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

基本信息

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
项目类别：
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

项目摘要

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.

概括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在配体相互作用时的结构和动态变化，以阐明跨膜信号传导。 G蛋白偶联受体（GPCR）在许多生物学中起着核心作用过程。重要的是，其中许多是对抗过敏的主要药物，精神分裂症，高血压，精神病，癌症或神经源性疼痛。在解决GPCR的初始X射线结构之后（Cherezov等人，2007年）已经报道了大约30个独特的GPCR晶体结构，〜100 具有各种配体的结构（Munk等，2016）。所有这些代表了不同活动的静态状态和非活性受体，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过渡。我们提出了大量的映射使用溶液NMR光谱法，GPCR的跨膜区域的动态状态。我们最初在这里专注于神经素受体NTR1。 13个残基神经素肽肽在多种疾病，例如帕金森氏症，精神分裂症，抗伤害感受，体温过低以及肺癌。它在整个中枢神经系统和肠道中表达，至少与三个结合不同的神经素受体（NTR）。 NTR1和NTR2是A类GPCR，而NTR3属于 Tortilin家族。神经素的大多数作用是通过NTR1介导的，其中肽的作用为激动剂，导致异三聚体G蛋白内GDP/GTP交换，然后磷脂酶C和腺苷酸环化酶的激活，它们在细胞质中产生第二个使者。我们提议将NTR1的进化HTGH4和TM86V形式的多面动态状态映射为揭示变构GPCR信号传导机制的模型系统。计划的研究是建立的关于我们共价循环的纳米盘的工程，可以大幅稳定并稳定磷脂双层斑块中的膜蛋白（Nasr等，2017）。此外，我们将依靠新的高级NMR，表达和标签技术，可以广泛表征骨干端侧链动力学。我们将追求三个具体目标：目标1：开发用于映射不同配体状态GPCR的动态景观的技术。 AIM 2：在胶束中实现动力学生成的NTR1的大量骨干和侧链分配纳米盘以获得探针的致密网络，以感知受体的动态状态。 AIM 3：表征没有配体的受体，这是主要但最少的状态，并且揭示结合激动剂，拮抗剂和异三体G的结构和动态变化蛋白质GI。尝试NMR表征异三聚体G蛋白的甲基信号。