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

## TL;DR

This study reveals how γ-carboxyglutamic acid (Gla) binds calcium, showing that two Gla residues are needed for strong binding and structural changes in proteins.

## Contribution

The study experimentally determines pKa and KD values for Gla calcium binding and shows cooperativity between two Gla residues.

## Key findings

- Gla residues have two pKa values (2.62 and 5.02) that decrease in the presence of calcium.
- Two Gla residues exhibit 25-fold increased calcium affinity due to cooperativity.
- Cooperative calcium binding increases α-helical content in model proteins.

## Abstract

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 (pKa and KD) 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 pKa 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 KD 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 pKa values of 2.62 ± 0.07 and 5.02 ± 0.05. In presence of calcium ions, pKa 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 pKa and KD values can be used for interpretation of binding interactions or for molecular dynamics simulations of Gla domains with unknown structure.

•γ-Carboxyglutamic acid (Gla) is an essential amino acid for coagulation and mineralization.•Biophysical characteristics of Gla are not completely known.•Gla has 2 different pKa values that decrease in presence of calcium.•Two Gla residues are required for optimal calcium binding.

γ-Carboxyglutamic acid (Gla) is an essential amino acid for coagulation and mineralization.

Biophysical characteristics of Gla are not completely known.

Gla has 2 different pKa values that decrease in presence of calcium.

Two Gla residues are required for optimal calcium binding.