1H, 13C and 15N assignments of EGF domains 8–11 of human Notch-1

Abstract

The Notch receptor is part of a core cell-cell signaling system crucial for development and tissue homeostasis in Metazoa. Structural information is available for the negative regulatory region, the ligand-binding region and the intracellular domain of Notch, but data for the remaining portions of the extracellular region which determine its overall shape at the cell surface are still lacking. This region consists of 36 EGF-like domains arranged as multiple tandem repeats. Most EGF-like domains near the ligand-binding domains EGF11 and 12 are of the calcium-binding type, with well-described, rigid and near-linear interdomain interfaces. However, EGF10 is a conserved, non-calcium-binding domain which may confer flexibility or a non-linear organization to the receptor. To probe this, we have expressed and purified a four-domain construct, EGF8-11, from human Notch-1, and report here the (1)H, (13)C and (15)N resonance assignments. Differences in EGF11 chemical shifts between this construct and a previously assigned construct, EGF11-13, confirm the presence of hydrophobic interdomain contacts between the hairpin turn of the major β-sheet in EGF11 and the conserved aromatic residue within the C-terminal region of EGF10. This suggests that the EGF10-11 interface is rigid.

Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies

“…Protein expression, isotopic labeling, refolding, and purification protocols have been described previously ( Muranyi et al., 2004 , Weisshuhn et al., 2015a , Weisshuhn et al., 2015b , Whiteman et al., 2014 ). Protein fragments were expressed in Escherichia coli BL21 cells transformed with a pQE30 (Qiagen)-based protein expression construct and a pREP4 plasmid for control of expression via the Lac Repressor.…”

“…Resonance assignments for EGF4-7, EGF8-11, and EGF11-13 have been described previously ( Muranyi et al., 2004 , Weisshuhn et al., 2015a , Weisshuhn et al., 2015b ) (Biological Magnetic Resonance Bank accession numbers 25172, 25533, 6031). 3D 15 N-edited total correlation spectroscopy-heteronuclear single quantum coherence (HSQC) and NOE spectroscopy (NOESY)-HSQC spectra were collected to assign the 1 H- 15 N HSQC spectrum of EGF7-9.…”

The Canonical Notch Signaling Pathway: Structural and Biochemical Insights into Shape, Sugar, and Force

“…However, X-ray structures of EGF repeats 4–7 from human Notch1 (Weisshuhn et al, 2015a) show a striking tilt angle of ~90° between repeats 5 and 6, which doesn’t bind Ca ++ (Figure 1). To analyze additional tilt/twist angles between EGF repeats, the authors used NMR spectroscopy with constructs corresponding to Notch1 EGF repeats 4–7, 7–9, 8–11, and 11–13 (Weisshuhn et al, 2015b). These studies confirmed that Ca ++ binding EGF repeats assume a rigid rod like structure.…”