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

对人白细胞介素-6(IL-6) c端区域的三个亮氨酸残基进行了定点诱变。与野生型IL-6相比，三突变体Leu168、175,182 -> - Val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。研究发现，Leu168-Met185和Ile88-Lys121肽具有显著的结合活性，但活性估计比完整的IL-6低10^4倍。这些结果表明，这些肽构成了受体结合区的一部分，并且存在于Leu168- met185区域的Leu168、Leu175和Leu182在受体结合中起重要作用。利用二维核磁共振波谱分析了肽Ile88-Lys121的溶液结构。结果表明，肽Ile88-Lys121在Glu107-Ser109区域形成两个α -螺旋棒状结构（Leu93-Phe106和Glu110-Ser119）。对野生型和突变型IL-6也进行了质子核磁共振研究。利用氘标记法结合定点诱变，确定了野生型IL-6中Leu152、168和175的甲基质子共振，以及突变IL-6中Val152、159、166、168和175的甲基质子共振。根据NOE数据和预测的IL-6二级结构，确定了Phe95、Val97、Ile167、Phe171和Phe174的质子共振。在这些信号分配的基础上，在B螺旋上的Phe95和D螺旋上的Phe171之间，以及B螺旋上的Tyr98和101以及C-D螺旋上的Leu152之间观察到两个远程NOE交叉峰。

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.