Protein-engineenng Studies on Unique Structures and Multi-functional Expression Mechanism of Plant-type Lectins

用X-射线晶体结构分析确定了美洲商陆根中5种凝集素的PL-C、PL-D1、PL-D2及其与三乙酰壳聚糖形成的复合物的三维结构。此外，通过比较PL-D1和PL-D2的结构探索了促有丝分裂活性的位点，根据PL-C的二聚体结构讨论了亚基间相互作用与功能的关系，并通过比较PL-C和PL-D2的碳水化合物复合物的结构讨论了蛋白质-蛋白质和蛋白质-碳水化合物相互作用的相似性。使用30%PEG 8000作为沉淀剂通过气相扩散法结晶PL-D1，使用18%PEG 8000作为沉淀剂通过分批法结晶PL-D2，使用2.0M硫酸铵作为沉淀剂和0.5%二氧六环作为添加剂通过气相扩散和宏观接种法结晶PL-C，和PL-D2的碳水化合物复合物通过使用20%PEG 8000作为沉淀剂的蒸气扩散法。x射线 ...更多信息 衍射数据在SR设备中被收集到高分辨率。PL-D_1、PL-D_2及配合物的结构由分子置换确定，PL-C的结构由双同晶置换确定。由于PL-D1和PL-D2的结构彼此非常相似，因此预期促有丝分裂活性的差异来自PL-D1中两个C-末端残基的延伸，并且PL-D与靶分子相互作用的位点可能存在于其C-末端附近。PL-C分子通过糖结合残基参与的亚基间相互作用以头对尾的方式采用独特的二聚体结构，从而通过覆盖糖结合位点而失去其糖结合活性。PL-D2的碳水化合物复合物采用独特的结构，其中两个PL-D2分子通过晶体学2_1-螺旋轴彼此相关，共享一个糖分子。PL-C中的蛋白质-蛋白质相互作用类似于PL-D2复合物中的蛋白质-碳水化合物相互作用。然而，前者的相互作用比后者更强。这两种相互作用之间的相似性表明，结构信息在设计用于调节凝集素功能的肽药物中是重要的。少

Of five lectins from the roots of pokeweed, three-dimensional structures of PL-C, PL-D1, PL-D2, and a complex of PL-D2 with tri-N-acetylchitotnose have been determined by X-ray crystal structure analysis. Furthermore, the site for the mitogenic activity has been explored by comparing the structures of PL-D1 and PL-D2, the relationship between intersubunit interaction and function of PL-C has been discussed on the basis of its dimer structure, and the similarity between protein-protein and protein-carbohydrate interactions has also been discussed by a structural comparison of PL-C and the carbohydrate complex of PL-D2. PL-D1 was crystallized by a vapor diffusion method using 30% PEG8000 as precipitant, PL-D2 by a batch method using 18% PEG8000 as precipitant, PL-C by a vapor diffusion and macro-seeding method using 2.0 M ammonium sulfate as precipitant and 0.5% dioxane as additive, and the carbohydrate complex of PL-D2 by a vapor diffusion method using 20% PEG8000 as precipitant. X-ray … More diffraction data were collected up to high resolution in a SR facility. The structures of PL-D1, PL-D2 and the complex were determined by molecular replacement and that of PL-C by double isomorphous replacement. Since the structures of PL-D1 and PL-D2 are very similar to each other, it has been expected that the difference in mitogenic activity comes from the extension of two C-terminal residues in PL-D1 and that the site for interaction of PL-D with target molecules may exist near its C-terminus. The PL-C molecule adopts a unique dimmer structure in a head-to-tail manner through intersubunit interactions involved by sugar-binding residues to loose its sugar-binding activity by covering the sugar-binding sites. The carbohydrate complex of PL-D2 adopts a unique structure in which two PL-D2 molecules, which are related to each other by a crystallographic 2_1-screw axis, share one sugar molecule. The protein-protein interaction in PL-C is similar to the protein-carbohydrate interaction in the complex of PL-D2. However, the former interaction is stronger than the latter. The similarity between these two kinds of interactions suggests structural information that is important in designing peptide medicines for modulating the function of lectins. Less