Enhanced Sampling of G-Protein-Coupled Receptor-G Protein Interactions

项目摘要 G蛋白偶联受体（GPCR）是人类膜蛋白的最大超家族， 目前市场上约1/3的药物的主要目标。腺苷受体的四种亚型，A1，A2 A，A2 B， 和A3介导广泛的生理功能。它们已成为重要的治疗靶点 用于治疗心脏缺血、神经性疼痛和癌症。在功能期间，A1和A3受体结合Gi/o 蛋白质，而A2 A和A2 B受体结合Gs蛋白。此外，GPCR-G蛋白相互作用是 由变构配体调节。这些配体结合到腺苷受体的假定细胞外位点， 显示出不同的序列和构象。与靶向高度依赖性细胞的传统激动剂相比， 保守的腺苷结合位点，并经常导致脱靶副作用，变构调节剂已经出现 作为选择性GPCR药物的有前途的候选者。到目前为止，腺苷受体是唯一的亚家族， GPCR具有与不同G蛋白复合确定的X射线或冷冻EM结构。虽然这些 结构提供了有价值的见解GPCR-G蛋白相互作用，他们是相当静态的图像， 蛋白质复合物目前的局限性包括：（1）目前尚不清楚柔性GPCR和G蛋白是如何在细胞内表达的。 动态识别对方。(2)特异性GPCR-G蛋白相互作用的决定因素仍不清楚。 (3)腺苷受体中别构调节剂结合的结构基础和机制仍然难以捉摸。 这些限制极大地阻碍了靶向腺苷受体的有效药物设计。为了 克服这些局限性，我们的具体目标包括：（1）发展一种新的计算方法， 一个强大的高斯加速分子动力学（GaMD）技术的成功，使所有原子模拟 蛋白质-蛋白质相互作用（PPIs），称为“PPI-GaMD”。(2)在广泛使用的开源中实现PPI-GaMD 模拟包。(3)测试PPI-GaMD模拟特定G蛋白与A1和A2 A的相互作用 受体。(4)应用PPI-GaMD模拟确定变构调节剂与A1结合的机制 和A2 A受体以及GPCR-G蛋白相互作用的变构调节。(5)体外模拟 通过诱变和结合试验，以及通过与领先的 GPCR实验组。反过来，模拟将帮助我们在原子理论上解释实验数据。 水平我们的长期目标是：（1）开发强大的计算方法来定量表征 疾病相关细胞信号通路中的生物分子识别和（2）设计有效的药物 靶向重要受体的分子。

Project Summary G-protein-coupled receptors (GPCRs) are the largest superfamily of human membrane proteins and serve as primary targets of about 1/3 of currently marketed drugs. Four subtypes of adenosine receptors, the A1, A2A, A2B, and A3, mediate a broad range of physiological functions. They have emerged as important therapeutic targets for treating cardiac ischemia, neuropathic pain and cancer. During function, the A1 and A3 receptors bind the Gi/o proteins, while the A2A and A2B receptors bind the Gs proteins. Moreover, the GPCR–G protein interactions are modulated by allosteric ligands. These ligands bind to a putative extracellular site of adenosine receptors, which exhibit divergent sequences and conformations. In contrast to traditional agonists that target at the highly conserved adenosine-binding site and often cause off-target side effects, allosteric modulators have emerged as promising candidates as selective GPCR drugs. To date, adenosine receptors are the sole subfamily of GPCRs that have X-ray or cryo-EM structures determined in complex with distinct G proteins. Although these structures provide valuable insights into the GPCR–G protein interactions, they are rather static images of the protein complexes. Current limitations include: (1) It remains unknown how the flexible GPCRs and G proteins dynamically recognize each other. (2) The determinants of specific GPCR–G protein interactions remain unclear. (3) The structural basis and mechanism of allosteric modulator binding in the adenosine receptors remain elusive. These limitations have greatly hindered effective drug design targeting the adenosine receptors. In order to overcome these limitations, our specific aims include: (1) Develop a new computational method based on recent success of a robust Gaussian accelerated molecular dynamics (GaMD) technique to enable all-atom simulations of protein-protein interactions (PPIs), called “PPI-GaMD”. (2) Implement PPI-GaMD in widely used open source simulation packages. (3) Test PPI-GaMD on simulations of specific G protein interactions with the A1 and A2A receptors. (4) Apply PPI-GaMD simulations to determine mechanisms of allosteric modulator binding to the A1 and A2A receptors and allosteric modulation of the GPCR–G protein interactions. (5) Validate simulations in vitro by mutagenesis and binding assays and in vivo by cellular functional assays through collaboration with a leading GPCR experimental group. In turn, the simulations will help us to interpret the experimental data at an atomistic level. Our long-term goals are (1) to develop robust computational methodologies to quantitatively characterize biomolecular recognition in disease-associated cellular signaling pathways and (2) to design effective drug molecules targeting important receptors.