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A site-directed mutagenesis study of yeast calmodulin
I Matsuura1, K Ishihara, Y Nakai
1Department of Chemistry, Faculty of Science, Hokkaido University.
Abstract:
A site-directed mutagenesis study was carried out in order to understand the regulatory mechanism of calmodulin. We started from the yeast (Saccharomyces cerevisiae) calmodulin gene since it has many differences in amino acid sequence and inferior functional properties compared with the vertebrate calmodulin. Recombinant yeast calmodulins were generated in Escherichia coli transformed by constructed expression plasmids. Three recombinant calmodulins were obtained. The first two were YCM61G, in which the Ca2(+)-binding site 2 (the four Ca2(+)-binding EF-hand structures in calmodulin were numbered from the N-terminus) was converted to the same as that in vertebrate calmodulin, and YCM delta 132-148, in which the C-terminal half sequence of site 4 was deleted. These two recombinant calmodulins had the same maximum Ca2+ binding (3 mol/mol) as yeast calmodulin, which indicates that site 4 of yeast calmodulin was the one losing Ca2+ binding capacity. YCM delta 132-148 could not activate target enzymes, whereas its Ca2+ binding profile was similar to those of yeast calmodulin and YCM61G. Therefore, the structure in site 4 which cannot bind Ca2+ is indispensable for the regulatory function of yeast calmodulin. The complete regulatory function of vertebrate calmodulin can be attained by the combination of 4 Ca2+ binding structures. The negative charge cluster in the central alpha-helix region is suggested to stabilize the active conformation of calmodulin, since the third yeast calmodulin mutant, YCM83E, which had the negative charge cluster, increased the maximum activation of myosin light chain kinase.
Insights
Site-directed mutagenesis revealed that yeast calmodulin's site 4 is crucial for its regulatory function, despite losing Ca2+ binding capacity. Modifying this site is essential for calmodulin
Area of Science:
- Molecular Biology
- Biochemistry
- Protein Engineering
Background:
- Calmodulin (CaM) is a vital calcium-binding protein regulating numerous cellular processes.
- Yeast (Saccharomyces cerevisiae) calmodulin exhibits distinct sequence and functional differences compared to vertebrate calmodulin.
- Understanding yeast CaM's regulatory mechanism offers insights into CaM evolution and function.
Purpose of the Study:
- To elucidate the regulatory mechanism of yeast calmodulin through site-directed mutagenesis.
- To identify specific structural elements in yeast CaM responsible for its functional properties.
- To compare the Ca2+ binding and enzymatic activation capabilities of wild-type and mutant yeast calmodulins.
Main Methods:
- Site-directed mutagenesis was employed to create recombinant yeast calmodulins in Escherichia coli.
- Three mutants were generated: YCM61G (site 2 altered), YCM delta 132-148 (site 4 deletion), and YCM83E (negative charge cluster).
- Ca2+ binding capacity and activation of target enzymes (myosin light chain kinase) were assessed for each mutant.
Main Results:
- Yeast calmodulin mutants YCM61G and YCM delta 132-148 exhibited reduced Ca2+ binding (3 mol/mol), indicating site 4's loss of binding capacity.
- The YCM delta 132-148 mutant, despite similar Ca2+ binding profiles, failed to activate target enzymes, highlighting site 4's structural importance.
- The YCM83E mutant, with an intact negative charge cluster, demonstrated enhanced myosin light chain kinase activation, suggesting its role in stabilizing active conformation.
Conclusions:
- The structural integrity of site 4 in yeast calmodulin is indispensable for its regulatory function, even with diminished Ca2+ binding.
- Achieving the full regulatory function of vertebrate calmodulin requires four functional Ca2+ binding sites.
- The negative charge cluster in the central alpha-helix region likely stabilizes the active conformation of calmodulin, enhancing enzymatic activity.