Conservative D133E mutation of calmodulin site IV drastically alters calcium binding and phosphodiesterase regulation.
Two calmodulin mutants, F92W and F92W/D133E, were prepared using site-specific cassette-mediated mutagenesis to examine the structure/calcium affinity relationships of cation chelating residues in calcium binding sites III and IV. The mutant, F92W, was prepared to produce a strong fluorescent label to follow the calcium-induced structural changes in the C-terminal domain of the protein. A second mutant, F92W/D133E, was prepared to destroy the calcium binding to site IV and thereby eliminate cooperativity between sites III and IV. The macroscopic calcium dissociation constants of the two sites in the C-terminal domain were derived from the calcium titration data that had been fitted to a two-site Hill equation. The calcium dissociation constants of site III and site IV in the F92W/D133E mutant were 335 microM and 2.76 mM, respectively. These values were significantly greater than the values of 14 and 1 microM for site III and site IV in F92W calmodulin, respectively. These results suggested that a very conservative D133E mutation in the +Z position of the site IV Ca2+-binding loop drastically decreased the calcium binding affinity of the site (2760-fold) and also significantly reduced that of site III in the same domain (24-fold). The D/E calmodulin mutant also had a 3-fold lower phosphodiesterase activation activity with a 25-fold lower affinity for this enzyme than that of F92W calmodulin in the presence of low calcium concentration (50 microM). However, the maximum phosphodiesterase activation activity of the F92W/D133E mutant and the affinity of this mutant for the enzyme were similar to those of F92W calmodulin in the presence of high calcium concentration (15 mM), suggesting that the D133E mutation altered calcium regulation of calmodulin mediated phosphodiesterase activity without affecting calmodulin interaction with the enzyme.
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Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
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D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D002118:Calcium