Calmodulin CaM is a multifunctional calcium transducer It fu

Calmodulin (CaM) is a multifunctional calcium transducer [8]. It functions as a central regulator of cellular metabolism in response to changes in cellular calcium levels by interacting with various targets [9]. Numerous studies have been devoted to the understanding CaM mechanism of action. Early models considered CaM as a simple off/on switch, in which apoCaM was inactive whereas the fully Ca2+ saturated protein allowed interaction with and activation of the various target enzymes. However both 1) the observation, namely with biophysical studies, that CaM Ca2+ BTZ043 Racemate were not equivalent and independent and 2) the steadily increasing number of target proteins and enzymes regulated by CaM, led to postulate a model in which CaM activity was regulated by the number of Ca2+ ions bound to it [10]. Further refinement of the model included spatio-temporal and genetic regulations [11], [12]. It is now generally admitted that CaM is highly flexible, this flexibility sustaining the multi-functionality of the protein [4]. Nevertheless, the precise mechanism underlying integration of a calcium signal by CaM into a quantitative biochemical process and a specific cellular response is still unclear. 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 [13]. 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 [14]. The electrostatic potential has been shown to play a major role in the stability and flexibility of CaM [15]. Investigations of CaM structures in complexes with different target peptides have highlighted the different modes of binding that illustrate the remarkable plasticity of CaM [10], [16], [17], [18], [19]. 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 [10], [14], [20], [21], the target specific Ca2+–CaM cooperative affinities [22], the diversity of CaM–target interaction interfaces [4] and the electrostatic character of the binding surfaces [23]. 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 [10], [23]. 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 [16], [17]. 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.