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

身体内的细胞通过释放被特定细胞表面受体识别的化学物质来相互交流。这种化学物质的一个例子是与P2X受体结合的ATP。在人类中，至少有13种不同类型的P2X受体，它们的性质各不相同，例如对ATP的敏感性。P2X受体在一系列正常身体功能中发挥重要作用，例如控制血压、凝血和味觉，以及治疗疼痛和神经退行性疾病（如阿尔茨海默病）的潜在药物靶点。P2X受体是一种膜蛋白，其部分位于细胞外部，可以识别ATP分子，一个穿过细胞壁的区域（与通道打开有关）和一个位于细胞内的区域（细胞内）。细胞内区域由不同的部分组成，这些部分在控制受体的活性时长方面起着重要作用。最近的研究显示了P2X受体在静止状态和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原理提供了重大进展。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结构的信息，或者它们如何调节受体的机制。这些信息对于理解与这一独特受体家族的活性相关的基本机制至关重要。受体的细胞内区域是由称为氨基酸的不同构建块的组合组成的，其中一些具有独特的性质，可以用来测量受体的分子距离。其中一种化学上独特的氨基酸是赖氨酸，其中几种存在于P2X1受体的细胞内区域。我们使用药物DSS来测定细胞内赖氨酸残基之间的距离。DSS分子可以被认为是一根两端有赖氨酸反应基团/球的棍子。当我们用DSS处理P2X1受体时，它会与赖氨酸残基结合，如果两个赖氨酸残基之间的距离相当于棒子末端两个球的距离，它们可以结合在一起（交联）。然后我们可以提取P2X1受体，并使用一种称为质谱的技术来确定哪些赖氨酸残基被DSS交联。这为我们提供了赖氨酸残基之间的距离约束，可用于计算机模拟，以提供受体胞内部分的3D结构模型。在这项研究中，我们将使用一系列化学交联剂（在棒状物的长度和两端基团的化学反应性上有所不同）作为分子标尺来确定受体细胞内部分的定义残基的接近程度。然后，这些距离将用于开发受体的3D模型，从而产生可以测试以验证和完善模型的预测。我们将在ATP缺失和存在的情况下测试交联剂的作用，以确定细胞内区域的相对位置是否存在与受体开启相关的运动。我们还将比较P2X1受体与P2X2受体之间的交联模式，前者在ATP的作用下会迅速关闭，而后者只要ATP存在就会表现出持续的反应。通过这种方式，我们将能够确定P2X受体细胞内部分的3D形状，以及它们在打开和关闭时是否会发生变化。这将为了解这些受体如何在分子水平上起作用提供基本的见解，并深入了解受体之间的差异，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失衡。

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.