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

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.

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.

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 Ca²⁺ binding were determined using Ca²⁺ nuclear magnetic resonance titrations. Secondary structure of peptides and proteins was assessed using circular dichroism and nuclear magnetic resonance.

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.

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.