Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B7-1
Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B7-1
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DOI：
10.1107/s0907444901001895
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发表时间：
2001-04-01
期刊：
影响因子：
--
通讯作者：
Stuart, DI
中科院分区：
文献类型：
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作者：
Davis, SJ;Ikemizu, S;Stuart, DI
B7-1与CD 28和CTLA-4的相互作用调节人类免疫应答的过程，使B7-1成为开发基于结构的治疗剂的重要靶标。然而，B7-1是在白细胞表面发现的最严重糖基化的蛋白质之一，使该分子的结构分析复杂化。描述了该分子的可溶性去糖基化形式的生产、结晶和硒代甲硫氨酸标记的方法。该蛋白质容易形成四面体板和双锥晶体，分别属于空间群I4（1）22和P4（1）22（或P4（3）22），晶胞参数a = B = 56.9，c = 298.7埃，晶胞参数a = B = 89.0，c = 261.9埃。I4（1）22和原始晶体形式分别为2.7和3.5埃。基于表面等离子体共振的测定表明，sB 7 -1的配体结合特性不受去糖基化的影响。由于没有一种方法依赖于任何特殊的结构特性的sB 7 -1，它建议，这种新的组合程序可以在原则上适用于许多其他糖蛋白的结构或功能的兴趣的系统分析。
The interactions of B7-1 with CD28 and CTLA-4 modulate the course of human immune responses, making B7-1 an important target for developing structure-based therapeutics. B7-1 is, however, one of the most heavily glycosylated proteins found at the leukocyte cell surface, complicating the structural analysis of this molecule. Methods for the production, crystallization and selenomethionine labelling of a soluble deglycosylated form of this molecule are described. The protein readily forms both tetragonal plate and bipyramidal crystals belonging to space groups I4(1)22, with unit-cell parameters a = b = 56.9, c = 298.7 Angstrom, and P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 89.0, c = 261.9 Angstrom, respectively. The I4(1)22 and primitive crystal forms diffract to 2.7 and 3.5 Angstrom, respectively. Surface plasmon resonance-based assays indicate that the ligand-binding properties of sB7-1 are unaffected by deglycosylation. Since none of the methods relied on any special structural properties of sB7-1, it is proposed that this novel combination of procedures could in principle be adapted to the systematic analysis of many other glycoproteins of structural or functional interest.