Rock F L, Li X, Chong P, Ida N, Klein M
Department of Biochemistry, University of Toronto, Ontario, Canada.
Biochemistry. 1994 May 3;33(17):5146-54. doi: 10.1021/bi00183a018.
Human IL-6 has two disulfide bonds linking Cys45 to Cys51 and Cys74 to Cys84, respectively. Previous site-directed mutagenesis studies have demonstrated that the Cys74-Cys84 bond is essential for full biological and receptor binding activities. To address the structural importance of these disulfide bonds in the formation and stabilization of IL-6 secondary and tertiary structures, we have generated a panel of disulfide bond-deficient rIL-6 analogs both by chemical reduction and alkylation as well as by site-directed mutagenesis. Conformational changes affecting these rIL-6 analogs were probed by circular dichroism spectroscopy, as well as reactivity with monoclonal antibodies, and correlated with changes in biological activities. We have shown that the first disulfide bridge (Cys45-Cys51) is highly sensitive to reduction and, therefore, more solvent-exposed or less thermodynamically stable. Contrary to previous reports, this bridge contributes, although minimally, to the full biological activity of the cytokine. However, no significant changes in secondary or tertiary structures were observed upon removal of this bond. In marked contrast, analogs lacking the disulfide bridge between Cys74 and Cys84 exhibited as little as 0.5% and 0.05% wild-type biological and receptor binding activities, respectively. These dramatic changes correlated with a slight reduction in alpha-helical content and a decreased reactivity with the neutralizing monoclonal antibody mAb8 which recognizes a conformational epitope associated with the active site. Our results suggest that the second disulfide bridge plays a critical role in maintaining the spatial relationship between the putative IL-6 A and D helices.
人白细胞介素-6有两个二硫键，分别将半胱氨酸45与半胱氨酸51以及半胱氨酸74与半胱氨酸84连接起来。先前的定点诱变研究表明，半胱氨酸74 - 半胱氨酸84键对于完整的生物学和受体结合活性至关重要。为了研究这些二硫键在白细胞介素-6二级和三级结构形成与稳定中的结构重要性，我们通过化学还原和烷基化以及定点诱变产生了一组二硫键缺陷型重组人白细胞介素-6类似物。通过圆二色光谱以及与单克隆抗体的反应性来探测影响这些重组人白细胞介素-6类似物的构象变化，并将其与生物活性变化相关联。我们已经表明，第一个二硫桥（半胱氨酸45 - 半胱氨酸51）对还原高度敏感，因此更多地暴露于溶剂中或热力学稳定性较低。与先前的报道相反，尽管这种桥对细胞因子的完整生物学活性贡献极小，但它确实有一定作用。然而，去除该键后未观察到二级或三级结构的显著变化。与之形成鲜明对比的是，缺乏半胱氨酸74和半胱氨酸84之间二硫桥的类似物分别仅表现出野生型生物学活性和受体结合活性的0.5%和0.05%。这些显著变化与α-螺旋含量的轻微降低以及与识别与活性位点相关的构象表位的中和单克隆抗体mAb8的反应性降低相关。我们的结果表明，第二个二硫桥在维持假定的白细胞介素-6 A螺旋和D螺旋之间的空间关系中起关键作用。
