Calcium binding to tandem repeats of EGF-like modules. Expression and characterization of the EGF-like modules of human Notch-1 implicated in receptor-ligand interactions

Protein Sci. 1997 Oct;6(10):2059-71. doi: 10.1002/pro.5560061002.


The Ca(2+)-binding epidermal growth factor (cbEGF)-like module is a structural component of numerous diverse proteins and occurs almost exclusively within repeated motifs. Notch-1, a fundamental receptor for cell fate decisions, contains 36 extracellular EGF modules in tandem, of which 21 are potentially Ca(2+)-binding. We report the Ca(2+)-binding properties of EGF11-12 and EGF10-13 from human Notch-1 (hNEGF11-12 and hNEGF10-13), modules previously shown to support Ca(2+)-dependent interactions with the ligands Delta and Serrate. Ca2+ titrations in the presence of chromophoric chelators, 5,5'-Br2BAPTA and 5-NBAPTA, gave two binding constants for hNEGF11-12, Kd1 = 3.4 x 10(-5) M and Kd2 > 2.5 x 10(-4) M. The high-affinity site was found to be localized to hNEGF12. Titration of hNEGF10-13 gave three binding constants, Kd1 = 3.1 x 10(-6) M, Kd2 = 1.6 x 10(-4) M, and Kd3 > 2.5 x 10(-4) M, demonstrating that assembly of EGF modules in tandem can increase Ca2+ affinity. The highest affinity sites in hNEGF11-12 and hNEGF10-13 had 10 to 100-fold higher affinity than reported for EGF32-33 and EGF25-31, respectively, from fibrillin-1, a connective tissue protein with 43 cbEGF modules. A model of hNEGF11-12 based on fibrillin-1 EGF32-33 demonstrates electronegative potential that could contribute to the higher affinity of the Ca(2+)-binding site in hNEGF12. These data demonstrate that the Ca2+ affinity of cbEGF repeats can be highly variable among different classes of cbEGF containing proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Calcium / metabolism*
  • Chelating Agents / metabolism
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / metabolism
  • Electrochemistry
  • Epidermal Growth Factor / chemistry*
  • Epidermal Growth Factor / genetics
  • Epidermal Growth Factor / metabolism
  • Gene Expression
  • Humans
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • Models, Molecular
  • Molecular Weight
  • Receptor, Notch1
  • Receptors, Cell Surface*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Repetitive Sequences, Nucleic Acid*
  • Serine Endopeptidases / metabolism
  • Transcription Factors*


  • 5-nitro-1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid
  • Chelating Agents
  • Membrane Proteins
  • NOTCH1 protein, human
  • Receptor, Notch1
  • Receptors, Cell Surface
  • Recombinant Proteins
  • Transcription Factors
  • Egtazic Acid
  • Epidermal Growth Factor
  • Serine Endopeptidases
  • lysyl endopeptidase
  • 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
  • Calcium