The role of dynamics in GPCR and arrestin allostery

项目摘要/摘要 GPCRs是最大的膜蛋白家族，也是治疗药物的靶标 超过30%的FDA批准的药物。这些药物的作用范围很广，从癌症到 炎症性、心血管、呼吸系统和胃肠道疾病。GPCR是典型的变构 将细胞外配体刺激转化为细胞内反应的蛋白质。与二进制数“On”相反 大多数信号分子的“或关”反应，GPCRs具有配体无关的基础活性，可以 在配体结合后增加或减少，然后受变构调节剂进一步调节。已激活 受体通过G蛋白和arrestin蛋白同等(平衡信号)或选择性地传递信号 (有偏差的信号)。综上所述，单个受体可能会特异性地识别几个配体并做出反应 每一个都是独一无二的，创造了一个复杂的构象景观。在过去的十年里，我们看到了近300次X射线 和低温EM结构极大地扩展了我们对GPCR体系结构和功能的看法；然而， 基础活性、部分激动性、偏向信号和变构调节的分子机制只能 部分是从这些结构衍生出来的。Monod-Wyman-Changeux(MWC)类的原理和 动态驱动(DD)变构可以显著增强我们对GPCR的理解和调整能力 发信号。而经典的MWC构象变构是相对简单的从X射线或冷冻-EM推断 对于结构模型，DD变构更难通过实验测量。核磁共振波谱研究进展 松弛测量经验表明，侧链甲基动力学可以作为一种 构象熵的代理(即DD变构)。此外，它仍然是唯一能够 在皮秒到秒的时间尺度上量化原子分辨率的运动，在许多情况下 检测填充仅为0.5%的州。我们提出了两个旨在探索变构的研究项目 MWC和DD变构机制在1)肽结合GPCRs和2)arrestin激活中的作用。 使用部分或偏向的激动剂调节受体信号的能力具有巨大的治疗潜力。 与完全的激动剂/拮抗剂形成对比--而且它实际上仍未被开发。我们的建议提供了更多- 需要对数十年的功能突变筛选、EPR、荧光标记和高分辨率进行补充 结构。从长远来看，我们研究计划的目标是描述构象转变和 不受晶体阻碍的GPCR超家族从非活化态到活化态的动力学 接触或稳定蛋白质。

PROJECT SUMMARY/ABSTRACT GPCRs form the largest membrane protein family and also dominate the therapeutic market as targets for more than 30% of FDA-approved drugs. These drugs act on a broad spectrum of indications from cancer to inflammatory, cardiovascular, respiratory and gastrointestinal disease. GPCRs are archetypical allosteric proteins that translate extracellular ligand stimulation into an intracellular response. In contrast to the binary “on or off” response of most signaling molecules, GPCRs possess a ligand-independent basal activity that can be increased or decreased upon ligand binding, and then further regulated by allosteric modulators. Activated receptors transduce signals through G protein and arrestin proteins equally (balanced signaling) or selectively (biased signaling). Taken together, a single receptor may specifically recognize several ligands and respond uniquely to each, creating a complex conformational landscape. The last decade has seen nearly 300 X-ray and cryo-EM structures have greatly expanded our view of GPCR architecture and function; however, the molecular mechanisms of basal activity, partial agonism, biased signaling, and allosteric modulation can only be partially derived from these structures. The principles of class Monod-Wyman-Changeux (MWC) and dynamically-drive (DD) allostery can significantly enhance our understanding of, and ability to tune, GPCR signaling. Whereas classic MWC conformational allostery is relatively simple to infer from X-ray or cryo-EM structural models, DD allostery is far more difficult to measure experimentally. Advances in NMR spectroscopy relaxation measurements have empirically-demonstrated that sidechain methyl dynamics can be used as a proxy for conformational entropy (i.e. DD allostery). In addition, it remains the only technique capable of quantifying atomic-resolution motions across the picosecond to second timescale and in many cases can detect states populated as little as 0.5%. We propose two research projects aimed at exploring the allosteric role of MWC and DD allosteric mechanisms in the activation of 1) peptide-binding GPCRs and 2) arrestin. There is immense therapeutic potential in the ability to tune receptor signaling using partial or biased agonists in contrast to full agonists/antagonists – and it remains virtually untapped. Our proposal provides a much- needed complement to decades of functional mutant screens, EPR, fluorescent labels, and high-resolution structures. In the long-term, the goal of our research program is to describe the conformational transitions and dynamics of the greater GPCR superfamily from the inactive state to the active state unhindered by crystal contacts or stabilizing proteins.