Abstract
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.
Highlights
Theyeast (Saccharomycescereuisiae) calmodulin is The structural basis of the enzyme activation mechanism unable to bind Ca2+
Further systematic studies are required to Calmodulin (CaM)' regulates a wide variety of eukaryotic understand how CaM recognizes a wide variety of target cellular functions by modulating the activity of Ca"-depend- proteins
CaM Calmodulin from the bakers' yeast (Saccharomyces cereuisis an acidic protein with a molecular mass of 16,700Da that iae) was found to have remarkably different properties from contains four EF-hand Ca2+-bindingdomains [1].X-ray crys- those of animal CaMs [17, 18].The amino acid sequence of tallographic analysis of vertebrate CaM has revealed a dumb- yeast CaM shares only 59% identity with vertebrate CaM
Summary
A ~ p ' ~ ~ - L yosf' ~ye~ast CaM. A BarnHI fragment of plasmid pCCMO(Cla1) containing chicken CaM cDNA was subcloned into a double strand replicative form DNA of MI3 mp phage. Since the introduced ClaI site used for recombination spans residues Ile' antdhaemino acid residues aftetrhe second position of the Ca2+ bindingloop of domain IV were replaced in each recombinantmutant.,the first residue of loop IV in mutant Y4C was Ser"' as inyeast CaM, andthefirst residue of the loop IV inmutants C4Y and. The difference in the structure of Retention times (RT)of CaM or mutant proteins on C8 reversedomain IV may directly relate to the higher order structure of CaM and itsCa2+-dependentchange Enzyme Activation-Activation of PDE and myosin light chain kinase by scallop CaM and domain IV-exchanged CaM phase columnwere measured inthe presence and the absencoef Ca2' as described under 'Experimental Procedures." ART is the difference in retention times observed in the aknce and in the presence of Caz+.Assays of PDE and myosin light chain kinase (MLCK) were performed as described under "Experimental Procedures." For both mutants were investigated.
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