Structural Basis of Cytochrome P450 Activity

细胞色素P450酶的一个子集在清除外源性蛋白质中执行第一步和限速步骤。 小分子药物和毒素从人体，而其他发挥关键作用的内源性途径。的 必要性，前者进化出结合和氧化广泛的小分子化学物质的灵活性， 支架，而后者似乎是灵活性较低，并具有更多的底物特异性。我们所知道的 所有这些膜蛋白的结构仅由X射线晶体学确定。这 一种方法提供了关于原子水平蛋白质/配体相互作用的详细信息，但尚未应用 在整个人类谱的P450酶，并没有捕获的构象范围，这些酶 能够与其他蛋白质相互作用。因此，应用横截面结构 技术是必不可少的，以提供所需的信息，以了解哪些P450酶结合哪些小 分子，它们如何结合，以及产物是什么。这些信息对于理解 药物/毒素代谢为活性或非活性形式，两种药物在 相同的P450活性位点和与多种疾病相关的内源性途径。 申请人的长期研究目标是促进对结构/功能原理的理解， 控制底物和抑制剂与P450酶的相互作用，以便利用这些信息， 更有效地预防和治疗人类疾病。这项建议的目的是建立新的结构， 人细胞色素P450酶与催化系统的关键组成部分：它们的配体·，氧化还原 伴侣蛋白，最终是细胞膜。许多人细胞色素P450酶不具有 结构可用，并且没有一个具有与其催化伴侣蛋白的结构。这些是我们提出的差距 利用我们以前的结构专业知识和20多名人力资源， 细胞色素P450酶，并与冷冻电子显微镜博士梅勒妮奥希合作。 具体目标1：确定人P450酶的结构。 在57种人类细胞色素P450酶中，只有大约一半存在结构。许多没有 结构参与关键的稳态途径，包括胆汁酸、脂肪酸、类二十烷酸和维生素， 阻碍了我们对一些相应疾病的了解。我们利用这个R37扩展 有机会提出一个小规模的结构基因组学项目，以“缩小差距”，通过确定结构， 尽可能多的P450酶正如我们成功地对许多其他人类P450所做的那样， 酶，我们将1）以通常产生P450全蛋白的方式设计合成基因，2）承担 表达和纯化试验，和3）使产生足够活性P450蛋白的那些进行结晶， X射线结构测定。我们将首先集中在那些负责胆汁酸和脂肪酸的合成 因为我们有更多的专业知识和工具来支持这些领域的成功。因为特定的配体可能是关键的 为了稳定和结晶，我们将使用一系列非特异性发光P450底物来筛选配体 与每个P450紧密结合，并可能促进结晶，如对CYP 1A 1的成功。在 产生蛋白质但未发生结晶的情况（CYP 2S 1、CYP 2 W1、CYP 3A 7、CYP 2F 我们将使用cryoEM启动结构。与Melanie博士合作的初步研究 Ohi很快就得到了一个CYP 3A 4的小数据集，其2D分辨率足以显示α 螺旋即使用特定的P450酶结晶是成功的，后一种方法也是独立的。 它是有价值的，因为它可能揭示P450“开放”构象，这种构象必须存在，但通常不能在晶体中获得。 具体目的2：确定细胞色素b和还原酶与人P450结合的结构效应 内切酶 没有b5或还原酶与任何人P450酶结合的结构。在上一个赠款期间 我们用核磁共振来鉴定两个b5。不同的细胞色素P450酶结合的表面。的 下一步建议是使用核磁共振来识别单个P450酶的相对表面及其影响 b5结合对这些P450酶的构象及其与配体的相互作用的影响。我们知道 根据我们之前的研究，当B5。结合CYP 17 A1，主要构象变化发生在相反方向 P450的表面，其通常参与膜结合和配体进入/离开活性位点。是 不清楚这是否普遍正确。先进的标记技术和底物定向核磁共振将被用来回答 这些问题。最后，我们将使用我们的P450/氧化还原伴侣融合构建体来阐明 使用cryoEM确定所选复合物的结构。 总的来说，我们的能力，探测P450结构使用正交技术，支持的初步数据 和专门知识已经开发下，这项赠款，独特的资格，我们扩大研究到这些领域。后 完成后，我们预计将显着扩展多种人类P450酶的结构 对于广泛的人类疾病具有重大意义。

A subset of cytochrome P450 enzymes perform the first and rate-limiting step in the clearance of foreign small molecule drugs and toxins from the human body, while others play key roles in endogenous pathways. Of necessity the former evolved the flexibility to bind and oxidize a broad range of small molecule chemical scaffolds, while the latter appear to be less flexible and have more substrate specificity. What we know about the structures of all of these membrane proteins has been determined solely by X-ray crystallography. This approach provides detailed information about atomic-level protein/ligand interactions, but has not been applied across the human spectrum of P450 enzymes and does not capture the range of conformations these enzymes are capable of adopting or their interactions with other proteins. Thus application of a cross-section of structural techniques is essential to provide the information needed to understand which P450 enzymes bind which small molecules, how they are bound, and what the products will be. This information is critical for understanding drug/toxin metabolism to forms that may be either active or inactive, adverse interactions of two drugs at the same P450 active site, and endogenous pathways related to diverse diseases. The applicant's long-term research goal is to promote understanding of the structure/function principles that control substrate and inhibitor interactions with P450 enzymes, in order that this information can be exploited to more effectively prevent and treat human disease. The objective of this proposal is to generate structures of new human cytochrome P450 enzymes with the critical components of the catalytic system: their ligands·, redox partner proteins, and eventually the membrane. A number of human cytochrome P450 enzymes do not have structures available and none have structures with their catalytic partner proteins. These are gaps we propose to bridge using the following approaches, building on our previous structural expertise with more than 20 human cytochrome P450 enzymes and a collaboration with cryo-electron microscopist Dr. Melanie Ohi. Specific aim 1: Determine structures of human P450 enzymes. A structure exists for only about half of the 57 human cytochrome P450 enzymes. Many of those without structures are involved in key homeostatic pathways involving bile acids, fatty acids, eicosanoids, and vitamins, impeding our understanding of a number of corresponding diseases. We take advantage of this R37 extension opportunity to propose a small-scale structural genomics project to "close the gap" by determining structures of as many of these P450 enzymes as possible. As we have done successfully for many other human P450 enzymes, we will 1) engineer synthetic genes in ways that usually produce P450 holoproteins, 2) undertake expression and purification trials, and 3) subject those yielding enough active P450 protein to crystallization for X-ray structure determination. We will initially focus on those responsible for bile acid and fatty acid synthesis as we have additional expertise and tools to support success in these areas. As specific ligands can be critical for stability and crystallization, we will use a range of nonspecific luminescent P450 substrates to screen ligands that bind each P450 tightly and are likely to facilitate crystallization, as has been successful for CYP1A 1. In cases where protein is generated but crystallization does not occur (CYP2S1, CYP2W1, CYP3A7, CYP2F currently in hand), we will initiate structures using cryoEM. Preliminary studies in collaboration with Dr. Melanie Ohi have very quickly yielded a small data set for CYP3A4 with 2D resolution good enough to visualize alpha helices. Even if crystallization is successful with a particular P450 enzyme, this latter approach is independently valuable as it is likely to reveal P450 "open" conformations that must exist but are usually not obtained in crystals. Specific aim 2: Determine structural effects of cytochrome bs and reductase binding to human P450 enzymes. There are no structures of b5 or reductase binding to any human P450 enzymes. Over the last grant period we used NMR to identify the two b5. surfaces that differentially bind individual cytochrome P450 enzymes. The next step proposed is to use NMR to identify the opposing surfaces on individual P450 enzymes and the effects that b5 binding has on the conformation of these P450 enzymes and their interactions with ligands. We know from our previous studies that when b5. binds CYP17 A1 that major conformational changes occur on the opposite face of the P450, which is normally involved in membrane binding and ligand entry/exit from the active site. It is unclear if this is generally true. Advanced labeling techniques and substrate-directed NMR will be used to answer these questions. Finally, we will employ our P450/redox partner fusion constructs to elucidate the feasibility of determining structures of selected complexes using cryoEM. Overall, our ability to probe P450 structure using orthogonal techniques, supported by the preliminary data and expertise already developed under this grant, uniquely qualify us to expand research into these areas. Upon completion, we expect to have significantly expanded the structures available for multiple human P450 enzymes of significant interest with respect to a broad range of human diseases.