Structure and Activation Mechanism of the Visual Pigment Rhodopsin

说明(申请人提供)：视紫红质的结构和激活机制视紫红质是一种在脊椎动物视杆细胞中发现的专门的G蛋白偶联受体(GPCR)。其11顺式视网膜生色团对光的吸收导致快速的光化学异构化和受体激活。由视网膜异构化引起的视紫红质胞外侧的结构变化与跨膜螺旋的运动相耦合，在受体的胞内侧产生一个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一个高分辨率的框架。由于与视紫红质激活有关的许多关键残基的高度保守性，新出现的模型表明，视觉受体不是唯一的，而是为理解A类GPCRs中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，针对受体的非活性和活性状态中的特定区域。其目的是从原子上详细了解特定的特征基序、基团保守基序和亚家族保守基序之间在视紫红质激活机制中的相互作用，并为其他GPCR的激活提供工作模型的基础。三个具体的目标涉及受体细胞外侧(目标1)、跨膜(TM)核心内(目标2)和受体细胞内侧(目标3)的区域的结构-功能问题。在目标1中，我们描述了两个氢键网络，它们将细胞外环2(EL2)拴在TM螺旋H5-H7的末端。我们建议通过核磁共振测量来量化EL2在激活时的位移，并确定这种位移是如何与螺旋运动耦合的。在目标2中，我们瞄准了视紫红质的保守稳定核心，该核心由连锁信号和基团保守残基组成。我们假设H6在转变为Meta I的过程中旋转，然后在视网膜Schiff碱基去质子化和EL2的相关运动时向外倾斜。在目标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。实验的目标是视紫红质与G1肽或G蛋白的非活性和活性复合体之间的结构转换。此外，我们的研究还阐述了特定的突变如何通过结构性激活、受体错误折叠或非功能受体构象的稳定而导致视网膜疾病。

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.