Calcium binding by γ-carboxyglutamic acid: it takes two to tether

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Vitamin K-dependent proteins feature γ-carboxyglutamic acid (Gla) residues crucial for calcium binding.
• Gla residues are vital for coagulation factors and mineralization processes.
• Incomplete biophysical data for Gla hinders molecular dynamics and structure predictions.

Purpose of the Study:

• To determine calcium binding characteristics (pKa and KD) of Gla in a protein environment.
• To investigate how Gla positioning influences cooperative calcium binding.
• To assess the impact of calcium binding on protein structure.

Main Methods:

• Quantified residue-based pKa of Gla carboxyl groups using pH-dependent NMR.
• Determined Ca2+ binding affinity (KD) via Ca2+ NMR titrations.
• Assessed peptide and protein secondary structure using circular dichroism and NMR.

Main Results:

• Gla exhibited two pKa values (2.62 and 5.02), decreasing upon Ca2+ binding.
• Single Gla affinity for Ca2+ was low (~15 mM), but cooperativity in two Gla residues increased affinity 25-fold (~0.6 mM).
• Cooperative calcium binding enhanced α-helical content in model proteins.

Conclusions:

• Vitamin K-dependent proteins leverage cooperative calcium binding by Gla residues.
• Experimentally determined pKa and KD values aid in interpreting Gla domain interactions.
• These findings support molecular dynamics simulations for Gla domains with unknown structures.