Calmodulin activation of target enzymes. Consequences of deletions in the central helix.
Calmodulin activation of target enzymes. Consequences of deletions in the central helix.
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DOI：
10.1016/s0021-9258(19)39658-9
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发表时间：
1990-03
期刊：
影响因子：
--
通讯作者：
M. VanBerkum;Samuel George;A. R. Means
中科院分区：
文献类型：
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作者：
M. VanBerkum;Samuel George;A. R. Means
钙调素（CaM）的中心螺旋区包括氨基酸65-92，用于分离两对Ca2（+）结合位点。该区域可能具有构象灵活性，并与靶蛋白相互作用。通过检测磷酸二酯酶、平滑肌肌球蛋白轻链（MLC）激酶和Ca2+/CaM依赖性蛋白激酶II （CaM激酶II）的激活来监测从中央螺旋中删除2、3、5或8个氨基酸的功能影响。CaMDM（-8）是一种钙调素缺失突变体，从中央螺旋中间缺失了8个氨基酸，在生理上显著浓度的激活剂下，CaMDM（-8）不能激活MLC激酶、磷酸二酯酶或CaM激酶II，但电泳迁移率和酪氨酸荧光特性也发生了改变，表明该突变体的结构发生了重大变化。缺失5个氨基酸（77-81）导致磷酸二酯酶（150倍）、CaM激酶II（25倍）和MLC激酶（5倍）的表观Kact相对于CaM增加。CaMDM也使CaM激酶II的最大自磷酸化活性降低了70%（-5）。对于磷酸二酯酶的激活，CaMDM（-2）的表观Kact值增加了15倍，而CaMDM（-3）的表观Kact值仅比天然CaM高3倍。相比之下，两个（79-80）和三个（79-81）氨基酸缺失突变体对MLC激酶的激活与天然CaM没有区别。CaMDM（-2）和CaMDM（-3）分别刺激原生CaM的85%和70%的CaM激酶II自磷酸化，而Kact的增加不到2倍。因此，CaM中心螺旋的所有缺失都降低了磷酸二酯酶的激活效率，这反映在Kact的大量改变上。然而，MLC激酶的激活对小的两个或三个氨基酸缺失相对不敏感。CaM激酶II与中心螺旋缺失突变体以复杂的方式相互作用，同时改变Kact和最大活性。数据表明，CaM的中心螺旋可能作为MLC激酶（以及较小程度上的CaM激酶II）的柔性系索，但从晶体结构预测的CaM的延伸构象可能需要磷酸二酯酶的激活。
The central helical region of calmodulin (CaM) includes amino acids 65-92 and serves to separate the two pairs of Ca2(+)-binding sites. This region may impart conformational flexibility and also interact with target proteins. The functional effects of deleting two, three, five, or eight amino acids from the central helix were monitored by examining the activation of phosphodiesterase, smooth muscle myosin light chain (MLC) kinase, and Ca2+/CaM-dependent protein kinase II (CaM kinase II). CaMDM(-8), a calmodulin-deletion mutant with 8 amino acids deleted from the middle of the central helix, failed to activate MLC kinase, phosphodiesterase, or CaM kinase II at physiologically significant concentrations of activator but also had altered electrophoretic mobility and tyrosine fluorescence properties suggesting major changes in the structure of this mutant. Deletion of five amino acids (77-81) resulted in an increase in apparent Kact for phosphodiesterase (150-fold), CaM kinase II (25-fold), and MLC kinase (5-fold) relative to CaM. The maximal autophosphorylation activity of CaM kinase II was also diminished 70% with CaMDM(-5). For phosphodiesterase activation, CaMDM(-2) has a 15-fold increase in apparent Kact while CaMDM(-3) had an apparent Kact value only 3-fold higher than native CaM. In contrast, the activation of MLC kinase by the two (79-80)- and three (79-81)-amino acid deletion mutants were indistinguishable from each other or native CaM. CaMDM(-2) and CaMDM(-3) stimulated CaM kinase II autophosphorylation to 85 and 70%, respectively, of native CaM with less than a 2-fold increase in Kact. Therefore, all deletions in the central helix of CaM reduce the efficiency of phosphodiesterase activation as reflected by substantial alterations in Kact. MLC kinase activation, however, is relatively insensitive to small two or three amino acid deletions. CaM kinase II interacts with the central helix deletion mutants in a complex manner with alterations in both the Kact and the maximum activity. The data suggest the central helix of CaM may serve as a flexible tether for MLC kinase (and to a lesser extent CaM kinase II) but that an extended conformation of CaM, as predicted from the crystal structure, may be required for phosphodiesterase activation.