The solution structure of the Mg2+ form of soybean calmodulin isoform 4 reveals unique features of plant calmodulins in resting cells

Soybean calmodulin isoform 4 (sCaM4) is a plant calcium‐binding protein, regulating cellular responses to the second messenger Ca²⁺. We have found that the metal ion free (apo‐) form of sCaM4 possesses a half unfolded structure, with the N‐terminal domain unfolded and the C‐terminal domain folded. This result was unexpected as the apo‐forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg²⁺ ions are always present at high concentrations in cells (0.5–2 mM), we suggest that Mg²⁺ should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg²⁺ the initially unfolded N‐terminal domain of sCaM4 folds into an α‐helix‐rich structure, similar to the Ca²⁺ form. We have used the NMR backbone residual dipolar coupling restraints ¹D_NH, ¹D_CαHα, and ¹D_C′Cα to determine the solution structure of the N‐terminal domain of Mg²⁺‐sCaM4 (Mg²⁺‐sCaM4‐NT). Compared with the known structure of Ca²⁺‐sCaM4, the structure of the Mg²⁺‐sCaM4‐NT does not fully open the hydrophobic pocket, which was further confirmed by the use of the fluorescent probe ANS. Tryptophan fluorescence experiments were used to study the interactions between Mg²⁺‐sCaM4 and CaM‐binding peptides derived from smooth muscle myosin light chain kinase and plant glutamate decarboxylase. These results suggest that Mg²⁺‐sCaM4 does not bind to Ca²⁺‐CaM target peptides and therefore is functionally similar to apo‐mCaM. The Mg²⁺‐ and apo‐structures of the sCaM4‐NT provide unique insights into the structure and function of some plant calmodulins in resting cells.