Human P2Y1 receptor:: Molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites
Human P2Y1 receptor:: Molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites
复制标题
DOI：
10.1021/jm970684u
复制
发表时间：
1998-04-23
影响因子：
7.3
通讯作者：
Jacobson, KA
中科院分区：
文献类型：
--
作者：
Moro, S;Guo, DP;Jacobson, KA
采用定点突变和分子模拟技术，探讨了新型竞争性拮抗剂2 ′-脱氧-N-6-甲基腺苷3 ′，5 ′-二磷酸（MRS 2179）被人P2 Y（1）受体识别的分子基础。这种拮抗剂的效力是在突变受体中测量的，其中跨膜螺旋结构域（TM）3、5、6和7中的关键残基被Ala或其他氨基酸取代。MRS 2179阻断由2-甲硫基腺苷5 '-二磷酸（2-MeSADP）促进的磷脂酶C刺激的能力在具有F226 A、K280 A或Q307 A突变的P2 Y（1）受体中丧失，表明这些残基对于拮抗剂分子的结合至关重要。残基His 132、Thr 222和Tyr 136的突变对MRS 2179阻断P2 Y（1）受体的能力具有中等影响。因此，这些位置似乎在识别这种拮抗剂中具有调节作用。F131 A、H277 A、T221 A、R310 K或S317 A突变体受体对PARS 2179表现出与野生型受体相似的表观亲和力。因此，Phe 131、Thr 221、His 277和Ser 317对于拮抗剂识别不是必需的。建立并分析了人类P2 Y（1）受体的计算机生成模型，以帮助解释这些结果。该模型是通过一级序列比较，二级结构预测，三维同源性建设，使用视紫红质作为模板，并与诱变研究获得的数据是一致的。我们已经引入了一个“交叉对接”的程序，以获得精力充沛的精细的配体-受体复合物的三维结构。交叉对接模拟由配体诱导的天然受体结构的重组。一个假定的核苷酸结合位点被定位，并用于预测哪些残基可能接近激动剂和拮抗剂。根据我们的模型，TM 6和TM 7靠近腺嘌呤环，TM 3和TM 6靠近核糖部分，TM 3、TM 6和TM 7靠近三磷酸链。
The molecular basis for recognition by human P2Y(1) receptors of the novel, competitive antagonist 2'-deoxy-N-6-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 P2Y(1) 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 P2Y(1) 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 PARS 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 P2Y(1) 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.