The phagocyte NADPH oxidase (NOX2) is an enzyme, crucial for innate immune defense, producing reactive oxygen species necessary for pathogen destruction. Its activation requires the assembly of soluble proteins (p47phox, p40phox, p67phox, and Rac) with the membrane-bound flavocytochrome b558 (cytb558). We combined circular-dichroism analyses, with decades of experimental data, to filter structural models of the NADPH oxidase complex generated by the artificial intelligence program AlphaFold2 (AF2). The predicted patterns tend to closely resemble the active states of the proteins, as shown by the compact structure of the cytb558, whose dehydrogenase domain is stabilized closer to the membrane. The modeling of the interaction of p47phox with cytb558, which is the initial assembly and activation steps of the NADPH oxidase, enables us to describe how the C-terminus of p47phox interacts with the cytb558. Combining the AF2 cytb558 -p47phox model and its classical molecular dynamics simulations, we highlighted new hydrophobic lipid insertions of p47phox, particularly at residues Trp80-Phe81 of its PX domain. The AF2 models also revealed the implications of intrinsically disordered regions, such as the fragment between the PX domain and the SH3 regions of p47phox, in ensuring distant protein-protein and membrane-protein interactions. Finally, the AF2 prediction of the cytb558-Trimera model highlighted the importance of leaving Rac1 as a separate protein to reach an active state of the NADPH oxidase complex. Altogether, our step-by-step approach provides a structural model of the active complex showing how disordered regions and specific lipid and protein interactions can enable and stabilize the multi-subunit assembly.

中文翻译：

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通过结合计算生物学和实验知识揭示全吞噬细胞 NADPH 氧化酶的结构图谱。


吞噬细胞 NADPH 氧化酶 （NOX2） 是一种酶，对先天免疫防御至关重要，可产生破坏病原体所需的活性氧。其激活需要可溶性蛋白（p47phox、p40phox、p67phox 和 Rac）与膜结合的黄素细胞色素 b558 （cytb558） 组装。我们将圆二色性分析与数十年的实验数据相结合，以过滤由人工智能程序 AlphaFold2 （AF2） 生成的 NADPH 氧化酶复合物的结构模型。预测的模式往往与蛋白质的活性状态非常相似，如 cytb558 的紧凑结构所示，其脱氢酶结构域在更靠近膜的位置稳定。p47phox 与 cytb558 相互作用的建模是 NADPH 氧化酶的初始组装和激活步骤，使我们能够描述 p47phox 的 C 端如何与 cytb558 相互作用。结合 AF2 cytb558 -p47phox 模型及其经典分子动力学模拟，我们强调了 p47phox 的新疏水脂质插入，特别是在其 PX 结构域的残基 Trp80-Phe81 处。AF2 模型还揭示了固有无序区域（例如 p47phox 的 PX 结构域和 SH3 区域之间的片段）在确保远距离蛋白质-蛋白质和膜-蛋白质相互作用方面的作用。最后，cytb558-Trimera 模型的 AF2 预测强调了将 Rac1 作为单独的蛋白质以达到 NADPH 氧化酶复合物活性状态的重要性。总而言之，我们的分步方法提供了活性复合物的结构模型，显示了无序区域以及特定的脂质和蛋白质相互作用如何实现和稳定多亚基组装。