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/calciu m affinity relationships of cation chelating residues in calcium bindi ng sites III and IV. The mutant, F92W, was prepared to produce a stron g fluorescent label to follow the calcium-induced structural changes i n the C-terminal domain of the protein. A second mutant, F92W/D133E, w as prepared to destroy the calcium binding to site IV and thereby elim inate 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 mu M and 2.76 mM, respectiv ely. These values were significantly greater than the values of 14 and 1 mu M for site III and site IV in F92W calmodulin, respectively. The se results suggested that a very conservative D133E mutation in the +Z position of the site IV Ca2+-binding loop drastically decreased the c alcium binding affinity of the site (2760-fold) and also significantly reduced that of site III in the same domain (24-fold). The D/E calmod ulin mutant also had a 3-fold lower phosphodiesterase activation activ ity with a 25-fold lower affinity for this enzyme than that of F92W ca lmodulin in the presence of low calcium concentration (50 mu M). Howev er, the maximum phosphodiesterase activation activity of the F92W/D133 E mutant and the affinity of this mutant for the enzyme were similar t o those of F92W calmodulin in the presence of high calcium concentrati on (15 mu M), suggesting that the D133E mutation altered calcium regul ation of calmodulin mediated phosphodiesterase activity without affect ing calmodulin interaction with the enzyme.