Blocking the Ca-induced Conformational Transitions in Calmodulin with Disulfide Bonds (*)
Blocking the Ca-induced Conformational Transitions in Calmodulin with Disulfide Bonds (*)
复制标题
用二硫键阻断钙调蛋白中 Ca 诱导的构象转变 (*)
DOI：
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发表时间：
1996
影响因子：
4.8
通讯作者：
Z. Grabarek
中科院分区：
文献类型：
--
作者：
R. Tan;Y. Mabuchi;Z. Grabarek
细胞内过程的钙依赖性调节是由蛋白质介导的，这些蛋白质在结合 Ca 时呈现出新的构象，这使得它们能够结合到其特定的靶蛋白并调节其功能。钙调蛋白 (CaM) 和肌钙蛋白 C 是两种最具特征的 Ca 调节蛋白，是利用螺旋-环-螺旋结构基序 (EF-hand) 的 Ca 结合蛋白家族的成员。 Herzberg, Moult 和 James (Herzberg, O., Moult, J., and James, M. N. G.(1986) J. Biol. Chem. 261, 2638-2644) 提出肌钙蛋白 C 中 Ca 诱导的构象转变涉及该蛋白 N 末端结构域中 α 螺旋片段之间的界面打开。在这里，我们测试了这样的假设：类似的转变是钙调蛋白中关键的 Ca 诱导的调节事件。使用定点诱变，我们分别用人肝钙调蛋白 N 端和 C 端结构域中的 Gln 和 Lys (CaM41/75) 或 Ile 和 Leu (CaM85/112) 替换半胱氨酸残基。根据分子模型，这些位置的半胱氨酸预计会在蛋白质的无 Ca 构象中形成分子内二硫键，从而阻止假定的 Ca 诱导的转变。我们发现，两种突变体都很容易形成分子内二硫键，导致对 Ca 的亲和力降低，并丧失激活靶酶、磷酸二酯酶和钙调神经磷酸酶的能力。在用二硫苏糖醇还原二硫键并通过羧酰胺甲基化或氰基化封闭半胱氨酸残基后，调节活性在 CaM41/75 中完全恢复，在 CaM85/112 中部分恢复。这些结果表明，Ca 诱导的 CaM 两个域中螺旋段之间的界面打开对于其与 Herzberg-Moult-James 模型一致的调节特性至关重要。
Calcium-dependent regulation of intracellular processes is mediated by proteins that on binding Ca assume a new conformation, which enables them to bind to their specific target proteins and to modulate their function. Calmodulin (CaM) and troponin C, the two best characterized Ca-regulatory proteins, are members of the family of Ca-binding proteins utilizing the helix-loop-helix structural motif (EF-hand). Herzberg, Moult, and James (Herzberg, O., Moult, J., and James, M. N. G.(1986) J. Biol. Chem. 261, 2638-2644) proposed that the Ca-induced conformational transition in troponin C involves opening of the interface between the α-helical segments in the N-terminal domain of this protein. Here we have tested the hypothesis that a similar transition is the key Ca-induced regulatory event in calmodulin. Using site-directed mutagenesis we have substituted cysteine residues for Gln and Lys (CaM41/75) or Ile and Leu (CaM85/112) in the N-terminal and C-terminal domains, respectively, of human liver calmodulin. Based on molecular modeling, cysteines at these positions were expected to form intramolecular disulfide bonds in the Ca-free conformation of the protein, thus blocking the putative Ca-induced transition. We found that intramolecular disulfide bonds are readily formed in both mutants causing a decrease in affinity for Ca and the loss of ability to activate target enzymes, phosphodiesterase and calcineurin. The regulatory activity is fully recovered in CaM41/75 and partially recovered in CaM85/112 upon reduction of the disulfide bonds with dithiotreitol and blocking the Cys residues by carboxyamidomethylation or cyanylation. These results indicate that the Ca-induced opening of the interfaces between helical segments in both domains of CaM is critical for its regulatory properties consistent with the Herzberg-Moult-James model.
鸡骨骼肌肌钙蛋白 C 的分子结构，分辨率为 3 埃。
DOI：
10.1126/science.3969570
发表时间：
1985
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Sundaralingam,M;Bergstrom,R;Strasburg,G;Rao,ST;Roychowdhury,P;Greaser,M;Wang,BC
通讯作者：
Wang,BC
DOI：
10.1093/protein/3.2.95
发表时间：
1989-11-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
SOWDHAMINI, R;SRINIVASAN, N;BALARAM, P
通讯作者：
BALARAM, P