Yamniuk Aaron P, Vogel Hans J
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
J Biol Chem. 2004 Feb 27;279(9):7698-707. doi: 10.1074/jbc.M310763200. Epub 2003 Dec 11.
The discovery that plants contain multiple calmodulin (CaM) isoforms having variable sequence identity to mammalian CaM has sparked a flurry of new questions regarding the intracellular role of Ca(2+) regulation in plants. To date, the majority of research in this field has focused on the differential enzymatic regulation of various mammalian CaM-dependent enzymes by the different plant CaM isoforms. However, there is comparatively little information on the structural recognition of target enzymes found exclusively in plant cells. Here we have used a variety of spectroscopic techniques, including nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy, to study the interactions of the most conserved and most divergent CaM isoforms from soybean, SCaM-1, and SCaM-4, respectively, with a synthetic peptide derived from the CaM-binding domain of cauliflower vacuolar calcium-ATPase. Despite their sequence divergence, both SCaM-1 and SCaM-4 interact with the calcium-ATPase peptide in a similar calcium-dependent, stoichiometric manner, adopting an antiparallel binding orientation with an alpha-helical peptide. The single Trp residue is bound in a solvent-inaccessible hydrophobic pocket on the C-terminal domain of either protein. Thermodynamic analysis of these interactions using isothermal titration calorimetry demonstrates that the formation of each calcium-SCaM-calcium-ATPase peptide complex is driven by favorable binding enthalpy and is very similar to the binding of mammalian CaM to the CaM-binding domains of myosin light chain kinases and calmodulin-dependent protein kinase I.
植物含有多种与哺乳动物钙调蛋白（CaM）具有不同序列一致性的钙调蛋白异构体，这一发现引发了一系列关于植物细胞内Ca(2+)调节作用的新问题。迄今为止，该领域的大多数研究都集中在不同植物CaM异构体对各种哺乳动物CaM依赖性酶的差异酶调节上。然而，关于仅在植物细胞中发现的靶酶的结构识别的信息相对较少。在这里，我们使用了多种光谱技术，包括核磁共振、圆二色性和荧光光谱，分别研究了大豆中最保守和最不同的CaM异构体SCaM-1和SCaM-4与源自花椰菜液泡钙-ATP酶CaM结合域的合成肽的相互作用。尽管它们的序列不同，但SCaM-1和SCaM-4都以类似的钙依赖性、化学计量方式与钙-ATP酶肽相互作用，与α-螺旋肽采用反平行结合方向。单个色氨酸残基结合在两种蛋白质C末端结构域上一个溶剂不可及的疏水口袋中。使用等温滴定量热法对这些相互作用进行的热力学分析表明，每个钙-SCaM-钙-ATP酶肽复合物的形成是由有利的结合焓驱动的，并且与哺乳动物CaM与肌球蛋白轻链激酶和钙调蛋白依赖性蛋白激酶I的CaM结合域的结合非常相似。
