Van Eijsden R R, De Pater B S, Kijne J W
Institute of Molecular Cytology, University of Amsterdam, The Netherlands.
Glycoconj J. 1994 Aug;11(4):375-80. doi: 10.1007/BF00731212.
Comparison of x-ray crystal structures of several legume lectins, co-crystallized with sugar molecules, showed a strong conservation of amino acid residues directly involved in ligand binding. For pea (Pisum sativum) lectin (PSL), these conserved amino acids can be classified into three groups: (I) D81 and N125, present in all legume lectins studied so far; (II) G99 and G216, conserved in almost all legume lectins; and (III) A217 and E218, which are only found in Vicieae lectins and are possibly determinants of sugar-binding specificity. Each of these amino acids in PSL was changed by site-directed mutagenesis, resulting in PSL molecules with single substitutions: for group I D81A, D81N, N125A; for group II G99R, G216L; and for group III A217L, E218Q, respectively. PSL double mutant Y124R; A126S was included as a control. The modified PSL molecules appeared not to be affected in their ability to form dimeric proteins, whereas the sugar-binding activity of each of the PSL mutants, with the exception of the control mutant (as shown by haemagglutination assays), was completely eliminated. These results confirm the model of the sugar-binding site of Vicieae lectins as deduced from X-ray analysis.
几种与糖分子共结晶的豆科植物凝集素的X射线晶体结构比较表明，直接参与配体结合的氨基酸残基具有很强的保守性。对于豌豆（Pisum sativum）凝集素（PSL），这些保守氨基酸可分为三组：（I）D81和N125，存在于迄今为止研究的所有豆科植物凝集素中；（II）G99和G216，在几乎所有豆科植物凝集素中保守；以及（III）A217和E218，仅在蚕豆凝集素中发现，可能是糖结合特异性的决定因素。通过定点诱变改变了PSL中的每一个氨基酸，产生了具有单取代的PSL分子：对于I组为D81A、D81N、N125A；对于II组为G99R、G216L；对于III组分别为A217L、E218Q。PSL双突变体Y124R；A126S作为对照。修饰后的PSL分子形成二聚体蛋白的能力似乎没有受到影响，而除对照突变体（如血凝试验所示）外，每个PSL突变体的糖结合活性完全消除。这些结果证实了从X射线分析推断出的蚕豆凝集素糖结合位点模型。
