MUTAGENESIS OF THE 4TH CALCIUM-BINDING DOMAIN OF YEAST CALMODULIN

The fourth calcium-binding domain (domain IV) of the yeast (Saccharomy ces cerevisiae) calmodulin is unable to bind Ca2+ (Matsuura, I., Ishih ara, K., Nakai, Y., Yazawa, M., Toda, H., and Yagi, K. (1991) J. Bioch em. (Tokyo) 109, 190-;197). The functional significance of Ca2+ bindin g to domain IV was investigated by site-directed mutagenesis or recomb inant DNA techniques. A recognition site for the restriction enzyme Cl aI was introduced at the homologous position of Ile130 in the nucleoti de sequence of chicken and yeast calmodulin cDNA, and chimeric protein s of the yeast and the vertebrate calmodulin were prepared. One of the resulting mutants named C4Y consisted of Ala1-Ile130 of chicken calmo dulin and Asp131-Lys148 of yeast calmodulin. The mutant C4Y showed the yeast-type feature, and its enzyme activation profiles were similar t o those of yeast calmodulin. A single substitution of Glu for Gln140 w as carried out in the mutant C4Y. The resulting mutant (C4Y140E) bound 4 mol of Ca2+ and showed the vertebrate-type enzyme activation. There fore, the alterations of 3 residues in the Ca2+-binding loop of the ye ast-type domain IV, Ser129 --> Asp, insertion of Ile130, and Gln140 -- > Glu, were enough for the recovery of Ca2+ binding and enzyme activat ion. Ca2+ binding to domain IV may induce the active conformation of t he C-terminal half-molecular domain.