STRUCTURES OF PEPTIDYL-PROLYL ISOMERASE/LIGAND COMPLEXES

肽基脯氨酰顺反异构酶（PPlases）是近年来发现的一种新的 可能调节信号转导途径的蛋白质家族的扩展 通过催化肽键的顺反异构化。 的 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PP酶（亲环素和FK-506结合蛋白） 其被认为参与T细胞活化的早期阶段。 另一种PPlase（来自果蝇的nina A基因产物）与 视觉传导通路的调节。 该项目的目标是表征这些蛋白质，并确定 PPase和PPase/配体复合物的三维结构 使用核磁共振光谱和X射线晶体学。 亲环素，FK-506 结合蛋白（FKBP），以及这些所需的其他PPase 结构研究将从天然来源中分离和纯化， 从过度表达蛋白质的细胞中， 分子生物学程序。 除了未标记的PP酶， 将制备用15 N和13 C同位素标记的蛋白质， 方便NMR研究。 环孢菌素A和子囊霉素（FK-1的类似物） 506)用13 C均匀标记的细胞将从产生 这些化合物在同位素标记的培养基上生长。 同位素标记 还将合成PPlase底物用于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NMR实验将用于 亲环素/环孢菌素A、子囊霉素/FKBP和其他的研究 PPase/配体复合物。 这些实验，可以快速进行， 并进行分析，以确定酶结合的构象 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， 活性部位 亲环素的完整三维结构， FKBP和PPlase/配体复合物将使用同位素测定。 标记的蛋白质和异构三维NMR光谱，如 以及通过X射线晶体学方法。 可以预期的是，实验得到的三维结构 PPlase和PPlase/配体复合物将有助于定义酶促 这类有趣的蛋白质的机制，并有助于设计 在临床上可用作免疫抑制剂或 阻断药理学和临床上感兴趣的其他生化过程 由这类蛋白质调控的基因。

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.