Regulation of P450 Activity by Cytochrome P450 Oxidoreductase

描述(由申请人提供)：本提案的总体目标是了解负责各种外源物质和内源性化合物氧化代谢的微粒体单加氧系统机制的结构和分子基础。该系统的两个关键组成部分是细胞色素P450(P450s)和NADPH-细胞色素P450氧化还原酶(CYPOR)。虽然我们对这两个伙伴各自的机制和结构都很了解，但他们之间的相互作用却知之甚少。在人类中，单一的CYPOR蛋白能够与~50个微粒体P450以及非P450酶，即血红素加氧酶(HO-1和HO-2)相互作用。此外，编码CYPOR的POR基因突变导致广泛的表型，从胚胎致死性、Antley-Bixler综合征(ABS)到轻微的类固醇激素性疾病。与以前获得的“闭合型”结构不同，我们最近获得的CYPOR的变体(CYPORTGEE)采用了一种开放的构象，能够还原P450，并与其伙伴形成稳定的络合物。我们将在EPR和结晶研究中使用CYPORTGEE，具体目标如下。目的1：用定点自旋标记EPR方法研究CYPOR与其电子转移配对结合后的构象变化及其相互作用。以野生型CYPOR和CYPORTGEE的晶体结构为指导，双半胱氨酸突变体(两个黄素结构域各一个)将被自旋标记，并测量自旋-自旋距离。自旋标记的CYP2B4和HO-1也将与单一标记的CYPOR一起使用，以测量复合体中两个伙伴之间的自旋-自旋距离。根据CYPOR的两个结构域之间的距离和两个配对分子之间的距离，可以构建出CYPOR-2B4/HO-1的络合物结构。由此得到的CYPOR及其合作伙伴复合体的模型结构将通过生化表征和定点突变相结合的方法进行验证。目的：确定CYPOR与细胞色素P450 D 6、细胞色素P3A4、细胞色素P450 B4和人HO-1的配合物的晶体结构。我们将使用人和大鼠的CYPORTGEE蛋白与合作伙伴形成稳定的复合体。可溶形式的CYP2B4、CYP3A4和CYP2D6以及可溶形式的HO-1将用于与CYPORTGEE的共结晶研究。上述研究的成功完成将对P450领域产生影响，因为它为该领域的核心问题提供了答案：识别的基础是什么，以及CYPOR与其氧化还原伙伴，包括P450和HO之间的电子转移机制是什么？这些研究的结果将分别为药物代谢和HO-1功能的机制提供更好的了解，从而用于药物开发和有效治疗的设计，并可能用于治疗导致类固醇合成异常的CYPOR缺陷的治疗方法的设计。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the structural and molecular basis for the mechanisms of the microsomal monooxygenation system that is responsible for the oxidative metabolism of various xenobiotics and endogenous compounds. The two key components of this system are cytochromes P450 (P450s) and NADPH-cytochrome P450 oxidoreductase (CYPOR). Although we know much about the mechanisms and structures of each of these two partners individually, their interactions with each other are poorly understood. In humans, the single CYPOR protein is capable of interacting with ~50 microsomal P450s, as well as the non-P450 enzymes, heme oxygenases (HO-1 and HO-2). Furthermore, mutations of the POR gene, encoding CYPOR, result in a wide range of phenotypes, from embryonic lethality, Antley-Bixler Syndrome (ABS) to mild steroidogenic disorders. In contrast to the previously obtained "closed form" structure of CYPOR, our recently obtained variant form of CYPOR (CYPORTGEE) adopts an open conformation that is capable of reducing P450s, and forms stable complexes with its partners. We will use CYPORTGEE in our EPR and crystallization studies as outlined in the following specific aims. Aim 1: To determine conformational changes of CYPOR upon binding to its electron transfer partners and the interactions between them by site-directed spin-labeling EPR methods. Using the crystal structures of wild type CYPOR and CYPORTGEE as guides, double cysteine mutants (one in each of the two flavin domains) will be spin labeled, and the spin-spin distances will be measured. Spin-labeled CYP2B4 and HO-1 will also be used with single labeled CYPOR to measure the spin- spin distances between the two partners in the complex. From the distances between the two domains of CYPOR and between the two partners, structures of the complexes of CYPOR-2B4/HO-1 will be constructed. The resulting model structures of complexes of CYPOR and its partners will be validated by biochemical characterization combined with site-specific mutagenesis. Aim 2: To determine the crystal structures of complexes of CYPOR with CYP2D6, CYP3A4, CYP2B4, and human HO-1. We will use both human and rat CYPORTGEE proteins that form stable complexes with the partners. Soluble forms of CYP2B4, CYP3A4, and CYP2D6 and the soluble form of HO-1 will be used in co-crystallization studies with CYPORTGEE. The successful completion of the above studies will impact the P450 field by providing the answer to the central question in the field: What is th basis for recognition and the mechanism of electron transfer between CYPOR and its redox partners, including P450s and HO? The results from these studies will be used for drug development and designing of effective therapies by providing a better understanding of drug metabolism and the mechanism of HO-1 functions, respectively, and possibly to the design of therapeutics for CYPOR deficiencies resulting in abnormal steroidogenesis.