Han C H, Roberts D M
Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville 37996-0840, USA.
Eur J Biochem. 1997 Mar 15;244(3):904-12. doi: 10.1111/j.1432-1033.1997.00904.x.
Calmodulin is trimethylated on Lys115 by a specific calmodulin methyltransferase. Previously, it was shown that the cam2 mutant (Ile136-->Thr) of Paramecium has a decreased level of methylated Lys115 [Lukas, T. J., Friedman, M. W., Kung, C. & Watterson, D. M. (1989) Proc. Natl Acad. Sci. USA 86, 7331-7335]. To investigate how this substitution affects calmodulin structure, function and recognition by the calmodulin methyltransferase, a calmodulin with a Thr136 substitution ([Thr136]calmodulin) was expressed in Escherichia coli in an unmethylated form for in vitro enzyme activator, calcium binding and methylation kinetic analyses. [Thr136]calmodulin was indistinguishable from wild-type calmodulin in saturating (1 mM) calcium in its ability to activate calmodulin-dependent enzymes and in its steady-state kinetic properties with isolated calmodulin methyltransferase. However, [Thr136]calmodulin did show two defects: a complete inability to be methylated in the absence of calcium; and defective calcium binding. As a result, an approximate 10-fold shift in the K0.5 values for calcium dependence of enzyme activation (shifted from 1.1 microM to 9.1 microM of Ca2+ for NAD kinase) and methylation (from 0.71 microM to 7.2 microM of Ca2+ in 0.15 M K+, 2 mM Mg2+) were observed. Non-denaturing electrophoresis and Tyr138 spectroscopic measurements suggest a difference in the conformation of the calcium-depleted structures of normal calmodulin and [Thr136]calmodulin. Overall, the results suggest that the mutation in this conserved position in the COOH-terminal hydrophobic core lowers calcium-binding affinity and alters the calcium-depleted structure leading to decreased methylation at physiological Ca2+ concentrations.
钙调蛋白在赖氨酸115位点被一种特定的钙调蛋白甲基转移酶三甲基化。此前研究表明，草履虫的cam2突变体（异亮氨酸136突变为苏氨酸）中赖氨酸115的甲基化水平降低[卢卡斯，T. J.，弗里德曼，M. W.，孔，C. & 沃特森，D. M.（1989年）《美国国家科学院院刊》86卷，7331 - 7335页]。为研究这种替换如何影响钙调蛋白的结构、功能以及被钙调蛋白甲基转移酶识别的情况，一种苏氨酸136替换的钙调蛋白（[苏氨酸136]钙调蛋白）在大肠杆菌中以未甲基化形式表达，用于体外酶激活、钙结合和甲基化动力学分析。[苏氨酸136]钙调蛋白在饱和（1 mM）钙浓度下激活钙调蛋白依赖性酶的能力以及与分离的钙调蛋白甲基转移酶的稳态动力学特性方面，与野生型钙调蛋白没有区别。然而，[苏氨酸136]钙调蛋白确实表现出两个缺陷：在没有钙的情况下完全无法被甲基化；以及钙结合存在缺陷。结果，观察到酶激活（对于NAD激酶，钙离子依赖性的K0.5值从1.1微摩尔转移到9.1微摩尔钙离子）和甲基化（在0.15 M钾离子、2 mM镁离子存在下，从0.71微摩尔钙离子转移到7.2微摩尔钙离子）的钙离子依赖性K0.5值大约有10倍的变化。非变性电泳和酪氨酸138光谱测量表明，正常钙调蛋白和[苏氨酸136]钙调蛋白的钙耗尽结构在构象上存在差异。总体而言，结果表明在COOH末端疏水核心中这个保守位置的突变降低了钙结合亲和力，并改变了钙耗尽结构，导致在生理钙离子浓度下甲基化减少。
