Cordle Jemima, Redfieldz Christina, Stacey Martin, van der Merwe P Anton, Willis Antony C, Champion Brian R, Hambleton Sophie, Handford Penny A
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
J Biol Chem. 2008 Apr 25;283(17):11785-93. doi: 10.1074/jbc.M708424200. Epub 2008 Feb 22.
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 wild-type 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.
Notch信号通路在包括细胞命运决定在内的众多细胞过程中发挥着关键作用。尽管对受体激活的下游后果进行了广泛研究，但关于Notch受体与其配体之间初始结合事件的分子数据却非常少。在本研究中，我们表达并纯化了来自人Notch-1的天然折叠野生型表皮生长因子样结构域（EGF）11-14构建体，并使用流式细胞术和表面等离子体共振分析来证明其与人类配体Delta样-1的钙依赖性相互作用。对Notch-（11-14）片段内三个钙结合位点进行定点诱变表明，只有与EGF12而非EGF11或EGF13的钙结合丧失才会消除配体结合。通过对Notch野生型和突变型片段进行有限蛋白酶解，进一步绘制该区域内的配体结合位点，结果表明主要的Delta样-1结合位点位于EGF12而非EGF11中。对扩展片段EGF-（10-14）（其中EGF11处于天然环境）的分析令人惊讶地表明配体结合减少，这表明EGF10通过限制配体的接近来调节结合。通过在EGF11中引入钙结合突变（使EGF-（10-11）模块界面解偶联）可以克服这种抑制作用。因此，本研究表明，在没有任何翻译后修饰的情况下，远距离钙依赖性结构扰动可以影响Notch对其配体的亲和力。
