Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK.
Department of Biochemistry, University of Oxford, Oxford, UK.
EMBO Rep. 2021 Oct 5;22(10):e52729. doi: 10.15252/embr.202152729. Epub 2021 Aug 4.
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.
精确的 Notch 信号对于发育和内稳态至关重要。 Notch 配体相互作用的微调对信号输出有重大影响。最近的结构研究在人类 Notch 配体中鉴定出一个保守的 N 端 C2 结构域，该结构域在体外赋予磷脂结合能力。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，其环区存在类似的变化，包括参与磷脂结合的所谓的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，我们在内源性 Delta 的β1-2 环中缺失了五个残基。引人注目的是，这种变化损害了配体的功能。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。由于修饰后的蛋白以正常数量存在于细胞膜表面，这些结果表明 C2 结构域磷脂结合对于体内稳健的信号转导是必要的，它可以微调跨膜和顺式配体-受体相互作用的平衡。
