Nakashima K, Maekawa H, Yazawa M
Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, Japan.
Biochemistry. 1996 Apr 30;35(17):5602-10. doi: 10.1021/bi952586l.
Various chimeric proteins were constructed from yeast (Saccharomyces cerevisiae) and chicken calmodulin (CaM), and regions essential for target activation and responsible for the specific features of the yeast CaM were identified. The chimeric CaMs were designed so that each Ca2+ binding site of the yeast CaM was replaced in series from the C-terminus. Resulting CaM proteins showed Ca2+ binding properties inherent to the original Ca2+ binding site. Cooperative Ca2+ binding and a suitable rearrangement of the two EF-hand sites in each half-molecular domain were shown to be important for high-affinity interaction with CaM-dependent cyclic nucleotide phosphodiesterase (PDE). Residues in chicken CaM sequences 129-148 and 88-128, respectively, were required for low values of Kact (the concentration of CaM required for the half-maximal activation) in the activation of PDE and myosin light chain kinase (skMLCK and smMLCK). The difference in the structural requirements indicated different manners of the interaction. While PDE was activated to similar levels by different chimeras, the maximum activity (Vmax) given by chicken CaMs was not achieved by any chimeric CaMs in MLCKs. Residues in chicken CaM sequences 1-50 and 88-129, in addition to Ca2+ binding to the fourth site, were important for high values of Vmax of skMLCK. On the other hand, Met51 and residues in chicken CaM sequence 88-129 were critical for the high Vmax of smMLCK. These residues may work to form the active structure of the catalytic site of each MLCK, while simple binding of CaM seems sufficient to expose the active site of PDE.
构建了多种由酵母（酿酒酵母）和鸡钙调蛋白（CaM）组成的嵌合蛋白，并鉴定了激活靶点所必需的区域以及酵母CaM特定特征的相关区域。设计嵌合CaM时，使酵母CaM的每个Ca²⁺结合位点从C端开始依次替换。所得的CaM蛋白表现出原始Ca²⁺结合位点固有的Ca²⁺结合特性。协同Ca²⁺结合以及每个半分子结构域中两个EF手型位点的适当重排对于与CaM依赖性环核苷酸磷酸二酯酶（PDE）的高亲和力相互作用很重要。在激活PDE和肌球蛋白轻链激酶（skMLCK和smMLCK）时，鸡CaM序列129 - 148和88 - 128中的残基分别是Kact（半最大激活所需的CaM浓度）低值所必需的。结构要求的差异表明了不同的相互作用方式。虽然不同的嵌合体将PDE激活到相似水平，但任何嵌合CaM在MLCKs中都未达到鸡CaMs给出的最大活性（Vmax）。除了Ca²⁺结合到第四个位点外，鸡CaM序列1 - 50和88 - 129中的残基对于skMLCK的高Vmax很重要。另一方面，Met51和鸡CaM序列88 - 129中的残基对于smMLCK的高Vmax至关重要。这些残基可能有助于形成每个MLCK催化位点的活性结构，而CaM的简单结合似乎足以暴露PDE的活性位点。
