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

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Abstract
The Ca2+‐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 Ca2+‐binding. We report the Ca2+‐binding properties of EGF11‐12 and EGF10‐13 from human Notch‐1 (hNEGF11‐12 and hNEGF10‐13), modules previously shown to support Ca2+‐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 × 10−5 M and Kd2 2.5 × 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 × 10−6 M, Kd2 = 1.6 × 10−4 M, and Kd3 > 2.5 × 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 Ca2+‐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.