α-actinin is a protein that, through its actin cross-linking activity, contributes to the organization of the actin cytoskeleton. The non-muscle α-actinin proteins, ACTN1 and ACTN4, contain a calmodulin-like domain (CaMD) at their C-terminus, composed of four EF-hand motifs. While Ca2+-binding to the CaMD is well described, the effect of Mg2+-ions on this domain is less well understood. The aim of this work was to investigate the interaction between Mg2+-ions and the recombinant CaMD domains of human α-actinins ACTN1 and ACTN4. Both proteins were expressed in a bacterial system and purified using a combination of chromatographic methods. The interaction between Mg2+-ions and each CaMD was studied by isothermal titration calorimetry (ITC), while the effect of Mg2+ and Ca2+-ions on protein thermal stability was assessed by DLS, nanoDSF and circular dichroism spectroscopy (CD). Based on structural and coordination differences between Ca2+ and Mg2+-ions, we hypothesized that Mg2+-binding to the CaMD of ACTN1 and ACTN4 would be weak or non-specific. ITC results did not show a clear binding isotherm that would allow reliable determination of thermodynamic binding parameters. The observed heat signals are most likely the result of non-specific electrostatic interactions between Mg2+-ions and the negatively charged protein surface. nanoDSF measurements showed a linear increase in the 350 nm/330 nm fluorescence ratio. This does not indicate protein denaturation, however, particle size measurements show a slight inflection, though at approximately the same temperature for all samples. CD measurements showed stabilization of the structure in the presence of Ca2+-ions. The results confirm the hypothesis that Mg2+-ions binds nonspecifically to the CaMD of α-actinin-1 and 4, with substantially lower affinity than Ca2+-ions.
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