Abstract
The discovery that plants contain multiple calmodulin (CaM) isoforms having variable sequence identity to mammalian CaM has sparked a flurry of new questions regarding the intracellular role of Ca(2+) regulation in plants. To date, the majority of research in this field has focused on the differential enzymatic regulation of various mammalian CaM-dependent enzymes by the different plant CaM isoforms. However, there is comparatively little information on the structural recognition of target enzymes found exclusively in plant cells. Here we have used a variety of spectroscopic techniques, including nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy, to study the interactions of the most conserved and most divergent CaM isoforms from soybean, SCaM-1, and SCaM-4, respectively, with a synthetic peptide derived from the CaM-binding domain of cauliflower vacuolar calcium-ATPase. Despite their sequence divergence, both SCaM-1 and SCaM-4 interact with the calcium-ATPase peptide in a similar calcium-dependent, stoichiometric manner, adopting an antiparallel binding orientation with an alpha-helical peptide. The single Trp residue is bound in a solvent-inaccessible hydrophobic pocket on the C-terminal domain of either protein. Thermodynamic analysis of these interactions using isothermal titration calorimetry demonstrates that the formation of each calcium-SCaM-calcium-ATPase peptide complex is driven by favorable binding enthalpy and is very similar to the binding of mammalian CaM to the CaM-binding domains of myosin light chain kinases and calmodulin-dependent protein kinase I.
Highlights
The calcium ion (Ca2ϩ) is one of the most important signaling molecules in both plant and animal cells
Because the majority of studies on the SCaMs have focused on their capacity to activate mammalian target enzymes, we chose to perform structural studies on a CaM-binding domain (CaMBD) derived from a plant protein, because it provides a more direct representation of the interactions with the plant CaM isoforms in vivo
Overall the results of our studies have demonstrated that the bindings of both Ca2ϩSCaM-1 and -4 to CATPp are very similar to each other, and that each one binds in a Ca2ϩ-dependent manner with a 1:1 molar stoichiometry. We showed that both lobes of each protein are involved in binding to CATPp, and that the Trp5 residue near the N terminus of the peptide binds into a solvent-inaccessible hydrophobic pocket on the C-domain of either protein
Summary
CaM, calmodulin; CATPp, calciumATPase peptide; mCaM, mammalian calmodulin; SCaM, soybean calmodulin; smMLCK, smooth muscle myosin light chain kinase; PMCA, plasma membrane calcium-ATPase; BCA1, cauliflower vacuolar calcium-ATPase; CaMKI, calmodulin-dependent protein kinase I; MALDI, matrix-assisted laser desorption ionization; ITC, isothermal titration calorimetry; DTT, dithiothreitol; TFE, trifluoroethanol; CaMBD, calmodulin-binding domain; HSQC, heteronuclear single quantum coherence. All of the information obtained by various spectroscopic techniques suggests that despite their sequence divergence, both SCaM-1 and -4 bind to CATPp in a structurally homologous manner, similar to Ca2ϩ-mCaM binding to many of its target peptides, including those from CaM-dependent protein kinase I (CaMKI) and the MLCKs
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