CALMODULIN REGULATION OF ANTHRAX AND ADENYLYL CYCLASES

环AMP （CAMP）和钙离子是两个关键的第二信使，传递大量的细胞外和细胞内信号，控制大量的生理反应，如学习和记忆，以及心率的控制。由这两个第二信使转导的两个信号之间存在显著的串扰。其中一个交叉点涉及钙调素，一种钙传感器介质，可以激活两类腺苷酸环化酶，这种酶可以合成cAMP。一类是致病菌分泌的毒素，如炭疽芽孢杆菌分泌的水肿因子；另一类是高级真核生物分泌的腺苷酸环化酶，如哺乳动物1型酶（AC1）。该项目的长期目标是阐明钙调蛋白调控细菌和哺乳动物腺苷酸环化酶的分子机制。水肿因子包括两个功能域。n端部分（28kda）介导与保护性抗原的关联，保护性抗原是炭疽杆菌产生的一种转运体，因此水肿因子可以被转运到真核细胞中。水肿因子的c端部分（60 kDa）具有较高的腺苷酸环化酶活性（周转数约为每秒1000次），其活性高度依赖于钙调素。我们表达并纯化了水肿因子的c端催化结构域，获得了水肿因子单独和与钙调素复合的衍射晶体。我们建议确定两种形式的酶的分子结构。然后，我们将使用这些结构来生成水肿因子激活的详细催化模型。我们将用生化、光谱学和额外的晶体学分析来测试这个模型。我们还将使用基于结构和基于基因的抑制剂筛选来寻找高亲和力的小分子和肽，以阻止钙调素的激活和水肿因子的催化。所有哺乳动物的膜结合腺苷酸环化酶都有一个共同的结构，包括两个高度保守的结构域（C1a和C2a），由保守度较低的C1b和跨膜结构域连接。C1a和C2a形成一种可溶酶，可被Gs的α亚基激活。AC1的C1b区由两亲性的α -螺旋区组成，这是钙调蛋白激活所必需的。突变分析表明，钙调素对AC1的激活与水肿因子的激活明显不同。我们建议利用AC1的c1和C2结构域及其同源物构建一种钙调素敏感的可溶性酶。我们将分析钙调素激活可溶性AC1的方式与我们分析水肿因子的方式相似。这项研究的成功不仅将增强我们对腺苷酸环化酶如何调节的认识，而且还将为钙调素如何调节其许多其他靶蛋白的活性提供重要的结构见解。此外，成功发现抑制水肿因子的先导化合物将为开发更好的药物来防御炭疽杆菌的感染提供手段。

Cyclic AMP (CAMP) and calcium ion are two key second messengers that transmit numerous extracellular and intracellular signals to control a plethora of physiological responses such as learning and memory, and control of heart rate. There is significant crosstalk between two signals transduced by these two second messengers. One of the intersection point involves calmodulin, a calcium sensor mediator that can activate two classes of adenylyl cyclase, the enzyme that synthesizes cAMP. One class is a toxin secreted from pathogenic bacteria such as edema factor from Bacillus anthracis and the second is adenylyl cyclase from higher eukaryotes such as mammalian type 1 enzyme (AC1). The long-term goal of this project is to elucidate the molecular mechanism that underlies the regulation of bacterial and mammalian adenylyl cyclases by calmodulin. Edema factor consists of two functional domains. The N-terminal portion (28 kDa) mediates association with protective antigen, a transporter produced by B. anthracis so that edema factor can be transported into eukaryotic cell. The C-terminal portion (60 kDa) of edema factor has high adenylyl cyclase activity (the turn over number is around 1,000 per sec) and the activity is highly dependent on calmodulin. We have expressed and purified the C-terminal catalytic domain of edema factor and have obtained diffracting crystals of edema factor alone and in complex with calmodulin. We propose to determine the molecular structures of both forms of the enzyme. We will then use these structures to generate a detailed catalytic model of edema factor activation. We will test this model with biochemical, spectroscopic, and additional crystallographic analyses. We will also use structure-based and genetic- based inhibitor screens to search for the high-affinity small molecules and peptides that block calmodulin activation and catalysis of edema factor. All mammalian membrane-bound adenylyl cyclases share a common structure, including two highly conserved domains (C1a and C2a) connected by the less conserved C1b and transmembrane domains. C1a and C2a form a soluble enzyme that can be activated by the alpha subunit of Gs. C1b region of AC1 consists of an amphipathic, alpha-helical region that is necessary for calmodulin activation. Mutational analysis suggests that activation of AC1 by calmodulin is distinctly different from that of edema factor. We propose to construct a calmodulin-sensitive soluble enzyme using C 1 and C2 domains of AC1 and its homologs. We will analyze calmodulin activates of the soluble AC1 in a manner similar to our analyses of edema factor. Success in this research will not only enhance our knowledge of how adenylyl cyclase is regulated, but also provide important structural insights into how calmodulin modulates the activities of its many other target proteins. In addition, success in finding a lead compound that inhibits edema factor would provide the means to develop better drugs to defend against the infection of B. anthracis.