Cross-linking and molecular modelling to determine the structure and dynamics of the intracellular regions of ATP gated P2X receptor ion channels

交联和分子建模以确定 ATP 门控 P2X 受体离子通道细胞内区域的结构和动力学

• 批准号: BB/M000990/1
• 负责人: Richard Evans
• 金额: $ 44.03万
• 依托单位: University of Leicester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM000990%2F1
• 关键词: Cross; linking; molecular; modelling; determine

Cells within the body communicate with one-another through the release of chemicals recognised by specific cell surface receptors. One example of such a chemical is ATP that binds to P2X receptors. In humans there are at least 13 different types of P2X receptor that vary in their properties, for example in terms of sensitivity to ATP. P2X receptors play an important role in a range of normal bodily functions e.g. in control of blood pressure, blood clotting and taste sensation, as well being potential drug targets for the treatment of pain and neurodegenerative diseases e.g. Alzheimer's disease. P2X receptors are membrane proteins with parts on the outside of the cell that recognize the ATP molecule, a region that passes through the cell wall (that is associated with the channel turning on) and a region that is inside the cell (intracellular). The intracellular region is made up of separate parts that play an important role in the controlling how long the receptor is active for. Recent studies have shown the 3D structure of a P2X receptor at rest and following ATP binding and this has provided a major advance in our understanding of how this novel family of receptors works. However to obtain these structures the intracellular parts of the receptor were removed, and therefore we have no information on the structure of the intracellular regions, or the mechanism of how they regulate the receptor. This information is essential to understand the fundamental mechanisms associated with the activity of this distinct family of receptors.The intracellular regions of the receptor are made up of a combination of different building blocks called amino acids, and some of these have unique properties that can be used to measure molecular distances in the receptor. One such chemically unique amino acid is lysine and several of these are present in the intracellular regions of the P2X1 receptor. We used a drug, DSS, to determine the distance between lysine residues in the intracellular region. The DSS molecule can be thought of as a stick with a lysine reactive group/ball at either end. When we treated the P2X1 receptor with DSS it will bind to lysine residues and two lysine residues can be bound together (cross-linked) if the distance between them is equivalent to that of the two balls at the end of the stick. We can then extract the P2X1 receptor and use a technique called mass spectrometry to identify which lysine residues have been cross-linked by the DSS. This provides us with a distance constraint between the lysine residues that can be used in computer simulations to provide a model of the 3D structure of the intracellular parts of the receptor. In this study we will use a range of chemical cross linkers (that vary in the length of the stick and the chemical reactivity of the groups at either end) as molecular rulers to determine how close defined residues in the intracellular parts of the receptor are. These distances will then be used to develop 3D models of the receptor that give rise to predictions that can be tested to validate and refine the model. We will test the effects of the cross-linker in the absence and presence of ATP to determine whether there is a movement in the relative positions of the intracellular regions that is associated with turning on of the receptor. We will also compare the pattern of cross-linking between the P2X1 receptor that turns off quickly in response to ATP and the P2X2 receptor that shows a sustained response as long as ATP is present. In this way we will be able to determine the 3D shape of the intracellular parts of P2X receptors and if these change when they are turned on and off. This will provide a fundamental insight into how these receptors work at the molecular level, and give insight into variations between receptors, and why genetic mutations affect receptor properties that can lead to imbalances in signalling and disease.

体内的细胞通过释放特定细胞表面受体识别的化学物质相互交流。这种化学物质的一个例子是与P2 X受体结合的ATP。在人类中，至少有13种不同类型的P2 X受体，它们的特性各不相同，例如对ATP的敏感性。P2 X受体在一系列正常身体功能中起重要作用，例如控制血压、血液凝固和味觉，以及作为治疗疼痛和神经退行性疾病（例如阿尔茨海默病）的潜在药物靶标。P2 X受体是一种膜蛋白，其部分位于细胞外部，识别ATP分子，一个穿过细胞壁的区域（与通道开启相关）和一个位于细胞内部的区域（细胞内）。细胞内区域由独立的部分组成，这些部分在控制受体的活性时间方面发挥重要作用。最近的研究显示了P2 X受体在静息状态和ATP结合后的3D结构，这为我们理解这种新型受体家族的工作方式提供了重大进展。然而，为了获得这些结构，受体的细胞内部分被移除，因此我们没有关于细胞内区域的结构或它们如何调节受体的机制的信息。这些信息对于理解与这种独特的受体家族的活性相关的基本机制至关重要。受体的细胞内区域由称为氨基酸的不同构建块的组合组成，其中一些具有独特的性质，可用于测量受体中的分子距离。一种这样的化学上独特的氨基酸是赖氨酸，其中几种存在于P2 X1受体的细胞内区域。我们使用了一种药物，DSS，以确定在细胞内区域赖氨酸残基之间的距离。DSS分子可以被认为是在任一端具有赖氨酸反应性基团/球的棒。当我们用DSS处理P2 X1受体时，它将与赖氨酸残基结合，并且如果两个赖氨酸残基之间的距离等于棒末端的两个球的距离，则两个赖氨酸残基可以结合在一起（交联）。然后，我们可以提取P2 X1受体，并使用一种称为质谱的技术来识别哪些赖氨酸残基已被DSS交联。这为我们提供了赖氨酸残基之间的距离约束，可以用于计算机模拟，以提供受体细胞内部分的3D结构模型。在这项研究中，我们将使用一系列化学交联剂（其在棒的长度和两端基团的化学反应性方面有所不同）作为分子标尺，以确定受体细胞内部分中定义的残基有多接近。然后，这些距离将用于开发受体的3D模型，这些模型可以产生预测，这些预测可以被测试以验证和改进模型。我们将在不存在和存在ATP的情况下测试交联剂的作用，以确定是否存在与受体开启相关的细胞内区域的相对位置的移动。我们还将比较P2 X1受体和P2 X2受体之间的交联模式，P2 X1受体对ATP的反应迅速关闭，而P2 X2受体只要ATP存在就显示出持续的反应。通过这种方式，我们将能够确定P2 X受体细胞内部分的3D形状，以及当它们被打开和关闭时这些形状是否会发生变化。这将为这些受体如何在分子水平上工作提供基本的见解，并深入了解受体之间的变化，以及为什么基因突变会影响受体特性，从而导致信号传导和疾病的不平衡。