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

Protein Sci. 2010 Mar;19(3):475-85. doi: 10.1002/pro.325.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Calmodulin / chemistry*
  • Glycine max / metabolism*
  • Hydrophobic and Hydrophilic Interactions
  • Manganese / chemistry
  • Molecular Sequence Data
  • Protein Folding
  • Protein Isoforms / chemistry
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Spectrometry, Fluorescence

Substances

  • Calmodulin
  • Protein Isoforms
  • Manganese