THE FIRST CRYSTAL STRUCTURE OF PROTEIN KINASE

蛋白激酶的第一个晶体结构

• 批准号: 3293185
• 负责人: JANUSZ M SOWADSKI
• 金额: $ 18.27万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3293185
• 关键词: X ray crystallography; active sites; adenosine triphosphate; chemical binding; computer simulation; conformation; crystallization; enzyme complex; enzyme inhibitors; enzyme structure; enzyme substrate complex; liquid crystal; molecular dynamics; molecular site; mutant; myristates; physical model; protein kinase A

The catalytic subunit of cAMP-dependent protein kinase represents the basic catalytic subunit that is conserved in every protein kinase and therefore, owing to the structural homology between protein kinases, may serve as a model for viewing the entire family of over one hundred protein kinases. Crystallization studies resulted in several different complexes in tow different space groups (P212121 and P6122). Structure solution of the catalytic subunit will be based on our best crystal, an orthorhombic crystal (P2121) of a binary complex of recombinant catalytic subunit with a specific 20-amino acid peptide inhibitor. A mercury derivative was used to identify two cysteine residues, Cys 199 and Cys 343. The "phasing power" (fr.m.s./Er.m.s.) of this derivative ranged from 3.13 to 1.91 within the resolution range of 10.15Angstrom to 3.61Angstrom. An electron density map to 2.7Angstrom resolution has been obtained by first generating a 3.5Angstrom resolution map using the B.C. Wang error filtering method and, second, resolution extension. The CdATP binding site was located using CdATP co-crystals of the ternary complex, with a specific peptide inhibitor. The identified ATP binding site appears in the immediate vicinity of Cys 199, as predicted from other biochemical studies. Within the elongated bilobal molecule, seven helices and nine beta-strands have been identified. Model building at the present stage consists of 90% alpha-carbons. Structure solution of the hexagonal crystal (P6122) of the pig heart catalytic subunit (myristic acid present) ternary complex with MgATP and a specific peptide inhibitor is based on the molecular envelope of the binary complex and the method of molecular replacement. The molecular orientation in the hexagonal unit cell was determined using the fast rotation function and data in the resolution range of 5.0Angstrom to 3.0Angstrom. The translational function applied to all Harker sections determined the molecular position and selected the correct space group. The real space orientation matrix is: 0.5331, -0.0356, 1.0000, 1.0504, - 0.0701 -0.0101, 0.0150, 0.2625, 0.0000 and the position of Hg-Cys 199 is: x= -0.082; y= -0.371; z= -0.033. This position corresponds to the position of Hg-Cys 199 x= 0.365, y= -0.133, z= 0.542 in the orthorhombic cell (P212121). Structure solution of two crystal forms will reveal not only the architecture of the catalytic subunit with and without myristic acid, but also the topology of the ATP and peptide binding sites. Furthermore, those structural studies will allow to proceed the crystallographic analysis of various mutants, the first of which has already been crystallized (Cys 343->Ser).

cAMP 依赖性蛋白激酶的催化亚基代表了基本的 每个蛋白激酶中都保守的催化亚基，因此， 由于蛋白激酶之间的结构同源性，可以作为 用于查看一百多个蛋白激酶的整个家族的模型。 结晶研究产生了两种不同的复合物 不同的空间群（P212121 和 P6122）。 结构解 催化亚基将基于我们最好的晶体，一种斜方晶系 重组催化亚基二元复合物的晶体（P2121） 特异性20个氨基酸肽抑制剂。 汞衍生物被用来 识别两个半胱氨酸残基，Cys 199 和 Cys 343。“定相能力” 该导数的 (fr.m.s./Er.m.s.) 范围为 3.13 至 1.91 分辨率范围为10.15埃至3.61埃。 电子密度图 至 2.7 埃的分辨率已通过首先生成 3.5埃分辨率地图使用B.C. Wang错误过滤方法和， 第二，决议延伸。 使用以下方法定位 CdATP 结合位点： 三元复合物的 CdATP 共晶，具有特定的肽 抑制剂。 识别出的 ATP 结合位点立即出现在 正如其他生化研究预测的那样，Cys 199 附近。 之内 细长的双叶分子，七个螺旋和九个β链 已被识别。 现阶段模型建设占90% α-碳。 六方晶体（P6122）的结构解 猪心催化亚基（存在肉豆蔻酸）三元复合物 MgATP 和特定的肽抑制剂基于分子包膜 二元配合物的结构和分子置换方法。 这 使用以下方法确定六方晶胞中的分子取向 快速旋转功能和数据分辨率范围为5.0埃至 3.0埃。 应用于所有 Harker 部分的平移功能 确定了分子位置并选择了正确的空间群。 真实空间方位矩阵为：0.5331、-0.0356、1.0000、1.0504、- 0.0701 -0.0101、0.0150、0.2625、0.0000，Hg-Cys 199 的位置为： x=-0.082； y=-0.371； z=-0.033。 这个位置对应的位置 斜方晶格中 Hg-Cys 199 x= 0.365，y= -0.133，z= 0.542 （P212121）。 两种晶型的结构解析不仅将揭示 有和没有肉豆蔻酸的催化亚基的结构， 还包括 ATP 和肽结合位点的拓扑结构。 此外， 这些结构研究将有助于进行晶体学研究 各种突变体的分析，其中第一个已经被 结晶（Cys 343->Ser）。