Ligand requirements for Ca2+ binding to EGF-like domains |
| |
Authors: | Mayhew Mark; Handford Penny; Baron Martin; Tse Albert G D; Campbell Iain D; Brownlee George G |
| |
Affiliation: | Sir William Dunn School of Pathology, University of Oxford South Parks Road, Oxford OX1 3RE, UK
1Department of Biochemistry, University of Oxford South Parks Road, Oxford, UK
2Present address: Howard Hughes Medical Institute, Department of Cell Biology and Biology, Yale Univeristy New Haven, CT 06511, USA
3Present address: Laboratory of Immunobiology, Dana-Farber Cancer Institute Boston, MA 02115, USA |
| |
Abstract: | Site-specific mutagenesis studies of the first epidermal growthfactor-like (EGF-like) domain of human clotting factor IX suggestthat the calcium-binding site present in this domain (dissociationconstant Kd=1.8 mM at pH 7.5 and ionic strength I=0.15) involvedthe carboxylate residues Asp47, Asp49 and Asp64. To furthercharacterize the ligands required for calcium binding to EGF-likedomains, two new mutations, Asp47 - Asn and Asp49 - Asn, wereintroduced into the domain by peptide synthesis. 1H-NMR spectroscopywas used to obtain the dissociation constants for calcium bindingto these mutations. Calcium binding to the Asp49- Asn modifieddomain is only mildly affected (Kd=6 mM, I=0.15), whereas bindingto the Asp47- Asn modified domain is severely reduced (Kd=42mM, I=0.15). From these data, it is proposed that the anionicoxygen atoms of the side chains of residues 47 and 64 are essentialfor calcium binding, whereas the side chain ligand for calciumat residue 49 can be a carboxyamide oxygen. As a control, theintroduction of the modification Glu78- Asp in a region of thedomain not believed to be involved in calcium binding had verylittle effect on the Kd for calcium (Kd=2.6 mM, I=0.15). Finally,the effect of an Asp47- Gly substitution found in the naturalhaemophilia B mutant, factor IXAlabama, was investigated. Thispeptide has a markedly reduced affinity for calcium (Kd=37 mM,I=0.15), suggesting that the defect in factor IXAlabama is dueto impaired calcium binding to its first EGF-like domain. |
| |
Keywords: | calcium binding/ EGF-like domains/ factor IXAlabama |
本文献已被 Oxford 等数据库收录! |
|