Localization of the delta-like-1-binding site in human notch-1 and its modulation by calcium affinity
Localization of the delta-like-1-binding site in human notch-1 and its modulation by calcium affinity
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DOI：
10.1074/jbc.m708424200
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发表时间：
2008-04-25
影响因子：
4.8
通讯作者：
Handford, Penny A.
中科院分区：
文献类型：
--
作者：
Cordle, Jemima;Redfield, Christina;Handford, Penny A.
Notch信号通路在包括细胞命运决定在内的无数细胞过程中起着关键作用。尽管对受体激活的下游后果进行了广泛的研究，但Notch受体与其配体之间的初始结合事件的分子数据很少。在这项研究中，我们从人类Notch-1中表达并纯化了一个天然折叠的野生型表皮生长因子样结构域（EGF） 11-14结构，并使用流式细胞术和表面等离子体共振分析证明了与人类配体Delta-like-1的钙依赖性相互作用。对Notch-(11-14)片段内三个钙结合位点的定点突变表明，只有钙与EGF12的结合丧失，而EGF11或EGF13则没有，从而取消了配体结合。通过对Notch野生型和突变体片段进行有限的蛋白水解，进一步绘制了该区域内的配体结合位点，表明EGF12而不是EGF11含有主要的delta -like-1结合位点。对一个扩展片段EGF-(10-14)的分析，其中EGF11被置于天然环境中，令人惊讶地显示了配体结合的减少，这表明EGF10通过限制配体的进入来调节结合。这种抑制可以通过在EGF11中引入钙结合突变来克服，该突变可以解耦EGF-(10-11)模块界面。因此，这项研究表明，在没有任何翻译后修饰的情况下，长期钙依赖的结构扰动可以影响Notch对其配体的亲和力。
The Notch signaling pathway plays a key role in a myriad of cellular processes, including cell fate determination. Despite extensive study of the downstream consequences of receptor activation, very little molecular data are available for the initial binding event between the Notch receptor and its ligands. In this study, we have expressed and purified a natively folded wildtype epidermal growth factor-like domain (EGF) 11-14 construct from human Notch-1 and have used flow cytometry and surface plasmon resonance analysis to demonstrate a calcium-dependent interaction with the human ligand Delta-like-1. Site-directed mutagenesis of three of the calcium-binding sites within the Notch-(11-14) fragment indicated that only loss of calcium binding to EGF12, and not EGF11 or EGF13, abrogates ligand binding. Further mapping of the ligand-binding site within this region by limited proteolysis of Notch wild-type and mutant fragments suggested that EGF12 rather than EGF11 contains the major Delta-like-1-binding site. Analysis of an extended fragment EGF-(10-14), where EGF11 is placed in a native context, surprisingly demonstrated a reduction in ligand binding, suggesting that EGF10 modulates binding by limiting access of ligand. This inhibition could be overcome by the introduction of a calcium binding mutation in EGF11, which decouples the EGF-(10-11) module interface. This study therefore demonstrates that long range calcium-dependent structural perturbations can influence the affinity of Notch for its ligand, in the absence of any post-translational modifications.