Molecular Analysis of the Interaction of Anthrax Adenylyl Cyclase Toxin, Edema Factor, with 2(3)-O-(N-(methyl)anthraniloyl)- Substituted Purine and Pyrimidine Nucleotides

The spore-forming bacterium Bacillus anthracis exerts its deleterious effects by production of three major exotoxins: EF, protective antigen, and lethal factor (Jedrzejas, 2002). EF and lethal factor enter host cells via a complex with membrane-associated protective antigen, which acts as a pH-dependent protein transporter. Lethal factor, a specific zinc-metalloprotease, inactivates mitogen-activated protein kinase (Hong et al., 2005). EF possesses ∼800 amino acid residues and an apparent molecular mass of ∼89 kDa and is a CaM-dependent AC (Drum et al., 2002). After entering host cells, EF forms a complex with CaM, a mammalian regulatory protein that mediates many aspects of calcium-regulated signaling (Shen et al., 2002). The binding of CaM induces a major conformational change in the catalytic domain of EF (Drum et al., 2002). This rearrangement renders EF highly efficient at catalyzing the conversion of ATP into cAMP, disrupting intracellular signaling pathways through excessive activation of cAMP-dependent signaling pathways (Shen et al., 2005). We resolved several crystal structures of nucleotide-EF-CaM complexes and characterized the amino acids that are important for binding of the substrate ATP and catalysis (Drum et al., 2002; Shen et al., 2002, 2005). In addition, we showed that mAC and bacterial AC toxins are potently inhibited by MANT-substituted nucleoside 5′-triphosphates (Gille et al., 2004; Mou et al., 2005, 2006; Gottle et al., 2007). MANT-nucleotides are fluorescent, and we exploited this property to suggest conformational changes associated with activation in purified catalytic subunits of mAC (Mou et al., 2005, 2006) and the Bordetella pertussis AC toxin CyaA (Gottle et al., 2007). In addition, by combining crystallographic and molecular modeling approaches, we developed a three-site pharmacophore model for mAC and CyaA, with binding domains for the base, the MANT group, and the polyphosphate chain (Gille et al., 2005; Mou et al., 2006; Gottle et al., 2007; Wang et al., 2007). Those studies revealed that the catalytic sites of mAC and CyaA exhibit substantial conformational flexibility, accommodating both purine and pyrimidine nucleotides. Despite this flexibility, the structure/activity relationships of MANT-nucleotides at mAC and CyaA are quite different, offering the opportunity to design potent and isoform-selective AC inhibitors. In contrast to mAC (Gille et al., 2004, 2005; Mou et al., 2005, 2006) and CyaA (Gottle et al., 2007), a detailed analysis of MANT-nucleotide/EF interactions has not yet been presented. Therefore, in the present study, we systematically examined the interactions of natural purine and pyrimidine nucleotides and several (M)ANT-substituted analogs with EF in terms of catalysis, fluorescence changes and molecular modeling.