Lundell D, Lunn C A, Senior M M, Zavodny P J, Narula S K
Department of Immunology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033.
J Biol Chem. 1994 Jun 10;269(23):16159-62.
Characterization of murine-human hybrid interferon-gamma (IFN-gamma) molecules suggests that substitution of the peptide connecting the A and B helices in human IFN-gamma with the murine sequence significantly blocks the protein's binding to the human interferon-gamma receptor. Mutagenesis showed that this effect is localized to the central part of this A-B loop peptide, particularly Ser20, Asp21, Val22, and Ala23. One mutant, IFN-gamma/A23E,D24E,N25K, was examined by NMR. This "EEK" mutation does not significantly alter the conformation of interferon-gamma, suggesting that the effects of these mutations are not the result of global conformational changes. The A-B loop is near histidine 111, a residue previously shown to be important in receptor-ligand interaction (Lunn, C. A., Fossetta, J., Dalgarno, D., Murgolo, N., Windsor, W., Zavodny, P. J., Narula, S. K., and Lundell, D. (1992) Protein Eng. 5, 253-257). We show that copper forms a complex between histidine 19 in the A-B loop and histidine 111. This metal complex lacks the ability to interact with the interferon-gamma receptor. These results suggest that the A-B loop contains important structural information needed for receptor-ligand binding and hence biological activity of human interferon-gamma.
对鼠-人杂交干扰素-γ（IFN-γ）分子的特性分析表明，用人干扰素-γ中连接A螺旋和B螺旋的肽段替换为鼠序列，会显著阻碍该蛋白与人干扰素-γ受体的结合。诱变实验表明，这种效应定位于该A-B环肽的中央部分，特别是Ser20、Asp21、Val22和Ala23。通过核磁共振（NMR）研究了一种突变体IFN-γ/A23E、D24E、N25K。这种“EEK”突变不会显著改变干扰素-γ的构象，这表明这些突变的效应不是全局构象变化的结果。A-B环靠近组氨酸111，先前已证明该残基在受体-配体相互作用中很重要（Lunn, C. A., Fossetta, J., Dalgarno, D., Murgolo, N., Windsor, W., Zavodny, P. J., Narula, S. K., and Lundell, D. (1992) Protein Eng. 5, 253 - 257）。我们发现铜在A-B环中的组氨酸19和组氨酸111之间形成了一种复合物。这种金属复合物缺乏与干扰素-γ受体相互作用的能力。这些结果表明，A-B环包含人干扰素-γ受体-配体结合以及生物活性所需的重要结构信息。
