Structural Basis of Cytochrome P450 Activity

细胞色素P450酶的一个子集执行清除外源基因的第一步，也是限速步骤 小分子药物和毒素来自人体，而其他的则在内源性途径中发挥关键作用。的 必要性前者进化出了结合和氧化多种小分子化学物质的灵活性。 支架，而后者似乎不那么灵活，具有更多的底物专一性。我们所知道的关于 所有这些膜蛋白的结构都是由X射线结晶学单独确定的。这 方法提供了有关原子水平的蛋白质/配体相互作用的详细信息，但尚未应用 跨越人类P450酶的光谱，并且没有捕捉到这些酶的构象范围 能够吸收或与其他蛋白质相互作用。因此，一种结构横截面的应用 技术是提供所需信息以了解哪些P450酶结合哪些小分子 分子，它们是如何结合的，以及产品将是什么。这些信息对于理解 药物/毒素代谢的形式可能是活跃的或不活跃的，两种药物在 相同的P450活性部位，以及与不同疾病相关的内源性途径。 申请人的长期研究目标是促进对以下结构/功能原理的理解 控制底物和抑制物与P450酶的相互作用，以便利用这些信息来 更有效地预防和治疗人类疾病。这项提议的目标是产生新的 人细胞色素P450酶与催化系统的关键成分：它们的配体·，氧化还原 配对蛋白质，最终是膜。许多人类细胞色素P450酶不具有 结构是可用的，并且没有一个结构与其催化伙伴蛋白有结构。这些是我们提出的差距 使用以下方法搭建桥梁，在我们之前的结构专业知识的基础上，与20多名 细胞色素P450酶以及与冷冻电子显微镜专家Melanie Ohi博士的合作。 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，只有大约一半的酶存在结构。其中许多人没有 结构涉及胆汁酸、脂肪酸、二十烷类化合物和维生素等关键的动态平衡途径。 阻碍了我们对一些相应疾病的了解。我们利用了R37扩展 有机会提出一个小规模的结构基因组学项目，通过确定 尽可能多地使用这些P450酶。就像我们对许多其他人类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)进行 表达和纯化试验，以及3)使那些产生足够活性的P450蛋白的人结晶 X射线结构测定。我们将首先关注那些负责胆汁酸和脂肪酸合成的人。 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至关重要的 为了稳定和结晶，我们将使用一系列非特定的发光P450底物来筛选配体 它将每个P450紧密结合在一起，并可能促进结晶，就像在CYP1A1中成功的那样。 产生蛋白质但不发生结晶的情况(CYP2S1、CYP2W1、CYP3A7、CYP2F 目前)，我们将使用CryoEM启动结构。与Melanie博士合作进行的初步研究 OHI已经非常迅速地产生了一个小数据集，用于CYP3A4，其2D分辨率足以可视化Alpha 螺旋。即使特定的P450酶结晶成功，后一种方法也是独立的 这很有价值，因为它很可能揭示P450的“开放”构象，这些构象肯定存在，但通常不会在晶体中获得。 特定目标2：确定细胞色素b和还原酶与人P450结合的结构效应 酵素。 没有b5或还原酶与任何人类P450酶结合的结构。在上一次授权期内 我们用核磁共振对两个b5进行了鉴定。以不同方式结合单个细胞色素P450酶的表面。这个 建议的下一步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酶的构象以及它们与配体的相互作用有影响。我们知道 根据我们之前的研究，当b5。与CYP17 A1结合，使其相反方向发生主要构象变化 P450的表面，通常参与膜结合和配体从活性部位进入/退出。它是 目前尚不清楚这是否普遍属实。将使用先进的标记技术和底物定向核磁共振来回答 这些问题。最后，我们将使用我们的P450/氧化还原伙伴融合构建来阐明 用低温电子显微镜测定选定的络合物的结构。 总体而言，我们使用正交技术探测P450结构的能力得到了初步数据的支持 根据这笔赠款已经开发的技术和专业知识，使我们有资格将研究扩展到这些领域。vt.在.的基础上 完成后，我们预计将显著扩展多种人类P450酶的可用结构 在广泛的人类疾病方面具有重大意义。

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.