Calcium-dependent and -independent binding of soybean calmodulin isoforms to the calmodulin binding domain of tobacco MAPK phosphatase-1.
The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.
Rainaldi
Mario
M
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Yamniuk
Aaron P
AP
Murase
Tomohiko
T
Vogel
Hans J
HJ
D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D054638:Dual Specificity Phosphatase 1; D017874:Immediate-Early Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D015398:Signal Transduction; D013025:Glycine max / Q000737:chemistry*; D014026:Nicotiana / Q000201:enzymology*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D010940:Plant Proteins; D020033:Protein Isoforms; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases; D002118:Calcium