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

## TL;DR

This study reveals that a specific region of the Notch receptor relies on calcium to maintain a rigid structure, which is important for proper signaling.

## Contribution

The study identifies a Ca2+-dependent structural organization in the EGF20-27 region of Notch-1 and its functional role in receptor activation.

## Key findings

- The EGF20-27 region of Notch-1 is rigid and elongated with high calcium affinity in most domains.
- Mutations in Ca2+-binding sites reduce trans-activation and cis-inhibition, showing the importance of calcium-stabilized structure.
- The Ax region's Ca2+-dependent structure influences Notch signaling efficiency and cis-inhibition balance.

## Abstract

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.

•hN-1 EGF20-27 is mainly rigid and elongated with high Ca2+ affinity in most domains•EGF21 has a weaker affinity for Ca2+ so the EGF20-21 interface may vary in rigidity•EGF24-25 Ca2+-binding site mutagenesis reduces trans-activation and cis-inhibition•This indicates the functional importance of the Ca2+-stabilized structure

hN-1 EGF20-27 is mainly rigid and elongated with high Ca2+ affinity in most domains

EGF21 has a weaker affinity for Ca2+ so the EGF20-21 interface may vary in rigidity

EGF24-25 Ca2+-binding site mutagenesis reduces trans-activation and cis-inhibition

This indicates the functional importance of the Ca2+-stabilized structure

The structure of the Notch ectodomain remains unsolved. Bo et al. identify an elongated Ca2+-dependent structure for EGF 20–27 which includes the Abruptex (Ax) region. Loss of Ca2+-dependent structure within this region leads to reduced trans-activation and cis-inhibition, indicating a role for rigidity in optimizing generation of the Notch signal.