CHIMERAS OF YEAST AND CHICKEN CALMODULIN DEMONSTRATE DIFFERENCES IN ACTIVATION MECHANISMS OF TARGET ENZYMES

Various chimeric proteins were constructed from yeast (Saccharomyces c erevisiae) and chicken calmodulin (CaM), and regions essential for tar get activation and responsible for the specific features of the yeast CaM were identified. The chimeric CaMs were designed so that each Ca2 binding site of the yeast CaM was replaced in series from the C-termi nus. Resulting CaM proteins showed Ca2+ binding properties inherent to the original Ca2+ binding site. Cooperative Ca2+ binding and a suitab le rearrangement of the two EF-hand sites in each half-molecular domai n were shown to be important for high-affinity interaction with CaM-de pendent cyclic nucleotide phosphodiesterase (PDE). Residues in chicken CaM sequences 129-148 and 88-128, respectively, were required for low values of K-act (the concentration of CaM required for the half-maxim al activation) in the activation of PDE and myosin light chain kinase (skMLCK and smMLCK). The difference in the structural requirements ind icated different manners of the interaction. While PDE was activated t o similar levels by different chimeras, the maximum activity (V-max) g iven by chicken CaMs was not achieved by any chimeric CaMs in MLCKs. R esidues in chicken CaM sequences 1-50 and 88-129, in addition to Ca2binding to the fourth site, were important for high values of V-max of skMLCK. On the other hand, Met51 and residues in chicken CaM sequence 88-129 were critical for the high V-max of smMLCK. These residues may work to form the active structure of the catalytic site of each MLCK, while simple binding of CaM seems sufficient to expose the active sit e of PDE.