Decoding Structural Determinants of Efficacy and Specificity in a GPCR Subfamily

解码 GPCR 亚家族中功效和特异性的结构决定因素

基本信息

批准号：
10572310
负责人：
Naomi Latorraca
金额：
$ 10.62万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10572310
关键词：
Address Adopted Affect Affinity Binding Biochemical Biophysics Cell membrane Clinic Communication Complex Data Deuterium Development Disease Doctor of Philosophy Drug Targeting Elements Environment Extracellular Domain FDA approved Family Fluorescence Resonance Energy Transfer G-Protein-Coupled Receptors Glutamates Goals Hydrogen Hydrogen Bonding Individual Investigation Knowledge Learning Leucine-Rich Repeat Ligand Binding Ligand Binding Domain Ligands Maps Mass Spectrum Analysis Measurement Membrane Proteins Mental Depression Mental disorders Mentors Metabotropic Glutamate Receptors Molecular Conformation Monitor Motion Nature Output Pathway interactions Pharmaceutical Preparations Physics Point Mutation Population Positioning Attribute Postdoctoral Fellow Protein Conformation Protein Family Proteins Receptor Activation Regulation Reporting Research Schizophrenia Signal Transduction Signaling Protein Specificity Stimulus Supervision Surface Synapses System Tertiary Protein Structure Testing Therapeutic Training Transmembrane Domain Variant Work academic preparation addiction biophysical techniques career computer studies cost design dimer drug discovery experimental study extracellular flexibility glutamatergic signaling leucine-rich repeat protein member molecular dynamics nervous system disorder novel novel strategies post-doctoral training programs rational design receptor response side effect simulation single molecule skills structural biology structural determinants temporal measurement theories therapeutic target transmission process

项目摘要

Project Summary G protein–coupled receptors (GPCRs), the largest family of membrane proteins and a major class of drug targets, convert extracellular stimuli into intracellular responses by undergoing conformational changes that enable them to bind and activate signaling proteins. Identifying the mechanisms that underly activation––the atomic-level rearrangements that propagate from the ligand-binding pocket to induce large-scale motions on the intracellular surface––can powerfully impact drug discovery but requires measurement of conformational change at multiple temporal and spatial scales. Thus, activation mechanisms for only a small number of GPCRs have been carefully mapped at the atomic level, hindering the rational design of therapeutics with high selectivity and few side effects. Uncovering these mechanisms for those therapeutically promising GPCRs that operate as multimers, with large extracellular domains, requires biophysical approaches that can bridge multiple scales. From my Ph.D., I have expertise in using physics-based simulations to capture conformational change in membrane proteins with high spatial and temporal resolution, but the computational cost of molecular dynamics (MD) limits accessible timescales and investigation of many members of a protein family. As a postdoctoral fellow at UC-Berkeley, I have pursued experimental studies to fill in the gaps associated with classical MD simulations. I have learned to carry out hydrogen-deuterium exchange–mass spectrometry (HDX-MS) under the supervision of Dr. Susan Marqusee, enabling me to quantify the local stability of structural elements in a protein. More recently, I have pursued an additional strategy, single-molecule Förster Resonance Energy Transfer (smFRET), to quantify the relative populations of states in a protein conformational landscape, under the guidance of Dr. Ehud Isacoff. I will pursue computational and experimental studies of a difficult-to-drug class of GPCRs, the metabotropic glutamate receptors (mGluRs). The mGluRs have a complex topology: the ligand-binding domains (LBD) of these GPCRs transmit a ligand's effects laterally, to the neighboring subunit, and intracellularly, to the transmembrane domain. In Aim 1, I will determine the mechanisms by which ligand binding affects conformational changes within a single subunit; across the dimer interface; and below, to the transmembrane domain. In Aim 2, I will investigate how sequence variation in the mGluR family leads to the strikingly broad range of glutamate affinity and efficacy previously observed for the eight mGluR subtypes. In Aim 3, I will investigate how endogenous extracellular binding partners modulate mGluRs to affect downstream activation. This work will provide a general strategy for investigating mechanisms of conformational change for multi-domain proteins and will enable discovery of allosteric ligands that can selectively target a particular receptor while eliciting a specific signaling output. The rich scientific environment of UC-Berkeley, along with the support of my outstanding mentors, will enable me to train in experimental biophysics while preparing for an academic career.

项目摘要 G蛋白 - 偶联受体（GPCR），最大的膜蛋白家族和主要类药物靶标，通过经历构象变化，将细胞外刺激转化为细胞内反应使它们能够结合并激活信号蛋白。确定在激活下的机制 - 原子级重排从配体结合口袋传播，以引起大规模运动细胞内表面 - 可以有力影响药物发现，但需要测量构象变化在多个临时和空间尺度上。那就是，只有少数GPCR的激活机制具有在原子水平上仔细映射，以高选择性和很少的副作用。发现这些机制的这些机制，用于那些以热的gpcr为单位的具有较大细胞外域的多聚体需要可以桥接多个尺度的生物物理方法。从我的博士学位上，我有使用基于物理的模拟来捕获构象变化的专业知识具有高空间和临时分辨率的膜蛋白，但分子动力学的计算成本（MD）限制了可访问的时间尺度和对蛋白质家族的许多成员的调查。作为博士后研究员在UC-Berkeley，我进行了实验研究，以填补与经典MD模拟相关的空白。我学会了在监督下执行氢 - 居民交换 - 质谱法（HDX-MS） Susan Marqusee博士使我能够量化蛋白质中结构元素的局部稳定性。更多的最近，我采用了另外的策略，即单分子förster共振能量转移（SMFRET），在蛋白质构象领域中量化状态的相对种群，在博士的指导下 Ehud Isacoff。我将购买一类GPCR类的计算和实验研究，代谢性谷氨酸受体（mglurs）。 mglurs具有复杂的拓扑：配体结合域这些GPCR的（LBD）将配体的效果横向传递到相邻的亚基，并细胞内传递到跨膜结构域。在AIM 1中，我将确定配体结合影响的机制单个亚基内的构象变化；跨二聚体接口；下面，到跨膜领域。在AIM 2中，我将研究MGLUR家族的序列变化如何导致惊人的广泛先前观察到八个mglur亚型的谷氨酸亲和力和效率的范围。在AIM 3中，我会研究内源性细胞外结合伴侣如何调节mglurs以影响下游激活。这项工作将为调查多域构象变化机制提供一般策略蛋白质并将启用可以选择可以选择性靶向特定接收器的变构配体引发特定的信号输出。 UC-Berkeley的丰富科学环境，以及我的支持杰出的导师将使我能够在为学术生涯做准备的同时培训实验性生物物理学。