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^1H-NMR study of the receptor-binding region of human IL-6

^1H-NMR 研究人 IL-6 受体结合区

基本信息

批准号：
04671322
负责人：
NISHIMURA Chiaki
金额：
$ 1.28万
依托单位：
Faculty of Pharmaceutical Sciences, University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04671322/
关键词：
IL-6 NMR site-directed mutagenesis stable isotope peptide fragment IL-6 receptor

项目摘要

Site-directed mutagenesis of three leucine residues in the C-terminal region of human interleukin-6(IL-6) was performed. Both receptor-binding and immunoglobulin-induction activities of a triple mutant Leu168, 175, 182->Val were only 1 % compared with those of wild-type IL-6. We also prepared the peptide fragments. A significant binding activity was observed for the peptides Leu168-Met185 and Ile88-Lys121, althouth the activity was estimated at 10^4-fold less than that of intact IL-6. These results suggest that these peptides compose a part of the receptor-binding region and that Leu168, Leu175, and Leu182 existing in the region of Leu168-Met185 play an important role in the receptor-binding. Solution structure of the peptide Ile88-Lys121 was analyzed by using two-dimensional nuclear magnetic resonance spectroscopy. The results indicate that the peptide Ile88-Lys121 forms two alpha-helical rods (Leu93-Phe106 and Glu110-Ser119) with a kink in the region of Glu107-Ser109. Proton nuclear magnetic resonance studies of wild-type and mutant IL-6 were also performed. Using a deuterium-labeling method in combination with the site-directed mutagenesis, the methyl proton resonances of Leu152, 168, and 175 in wild-type IL-6 and Val152, 159, 166, 168, and 175 in mutated IL-6 were assigned. On the basis of NOE data and predicted secondary structure of IL-6, the proton resonances of Phe95, Val97, Ile167, Phe171, and Phe174 were assigned. On the basis of these signal assignments, two long-range NOE cross-peaks were observed between Phe95 in the helix B and Phe171 in the helix D, and between Tyr98 and 101 in the helix B and Leu152 in the helix C-D loop.

对人白细胞介素-6（IL-6）C-末端区域的三个亮氨酸残基进行了定点突变。与野生型IL-6相比，三重突变体Leu 168，175，182->瓦尔的受体结合和免疫球蛋白诱导活性仅为1%。我们还制备了肽片段。观察到肽Leu 168-Met 185和Ile 88-Lys 121具有显著的结合活性，尽管该活性估计为完整IL-6的10^4倍。这些结果表明，这些肽组成的受体结合区的一部分，Leu 168，Leu 175，和Leu 182存在于Leu 168-Met 185区域中发挥重要作用的受体结合。通过使用二维核磁共振光谱分析肽Ile 88-Lys 121的溶液结构。结果表明，肽Ile 88-Lys 121形成两个α-螺旋杆（Leu 93-Phe 106和Glu 110-Ser 119），在Glu 107-Ser 109区域中具有扭结。还进行了野生型和突变型IL-6的质子核磁共振研究。使用氘标记方法结合定点诱变，对野生型IL-6中Leu 152、168和175以及突变型IL-6中Val 152、159、166、168和175的甲基质子共振进行了归属。基于NOE数据和预测的IL-6二级结构，对Phe 95、Val 97、Ile 167、Phe 171和Phe 174的质子共振峰进行了归属。基于这些信号分配，在螺旋B中的Phe 95和螺旋D中的Phe 171之间，以及螺旋B中的Tyr 98和101与螺旋C-D环中的Leu 152之间观察到两个长程NOE交叉峰。