known to self-aggregate in a steric zipper arrangement. Results on mixed [(15)N:(13)C]-labeled K19 fibrils show that β-strands are stacked in a parallel, in-register manner. Disulfide bridges formed between C322 residues of different molecules lead to a disturbance of the β-sheet structure, and polymorphism in ssNMR spectra is observed. In particular, residues K321-S324 exhibit two sets of resonances. Experiments on K19 C322A PHFs further confirm the influence of disulfide bond formation on the core structure. Our structural data are supported by H/D exchange NMR measurements on K19 as well as a truncated four-repeat isoform of tau (K18). Site-directed mutagenesis studies show that single-point mutations within the three different β-strands result in a significant loss of PHF aggregation efficiency, highlighting the importance of the β-structure-rich regions for tau aggregation.
阿尔茨海默病的特征之一是微管相关蛋白 tau 自身装配成称为“双螺旋丝纤维”（PHF）的纤维。然而，PHF 组装的原子细节的结构基础在很大程度上仍是未知的。在这里，我们应用固态核磁共振（ssNMR）光谱法研究了来自截断三重复 tau 异构体（K19）的体外组装的 PHF，K19 代表 PHF 的核心。我们发现，纤维的刚性核心由残基 V306 到 S324 的 18 个氨基酸组成，99 个残基中仅有 18 个，包含三个通过两个短弯曲连接的 β-链。第一个 β-链由众所周知的六肽基序 VQIVYK 组成，该基序以空间拉链排列的方式自聚集。关于混合 [(15)N:(13)C]-标记 K19 纤维的结果表明，β-链以平行、对齐的方式堆叠。不同分子的 C322 残基之间形成的二硫键导致 β-片层结构的扰乱，并观察到 ssNMR 光谱中的多态性。特别是，残基 K321-S324 表现出两组共振。K19 C322A PHF 的实验进一步证实了二硫键形成对核心结构的影响。我们的结构数据得到了 K19 以及截断四重复 tau 异构体（K18）的 H/D 交换 NMR 测量的支持。定点突变研究表明，三个不同 β-链内的单点突变会导致 PHF 聚集效率显著降低，这突出了富含 β-结构区域对 tau 聚集的重要性。
