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

Background: The small family of vitamin K-dependent proteins are characterized by posttranslational modification of specific glutamic acid residues to yield γ-carboxyglutamic acid (Gla). Gla residues give these proteins calcium ion-binding properties, which are essential for a number of coagulation factors and mineralization processes. Biophysical characteristics of Gla are, however, incomplete, hindering molecular dynamics simulations and protein structure predictions. Objectives: This study aimed to elucidate the general biophysical characteristics (pK _a and K _D) of calcium binding to γ-carboxyglutamic acid in a protein environment and determine how positioning of γ-carboxyglutamic acid influences cooperative calcium binding and protein structure. Methods: Residue-based pK _a of Gla carboxyl groups in model peptides was individually determined by measuring ¹H and ¹³C nuclear magnetic resonance chemical shift changes as a function of pH. In addition, residue-based K _D values of Ca ²⁺ binding were determined using Ca ²⁺ nuclear magnetic resonance titrations. Secondary structure of peptides and proteins was assessed using circular dichroism and nuclear magnetic resonance. Results: Carboxylic acid groups present on Gla residues have 2 different pK _a values of 2.62 ± 0.07 and 5.02 ± 0.05. In presence of calcium ions, pK _a values drop to 2.54 ± 0.02 and 4.55 ± 0.04. Affinity of a single Gla residue for calcium is low (∼15 mM); 2 Gla residues show cooperativity, resulting in a 25-fold increased affinity for calcium ions (0.6 mM). Finally, cooperative calcium ion binding led to increased α-helical content in model proteins. Conclusion: Vitamin K-dependent proteins present Gla residues in a different manner but benefit from cooperative calcium ion binding. Experimentally determined pK _a and K _D values can be used for interpretation of binding interactions or for molecular dynamics simulations of Gla domains with unknown structure.