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以控制大量的生理反应，例如学习和记忆以及心率控制。这两个第二信使传递的两个信号之间存在显着的串扰。其中一个交叉点涉及钙调蛋白，这是一种钙传感器介体，可以激活两类腺苷酸环化酶，即合成 cAMP 的酶。一类是病原菌分泌的毒素，例如炭疽杆菌的水肿因子，第二类是高等真核生物的腺苷酸环化酶，例如哺乳动物1型酶（AC1）。该项目的长期目标是阐明钙调蛋白调节细菌和哺乳动物腺苷酸环化酶的分子机制。水肿因子由两个功能域组成。 N 末端部分 (28 kDa) 介导与保护性抗原（炭疽芽孢杆菌产生的转运蛋白）的结合，从而使水肿因子可以转运到真核细胞中。水肿因子的C末端部分（60 kDa）具有高腺苷酸环化酶活性（周转数约为每秒1,000次），并且该活性高度依赖于钙调蛋白。我们表达并纯化了水肿因子的C端催化结构域，并获得了水肿因子单独和与钙调蛋白复合物的衍射晶体。我们建议确定两种形式酶的分子结构。然后，我们将使用这些结构生成水肿因子激活的详细催化模型。我们将通过生化、光谱和其他晶体学分析来测试该模型。我们还将使用基于结构和基于遗传的抑制剂筛选来寻找阻断钙调蛋白激活和水肿因子催化的高亲和力小分子和肽。所有哺乳动物膜结合腺苷酸环化酶都具有共同的结构，包括两个高度保守的结构域（C1a 和 C2a），由不太保守的 C1b 和跨膜结构域连接。 C1a 和 C2a 形成可溶性酶，可被 Gs 的 α 亚基激活。 AC1 的 C1b 区域由钙调蛋白激活所必需的两亲性 α 螺旋区域组成。突变分析表明钙调蛋白对 AC1 的激活与水肿因子的激活明显不同。我们建议使用 AC1 及其同源物的 C 1 和 C2 结构域构建钙调蛋白敏感的可溶性酶。我们将以类似于水肿因子分析的方式分析可溶性 AC1 的钙调蛋白激活。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了解，而且还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，成功找到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.