Wu X, Reid R E
Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Biochemistry. 1997 Mar 25;36(12):3608-16. doi: 10.1021/bi962149m.
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.
利用位点特异性盒式诱变技术制备了两种钙调蛋白突变体F92W和F92W/D133E，以研究钙结合位点III和IV中阳离子螯合残基的结构/钙亲和力关系。制备突变体F92W是为了产生一个强荧光标记，以跟踪蛋白质C端结构域中钙诱导的结构变化。制备第二个突变体F92W/D133E是为了破坏钙与位点IV的结合，从而消除位点III和IV之间的协同作用。C端结构域中两个位点的宏观钙解离常数来自拟合到双位点希尔方程的钙滴定数据。F92W/D133E突变体中位点III和位点IV的钙解离常数分别为335 μM和2.76 mM。这些值分别显著高于F92W钙调蛋白中位点III和位点IV的14 μM和1 μM。这些结果表明，位点IV钙结合环+Z位置上一个非常保守的D133E突变极大地降低了该位点的钙结合亲和力（2760倍），同时也显著降低了同一结构域中位点III的钙结合亲和力（24倍）。在低钙浓度（50 μM）下，D/E钙调蛋白突变体的磷酸二酯酶激活活性也比F92W钙调蛋白低3倍，对该酶的亲和力低25倍。然而，在高钙浓度（15 mM）下，F92W/D133E突变体的最大磷酸二酯酶激活活性和该突变体对该酶的亲和力与F92W钙调蛋白相似，这表明D133E突变改变了钙调蛋白介导的磷酸二酯酶活性的钙调节，而不影响钙调蛋白与该酶的相互作用。
