An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis
An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis
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DOI：
10.1074/jbc.m004778200
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发表时间：
2000-11-17
影响因子：
4.8
通讯作者：
Tang, WJ
中科院分区：
文献类型：
--
作者：
Drum, CL;Yan, SZ;Tang, WJ
炭疽杆菌产生的水肿因子外毒素是一种腺苷酸环化酶，在受感染细胞的静息态钙浓度下，该酶被钙调素 (CaM) 激活。克隆了对应于水肿因子 (EF3) 催化结构域的 C 端 60 kDa 片段，在大肠杆菌中过表达并纯化。 EF3 的 N 端 43 kDa 结构域 (EF3-N) 是与百日咳博德特氏菌和铜绿假单胞菌腺苷酸环化酶同源的水肿因子的唯一结构域，对蛋白酶消化具有高度抵抗力。 EF3 的 C 端 160 个氨基酸结构域 (EF3-C) 在缺乏 CaM 的情况下对蛋白水解敏感。添加 CaM 可保护 EF3-C 不被蛋白酶消化。 EF3-N 和 EF3-C 分别表达，并且两个片段都需要重建完整的 CaM 敏感酶活性。使用双标记 CaM 分子进行荧光共振能量转移实验，结果表明 CaM 在与 EF3 结合后采用扩展构象。这与 CaM 在结合肌球蛋白轻链激酶和 CaM 依赖性蛋白激酶 II 型时采用的紧凑构象形成鲜明对比。检查了 CaM 四个钙结合位点中每一个的突变对 EF3 激活的影响。发现位点 3 和 4 对于激活至关重要，CaM 的 N 端结构域和 C 端结构域单独都无法激活 EF3。基于水肿因子家族同源性的 EF3 功能丧失突变和定点突变的基因筛查揭示了一对保守的天冬氨酸残基和一个精氨酸，它们对于催化很重要。类似的残基对于哺乳动物腺苷酸环化酶以及 DNA 聚合酶和核苷酸转移酶家族中的二金属介导的催化作用至关重要。这表明水肿因子可能利用类似的催化机制。
The edema factor exotoxin produced by Bacillus anthracis fs an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed in Escherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis and Pseudomonas aeruginosa, is highly resistant to protease digestion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM-dependent protein kinase type II. Mutations in each of the four calcium binding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic screen probing loss-of-function mutations of EF3 and site-directed mutations based on the:homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that: are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.