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

P2X受体是一类同源或异源三聚体配基门控离子通道，在细胞外ATP结合时介导阳离子去极化流入细胞内，是新药开发的重要靶点。在过去的三年里，我们对P2XRs的分子功能、调控和结构的了解取得了重大进展。我们确定了(I)一种离子锁定机制，它稳定了通过ATP结合释放的P2X2R激动剂结合部位的闭合构象；(Ii)控制了P2X3R的构象变化；(Iii)配体与P2XRs之间的亚型特异性相互作用。最近公布的第一批哺乳动物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。首次包括细胞内结构域的开放、封闭和钝化状态的hP2X3R结构提供了一个前所未有的机会，通过广泛的全原子分子动力学(MD)模拟，可靠地将这些结构与功能测量联系起来。在第一个授权期，我们证明了结构导向诱变和电生理分析相结合是检测和验证分子相互作用的一种高效方法。我们期望MD刺激将极大地改善电生理实验中定点突变的基于结构的指导。在第一个授权期的继续，第二个阶段的主要目标是确定配体激活和门控的动态分子机制，以及负责离子渗透、电导和选择性的分子机制。一个相关的目标是确定亚型特定配体相互作用的分子决定因素，以了解药物如何选择性地靶向P2XRs。为此，我们希望综合使用MD模拟、配体对接和虚拟筛选模拟。从这些分析得出的假说将通过定点突变和宏观和单通道水平的电生理分析相结合来验证。电生理数据将为采用MD模拟提供迭代反馈，目的是详细了解P2XRs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.