Aimeur Sana, Fas Burcu Aykac, Serfaty Xavier, Santuz Hubert, Sacquin-Mora Sophie, Bizouarn Tania, Taly Antoine, Baciou Laura
Institut de Chimie Physique, UMR 8000, CNRS, Université Paris Saclay, Orsay, France.
Laboratoire de Biochimie Théorique, CNRS, UPR 9080, Université Paris-Cité, Paris, France.
J Biol Chem. 2024 Dec;300(12):107943. doi: 10.1016/j.jbc.2024.107943. Epub 2024 Oct 29.
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 (p47, p40, p67, and Rac) with the membrane-bound flavocytochrome b (cytb). 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 cytb, whose dehydrogenase domain is stabilized closer to the membrane. The modeling of the interaction of p47 with cytb, which is the initial assembly and activation steps of the NADPH oxidase, enables us to describe how the C terminus of p47 interacts with the cytb. Combining the AF2 cytb-p47 model and its classical molecular dynamics simulations, we highlighted new hydrophobic lipid insertions of p47, 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 p47, in ensuring distant protein-protein and membrane-protein interactions. Finally, the AF2 prediction of the cytb-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 multisubunit assembly.
吞噬细胞NADPH氧化酶（NOX2）是一种对先天性免疫防御至关重要的酶，可产生活化病原体所需的活性氧。其激活需要可溶性蛋白（p47、p40、p67和Rac）与膜结合的黄素细胞色素b（cytb）组装在一起。我们将圆二色性分析与数十年的实验数据相结合，以筛选由人工智能程序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，cytb的紧密结构就表明了这一点，其脱氢酶结构域更靠近膜稳定。对p47与cytb相互作用的建模，即NADPH氧化酶的初始组装和激活步骤，使我们能够描述p47的C末端如何与cytb相互作用。结合AF2 cytb - p47模型及其经典分子动力学模拟，我们突出了p47新的疏水脂质插入，特别是在其PX结构域的Trp80 - Phe81残基处。AF2模型还揭示了内在无序区域的影响，例如p47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 - 蛋白质相互作用中的作用。最后，AF2对cytb - Trimera模型的预测突出了将Rac1作为单独蛋白质保留以达到NADPH氧化酶复合物活性状态的重要性。总之，我们的逐步方法提供了活性复合物的结构模型，展示了无序区域以及特定的脂质和蛋白质相互作用如何促成并稳定多亚基组装。
