The fourth calcium-binding domain (domain IV) of the yeast (Saccharomyces cerevisiae) calmodulin is unable to bind Ca2+ (Matsuura, I., Ishihara, K., Nakai, Y., Yazawa, M., Toda, H., and Yagi, K. (1991) J. Biochem. (Tokyo) 109, 190-197). The functional significance of Ca2+ binding to domain IV was investigated by site-directed mutagenesis or recombinant DNA techniques. A recognition site for the restriction enzyme ClaI was introduced at the homologous position of Ile130 in the nucleotide sequence of chicken and yeast calmodulin cDNA, and chimeric proteins of the yeast and the vertebrate calmodulin were prepared. One of the resulting mutants named C4Y consisted of Ala1-Ile130 of chicken calmodulin and Asp131-Lys148 of yeast calmodulin. The mutant C4Y showed the yeast-type feature, and its enzyme activation profiles were similar to those of yeast calmodulin. A single substitution of Glu for Gln140 was carried out in the mutant C4Y. The resulting mutant (C4Y140E) bound 4 mol of Ca2+ and showed the vertebrate-type enzyme activation. Therefore, the alterations of 3 residues in the Ca(2+)-binding loop of the yeast-type domain IV, Ser129-->Asp, insertion of Ile130, and Gln140-->Glu, were enough for the recovery of Ca2+ binding and enzyme activation. Ca2+ binding to domain IV may induce the active conformation of the C-terminal half-molecular domain.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|