Structural and functional studies of the EGF20-27 region reveal new features of the human Notch receptor important for optimal activation

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.