A general strategy to characterize calmodulin–calcium complexes involved in CaM–target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin–calcium complexes

Abstract

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four "EF hand" type Ca(2+) binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca(2+) ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca(n) (n=0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

The adaptors Grb10 and Grb14 are calmodulin‐binding proteins

“…As we have previously described, the minimum number of Ca 2+ bound to CaM required to induce an effect depends on the target protein and might vary from protein to protein, offering extra regulatory mechanisms . We found specific recognition of CaM for the CaM‐BD peptides of Grb7, Grb10 and Grb14, when CaM had a single Ca 2+ bound, suggesting that although the affinity for CaM is different, the recognition mechanism appears to be very similar.…”

Synergetic Effect of Recoverin and Calmodulin on Regulation of Rhodopsin Kinase

“…These processes can be approximated by the Hill equation based model (Scheme [2]), which is widely used for simplified description of such complex processes. Alternatively, the Ca 2+ -dependence of interaction between calmodulin and its target could be described by the model of sequential filling of EF-hands (Dagher et al, 2011). However, this approach requires the introduction into the Scheme [2] of additional chemical equilibria, which would drastically complicate modeling of the system.…”

“…Different approaches could be applied to modeling of equilibria between RK, recoverin, calmodulin, and calcium ions. Full description of the system would require characterization of each step of the calcium-binding to the calcium-sensing proteins (Dagher et al, 2011), as well as characterization of all protein–protein interactions in the system. Considering that membranes are known to affect the distribution of RK and recoverin between soluble and membrane-bound fractions, as well as to modulate calcium affinity of recoverin (Zozulya and Stryer, 1992; Senin et al, 1995; Sanada et al, 1996), it can be concluded that the total number of chemical equilibria is significantly higher (see Modeling of Equilibrium Between RK, Recoverin, Calmodulin, and Calcium Ions in Appendix).…”

Significance of Calcium Binding, Tyrosine Phosphorylation, and Lysine Trimethylation for the Essential Function of Calmodulin in Vertebrate Cells Analyzed in a Novel Gene Replacement System

“…However, CaM can activate some of its targets in a Ca 2ϩ -independent manner (15). Moreover, different CaM target proteins appear to require a distinct number of calcium ions in the Ca 2ϩ -CaM complex to attain maximum binding efficiency (16). Upon Ca 2ϩ binding, CaM undergoes large conformational changes.…”