Rand M D, Lindblom A, Carlson J, Villoutreix B O, Stenflo J
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden.
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.
钙离子结合表皮生长因子（cbEGF）样结构域是众多不同蛋白质的结构组成部分，几乎仅存在于重复基序中。Notch-1是细胞命运决定的基本受体，串联包含36个细胞外EGF结构域，其中21个可能结合钙离子。我们报告了人Notch-1的EGF11-12和EGF10-13（hNEGF11-12和hNEGF10-13）的钙离子结合特性，这些结构域先前已显示支持与配体Delta和Serrate的钙离子依赖性相互作用。在发色螯合剂5,5'-Br2BAPTA和5-NBAPTA存在下进行钙离子滴定，得到hNEGF11-12的两个结合常数，Kd1 = 3.4 x 10(-5) M和Kd2 > 2.5 x 10(-4) M。发现高亲和力位点定位于hNEGF12。hNEGF10-13的滴定给出三个结合常数，Kd1 = 3.1 x 10(-6) M，Kd2 = 1.6 x 10(-4) M，和Kd3 > 2.5 x 10(-4) M，表明EGF结构域串联组装可增加钙离子亲和力。hNEGF11-12和hNEGF10-13中最高亲和力位点的亲和力分别比来自具有43个cbEGF结构域的结缔组织蛋白原纤维蛋白-1的EGF32-33和EGF25-31报道的亲和力高10至100倍。基于原纤维蛋白-1 EGF32-33的hNEGF11-12模型显示出负电势，这可能有助于hNEGF12中钙离子结合位点的更高亲和力。这些数据表明，cbEGF重复序列的钙离子亲和力在不同类别的含cbEGF蛋白质中可能有很大差异。
