The EF-hand protein S100A2 is a cell cycle regulator involved in tumorigenesis, acting through regulation of the p53 activation state. Metal ion-free S100A2 is homodimeric and contains two Ca(2+)-binding sites and two Zn(2+)-binding sites per subunit, whereby the Zn(2+) ion binding to one of the sites is coordinated by residues from two homodimers. The effect of selective binding of these metal ions was investigated using site-specific mutants which lacked one or both zinc sites. CD analysis of secondary structure changes on metallation showed that Zn(2+) binding was associated with a decrease in the secondary structure content, whereas Ca(2+) had the opposite effect in two of the three S100A2 mutants studied. The energy of unfolding (DeltaG(U)) of the apo wild-type S100A2 was determined to be 89.9 kJ mol(-1), and the apparent midpoint transition temperature (T(m)(app))) was 58.4 degrees C. In addition, a detailed study of the urea and thermal unfolding of the S100A2 mutants in different metallation states (apo, Zn(2+) and Ca(2+)) was performed. Thermal denaturation experiments showed that Zn(2+) acts as a destabilizer and Ca(2+) as a stabilizer of the protein conformation. This suggests a synergistic effect between metal binding, protein stability and S100A2 biological activity, according to which Ca(2+) activates and stabilizes the protein, the opposite being observed on Zn(2+) binding.