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 Ca(2+). 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 Mg(2+) ions are always present at high concentrations in cells (0.5-2 mM), we suggest that Mg(2+) should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg(2+) the initially unfolded N-terminal domain of sCaM4 folds into an alpha-helix-rich structure, similar to the Ca(2+) form. We have used the NMR backbone residual dipolar coupling restraints (1)D(NH), (1)D(C alpha H alpha), and (1)D(C'C alpha) to determine the solution structure of the N-terminal domain of Mg(2+)-sCaM4 (Mg(2+)-sCaM4-NT). Compared with the known structure of Ca(2+)-sCaM4, the structure of the Mg(2+)-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 Mg(2+)-sCaM4 does not bind to Ca(2+)-CaM target peptides and therefore is functionally similar to apo-mCaM. The Mg(2+)- and apo-structures of the sCaM4-NT provide unique insights into the structure and function of some plant calmodulins in resting cells.
Huang
Hao
H
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
Ishida
Hiroaki
H
Vogel
Hans J
HJ
D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013025:Glycine max / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D020033:Protein Isoforms; D008345:Manganese