β-Sheet Core of Tau Paired Helical Filaments Revealed by Solid-State NMR
β-Sheet Core of Tau Paired Helical Filaments Revealed by Solid-State NMR
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DOI：
10.1021/ja305470p
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发表时间：
2012-08-29
影响因子：
15
通讯作者：
Lange, Adam
中科院分区：
文献类型：
--
作者：
Daebel, Venita;Chinnathambi, Subashchandrabose;Lange, Adam
阿尔茨海默病的标志之一是微管相关蛋白tau自组装成称为“成对螺旋丝”（PHF）的纤维。然而，PHF组装在原子细节上的结构基础在很大程度上尚不清楚。在这里，我们应用固态核磁共振（ssNMR）光谱研究在体外组装的PHFs从一个截断的三重复tau亚型（K19），代表核心的PHFs。我们发现原纤维的刚性核心由氨基酸V306至S324形成，仅为99个残基中的18个，并且包括由两个短扭结连接的三条β链。第一条β链由充分研究的六肽基序VQIVYK形成，已知该基序以空间拉链排列自聚集。混合的[N-15：C-13]标记的K19原纤维的结果显示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桥导致β-折叠结构的干扰，并且观察到ssNMR谱的多态性。特别地，残基K321-S324表现出两组共振。对K19 C322 A PHFs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tau（K18）的截短四重复异构体的H/D交换NMR测量的支持。定点诱变研究表明，三种不同β链内的单点突变导致PHF聚集效率的显著损失，突出了富含β结构的区域对于tau聚集的重要性。
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 beta-strands connected by two short kinks. The first beta-strand is formed by the well-studied hexapeptide motif VQIVYK that is known to self-aggregate in a steric zipper arrangement. Results on mixed [N-15:C-13]-labeled K19 fibrils show that beta-strands are stacked in a parallel, in-register manner. Disulfide bridges formed between C322 residues of different molecules lead to a disturbance of the beta-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 beta-strands result in a significant loss of PHF aggregation efficiency, highlighting the importance of the beta-structure-rich regions for tau aggregation.