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

GPCR 结构、动力学和功能的下一代解决方案 NMR 技术

• 批准号: 9768515
• 负责人: GERHARD WAGNER
• 金额: $ 50.85万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9768515
• 关键词: Adenylate Cyclase; Agonist; Amides; Amino Acids; Binding; Binding Proteins; Biological Models; Biological Process; Biophysics; Cells; Complex; Crystallization; Culture Media; Cytosol; Data; Disease; Drug Targeting; Engineering; Environment; Escherichia coli; Event; Exhibits; Face; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Goals; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Hypersensitivity; Hypertension; Inhibitory G-Protein Gi; Insecta; Integral Membrane Protein; Label; Ligands; Malignant Neoplasms; Malignant neoplasm of lung; Maps; Measures; Mediating; Membrane; Membrane Proteins; Micelles; Molecular; Molecular Conformation; Monitor; Motion; NMR Spectroscopy; Neuraxis; Neurotensin; Neurotensin Receptors; Nucleotides; Pain; Parkinson Disease; Peptides; Pharmaceutical Preparations; Phospholipase C; Phospholipids; Play; Protocols documentation; Psychotic Disorders; Relaxation; Reporter; Reporting; Research; Resolution; Roentgen Rays; Role; Sampling; Schizophrenia; Second Messenger Systems; Shapes; Side; Signal Transduction; Signaling Protein; Solubility; Structure; System; Techniques; Technology; Transmembrane Domain; Vertebral column; antagonist G; cell growth; experimental study; fighting; insight; nanodisk; natural hypothermia; next generation; overexpression; receptor; reconstruction; sortilin

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.

总结 我们建议开发和应用新的溶液NMR和生物物理实验，以揭示 GPCR在配体相互作用后的结构和动力学变化，以阐明 跨膜信号G蛋白偶联受体（GPCRs）在许多生物学过程中起着重要作用。 流程.重要的是，它们中的许多是对抗过敏，精神分裂症， 高血压、精神病、癌症或神经性疼痛。在解决了GPCR的初始X射线结构之后， （Cherezov等人，2007年）已报告了约30种独特的GPCR晶体结构， 具有各种配体的结构（Munk等人，2016年）。所有这些都代表了不同活动的静态 和非活性受体，但在阐明信号传导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。我们提出了一个广泛的映射， GPCR跨膜区的动态使用溶液NMR光谱。在这里，我们最初 重点关注神经降压素受体NTR 1。13个残基的神经降压肽在神经系统中起重要作用， 多种疾病，如帕金森病、精神分裂症、抗伤害感受、体温过低和肺癌。 它在整个中枢神经系统和肠道中表达，在肠道中它与至少三种 不同的神经降压素受体（NTRs）。NTR 1和NTR 2是A类GPCR，而NTR 3属于A类GPCR。 sortilin家族神经降压素的大多数作用是通过NTR 1介导的，其中该肽充当 一种激动剂，导致异源三聚体G蛋白内的GDP/GTP交换，随后导致 磷脂酶C和腺苷酸环化酶的激活，在胞质溶胶中产生第二信使。我们 建议映射NTR 1的进化HTGH 4和TM 86 V形式的多方面动态状态， 用于揭示变构GPCR信号传导机制的模型系统。计划的研究是建立在 在我们的共价环化纳米盘工程，它可以封闭和显着稳定， 磷脂双层片中的膜蛋白（Nasr等，2017年）。此外，我们将依靠 新的先进的NMR，表达和标记技术，将允许广泛的表征， 主链末端侧链动力学。我们将追求三个具体目标： 目标1：开发绘制不同配体状态下GPCR动态图的技术。 目的2：实现胶束中激动剂结合NTR 1的许多主链和侧链分配 和纳米盘以获得感测受体动态的探针的密集网络。 目的3：表征无配体受体，这是主要但表征最少的状态， 揭示了结合激动剂、拮抗剂和异源三聚体G Gi蛋白尝试NMR表征异源三聚体G蛋白的甲基信号。