The conserved C2 phospholipid-binding domain in Delta contributes to robust Notch signalling.
The conserved C2 phospholipid-binding domain in Delta contributes to robust Notch signalling.
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DOI：
10.15252/embr.202152729
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发表时间：
2021-10-05
期刊：
影响因子：
7.7
通讯作者：
Bray SJ
中科院分区：
文献类型：
--
作者：
Martins T;Meng Y;Korona B;Suckling R;Johnson S;Handford PA;Lea SM;Bray SJ
准确的 Notch 信号对于发育和体内平衡至关重要。 Notch-配体相互作用的微调对信号输出具有重大影响。最近的结构研究发现，人 Notch 配体中存在一个保守的 N 末端 C2 结构域，可在体外赋予磷脂结合作用。在这里，我们表明果蝇配体 Delta 和 Serrate 采用相同的 C2 结构域结构，但环区域有类似的变化，包括参与磷脂结合的所谓 β1-2 环。 Delta C2 结构域的 β1-2 环中的突变保留了 Notch 结合，但在体外与磷脂相互作用的能力受损。为了研究其在体内的作用，我们删除了内源 Delta β1-2 环内的 5 个残基。引人注目的是，这种变化损害了配体功能。修饰后的 Delta 增强了 Delta 功能丧失等位基因产生的表型，并抑制了 Notch 等位基因的表型。由于修饰蛋白以正常量存在于细胞表面，这些结果表明 C2 结构域磷脂结合对于体内微调反式和顺式配体-受体相互作用的平衡的稳健信号传导是必要的。内源性 Delta 和 Serrate 的保守 N 端 C2 结构域的工程突变表明，干扰赋予磷脂结合的突出环之一会导致体内 Notch 信号传导缺陷，从而证实了其对正常配体活性的贡献。
Accurate Notch signalling is critical for development and homeostasis. Fine‐tuning of Notch–ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N‐terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so‐called β1‐2 loop that is involved in phospholipid binding. Mutations in the β1‐2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the β1‐2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss‐of‐function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine‐tuning the balance of trans and cis ligand–receptor interactions. Engineered mutations in the conserved N‐terminal C2 domains of endogenous Delta and Serrate show that perturbing one of the protruding loops conferring phospholipid binding leads to defective Notch signalling in vivo, confirming its contribution to normal ligand activity.