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

Abstract

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.