The three dimensional crystal structure of Ca
The three-dimensional crystal structure of Ca2+ loaded CaM reveals a dumbbell-shaped molecule with two roughly globular lobes, the N- and C-terminal lobes linked by a long solvent-exposed helix, which has been shown by NMR to be non-helical in its central part and flexible in solution . Each globular lobe contains two coupled EF-hand motifs. The two lobes of CaM share a high sequence homology (75%) with significant differences in their electrostatic potential surfaces that confer to these two regions of CaM distinct biochemical properties. Besides these fundamental differences between the two lobes, the flexible linker that separates them, allows for numerous orientations and therefore provides a mean to specifically recognize a large number of distinct peptides used to characterize different CaM/target proteins interactions . The electrostatic potential has been shown to play a major role in the stability and flexibility of CaM . Investigations of CaM structures in complexes with different target peptides have highlighted the different modes of binding that illustrate the remarkable plasticity of CaM , , , , . The selective Ca2+–CaM dependent-target regulation is likely to be due to the order of association of Ca2+, CaM and its target, as well as to the number of calcium ions bound , , , , the target specific Ca2+–CaM cooperative affinities , the M 1145 of CaM–target interaction interfaces  and the electrostatic character of the binding surfaces . CaM exhibits four EF-hand Ca2+-binding sites, numbered I to IV starting at the N-terminal part of the protein. Calcium binding to CaM is best described by a sequential and ordered model (or preferential pathway binding model) which assumes strong coupling factors between the different sites of the molecule , . The sequential model implies a striking asymmetry of the molecule in the apo form (only site III has a high affinity for Ca2+). Calcium binding to the first site then triggers conformational changes allowing the second site to bind Ca2+ with high affinity and so one, with the following order of sites occupancy: site III→site IV→site I→site II , . We assume that this particular Ca2+ binding property partly sustains CaM mechanism of action in that it allows the protein to adopt specific conformations as a function of the number of Ca2+ bound. These specific conformations enable CaM to interact with a given target protein or set of target proteins. In order to further dissect the features underlying partner recognition by CaM, we undertook a quantitative study aimed at characterizing the complexes CaM–Can (n=0–4) involved in target interaction. Using fluorescence polarization, we analyzed the interaction between SynCaM, the product of the synthetic gene coding for a protein hybrid of mammalian and plant CaM able to activate the Ca2+–CaM-dependent enzymes from mammalian and plant cells  and four targets, one being a fluorescent probe previously shown to interact with CaM in a Ca2+-dependent manner , the second two corresponding to a peptide analog of death associated kinase (DAPK) CaM regulatory site in its phosphorylated and unphosphorylated form and the last one, the CaM binding domain of the EGFR receptor . DAPK is a member of the Ser/Thr-kinases, shown to be implicated in the regulation of programmed cell death. When the gene coding for DAPK was discovered, a CaM-binding domain could be identified in the C-terminal part of the kinase domain based on sequence . Yeast two hybrid and CaM overlay assays subsequently confirmed the physical interaction of CaM with its predicted binding domain on DAPK. The presence of Ca2+-bound CaM enhances the kinase\'s enzymatic activity  and CaM/DAPK interaction was shown to be modulated by phosphorylation of the CaM-binding domain. EGFR is a membrane tyrosine kinase receptor of paramount importance in cell proliferation and differentiation. CaM appears to regulate EGFR kinase activity in a Ca2+ dependent manner .