The Notch receptor is activated by the Delta/Serrate/Lag-2 (DSL) family of ligands. The organization of the extracellular signaling complex is unknown, although structures of Notch/ligand complexes comprising the ligand-binding region (LBR), and negative regulatory region (NRR) region, have been solved. Here, we investigate the human Notch-1 epidermal growth factor-like (EGF) 20-27 region, located between the LBR and NRR, and incorporating the Abruptex (Ax) region, associated with distinctive Drosophila phenotypes. Our analyses, using crystallography, NMR and small angle X-ray scattering (SAXS), support a rigid, elongated organization for EGF20-27 with the EGF20-21 linkage showing Ca²⁺-dependent flexibility. In functional assays, Notch-1 variants containing Ax substitutions result in reduced ligand-dependent trans-activation. When cis-JAG1 was expressed, Notch activity differences between WT and Ca²⁺-binding Ax variants were less marked than seen in the trans-activation assays alone, consistent with disruption of cis-inhibition. These data indicate the importance of Ca²⁺-stabilized structure and suggest the balance of cis- and trans-interactions explains the effects of Drosophila Ax mutations.

中文翻译：

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EGF20-27 区域的结构和功能研究揭示了人类 Notch 受体的新特征，对最佳激活很重要


Notch 受体被 Delta/Serrate/Lag-2 （DSL） 配体家族激活。细胞外信号复合物的组织尚不清楚，尽管已经解决了包含配体结合区 （LBR） 和负调节区 （NRR） 区域的 Notch/配体复合物的结构。在这里，我们研究了人类 Notch-1 表皮生长因子样 （EGF） 20-27 区域，位于 LBR 和 NRR 之间，并结合了与独特的果蝇表型相关的 Abruptex （Ax） 区域。我们使用晶体学、NMR 和小角 X 射线散射 （SAXS） 进行分析，支持 EGF20-27 的刚性、细长组织，EGF20-21 键显示出 Ca²⁺ 依赖性柔韧性。在功能测定中，含有 Ax 取代的 Notch-1 变体导致配体依赖性反式激活减少。当 cis-JAG1 表达时，WT 和 Ca²⁺ 结合 Ax 变体之间的 Notch 活性差异不如单独反式激活测定中观察到的明显，这与顺式抑制的破坏一致。这些数据表明了 Ca²⁺ 稳定结构的重要性，并表明顺式和反式相互作用的平衡解释了果蝇 Ax 突变的影响。