Molecular Dynamics Guided Analysis of Ligand Activation, Ion Permeation and Subtype-specific Ligand Interactions in P2X Receptors

P2X受体代表了同源或异源三聚体配体门控离子通道家族（P2X1R-P2X7R），通过与细胞外ATP结合介导阳离子进入细胞的去极化，是新药的有趣靶点。在过去的三年中，我们对p2xr的分子功能、调制和结构的理解取得了重大进展。我们确定了(i)一种离子锁定机制，该机制稳定了P2X2R激动剂结合位点的封闭状态构象，该构象通过ATP结合释放；（ii）控制P2X3R的构象变化；（iii）配体与P2XRs之间的亚型特异性相互作用。最近发布的首个哺乳动物p2xr、人类P2X3R和熊猫P2X7R的高分辨率x射线晶体结构对该项目的继续至关重要。开放，封闭和脱敏状态的hP2X3R结构首次包括细胞内结构域，通过广泛的全原子分子动力学（MD）模拟，为可靠地桥接这些结构提供了前所未有的机会。在第一个资助期，我们证明了结构引导诱变和电生理分析的结合是一种检测和验证分子相互作用的高效方法。我们期望MD刺激能够极大地改善电生理实验中基于结构的定点诱变指导。作为第一个资助期的延续，第二个资助期的主要目标是确定配体激活和门控的动态分子机制，以及离子渗透、电导和选择性的分子机制。一个相关的目标是确定亚型特异性配体相互作用的分子决定因素，以了解p2xr如何被药物选择性靶向。为此，我们要综合运用MD仿真、配体对接和虚拟筛选仿真。从这些分析中推断出的假设将通过位点定向诱变与宏观和单通道水平的电生理分析相结合来验证。电生理数据将为适应MD模拟提供迭代反馈，目的是在原子细节上理解p2xr中配体结合耦合离子流的功能动力学。

P2X receptors represent a family (P2X1R–P2X7R) of homo- or heterotrimeric ligand-gated ion channels that mediate the depolarizing influx of cations into cells upon binding of extracellular ATP and are interesting targets for new drugs. In the past 3 years, significant advances in our understanding of the molecular function, modulation, and structure of the P2XRs have been made. We identified (i) an ionic-lock mechanism that stabilizes the closed-state conformation of the agonist-binding site of the P2X2R, which is released by ATP binding; (ii) gating conformational changes of the P2X3R; and (iii) subtype-specific interactions between ligands and P2XRs. The recent releases of the first high-resolution X-ray crystal structures of mammalian P2XRs, the human P2X3R and panda P2X7R are crucial for the continuation of the project. The open-, closed-, and desensitized-state hP2X3R structures that for the first time include intracellular domains, provide an unprecedented opportunity to reliably bridge these structures with functional measurements via extensive all-atom molecular dynamics (MD) simulations. In the first grant period, we proved that the combination of structure-guided mutagenesis and electrophysiological analysis is a highly efficient way of detecting and validating molecular interactions. We expect MD stimulations to greatly improve the structure-based guidance of site-directed mutagenesis for electrophysiological experiments. In continuation of the first grant period, main objectives of the second period are to identify the dynamic molecular mechanisms of ligand activation and gating, along with the molecular mechanisms responsible for ion permeation, conductance, and selectivity. A related objective is to identify the molecular determinants of subtype-specific ligand interactions to understand how P2XRs can be selectively targeted by drugs. To this end, we want to comprehensively use MD simulations, ligand-docking, and virtual screening simulations. Hypotheses deduced from these analyses will be validated by site-directed mutagenesis in combination with electrophysiological analysis at the macroscopic and single channel levels. Electrophysiological data will provide iterative feedback for adapting MD simulations with the aim of understanding the functional dynamics of ligand binding-coupled ion flow in P2XRs in atomic detail.