Mutagenesis of the fourth calcium-binding domain of yeast calmodulin.
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.
Matsuura
I
I
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan.
Kimura
E
E
Tai
K
K
Yazawa
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002645:Chickens; D004789:Enzyme Activation; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D009838:Oligodeoxyribonucleotides; D009219:Myosin-Light-Chain Kinase; D010727:Phosphoric Diester Hydrolases; D002118:Calcium