Coupling of calmodulin's (CaM) calcium binding and enzyme recognition properties: Effects of a point mutation at the CaMipeptide interface

In studies of how calmodulin (CaM) transduces cellular Ca²⁺ signals into biological responses through enzyme regulation, we investigated the effects of CaM mutations on its Ca²⁺ binding properties in the presence and absence of a protein or peptide target. IIE84 of CaM is invariant among all known CaMs and is important for CaM function m vitro (Shoemaker et al (1990), J. Cell Biol. Ill, 1107-1125) and in vivo (Harris et ai (1994], J. Cell Sci. 107, 3235-3249). Charge perturbation mutation of E84 by site directed mutagenesis affects activation of several enzymes including smooth muscle/nonmusle myosin light chain kinase (MLCK). A peptide (RS20) based upon the CaM binding domain of MLCK enhances the Ca²⁺ binding properties of CaM similar to the whole enzyme. E84K-CaM exhibits dramatically altered Ca²⁺ activation of MLCK and altered Ca²⁺ binding by the E84K-CaM:RS20 complex although E84 is more than 20 angstroms from a Ca²⁺ ion. However, the small differences in peptide binding affinity in the presence or absence of Ca²⁺ do not account for the loss in Ca²⁺ binding to the complex. RS20 binding to wild-type and E84K-CaM was analyzed by fluorescence and microcalorimetry titrations. The data indicate that the enthalpic and entropie components of the Ca²⁺-dependent binding of E84K-CaM to RS20 are different from wild-type CaM. These results support a new model of Ca²⁺ binding to CaM complexes that allows for both sequential Ca²⁺ occupancy and partitioning of CaM states that are differentially selected, Supported in part by NI H grant GM30861.