Identification and analysis of functionally important amino acids in human purinergic 12 receptor using a Saccharomyces cerevisiae expression system
Identification and analysis of functionally important amino acids in human purinergic 12 receptor using a Saccharomyces cerevisiae expression system
复制标题
DOI：
10.1111/j.1742-4658.2011.08410.x
复制
发表时间：
2012-01-01
期刊：
影响因子：
5.4
通讯作者：
Klovins, Janis
中科院分区：
文献类型：
--
作者：
Ignatovica, Vita;Megnis, Kaspars;Klovins, Janis
嘌呤能12受体（P2 Y12）是抗凝治疗的主要药物靶点，但对该受体的配体结合和激活所涉及的区域知之甚少。我们生成了四个随机化的P2 Y12文库，并研究了它们的配体结合特性。在酿酒酵母模型系统中表达P2 Y12。用在位置181、256、265和280处的随机化氨基酸产生四个文库。使用天然P2 Y12配体ADP在酵母中筛选突变体变体的功能活性。活化结果进行了研究，使用定量构效关系（QSAR）模型和配体受体对接。我们通过用具有不同理化性质的氨基酸取代来筛选P2 Y12中的四个位置的功能活性。该分析表明，位置E181、R256和R265以特定方式改变P2 Y12的功能活性。E181和R256突变体库的QSAR模型强烈支持实验数据。氨基酸K280的所有取代都是完全失活的，突出了该残基在P2 Y12功能中的关键作用。配体-受体对接表明K280可能是P2 Y12配体结合口袋中的关键元件。本研究的结果表明，P2 Y12的位置181，256，265和280对于受体的功能完整性是重要的。此外，K280似乎是P2 Y12配体结合口袋的关键特征。这些结果对合理设计新型抗血小板药物具有重要意义。
The purinergic 12 receptor (P2Y12) is a major drug target for anticoagulant therapies, but little is known about the regions involved in ligand binding and activation of this receptor. We generated four randomized P2Y12 libraries and investigated their ligand binding characteristics. P2Y12 was expressed in a Saccharomyces cerevisiae model system. Four libraries were generated with randomized amino acids at positions 181, 256, 265 and 280. Mutant variants were screened for functional activity in yeast using the natural P2Y12 ligand ADP. Activation results were investigated using quantitative structureactivity relationship (QSAR) models and ligandreceptor docking. We screened four positions in P2Y12 for functional activity by substitution with amino acids with diverse physiochemical properties. This analysis revealed that positions E181, R256 and R265 alter the functional activity of P2Y12 in a specific manner. QSAR models for E181 and R256 mutant libraries strongly supported the experimental data. All substitutions of amino acid K280 were completely inactive, highlighting the crucial role of this residue in P2Y12 function. Ligandreceptor docking revealed that K280 is likely to be a key element in the ligand-binding pocket of P2Y12. The results of this study demonstrate that positions 181, 256, 265 and 280 of P2Y12 are important for the functional integrity of the receptor. Moreover, K280 appears to be a crucial feature of the P2Y12 ligand-binding pocket. These results are important for rational design of novel antiplatelet agents.