CALMODULIN REGULATION OF ANTHRAX AND ADENYLYL CYCLASES

环磷酸腺苷(CAMP)和钙离子是两个关键的第二信使，它们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，以及控制心率。在由这两个第二信使转换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蛋白，这是一种钙感应媒介，可以激活两种类型的腺苷环化酶，腺苷环化酶是合成cAMP的酶。一类是由致病细菌分泌的毒素，如炭疽芽孢杆菌的水肿因子；另一类是来自高等真核生物如哺乳动物1型酶(AC1)的腺酰环化酶。该项目的长期目标是阐明钙调蛋白调节细菌和哺乳动物腺酰环化酶的分子机制。水肿因子由两个功能结构域组成。N-末端(28 KDa)与炭疽杆菌产生的保护性抗原结合，从而使水肿性因子进入真核细胞。水肿因子的C末端(60 KDa)具有较高的腺苷环化酶活性(翻转次数约为1,000次/秒)，其活性高度依赖于钙调蛋白。我们表达和纯化了水肿因子的C端催化结构域，并获得了单独的和与钙调蛋白形成复合体的衍射晶。我们建议确定这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一个详细的水肿因子激活催化模型。我们将用生化、光谱分析和额外的结晶学分析来测试这个模型。我们还将使用基于结构和遗传的抑制剂筛选来寻找高亲和力的小分子和多肽，这些小分子和多肽可以阻断钙调蛋白的激活和对水肿因子的催化。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)，它们由较不保守的C1b结构域和跨膜结构域连接。C1a和C2a形成一种可溶性酶，可被Gs的阿尔法亚单位激活。AC1的C1b区由钙调素激活所必需的两亲性α-螺旋区域组成。突变分析表明，钙调素对AC1的激活与对水肿因子的激活明显不同。我们建议利用AC1及其同源物的C_1和C_2结构域构建钙调素敏感的可溶酶。我们将以类似于我们对水肿因子的分析的方式来分析可溶性AC1的钙调蛋白活性。这项研究的成功不仅将加强我们对腺酰环化酶如何调控的了解，还将为钙调蛋白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，成功地找到一种抑制浮肿因子的先导化合物将为开发更好的药物来预防炭疽杆菌感染提供手段。

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.