Engineering the C-region of human insulin-like growth factor-1: Implications for receptor binding
Engineering the C-region of human insulin-like growth factor-1: Implications for receptor binding
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DOI：
10.1093/protein/9.11.1011
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发表时间：
1996-11-01
期刊：
影响因子：
--
通讯作者：
Wood, S
中科院分区：
文献类型：
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作者：
Gill, R;Wallach, B;Wood, S
从酵母中分泌并纯化重组野生型人IGF-1和C区突变体，其中残基28-37已被4-甘氨酸桥取代(4-Gly IGF-1)。通过定点诱变产生并表达了 C 区残基 29-41 已被删除的 IGF-1 类似物（微型 IGF-1）。所有三种蛋白质均采用圆二色性测定的胰岛素倍数。微型 IGF-1 表达水平显着升高，可以记录二维 NMR 谱。 4-Gly IGF-1对IGF-1受体的亲和力比野生型IGF-1低100倍，对胰岛素受体的亲和力比野生型IGF-1低10倍，Mini IGF-1对任一受体均无亲和力。 IGF-1 的 C 区不仅直接贡献与 IGF-1 受体结合的自由能，而且该区域缺乏灵活性，完全消除了结合。正如胰岛素与其自身受体结合的假设一样，IGF-1 与 IGF-1 受体的结合还涉及构象变化，其中 C 端 B 区残基从分子体上分离，露出下面的 A 区残基。
Recombinant wild-type human IGF-1 and a C-region mutant in which residues 28-37 have been replaced by a 4-glycine bridge (4-Gly IGF-1) were secreted and purified from yeast. An IGF-1 analogue in which residues 29-41 of the C-region have been deleted (mini IGF-1) was created by site-directed mutagenesis and also expressed. All three proteins adopted the insulin-fold as determined by circular dichroism. The significantly raised expression levels of mini IGF-1 allowed the recording of two-dimensional NMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 receptor was similar to 100-fold lower than that of wild-type IGF-1 and the affinity for the insulin receptor was similar to 10-fold lower Mini IGF-1 showed no affinity for either receptor. Not only does the C-region of IGF-1 contribute directly to the free energy of binding to the IGF-1 receptor, but also the absence of flexibility in this region eliminates binding altogether. As postulated for the binding of insulin to its own receptor, it is proposed that binding of IGF-1 to the IGF-1 receptor also involves a conformational change in which the C-terminal B-region residues detach from the body of the molecule to expose the underlying A-region residues.