Probing Receptor Specificity by Sampling the Conformational Space of the Insulin-like Growth Factor II C-domain.
Probing Receptor Specificity by Sampling the Conformational Space of the Insulin-like Growth Factor II C-domain.
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DOI：
10.1074/jbc.m116.741041
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发表时间：
2016-09-30
期刊：
影响因子：
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通讯作者：
Žáková L
中科院分区：
文献类型：
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作者：
Hexnerová R;Křížková K;Fábry M;Sieglová I;Kedrová K;Collinsová M;Ullrichová P;Srb P;Williams C;Crump MP;Tošner Z;Jiráček J;Veverka V;Žáková L
胰岛素和胰岛素样生长因子 I 和 II 是密切相关的蛋白质激素。它们独特的进化导致了不同但重叠的生物学功能，胰岛素成为代谢的关键调节剂，而胰岛素样生长因子（IGF）-I/II 是主要的生长因子。胰岛素和 IGF 以不同的亲和力与密切相关的胰岛素受体亚型 A 和 B（IR-A 和 IR-B）以及胰岛素样生长因子 I 型受体 (IGF-1R) 交叉结合。鉴定 IGF 和胰岛素中触发其特定信号通路的结构决定因素对于设计具有潜在治疗应用的受体特异性类似物越来越重要。在这里，我们开发了一种用于生产重组 IGF-II 的简单方案，并制备了六种具有 IGF-I 样突变的 IGF-II 类似物。所有修饰分子均表现出对 IR-A 的亲和力显着降低，特别是在 C 结构域中插入了 Pro-Gln 的类似物。此外，由于 Pro-Gln 插入和 S29N 点突变的协同效应，其中一种类似物对 IGF-1R 的结合亲和力增强。因此，与天然 IGF-II 相比，该类似物的 IGF-1R/IR-A 结合特异性几乎高出 10 倍。已建立的 IGF-II 纯化方案可实现 IGF-II 类似物详细 NMR 结构表征所需的经济有效的同位素标记，揭示所选类似物改变的结合行为与其 C 结构域构象重排之间的联系。
Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains.