STRUCTURES OF PEPTIDYL-PROLYL ISOMERASE/LIGAND COMPLEXES

肽基-脯氨酰异构酶/配体复合物的结构

基本信息

批准号：
2183102
负责人：
STEPHEN W FESIK
金额：
$ 15.79万
依托单位：
ABBOTT LABORATORIES
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-07-01 至 1995-06-30
项目状态：
已结题

项目摘要

Peptidyl-prolyl cis-trans isomerases (PPlases) represent a new and rapidly expanding family of proteins that may regulate signal transduction pathways by catalyzing the cis-trans isomerization of peptide bonds. The immunosuppressive agents, cyclosporin A and FK-506 bond to and inhibit two different cytosolic PPlases (cyclophilin and the FK-506 binding protein) which are believed to be involved in the early stages of T-cell activation. Another PPlase (nina A gene product from Drosophila) has been implicated in the regulation of the visual transduction pathway. The goal of this project is to characterize these proteins and to determine the three-dimensional structures of the PPlases and PPlase/ligand complexes using NMR spectroscopy and X-ray crystallography. Cyclophilin, the FK-506 binding protein (FKBP), and the other PPlases that are required for these structural studies will be isolated and purified from natural sources or from cells that overexpress the proteins engineered in-house using molecular biological procedures. In addition to unlabeled PPlases, proteins isotopically labeled with 15N and 13C will be prepared to facilitate the NMR studies. Cyclosporin A and ascomycin (an analog of FK- 506) uniformly labeled with 13C will be isolated from cells that produce these compounds grown on isotopically labeled media. Isotopically labeled PPlase substrates will also be synthesized for the NMR studies. Using the isotopically labeled inhibitors (cyclosporin A and ascomycin) and substrates, isotope-edited proto NMR experiments will be employed in studies of cyclophilin/cyclosporin A, ascomycin/FKBP, and other PPlase/ligand complexes. These experiments, which can be rapidly performed and analyzed will be used to determine the enzyme-bound conformations of the ligands, identify the portions of the ligands involved in the interaction with the enzyme, and provide structural information on the active site. The complete three-dimensional structures of cyclophilin, FKBP, and PPlase/ligand complexes will be determined using isotopically labeled proteins and heteronuclear three-dimensional NMR spectroscopy, as well as by x-ray crystallographic methods. It is expected that the experimentally-derived three-dimensional structures of PPlases and PPlase/ligand complexes will help define the enzymatic mechanism(s) of this interesting class of proteins and aid in the design of PPlase inhibitors that are clinically useful as immunosuppressants or that block other biochemical processes of pharmacological and clinical interest that are regulated by this class of proteins.

肽基 - 丙基顺式传播异构酶（PPLASE）代表了一种新的和快速的扩大可能调节信号转导途径的蛋白质家族通过催化肽键的顺式传播异构化。这免疫抑制剂，环孢菌素A和FK-506键与两个不同的胞质pplase（环磷脂和FK-506结合蛋白）据信这与T细胞激活的早期阶段有关。另一个PPLASE（Nina A基因产物来自果蝇的基因产物）已与视觉转导途径的调节。该项目的目的是表征这些蛋白质并确定 Pplase和Pplase/配体配合物的三维结构使用NMR光谱和X射线晶体学。环磷脂，FK-506 结合蛋白（FKBP）和这些所需的其他PPLASE 结构研究将被隔离并从天然来源或从过表达蛋白质内部设计的细胞分子生物学程序。除了未标记的pplase，用15N和13C标记的同位素标记的蛋白质将准备好促进NMR研究。环孢菌素A和commycin（FK-的类似物 506）将从产生的细胞中分离出13C的标记这些化合物在同位素标记的介质上生长。同位素标记 NMR研究也将合成PPLASE底物。使用同位素标记的抑制剂（环孢菌素A和子霉素）和基材，同位素编辑的原始NMR实验将用于环胞素/环孢菌素A，子霉素/FKBP和其他的研究 PPLASE/配体配合物。这些实验可以迅速进行分析将用于确定酶结合的构象配体，确定涉及的配体的部分与酶相互作用，并提供有关该酶的结构信息活性站点。环糖蛋白的完整三维结构， FKBP和PPLASE/配体配合物将使用同位素确定标记的蛋白质和异核三维NMR光谱学为以及X射线晶体学方法。预计实验衍生的三维结构 Pplase和Pplase/Pplase/配体配合物将有助于定义酶促这种有趣的蛋白质的机制，并有助于设计在临床上用作免疫抑制剂的PPLASE抑制剂或阻止药理和临床兴趣的其他生化过程由这类蛋白质调节。