Fesik S W, Gampe R T, Eaton H L, Gemmecker G, Olejniczak E T, Neri P, Holzman T F, Egan D A, Edalji R, Simmer R
Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, Illinois 60064.
Biochemistry. 1991 Jul 2;30(26):6574-83. doi: 10.1021/bi00240a030.
Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin. The 1H and 13C NMR signals of cyclosporin A in the bound state have been assigned, and from a quantitative interpretation of the 3D NOE data, the bound conformation of CsA has been determined. Three-dimensional structures of CsA calculated from the NOE data by using a distance geometry/simulated appealing protocol were found to be very different from previously determined crystalline and solution conformations of uncomplexed CsA. In addition, from CsA/CyP NOEs, the portions of CsA that interact with cyclophilin were identified. For the most part, those CsA residues with NOEs to cyclophilin were the same residues important for cyclophilin binding and immunosuppressive activity as determined from structure/activity relationships. The structural information derived in this study together with the known structure/activity relationships for CsA analogues may prove useful in the design of improved immunosuppressants. Moreover, the approach that is described for obtaining the structural information is widely applicable to the study of small molecule/large molecule interactions.
环孢菌素A（CsA）是一种强效免疫抑制剂，已知它能与亲环蛋白（CyP）高度特异性结合，亲环蛋白是一种具有肽基脯氨酰异构酶活性的17.7 kDa蛋白质。为了研究CsA/CyP复合物的三维结构，我们应用了多种多维核磁共振方法来研究与亲环蛋白结合的均匀13C标记的CsA。已确定结合状态下环孢菌素A的1H和13C NMR信号，并通过对3D NOE数据的定量解释，确定了CsA的结合构象。利用距离几何/模拟退火协议从NOE数据计算得到的CsA三维结构与先前确定的未复合CsA的晶体结构和溶液构象非常不同。此外，通过CsA/CyP NOE，确定了CsA与亲环蛋白相互作用的部分。在很大程度上，那些与亲环蛋白有NOE的CsA残基与根据结构/活性关系确定的对亲环蛋白结合和免疫抑制活性重要的残基相同。本研究中获得的结构信息以及CsA类似物已知的结构/活性关系可能对设计改进的免疫抑制剂有用。此外，所描述的获取结构信息的方法广泛适用于小分子/大分子相互作用的研究。
