Ruan Cheng-Huai, Wu Jaixin, Ruan Ke-He
Vascular Biology Research Center, Division of Hematology, Department of Internal Medicine, The University of Texas Health Science Center, Houston, Texas 77030, USA.
BMC Biochem. 2005 Nov 4;6:23. doi: 10.1186/1471-2091-6-23.
Prostacyclin receptor (IP) and thromboxane A2 receptor (TP) belong to rhodopsin-type G protein-coupling receptors and respectively bind to prostacyclin and thromboxane A2 derived from arachidonic acid. Recently, we have determined the extracellular loop (eLP) structures of the human TP receptor by 2-D 1H NMR spectroscopy using constrained peptides mimicking the individual eLP segments. The studies have identified the segment along with several residues in the eLP domains important to ligand recognition, as well as proposed a ligand recognition pocket for the TP receptor.
The IP receptor shares a similar primary structure in the eLPs with those of the TP receptor. Forty percent residues in the second eLPs of the receptors are identical, which is the major region involved in forming the ligand recognition pocket in the TP receptor. Based on the high homology score, the eLP domains of the IP receptor were constructed by the homology modeling approach using the NMR structures of the TP eLPs as templates, and then configured to the seven transmembrane (TM) domains model constructed using the crystal structure of the bovine rhodopsin as a template. A NMR structure of iloprost was docked into the modeled IP ligand recognition pocket. After dynamic studies, the segments and residues involved in the IP ligand recognition were proposed. A key residue, Arg173 involved in the ligand recognition for the IP receptor, as predicted from the modeling, was confirmed by site-directed mutagenesis.
A 3-D model of the human IP receptor was constructed by homology modeling using the crystal structure of bovine rhodopsin TM domains and the NMR structures of the synthetic constrained peptides of the eLP domains of the TP receptor as templates. This strategy can be applied to molecular modeling and the prediction of ligand recognition pockets for other prostanoid receptors.
前列环素受体（IP）和血栓素A2受体（TP）属于视紫红质型G蛋白偶联受体，分别与源自花生四烯酸的前列环素和血栓素A2结合。最近，我们使用模拟各个细胞外环（eLP）片段的受限肽，通过二维1H NMR光谱法确定了人TP受体的细胞外环结构。这些研究确定了eLP结构域中对配体识别重要的片段以及几个残基，并提出了TP受体的配体识别口袋。
IP受体在eLP中的一级结构与TP受体相似。受体第二个eLP中40%的残基是相同的，这是TP受体中参与形成配体识别口袋的主要区域。基于高同源性得分，以TP eLP的NMR结构为模板，通过同源建模方法构建IP受体的eLP结构域，然后将其配置到以牛视紫红质晶体结构为模板构建的七跨膜（TM）结构域模型中。将依洛前列素的NMR结构对接至模拟的IP配体识别口袋中。经过动力学研究，提出了参与IP配体识别的片段和残基。通过定点诱变证实了建模预测的参与IP受体配体识别的关键残基Arg173。
以牛视紫红质TM结构域的晶体结构和TP受体eLP结构域的合成受限肽的NMR结构为模板，通过同源建模构建了人IP受体的三维模型。该策略可应用于其他前列腺素受体的分子建模和配体识别口袋的预测。
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