Structural investigation into the differential target enzyme regulation displayed by plant calmodulin isoforms.
The conserved calmodulin (CaM) isoform SCaM-1 and the divergent SCaM-4 from soybean bind to many of the same target enzymes, but differentially activate or competitively inhibit them. Class 1 target enzymes are activated by both calcium (Ca(2+))-bound SCaM-1 (Ca(2+)-SCaM-1) and Ca(2+)-bound SCaM-4 (Ca(2+)-SCaM-4), while class 2 enzymes are activated by Ca(2+)-SCaM-1 but competitively inhibited by Ca(2+)-SCaM-4, and class 3 enzymes are activated by Ca(2+)-SCaM-4 but competitively inhibited by Ca(2+)-SCaM-1. To determine whether these differences can be attributed to unique interactions with the CaM-binding domains (CaMBD) of these enzymes, we have studied the binding of each protein to peptides derived from the CaMBD of a representative target enzyme from each of these three classes. Using a combination of NMR spectroscopy and isothermal titration calorimetry, we demonstrate that the N- and C-domains of either Ca(2+)-SCaM bind to each peptide to form structurally compact complexes driven by the burial of hydrophobic surfaces. Interestingly, the interactions with the CaMBD peptides from classes 1 and 2 are similar for the two proteins; however, binding to the peptide from class 3 is structurally and thermodynamically distinct for Ca(2+)-SCaM-1 and -4. We also demonstrate that both calcium-free SCaM-1 (apo-SCaM-1) and calcium-free SCaM-4 (apo-SCaM-4) bind to the CaMBD from cyclic nucleotide phosphodiesterase, and that the interactions are similar to each other and to the interactions with apo-mammalian CaM. Therefore, the apo-SCaMs are also capable of binding to the same target enzymes, which could provide an additional mechanism for CaM-dependent signaling in plants.
Yamniuk
Aaron P
AP
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4.
Vogel
Hans J
HJ
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D002151:Calorimetry; D017124:Conserved Sequence; D019277:Entropy; D004798:Enzymes / Q000378:metabolism*; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D013025:Glycine max / Q000378:metabolism; D013816:Thermodynamics
D002147:Calmodulin; D004798:Enzymes; D010940:Plant Proteins; D020033:Protein Isoforms; D002118:Calcium