Calcium-dependent and -independent Binding of Soybean Calmodulin Isoforms to the Calmodulin Binding Domain of Tobacco MAPK Phosphatase-1

Abstract

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.

Structural Studies of Soybean Calmodulin Isoform 4 Bound to the Calmodulin-binding Domain of Tobacco Mitogen-activated Protein Kinase Phosphatase-1 Provide Insights into a Sequential Target Binding Mode

“…10, b and c). This result agrees with the binding constant derived by ITC that is very similar to the previously reported binding constant of NtMKP1pA with the N-lobe fragment of SCaM isoforms (26). On the other hand, a few relatively small CSPs were observed for the C-lobe of SCaM4.…”

“…6d). Because all the side chains of the Met residues in CaM (except for Met 76 in the central linker) form direct hydrophobic contacts with the CaMBDs in most Ca 2ϩ -CaM-target complexes, the methyl signals of the Met residue have been utilized extensively as a sensitive probe to monitor Ca 2ϩ -CaM-target interactions (26,27,47,48). For essentially all the methyl signals of SCaM4CT-Nt-MKP1, the corresponding signals are found in almost the same positions in the spectrum of the noncovalent SCaM4/ All experiments were performed at 30°C, 100 mM KCl, 2 mM CaCl 2 , 20 mM HEPES (pH 7.5).…”

“…. On the other hand, the binding of a second CaMBD of NtMKP1 occurs only through the N-lobe of Ca 2ϩ -CaM, and the binding constant was around 10 5 M Ϫ1 (26). To date, structural information about the manner in which Ca 2ϩ -CaM binds sequentially to the unusual amino acid sequence of the CaMBD of NtMKP1 is unavailable.…”

Structural Insights into Calmodulin-regulated L-selectin Ectodomain Shedding

“…From these titrations, the requirement for Ca 2ϩ as well as a portion of L-selectin predicted to be in the membrane-spanning region becomes apparent. The binding curve of LSELl with Ca 2ϩ -CaM is characterized by a two-step process, similar to that reported for other CaM-binding peptides (32,33). The first, which describes the binding of the first peptide to CaM, has a dissociation constant (K d ) on the order of 10 Ϫ9 M and is driven predominantly by entropic factors (Fig.…”

Calmodulin has the Potential to Function as a Ca2+-Dependent Adaptor Protein

“…21 Using several biophysical techniques we recently characterized the interaction between CaM isoforms (mammalian CaM, soybean CaM isoforms SCaM-1 and SCaM-4) and a novel CaMBD derived from the Nicotiana tabacum mitogen-activated protein kinase phosphatase (NtMKP1). 22 The NtMKP1 protein was initially identified as a CaM-binding protein by Ohashi and coworkers, 23 and the same group recently showed that CaM-binding NtMKP1 homologs are also present in other plant species as well. 24 We found that each CaM isoform was capable of binding to the NtMKP1 CaMBD in the absence of Ca 2+ using only the apo-C-lobe, with the primary binding site consisting of NtMKP1 residues N438 -S449, and additional C-terminal residues G450 -K460 enhancing the overall binding affinity (K d ~10 -5 M).…”