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Human P2Y1 Receptor: Molecular Modeling and Site-Directed Mutagenesis as Tools To Identify Agonist and Antagonist Recognition Sites

机译：人类P2Y1受体：分子建模和定点诱变作为识别激动剂和拮抗剂识别位点的工具。

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摘要

The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5′-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a “cross-docking” procedure to obtain energetically refined 3D structures of the ligand–receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.
机译：通过定点诱变和人为探测，探索了人类P2Y1受体识别新型竞争性拮抗剂2'-脱氧-N 6 -甲基腺苷3'，5'-二磷酸酯（MRS 2179）的分子基础。分子建模。在突变受体中测量该拮抗剂的效力，在突变受体中，跨膜螺旋结构域（TMs）3、5、6和7中的关键残基被Ala或其他氨基酸取代。在具有F226A，K280A或Q307A突变的P2Y1受体中，MRS 2179阻止了由2-甲基硫代腺苷5'-二磷酸（2-MeSADP）促进的磷脂酶C刺激的能力，这表明这些残基对于结合蛋白至关重要。拮抗剂分子。残基His132，Thr222和Tyr136的突变对MRS 2179阻断P2Y1受体的能力具有中等影响。因此，这些位置似乎在识别该拮抗剂中具有调节作用。 F131A，H277A，T221A，R310K或S317A突变受体对MRS 2179的表观亲和力与野生型受体相似。因此，Phe131，Thr221，His277和Ser317对于拮抗剂识别不是必需的。建立并分析了计算机生成的人P2Y1受体模型，以帮助解释这些结果。该模型通过视紫红质作为模板，通过一级序列比较，二级结构预测和三维同源性构建而获得，与诱变研究获得的数据一致。我们引入了一种“跨接库”程序，以获取经能量精炼的配体-受体复合物的3D结构。交叉停靠模拟由配体诱导的天然受体结构的重组。推定的核苷酸结合位点被定位并用于预测哪些残基可能与激动剂和拮抗剂接近。根据我们的模型，TM6和TM7靠近腺嘌呤环，TM3和TM6靠近核糖部分，TM3，TM6和TM7靠近三磷酸链。

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