Structural basis for detoxification of environmental pollutants by native complexes of CYP2B family with its redox partners

摘要 多氯联苯和有机磷农药是普遍存在的环境污染物 污染物这些污染物在人体组织中的生物累积与癌症有关， 神经毒性它们优选通过CYP 2B亚家族酶代谢。必不可少的 这些污染物的解毒是CYP 2B酶与其氧化还原的相互作用 细胞色素P450氧化还原酶（POR）和细胞色素b5（cyt b5）。但这些 内质网（ER）膜的背景下的相互作用仍然知之甚少。 特别是，细胞色素b5在微粒体P450系统中的作用是神秘的。它已经被讨论过了 几十年来，细胞色素b5是否作为电子供体或变构效应物影响P450活性， 两者在细胞色素b5在P450活性中的重要作用和 我们对其机制的理解。缺乏理解的部分原因是缺乏3D 天然P450：b5复合物的结构。通过以下方式获得这样的结构是非常具有挑战性的： 常规方法如X射线晶体学或NMR光谱学。单粒子冷冻电镜 已成为多单元膜结构测定的首选方法 衍射晶体不可用的蛋白质复合物。我们假设 天然CYP 2B 4-cyt b5复合物的3D结构的测定可以使用 单颗粒冷冻EM结合新的样品制备。我们建议测试 假设和开发急需的冷冻EM方法在两个特定的目标。在目标1中，我们 在新型纳米颗粒中制备CYP 2B 4-cyt b5复合物，然后确定其3D结构 通过Aim 2中的单粒子冷冻EM。这两个目标的成功实现， 关键的知识，了解细胞色素b5在CYP 2B 4活性的作用，而且还制定了一个 一种急需的用于分析cyt b5和POR天然复合物结构的方法 与许多其他P450酶。建立这种方法和提供这些方法 本地复合体将是开创性的，并提供了一个路线图，以询问 微粒体P450系统解毒环境污染物的机制。

ABSTRACT Polychlorinated biphenyls and organophosphorus pesticides are ubiquitous environmental pollutants. Bioaccumulation of these pollutants in human tissues is associated with cancer and neurotoxicity. They are preferably metabolized by CYP2B subfamily enzymes. Essential to the detoxification of these pollutants are the interactions of CYP2B enzymes with their redox partners, cytochrome P450 oxidoreductase (POR) and cytochrome b5 (cyt b5). However, these interactions in the context of endoplasmic reticulum (ER) membrane remain poorly understood. In particular, the role of cyt b5 in the microsomal P450 system is enigmatic. It has been debated for decades whether cyt b5 affects P450 activity as an electron donor or allosteric effector or both. There is a clear knowledge gap between the important role of cyt b5 in P450 activity and our understanding of its mechanism. Lack of understanding is in part due to the absence of 3D structure of a native P450:b5 complex. It is highly challenging to obtain such a structure by conventional methods like X-ray crystallography or NMR spectroscopy. Single-particle cryo-EM has emerged as the method of choice for structural determination of multi-unit membrane protein complexes where diffracting crystals are not available. We hypothesize that determination of the 3D structure of a native CYP2B4-cyt b5 complex can be achieved using single-particle cryo-EM in conjunction with novel sample preparations. We propose to test the hypothesis and develop a much-needed cryo-EM method in two specific aims. In Aim 1, we will prepare the CYP2B4-cyt b5 complex in novel nanoparticles and then determine its 3D structure by single-particle cryo-EM in Aim 2. Successful completion of the two aims will not only provide critical knowledge for understanding the role of cyt b5 in CYP2B4 activity, but also develop a much-needed methodology for structural analysis of the native complexes of cyt b5 and POR with many other P450 enzymes. Establishment of this methodology and availability of these native complexes will be groundbreaking and provide a roadmap to interrogate the mechanism(s) by which the microsomal P450 system detoxifies environmental pollutants.