Next Generation Solution NMR Techniques for GPCR Structure, Dynamics and Function

摘要 我们建议开发和应用新的溶液核磁共振和生物物理实验，以揭示 GPCRs的结构和动态变化对配体相互作用的影响 跨膜信号。G蛋白偶联受体(GPCRs)在许多生物学领域发挥着重要作用 流程。重要的是，它们中的许多都是对抗过敏、精神分裂症、 高血压、精神病、癌症或神经源性疼痛。在解决了GPCR的初始X射线结构之后 (Cherezov等人)2007)已经报道了大约30个独特的GPCR晶体结构和~100个 具有各种配体的结构(Munk等人，2016)。所有这些都代表了不同活动的静态 和不活跃的受体，但在阐明信号的动态机制方面受到限制 涉及许多不同动态状态之间的转换。我们提出了一种广泛的映射 用溶液核磁共振波谱研究GPCRs跨膜区的动态。在这里，我们最初 关注神经降压素受体NTR1。13个残基的神经降压素肽在 多种疾病，如帕金森、精神分裂症、抗伤害性感觉、体温过低和肺癌。 它在整个中枢神经系统和肠道中都有表达，在那里它至少与三个 不同的神经降压素受体。NTR1和NTR2是A类GPCR，而NTR3属于 山梨素家族。神经降压素的大部分作用是通过NTR1介导的，在NTR1中，多肽起着 一种激动剂，导致异三聚体G蛋白内的GDP/GTP交换，随后导致 激活磷脂酶C和腺苷环化酶，它们在胞质中产生第二信使。我们 建议将NTR1的HTGH4和TM86V进化形式的多方面动态映射为 揭示变构gpr信号机制的模型系统。计划中的研究是建立起来的 关于我们的共价循环纳米盘的工程，它可以封闭并显著稳定 磷脂双层斑块中的膜蛋白(Nasr等人，2017年)。此外，我们将依靠 新的先进的核磁共振、表达和标记技术，将允许广泛的表征 主干末端侧链动力学。我们将追求三个具体目标： 目的1：开发绘制不同配基状态下GPCRs动态地形图的技术。 目标2：在胶束中实现激动剂结合的NTR1的大量主链和侧链分配 和纳米盘，以获得检测受体动态状态的密集的探针网络。 目的3：表征非配体受体，这是最主要但最不典型的状态 揭示结合激动剂、拮抗剂和异三聚体G的结构和动态变化 蛋白质Gi。尝试核磁共振表征异源三聚体G蛋白的甲基信号。

Summary We propose to develop and apply new solution NMR and biophysical experiments tailored to reveal structural and dynamic changes of GPCRs upon ligand interactions in order to elucidate mechanisms of transmembrane signaling. G protein-coupled receptors (GPCRs) play central roles in many biological processes. Importantly, many of them are major drug targets for fighting allergies, schizophrenia, hypertension, psychosis, cancer or neurogenic pain. After the initial X-ray structure of a GPCR was solved (Cherezov et al. 2007) approximately 30 unique GPCR crystal structures have been reported and ~ 100 structures with various ligands (Munk et al., 2016). All of these represent static states of different active and inactive receptors but are limited in elucidating the dynamic mechanisms of signaling which is thought to involve transitions between many different dynamic states. We propose an extensive mapping of the dynamic states of the transmembrane region of GPCRs using solution NMR spectroscopy. Here we initially focus on the neurotensin receptor NTR1. The 13-residue neurotensin peptide plays important roles in multiple diseases, such as Parkinson, schizophrenia, antinociception, and hypothermia and in lung cancer. It is expressed throughout the central nervous system and in the gut, where it binds to at least three different neurotensin receptors (NTRs). NTR1 and NTR2 are class A GPCRs whereas NTR3 belongs to the sortilin family. Most of the effects of neurotensin are mediated through NTR1, where the peptide acts as an agonist, leading to GDP/GTP exchange within heterotrimeric G proteins and subsequently to the activation of phospholipase C and adenylyl cyclase, which produce second messengers in the cytosol. We propose to map the multifaceted dynamic states of the evolved HTGH4 and TM86V forms of NTR1 as model systems for revealing mechanisms of allosteric GPCR signaling. The planned research is founded on our engineering of covalently circularized nanodiscs, which can enclose and dramatically stabilize membrane proteins in patches of phospholipid bilayers (Nasr et al., 2017). Furthermore, we will rely on new advanced NMR, expression and labeling techniques that will allow extensive characterization of backbone end side chain dynamics. We will pursue three Specific Aims: Aim 1: Develop technologies for mapping the dynamic landscape of GPCRs in different ligand states. Aim 2: Achieve numerous backbone and side chain assignments for agonist-bound NTR1 in micelles and nanodiscs to obtain a dense network of probes sensing the dynamic state of the receptor. Aim 3: Characterize the ligand-free receptor, which is the main but least characterized state and reveal structural and dynamic changes upon binding agonists, antagonists and the heterotrimeric G protein Gi. Attempt NMR characterization of methyl signals of the heterotrimeric G protein.