Calcium sensors in plants: biochemical and biophysical study of Arabidopsis thaliana calmodulin-like proteins 14 and 36

Calcium (Ca2+) regulates a wide range of physiological responses to external stimuli in plants. Besides conserved Calmodulins (CaMs), plants possess a unique family of 50 calmodulin-like proteins (CMLs) playing important roles in plant development and stress responses. However, to date, only the Ca2+ binding properties of few CMLs have been experimentally determined. The present study reports a detailed biochemical and biophysical in vitro characterization of two recombinant CMLs, namely, CML14 and CML36 from Arabidopsis thaliana, undertaken in order to clarify whether these proteins may be endowed with the typical features of the Ca2+ sensors. CML14 possesses three EF-hand motifs, but only one is predicted to be a functional Ca2+ binding site, whereas CML36 is predicted to have three functional Ca2+ binding sites despite the presence of four EF-hand motifs. We applied isothermal titration calorimetry (ITC) to analyse the energetics of Ca2+ and Mg2+ binding to CML14 and CML36. In addition nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy, size exclusion chromatography (SEC) were used together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry (DSC) and limited proteolysis were used to characterize proteins thermal and local stability. Our results demonstrated that CML14 binds one Ca2+ ion with a micromolar affinity (Kd ~ 12 µM) while, the presence of Mg2+ decreases the Ca2+ affinity by ~ 5-fold. Although binding of Ca2+ to CML14 increased thermal and local stability, it does not result in a more hydrophobic protein surface and does not induce a large conformational rearrangement. However Ca2+ binding causes only localized structural changes in the unique functional EF-hand. Regarding the CML36 protein, our data revealed that the binding of Mg2+ and Ca2+ induce significant and distinct conformational changes throughout the protein. In particular, the binding of Ca2+ to CML36 increased the local stability and in line with a Ca2+ sensor function, the protein underwent a Ca2+- dependent increase in surface-exposed hydrophobicity while Mg2+ dependent effect was not noted. In addition, our findings enabled us to speculate on the apo- form of CML36 as a flexible molten globule state. CML36 binds three Ca2+ ions in the absence of Mg2+ with a high affinity binding site (Kd ~ 0.7 µM) and two lower affinity sites (Kd ~ 47 µM), and two Ca2+ ions in the presence of Mg2+ (Kd1 ~ 0.7 µM and Kd2 ~ 5 µM). These data suggest that, under physiological conditions, one EF-hand must be constitutively bound to Mg2+ while Ca2+ binds functionally only to two EF-hands. Together our results suggested that CML14 does not appear to be endowed with the characteristics of a classical Ca2+ sensor and, in contrast to the typical switch-like role controlled by protein exposure of the hydrophobic patches, indicate a different role of Ca2+ for target interactions of CML14. Instead CML36 showed the characteristics of a typical Ca2+ sensor. In conclusion our work provides new and interesting insights into the biochemical properties of Arabidopsis CML14 and CML36 that may be useful to future studies aimed at elucidating their precise physiological role.