Meyer D F, Mabuchi Y, Grabarek Z
Muscle Research Group, Boston Biomedical Research Institute, Massachusetts 02114, USA.
J Biol Chem. 1996 May 10;271(19):11284-90. doi: 10.1074/jbc.271.19.11284.
Recent studies have shown that substitution of Ala for one or more Phe residues in calmodulin (CaM) imparts a temperature-sensitive phenotype to yeast (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). The Phe residue immediately preceding the first Ca(2+) ligand in site III of CaM (Phe-92) was found to be of particular importance because the mutation at this position alone was sufficient to induce this phenotype. In the present work we have studied the functional and structural consequences of the Phe-92 --> Ala mutation in human liver calmodulin. We found that the mutant (CaMF92A) is incapable of activating phosphodiesterase, and the maximal activation of calcineurin is reduced by 40% as compared with the wild type CaM. Impaired regulatory properties of CaMF92A are accompanied by an increase in affinity for Ca(2+) at the C-terminal domain. To investigate the structural consequences of the F92A mutation, we constructed four recombinant C-terminal domain fragments (C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW); 2) Ala substituted for Phe-92 (C-CaMF92A); 3) cysteine residues introduced at position 85 and 112 to lock the domain with a disulfide bond in the Ca(2+)-free (closed) conformation (C-CaM85/112); and 4) mutations 2 and 3 combined (C-CaM85/112F92A). The Cys-containing mutants readily form intramolecular disulfide bonds regardless whether Phe or Ala is present at position 92. The F92A mutation causes a decrease in stability of the domain in the absence of Ca(2+) as indicated by an 11.8 degree C shift in the far UV circular dichroism thermal unfolding curve. This effect is reversed by the disulfide bond in the C-CaM85/112F92A mutant. The C-CaMW peptide shows a characteristic Ca(2+)-dependent increase in solvent-exposed hydrophobic surface which was monitored by an increase in the fluorescence of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid. The fluorescence increase induced by C-CaMF92A is approximately 45% lower than that induced by C-CaMW suggesting that the F92A mutation causes a decrease in the accessibility of several hydrophobic side chains in the C-terminal domain of CaM in the presence of Ca(2+). The Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fold decrease in Ca(2+) affinity depending on whether Phe or Ala is present at position 92, respectively, suggesting that coupling between Ca(2+) binding and the conformational transition is weaker in the absence of the phenyl ring at position 92. Our results indicate that Phe-92 makes an important contribution to the Ca(2+)-induced transition in the C-terminal domain of CaM. This is most likely the reason for the severely impaired regulatory properties of the CaM mutants having Ala substituted for Phe-92.
最近的研究表明，在钙调蛋白（CaM）中用丙氨酸替代一个或多个苯丙氨酸残基会赋予酵母温度敏感型表型（Ohya，Y.，和Botstein，D.（1994年）《科学》263卷，963 - 966页）。发现CaM位点III中第一个Ca(2+)配体之前紧邻的苯丙氨酸残基（Phe - 92）尤为重要，因为仅该位置的突变就足以诱导这种表型。在本研究中，我们研究了人肝脏钙调蛋白中Phe - 92→Ala突变的功能和结构后果。我们发现突变体（CaMF92A）无法激活磷酸二酯酶，与野生型CaM相比，钙调神经磷酸酶的最大激活程度降低了40%。CaMF92A调节特性受损的同时，其C末端结构域对Ca(2+)的亲和力增加。为了研究F92A突变的结构后果，我们构建了钙调蛋白（残基78 - 148）的四个重组C末端结构域片段（C - CaM）：1）野生型（C - CaMW）；2）用丙氨酸替代Phe - 92（C - CaMF92A）；3）在第85和112位引入半胱氨酸残基以通过二硫键在无Ca(2+)（闭合）构象下锁定该结构域（C - CaM85/112）；4）将突变2和3组合（C - CaM85/112F92A）。含半胱氨酸的突变体无论第92位是苯丙氨酸还是丙氨酸都能轻易形成分子内二硫键。F92A突变导致在无Ca(2+)时该结构域稳定性降低，如远紫外圆二色性热变性曲线有11.8℃的位移所示。此效应在C - CaM85/112F92A突变体中被二硫键逆转。C - CaMW肽显示出溶剂暴露疏水表面的特征性Ca(2+)依赖性增加，这通过疏水探针1,1'-双（4 - 苯胺基）-萘 - 5,5'-二磺酸荧光增加来监测。C - CaMF92A诱导的荧光增加比C - CaMW诱导的低约45%，表明F92A突变导致在有Ca(2+)存在时CaM C末端结构域中几个疏水侧链的可及性降低。Cys - 85 - Cys - 112二硫键使Ca(2+)亲和力分别降低10倍或5.9倍，这取决于第92位是苯丙氨酸还是丙氨酸，表明在第92位没有苯环时Ca(2+)结合与构象转变之间的偶联较弱。我们的结果表明Phe - 92对CaM C末端结构域中Ca(2+)诱导的转变有重要贡献。这很可能是用丙氨酸替代Phe - 92的CaM突变体调节特性严重受损的原因。
