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

• 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
• 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

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（GPCRs）是最大的膜蛋白家族，也是一类主要的药物 靶点，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。识别激活的机制-- 从配体结合口袋传播的原子级重排诱导了 细胞内表面-可以有力地影响药物发现，但需要测量构象变化 在多个时空尺度上。因此，仅少数GPCR的激活机制具有 在原子水平上被仔细绘制，阻碍了具有高选择性的治疗方法的合理设计， 副作用少。揭示这些机制，为那些有治疗前景的GPCR，作为 具有大的胞外结构域的多聚体需要能够桥接多个尺度的生物物理方法。 从我的博士学位，我擅长使用基于物理的模拟来捕捉构象变化， 膜蛋白具有高的空间和时间分辨率，但分子动力学的计算成本 (MD)限制了可获得的时间尺度和对蛋白质家族的许多成员的研究。担任博士后研究员 在加州大学伯克利分校，我进行了实验研究，以填补与经典MD模拟相关的空白。 在老师的指导下，我学会了氢氘交换质谱法 Susan Marqusee博士的研究，使我能够量化蛋白质中结构元素的局部稳定性。更 最近，我采取了一种额外的策略，单分子福斯特共振能量转移（smFRET）， 为了量化蛋白质构象景观中状态的相对种群，在Dr. 埃胡德·伊萨科夫我将继续对一种难以给药的GPCR进行计算和实验研究， 代谢型谷氨酸受体（mGluRs）。mGluR具有复杂的拓扑结构：配体结合结构域 (LBD)这些GPCR的横向传递配体的影响，相邻的亚基，和细胞内， 跨膜结构域在目标1中，我将确定配体结合影响 在单个亚基内的构象变化;穿过二聚体界面;和下面，到跨膜 域在目标2中，我将研究mGluR家族中的序列变异如何导致惊人的广泛的 先前对八种mGluR亚型观察到的谷氨酸亲和力和功效的范围。在目标3中，我将 研究内源性细胞外结合配偶体如何调节mGluRs以影响下游激活。 这一工作将为研究多结构域构象变化机制提供一个通用的策略 蛋白质，并将使发现变构配体，可以选择性地靶向特定的受体， 引发特定的信号输出。加州大学伯克利分校丰富的科学环境，沿着我的支持， 优秀的导师，将使我能够在实验生物物理学方面的培训，同时为学术生涯做准备。