Structure Function Studies on the Mechanisms of P2X3 Receptor Desensitization

描述(申请人提供)：嘌呤能受体(P2X)是被三磷酸腺苷和腺苷结合激活的蛋白质。由于它们在心血管、神经和免疫系统中都有活跃的表达，因此这些受体发挥作用的结构机制对于了解它们如何调节诸如平滑肌收缩、血小板激活、突触传递和炎症等过程非常重要。因此，它们已成为心血管、神经和炎症性疾病的关键潜在药理靶点。尽管是一个积极追求的药理靶点，但对于任何一种嘌呤能受体亚型，只有两个高分辨率结构存在，而且都不是在人类同系物上。斑马鱼P2X4受体的脱敏关闭状态和ATP结合状态的结构已经为受体的激活提供了一些结构性的见解；然而，围绕脱敏(快速和缓慢)的机制仍然没有答案。由于P2X3受体表现出快速的脱敏动力学，而P2X4则表现出缓慢的脱敏动力学，因此这两种受体之间的结构差异将有助于深入了解脱敏的机制。这项建议概述了利用X射线结晶学来确定脱敏状态下的人P2X3受体高分辨率结构的方法。首先，我将确定一个人P2X3的“最小功能结构”，它表现出与野生型相同的激活和脱敏特性，但对于哺乳动物表达系统的表达和纯化是最佳的。通过这个过程，我将确定在人P2X3的N-末端和C-末端结构域上对激活和脱敏至关重要的关键残基。结晶试验将不仅限于经典的uSig洗涤剂/蛋白质胶束方法，还将包括允许在富含脂质的环境中结晶的新技术，如双孔结晶和脂类立方相结晶。为了增加获得减敏状态的结构的可能性，将执行共结晶 在存在两种已知可以稳定脱敏状态的毒素的情况下，以及与来自构象特异性抗体的Fab片段的结合。这项工作的结果将大大有助于理解嘌呤能受体激活和随后的脱敏机制。

DESCRIPTION (provided by applicant): Purinergic receptors (P2X) are proteins that are activated by the binding of ATP and adenosine. Because they are actively expressed in the cardiovascular, nervous, and immune systems, the structural mechanisms by which these receptors function are important to understand how they modulate processes such as smooth muscle contraction, platelet activation, synaptic transmission and inflammation. As such, they have become a key potential pharmacologic target for cardiovascular, neuronal and inflammatory diseases. Despite being an actively pursued pharmacologic target, only two high-resolution structures exist for any of the purinergic receptor subtypes and neither is on a human homolog. Structures of an apo-closed state as well as an ATP-bound state of zebra fish P2X4 receptor have provided some structural insight into receptor activation; however, the mechanisms surrounding desensitization (rapid vs. slow) remain unanswered. Because P2X3 receptor displays rapid desensitization kinetics, as opposed to P2X4, which displays slow desensitization kinetics, structural differences between the two receptors will give insight into mechanisms of desensitization. This proposal outlines the use of proven strategies to determine a high-resolution structure of human P2X3 receptor in the desensitized state using X-ray crystallography. First, I will identify a "minimal functional construct" of human P2X3 which displays activation and desensitization properties identical to wild type yet is optimal for expression and purification in a mammalian expression system. Through this process, I will identify key residues in the N- and C-terminal domains of human P2X3 that are important for activation and desensitization. Crystallization trials will not be limited to classical methods usig detergent/protein micelles but will also include new techniques that allow crystallization in a lipid-rich environment, such as bicelle and lipidic cubic phase crystallization. To increase the likelihood of obtaining a structure of the desensitized state, co-crystallization will be performed in the presence of two toxins that are known to stabilize the desensitized state as well as with Fab fragments from conformation-specific antibodies. The results of this work will substantially contribute to the understanding of the mechanisms for purinergic receptor activation and subsequent desensitization.