Identification of core amino acids stabilizing rhodopsin
Identification of core amino acids stabilizing rhodopsin
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DOI：
10.1073/pnas.0401429101
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发表时间：
2004-05-11
影响因子：
11.1
通讯作者：
Klein-Seetharaman, J
中科院分区：
文献类型：
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作者：
Rader, AJ;Anderson, G;Klein-Seetharaman, J
视紫红质是已知其313结构的唯一G蛋白偶联受体，因此，它是研究G蛋白偶联受体家族的原型。视紫红质功能障碍与错误折叠有关，错误折叠是由影响C110和C187之间自然产生的二硫键的化学修饰引起的。在这里，我们通过对视紫红质结构的计算分析和与先前体外突变研究的数据比较，确定稳定视紫红质的结构元件。我们使用最近发展的软包裹体和刚性亚结构拓扑学(First)方法[Jacobs，D.J.，Rader，A.J.，Kuhn，L.A.&Thorpe，M.F.(2001)Proteins 44,150-165]，通过按相对强度顺序破坏自然态氢键来模拟视紫红质的热展开。在热变性下最稳定的残基是核心的一部分，这被认为对折叠视紫红质的形成和稳定性是重要的。这个核心包括位于残基中心的C110-C187二硫键，形成跨膜和视网膜结合口袋附近的细胞外域之间的界面。利用高斯网络模型对视紫红质进行快速模式分析还发现，二硫键和视网膜配体结合袋是视紫红质中最坚硬的区域。实验证实，第一种方法预测的核心氨基酸中有90%是在突变时错误折叠的。在所有GPCR类中观察到的这种二硫键的高度保守性(78.9%)表明它对GPCRs的稳定性和功能至关重要。
Rhodopsin is the only G protein-coupled receptor (GPCR) whose 313 structure is known; therefore, it serves as a prototype for studies of the GPCR family of proteins. Rhodopsin dysfunction has been linked to misfolding, caused by chemical modifications that affect the naturally occurring disulfide bond between C110 and C187. Here, we identify the structural elements that stabilize rhodopsin by computational analysis of the rhodopsin structure and comparison with data from previous in vitro mutational studies. We simulate the thermal unfolding of rhodopsin by breaking the native-state hydrogen bonds sequentially in the order of their relative strength, using the recently developed Floppy Inclusion and Rigid Substructure Topography (FIRST) method [Jacobs, D. J., Rader,A.J., Kuhn, L.A. & Thorpe, M. F. (2001) Proteins 44,150-165]. Residues most stable under thermal denaturation are part of a core, which is assumed to be important for the formation and stability of folded rhodopsin. This core includes the C110-C187 disulfide bond at the center of residues forming the interface between the transmembrane and the extracellular domains near the retinal binding pocket. Fast mode analysis of rhodopsin using the Gaussian network model also identifies the disulfide bond and the retinal ligand binding pocket to be the most rigid region in rhodopsin. Experiments confirm that 90% of the amino acids predicted by the FIRST method to be part of the core cause misfolding upon mutation. The observed high degree of conservation (78.9%) of this disulfide bond across all GPCR classes suggests that it is critical for the stability and function of GPCRs.