Structure and Activation Mechanism of the Visual Pigment Rhodopsin

视色素视紫红质的结构和激活机制

• 批准号: 8727569
• 负责人: STEVEN Owen SMITH
• 金额: $ 43.07万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8727569
• 关键词: 11 cis Retinal; Address; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; Chemicals; Complex; Coupled; Coupling; DNA Sequence Rearrangement; Data; Detergents; Disease; Family; Family member; Fluorescence Spectroscopy; Foundations; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Heterotrimeric GTP-Binding Proteins; Hydrogen Bonding; Indium; Lead; Leber' s disease; Ligand Binding; Ligands; Light; Link; Lipids; Measurement; Membrane; Methods; Micelles; Modeling; Molecular; Molecular Conformation; Motion; Mutation; NMR Spectroscopy; Night Blindness; Opsin; Optics; Peptides; Pharmacologic Substance; Photoreceptors; Positioning Attribute; Protein Binding; Receptor Activation; Research; Resolution; Retina; Retinal; Retinal Diseases; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Role; Rotation; Schiff Bases; Side; Signal Transduction; Site; Sodium Chloride; Spectroscopy, Fourier Transform Infrared; Structure; Subcellular structure; Surface; Water; Work; absorption; base; chromophore; deprotonation; drug development; extracellular; human disease; mutant; peptide G; protein activation; receptor; research study; retinal rods; solid state; solid state nuclear magnetic resonance; working group

DESCRIPTION (provided by applicant): Structure and Activation Mechanism of the Visual Pigment Rhodopsin Rhodopsin is a specialized G protein-coupled receptor (GPCR) found in vertebrate rod cells. Absorption of light by its 11-cis retinal chromophore leads to rapid photochemical isomerization and receptor activation. Structural changes on the extracellular side of rhodopsin induced by the retinal isomerization are coupled to motion of the membrane-spanning helices to create a G-protein binding pocket on the intracellular side of the receptor. The existing crystal structures of rhodopsin provide a high-resolution framework to study in detail the role of specific residues and motifs in receptor activation. Because of the high conservation of many of the key residues involved activation of rhodopsin, the emerging model indicates that rather than being unique, the visual receptors provide a basis for understanding the common structural and dynamic elements in the class A GPCRs. The general experimental strategy is to use solid-state NMR spectroscopy in combination with mutational, optical and biochemical methods to target specific regions in the inactive and active states of the receptor. The goal is to understand in atomic detail the interplay between specific signature, group-conserved and subfamily-conserved motifs in the activation mechanism of rhodopsin and derive the basis of a working model for the activation of other GPCRs. Three specific aims address structure-function questions involving regions on the extracellular side of the receptor (Aim 1), within the transmembrane (TM) core (Aim 2) and on the intracellular side of the receptor (Aim 3). In Aim 1, we describe two hydrogen-bonding networks that tether extracellular loop 2 (EL2) to the ends of the TM helices H5-H7. We propose NMR measurements to quantify the displacement of EL2 upon activation and to establish how this displacement is coupled to helix motion. In Aim 2, we target the conserved stable core of rhodopsin composed of interlocking signature and group-conserved residues. We hypothesize that H6 rotates in the conversion to Meta I and then tilts outward upon deprotonation of the retinal Schiff base and associated motion of EL2. In Aim 3, we focus on the G-protein and its interactions with residues on the intracellular surface of Meta I and Meta II. The experiments target the structural transitions between inactive and active complexes of rhodopsin with G1 peptide or G-protein. In addition, our studies address how specific mutations lead to retinal diseases through constitutive activation, receptor misfolding or stabilization of non-functional receptor conformations.

视紫红质是一种特殊的G蛋白偶联受体（GPCR），存在于脊椎动物的杆状细胞中。通过其11-顺式视网膜发色团吸收光导致快速光化学异构化和受体激活。视网膜异构化引起的视紫红质细胞外侧的结构变化与膜跨越螺旋的运动相结合，在受体的细胞内侧形成g蛋白结合袋。现有的视紫红质晶体结构为详细研究特定残基和基序在受体激活中的作用提供了高分辨率的框架。由于许多涉及视紫红质活化的关键残基的高度保守性，新出现的模型表明，视觉受体不是独一无二的，而是为理解a类gpcr的共同结构和动态元件提供了基础。一般的实验策略是使用固态核磁共振波谱结合突变、光学和生化方法来靶向受体非活性和活性状态下的特定区域。目的是在原子细节上了解视紫红质激活机制中特定特征基序、基团保守基序和亚家族保守基序之间的相互作用，并为其他gpcr的激活建立工作模型的基础。三个具体目标涉及受体细胞外侧（Aim 1）、跨膜（TM）核心（Aim 2）和受体细胞内侧（Aim 3）的结构-功能问题。在Aim 1中，我们描述了将细胞外环2 （EL2）连接到TM螺旋H5-H7末端的两个氢键网络。我们提出核磁共振测量来量化激活后EL2的位移，并确定这种位移如何与螺旋运动耦合。在Aim 2中，我们瞄准了由互锁特征和基团保守残基组成的视紫红质的保守稳定核心。我们假设H6在转化为Meta I的过程中旋转，然后在视网膜希夫碱的去质子化和EL2的相关运动时向外倾斜。在Aim 3中，我们重点研究了g蛋白及其与Meta I和Meta II细胞内表面残基的相互作用。实验的目标是紫红质与G1肽或g蛋白的无活性复合物和活性复合物之间的结构转变。此外，我们的研究解决了特定突变如何通过组成激活、受体错误折叠或非功能性受体构象的稳定导致视网膜疾病。

项目成果

Expression and purification of rhodopsin and its mutants from stable mammalian cell lines: application to NMR studies.

稳定哺乳动物细胞系中视紫红质及其突变体的表达和纯化：在 NMR 研究中的应用。

• DOI: 10.1042/bst0270950
• 发表时间: 1999
• 期刊: Biochemical Society transactions
• 影响因子: 3.9
• 作者: Reeves,PJ;Klein-Seetharaman,J;Getmanova,EV;Eilers,M;Loewen,MC;Smith,SO;Khorana,HG
• 通讯作者: Khorana,HG

Localization of the retinal protonated Schiff base counterion in rhodopsin.

视紫红质中视网膜质子化席夫碱抗衡离子的定位。

• DOI: 10.1016/s0006-3495(93)81117-2
• 发表时间: 1993
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Han,M;DeDecker,BS;Smith,SO
• 通讯作者: Smith,SO

Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation.

• DOI: 10.1038/nsmb.1549
• 发表时间: 2009-02
• 期刊: Nature structural & molecular biology
• 影响因子: 16.8

Synthesis of [19, 35, 36-(13)C(3)]-labeled TAK779 as a molecular probe.

合成[19,35,36-(13)C(3)]标记的TAK779作为分子探针。

• DOI: 10.1016/j.bmc.2009.07.026
• 发表时间: 2009
• 期刊: Bioorganic & medicinal chemistry
• 影响因子: 3.5
• 作者: Konno,Hiroyuki;Aimoto,Saburo;Smith,StevenO;Nosaka,Kazuto;Akaji,Kenichi
• 通讯作者: Akaji,Kenichi

High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

组成型活性血栓素 A2 受体多态性变体的高水平表达、纯化和表征。

• DOI: 10.1371/journal.pone.0076481
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Xu,Bing;Chakraborty,Raja;Eilers,Markus;Dakshinamurti,Shyamala;O'Neil,JoeD;Smith,StevenO;Bhullar,RajinderP;Chelikani,Prashen
• 通讯作者: Chelikani,Prashen