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 alpha-helix-rich structure, similar to the Ca2+ form. We have used the NMR backbone residual dipolar coupling restraints D-1(NH), D-1(C alpha H alpha), and D-1(c'c alpha) 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 Mg(2+)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.