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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Flicker, Paula F
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Chicago
Internal Medicine/Medicine
Schools of Medicine
United States
Zip Code
Mones, Letif; Tang, Wei-Jen; Florián, Jan (2013) Empirical valence bond simulations of the chemical mechanism of ATP to cAMP conversion by anthrax edema factor. Biochemistry 52:2672-82
Schuler, Dominik; Lübker, Carolin; Lushington, Gerald H et al. (2012) Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: comparison with membranous adenylyl cyclase I. Biochem Pharmacol 83:839-848
Gottle, Martin; Dove, Stefan; Kees, Frieder et al. (2010) Cytidylyl and uridylyl cyclase activity of bacillus anthracis edema factor and Bordetella pertussis CyaA. Biochemistry 49:5494-503
Bishop, Brian L; Lodolce, James P; Kolodziej, Lauren E et al. (2010) The role of anthrolysin O in gut epithelial barrier disruption during Bacillus anthracis infection. Biochem Biophys Res Commun 394:254-9
Bourdeau, Raymond W; Malito, Enrico; Chenal, Alexandre et al. (2009) Cellular functions and X-ray structure of anthrolysin O, a cholesterol-dependent cytolysin secreted by Bacillus anthracis. J Biol Chem 284:14645-56
Tang, Wei-Jen; Guo, Qing (2009) The adenylyl cyclase activity of anthrax edema factor. Mol Aspects Med 30:423-30
Suryanarayana, Srividya; Wang, Jenna L; Richter, Mark et al. (2009) Distinct interactions of 2'- and 3'-O-(N-methyl)anthraniloyl-isomers of ATP and GTP with the adenylyl cyclase toxin of Bacillus anthracis, edema factor. Biochem Pharmacol 78:224-30
Taha, Hesham M; Schmidt, Jennifer; Göttle, Martin et al. (2009) Molecular analysis of the interaction of anthrax adenylyl cyclase toxin, edema factor, with 2'(3')-O-(N-(methyl)anthraniloyl)-substituted purine and pyrimidine nucleotides. Mol Pharmacol 75:693-703
Szarowicz, Sarah E; During, Russell L; Li, Wei et al. (2009) Bacillus anthracis edema toxin impairs neutrophil actin-based motility. Infect Immun 77:2455-64
Guo, Qing; Jureller, Justin E; Warren, Julia T et al. (2008) Protein-protein docking and analysis reveal that two homologous bacterial adenylyl cyclase toxins interact with calmodulin differently. J Biol Chem 283:23836-45

Showing the most recent 10 out of 31 publications