Protein Crystallography Group, Leibniz Institute for Age Research, Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, 07745 Jena, Germany.
Protein Crystallography Group, Leibniz Institute for Age Research, Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, 07745 Jena, Germany.
J Mol Biol. 2015 Jan 30;427(2):433-42. doi: 10.1016/j.jmb.2014.12.005. Epub 2014 Dec 17.
The amyloid precursor protein (APP) and its proteolytic cleavage product Aβ are widely believed to be central to the etiology of Alzheimer's disease (AD). APP and its family members are also essential for proper neuronal development and homeostasis. APP is located at the cell surface and within intracellular compartments, cellular regions that exhibit different pH values. The AD-associated amyloidogenic processing of APP is initiated predominantly in intracellular acidic compartments, whereas its non-amyloidogenic cleavage is initiated at the cell surface at slightly basic pH. We analyzed the influence of pH on the APP-E1 domain and found that its two constituting subdomains, GFLD and CuBD, interact with each other in a pH-dependent manner. Dynamic light scattering showed that APP-E1 represents a more open conformation at neutral pH and a more closed conformation at acidic pH. Analyzing a 1.4 Å, high-resolution X-ray structure of E1 derived from merohedrally twinned crystals resulted in the identification of individual residues that are responsible for these pH-dependent interactions. Mutational studies and dynamic light scattering measurements further proved that specific hydrogen bonds between the two carboxylates of D177 and E87, as well as between N89 and H147, are major determinants of this pH-driven conformational switch in APP-E1. These findings show how APP can adopt different conformations depending on pH and suggest that the protein fulfils different functions at distinct localizations within the cell. Additionally, our data suggest a novel strategy for treating AD based on regulating the amyloidogenic processing of APP by the specific interruption of the interaction between the APP-E1 subdomains.
淀粉样前体蛋白（APP）及其蛋白水解产物 Aβ 被广泛认为是阿尔茨海默病（AD）发病机制的核心。APP 及其家族成员对于正常神经元的发育和稳态也是必不可少的。APP 位于细胞表面和细胞内隔室，这些细胞区域具有不同的 pH 值。AD 相关的淀粉样前体蛋白的淀粉样生成处理主要在细胞内酸性隔室中起始，而其非淀粉样生成切割则在细胞表面在稍碱性 pH 下起始。我们分析了 pH 值对 APP-E1 结构域的影响，发现其两个组成亚结构域，GFLD 和 CuBD，以 pH 依赖性方式相互作用。动态光散射表明，APP-E1 在中性 pH 下呈现更开放的构象，在酸性 pH 下呈现更封闭的构象。分析来自于具有merohedrally 孪晶的晶体的 E1 的 1.4 Å，高分辨率 X 射线结构导致确定了负责这些 pH 依赖性相互作用的单个残基。突变研究和动态光散射测量进一步证明了 D177 和 E87 的两个羧酸盐之间以及 N89 和 H147 之间的特定氢键是 APP-E1 中这种 pH 驱动构象转换的主要决定因素。这些发现表明 APP 如何根据 pH 值采用不同的构象，并表明该蛋白在细胞内的不同定位处发挥不同的功能。此外，我们的数据表明了一种基于通过特定中断 APP-E1 亚结构域之间的相互作用来调节 APP 的淀粉样生成处理的治疗 AD 的新策略。
