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.
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.
Rand
M D
MD
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden.
Lindblom
A
A
Carlson
J
J
Villoutreix
B O
BO
Stenflo
J
J
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004563:Electrochemistry; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D014157:Transcription Factors
C109928:5-nitro-1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid; D002614:Chelating Agents; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014157:Transcription Factors; D004533:Egtazic Acid; D004815:Epidermal Growth Factor; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium