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.

肽基脯氨酰顺反异构酶（PPlases）代表了一种新的、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反异构化。这免疫抑制剂，环孢菌素 A 和 FK-506 结合并抑制两种不同的胞质 PPlases（亲环蛋白和 FK-506 结合蛋白）据信它们参与了 T 细胞激活的早期阶段。另一种 PPlase（来自果蝇的 nina A 基因产物）与视觉传导途径的调节。该项目的目标是表征这些蛋白质并确定 PPlase 和 PPlase/配体复合物的三维结构使用核磁共振波谱法和 X 射线晶体学。亲环蛋白，FK-506 结合蛋白 (FKBP) 以及这些所需的其他 PPlases 结构研究将从天然来源中分离和纯化或来自过度表达内部工程蛋白质的细胞分子生物学程序。除了未标记的 PPlas 之外，将制备用 15N 和 13C 同位素标记的蛋白质促进 NMR 研究。环孢素 A 和子囊霉素（FK-的类似物） [506] 统一用 13C 标记的细胞将从产生的细胞中分离出来这些化合物在同位素标记的培养基上生长。同位素标记 PPlase 底物也将被合成用于 NMR 研究。使用同位素标记的抑制剂（环孢菌素 A 和子囊霉素）和底物，同位素编辑的原核磁共振实验将用于亲环蛋白/环孢菌素 A、子囊霉素/FKBP 等的研究 PPlase/配体复合物。这些实验可以快速进行并进行分析将用于确定酶结合构象配体，识别参与配体的部分与酶的相互作用，并提供结构信息活跃站点。亲环蛋白的完整三维结构， FKBP 和 Pplase/配体复合物将使用同位素测定标记蛋白质和异核三维核磁共振波谱，如以及 X 射线晶体学方法。预计实验得出的三维结构 PPlase 和 PPlase/配体复合物的研究将有助于定义酶促这一类有趣的蛋白质的机制并有助于设计 PPlase 抑制剂在临床上可用作免疫抑制剂或阻断具有药理学和临床意义的其他生化过程受此类蛋白质的调节。