The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand, Jagged1, to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical, and structural techniques on the full extracellular complex and targeted sites, we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network. The Notch1 membrane–proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8–EGF13 and Jagged1 C2–EGF3 activation sites as well as to two additional Jagged1 regions, EGF8–EGF11 and cysteine-rich domain. XL-MS and quantitative interaction experiments show that the three Notch1-binding sites on Jagged1 also engage intramolecularly. These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility, determined by small-angle X-ray scattering, support the formation of nonlinear architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal but engage directly to control Notch1 signaling, thereby redefining the Notch1–Jagged1 activation mechanism and indicating routes for therapeutic applications.

Notch信号系统将细胞命运与其邻居的命运联系起来，驱动后生动物的增殖、凋亡和细胞分化，而功能障碍则导致衰弱性发育障碍和癌症。除了 5 x 5 结构域复合体之外，目前还不清楚 Notch1 受体的 40 个胞外结构域如何共同结合其经典配体 Jagged1 的 19 个结构域来激活 Notch1 信号传导。在这里，我们对完整的细胞外复合物和目标位点使用交联质谱 (XL-MS)、生物物理和结构技术，确定了五个不同的区域，两个在 Notch1 上，三个在 Jagged1 上，形成了一个相互作用网络。 Notch1 近膜调节区分别与已建立的 Notch1 表皮生长因子 (EGF) 8-EGF13 和 Jagged1 C2-EGF3 激活位点以及两个额外的 Jagged1 区域、EGF8-EGF11 和富含半胱氨酸的结构域结合。 XL-MS 和定量相互作用实验表明，Jagged1 上的三个 Notch1 结合位点也在分子内结合。这些相互作用与由小角度 X 射线散射决定的 Notch1 和 Jagged1 胞外域尺寸和灵活性一起支持非线性结构的形成。综合起来，数据表明关键的 Notch1 和 Jagged1 区域并不位于远端，而是直接参与控制 Notch1 信号传导，从而重新定义了 Notch1-Jagged1 激活机制并指明了治疗应用的途径。