Ruan Ke-He, Wu Jiaxin, So Shui-Ping, Jenkins Lori A
Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, The University of Texas Health Science Center, Houston, TX 77030, USA.
Arch Biochem Biophys. 2003 Oct 1;418(1):25-33. doi: 10.1016/s0003-9861(03)00401-6.
In previous studies, we have determined the solution structure of the second extracellular loop (eLP(2)) of the human thromboxane A(2) receptor (TP) and identified the residues in the eLP(2) domain involved in ligand recognition, by using a combination of approaches including a constrained synthetic peptide, 2D NMR spectroscopy, and recombinant proteins. These findings led us to hypothesize that the specific ligand recognition sites may be localized in the eLP(2) for all the prostanoid receptors. To test this hypothesis, we have investigated the ligand recognition site for another prostanoid receptor, the prostacyclin receptor (IP), which mediates an opposite biological function compared to that of the TP receptor. The identification of the interaction between the IP receptor and its agonist, iloprost, was achieved with a constrained synthetic peptide mimicking the eLP(2) region of the receptor. The IP eLP(2) segment was designed and synthesized to form a constrained loop, using a homocysteine disulfide bond connecting the ends of the peptide, based on the distance predicted from the IP receptor model created by homology modeling using the crystal structure of bovine rhodopsin as a template. The evidence of the constrained IP eLP(2) interaction with iloprost was found by the identification of the conformational changes of the eLP(2) induced by iloprost using fluorescence spectroscopy, and was further confirmed by 1D and 2D 1H NMR experiments. In addition, the IP eLP(2)-induced structure of iloprost in solution was elucidated through a complete assignment of the 2D 1H NMR spectra for iloprost in the presence of the IP eLP(2) segment. In contrast, no ordered structure was observed in the 2D 1H NMR experiments for iloprost alone in solution. These studies not only identified that the eLP(2) segment of the IP receptor is involved in ligand recognition, but also solved the 3D solution structure of the bound-form of iloprost, which could be used to study the receptor-ligand interaction in structural terms.
在先前的研究中，我们已确定人血栓素A2受体（TP）第二个细胞外环（eLP(2)）的溶液结构，并通过使用包括约束合成肽、二维核磁共振光谱和重组蛋白在内的多种方法，鉴定了eLP(2)结构域中参与配体识别的残基。这些发现使我们推测，所有前列腺素受体的特异性配体识别位点可能都位于eLP(2)中。为了验证这一假设，我们研究了另一种前列腺素受体——前列环素受体（IP）的配体识别位点，该受体介导与TP受体相反的生物学功能。通过模拟该受体eLP(2)区域的约束合成肽，实现了IP受体与其激动剂伊洛前列素之间相互作用的鉴定。基于以牛视紫红质晶体结构为模板通过同源建模创建的IP受体模型预测的距离，设计并合成了IP eLP(2)片段，使其形成一个约束环，使用同型半胱氨酸二硫键连接肽的两端。通过荧光光谱法鉴定伊洛前列素诱导的eLP(2)构象变化，发现了约束IP eLP(2)与伊洛前列素相互作用的证据，并通过一维和二维1H NMR实验进一步证实。此外，通过对存在IP eLP(2)片段时伊洛前列素的二维1H NMR谱进行完全归属，阐明了溶液中IP eLP(2)诱导的伊洛前列素结构。相比之下，在溶液中单独对伊洛前列素进行的二维1H NMR实验中未观察到有序结构。这些研究不仅确定了IP受体的eLP(2)片段参与配体识别，还解析了伊洛前列素结合形式的三维溶液结构，可用于从结构角度研究受体-配体相互作用。
