BINDING INTERACTIONS OF HUMAN INTERLEUKIN-5 WITH ITS RECEPTOR-ALPHA SUBUNIT - LARGE-SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL, STUDIES OF DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS
BINDING INTERACTIONS OF HUMAN INTERLEUKIN-5 WITH ITS RECEPTOR-ALPHA SUBUNIT - LARGE-SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL, STUDIES OF DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS
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DOI：
10.1074/jbc.270.16.9459
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发表时间：
1995-04-21
影响因子：
4.8
通讯作者：
CHAIKEN, I
中科院分区：
文献类型：
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作者：
JOHANSON, K;APPELBAUM, E;CHAIKEN, I
人类白细胞介素5 （hIL5）及其受体α亚基的可溶性形式在果蝇细胞中表达并纯化到均匀性，允许详细的结构和功能分析，B细胞增殖证实hIL5具有生物活性。去糖基化的hTL5保持活性，而类似的去糖基化受体α亚基失去活性。去糖基化的hIL5的晶体结构被确定为2.6埃的分辨率，发现与大肠杆菌中产生的蛋白质相似。人IL5通过超离心分析显示与hIL5受体α亚基（shIL5R α）的可溶性结构域形成1:1的复合物。此外，hIL5中配体和受体的相对丰度。通过滴定量热法和sds -聚丙烯酰胺凝胶电泳分析，确定shIL5R α配合物为1:1。滴定微热法测得hIL5与shIL5R α结合的平衡解离常数分别为3.1 nM和2.0 nM， hIL5与含有shIL5R α的嵌合形式的受体融合到免疫球蛋白Fc结构域（shIL5R α -Fc）。对IL5及其可溶性受体的结合热力学分析表明，构象变化与结合反应相耦合，使用表面等离子体共振的动力学分析得出的数据与量热法的K-d值一致，也与hIL5与受体α亚基相互作用的构象异构化的可能性一致。在果蝇细胞膜中发现hIL5与全长hIL5R α的亲和力为6 nM，与可溶性受体形式的亲和力一致，因此α受体的大部分结合能由可溶性结构域提供。结合hIL5结构和生物活性的其他方面，获得的数据可以预测1:1的化学计量和构象变化如何导致hIL5受体α - β复合物的形成和信号转导。
Human interleukin 5 (hIL5) and soluble forms of its receptor alpha subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis, B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hTL5 remained active, while similarly deglycosylated receptor alpha subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-Angstrom resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor alpha subunit (shIL5R alpha). Additionally, the relative abundance of ligand and receptor in the hIL5 . shIL5R alpha complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 nM, respectively, for the binding of hIL5 to shIL5R alpha and to a chimeric form of the receptor containing shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction, Kinetic analysis using surface plasmon resonance yielded data consistent with the K-d values from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit, Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R alpha in Drosophila membranes was found to be 6 nM, in accord with the affinities measured for the soluble receptor forms, Hence, most of the binding energy of the alpha receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5-receptor alpha beta complex and signal transduction.