Ligand requirements for Ca2+ binding to EGF-like domains

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• The first epidermal growth factor-like (EGF-like) domain of human clotting factor IX plays a critical role in calcium binding.
• Previous studies suggested that carboxylate residues Asp47, Asp49, and Asp64 are involved in this calcium-binding site.

Purpose of the Study:

• To further characterize the specific amino acid residues and their side chains essential for calcium binding within the EGF-like domain of human factor IX.
• To investigate the impact of specific mutations on calcium binding affinity and to understand the molecular basis of factor IX Alabama's impaired function.

Main Methods:

• Site-specific mutagenesis was employed to introduce targeted mutations (Asp47Asn, Asp49Asn, Glu78Asp) into the EGF-like domain of human factor IX.
• Peptide synthesis was used to create the modified protein domains.
• Proton nuclear magnetic resonance (1H-NMR) spectroscopy was utilized to determine the dissociation constants (Kd) for calcium binding to the wild-type and mutant peptides.

Main Results:

• The Asp49Asn mutation showed a mild effect on calcium binding (Kd = 6 mM), while the Asp47Asn mutation significantly reduced calcium binding affinity (Kd = 42 mM).
• A control mutation (Glu78Asp) in a non-calcium-binding region had minimal impact on calcium binding (Kd = 2.6 mM).
• The Asp47Gly substitution, mimicking the factor IX Alabama mutation, resulted in markedly reduced calcium binding (Kd = 37 mM).

Conclusions:

• Anionic oxygen atoms from the side chains of Asp47 and Asp64 are essential for calcium binding to the first EGF-like domain of factor IX.
• The residue at position 49 can accommodate a carboxyamide oxygen as a ligand, indicating greater flexibility.
• Impaired calcium binding to the first EGF-like domain is the likely cause of the defect in the factor IX Alabama (haemophilia B) mutant.