Mutagenesis of Ca²⁺-coordinating residues abolishes the stabilizing effect of Ca²⁺ in A2VicCC. DSF measurements were carried out in the presence of 5 mM CaCl₂ (black) or 1 mM EDTA (gray) with the WT A2VicCC construct and Ca²⁺-binding site mutants D1498A (A), D1596A (B), N1602A (C), D1498A/D1596A (D), D1498A/N1602A (E), D1596A/N1602A (F), and D1498A/D1596A/N1602A (triple, G). Results are means ± SEM of at least 3 independent experiments.