title: Chimeras of yeast and chicken calmodulin demonstrate differences in activation mechanisms of target enzymes.
abstract: Various chimeric proteins were constructed from yeast (Saccharomyces cerevisiae) and chicken calmodulin (CaM), and regions essential for target 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-terminus. Resulting CaM proteins showed Ca2+ binding properties inherent to the original Ca2+ binding site. Cooperative Ca2+ binding and a suitable rearrangement of the two EF-hand sites in each half-molecular domain were shown to be important for high-affinity interaction with CaM-dependent cyclic nucleotide phosphodiesterase (PDE). Residues in chicken CaM sequences 129-148 and 88-128, respectively, were required for low values of Kact (the concentration of CaM required for the half-maximal activation) in the activation of PDE and myosin light chain kinase (skMLCK and smMLCK). The difference in the structural requirements indicated different manners of the interaction. While PDE was activated to similar levels by different chimeras, the maximum activity (Vmax) given by chicken CaMs was not achieved by any chimeric CaMs in MLCKs. Residues in chicken CaM sequences 1-50 and 88-129, in addition to Ca2+ binding to the fourth site, were important for high values of Vmax of skMLCK. On the other hand, Met51 and residues in chicken CaM sequence 88-129 were critical for the high Vmax 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 site of PDE.
authors.affiliation: Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Nakashima
authors.forename: H
authors.initials: H
authors.lastname: Maekawa
authors.forename: M
authors.initials: M
authors.lastname: Yazawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry*; D002645:Chickens; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D009219:Myosin-Light-Chain Kinase; D002118:Calcium