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

Anthrax disease is caused by the spore-forming bacterium, Bacillus anthracis. B. anthracis produces a calmodulin-activated adenylyl cyclase (AC) toxin, edema factor (EF). Through excessive cAMP accumulation EF disrupts host defence. In a recent study [Taha HM, Schmidt J, Göttle M, Suryanarayana S, Shen Y, Tang WJ, et al. 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 2009;75:693–703] we showed that various 2′(3′)-O-N-(methyl)anthraniloyl (MANT)-substituted nucleoside 5′-triphosphates are potent inhibitors (Ki values in the 0.1–5 μM range) of purified EF. Upon interaction with calmodulin we observed efficient fluorescence resonance energy transfer (FRET) between tryptophan and tyrosine residues of EF and the MANT-group of MANT-ATP. Molecular modelling suggested that both the 2′- and 3′-MANT-isomers can bind to EF. The aim of the present study was to examine the effects of defined 2′- and 3′-MANT-isomers of ATP and GTP on EF. 3′-MANT-2′-deoxy-ATP inhibited EF more potently than 2′-MANT-3′-deoxy-ATP, whereas the opposite was the case for the corresponding GTP analogs. Calmodulin-dependent direct MANT fluorescence and FRET was much larger with 2′-MANT-3′-deoxy-ATP and 2′-MANT-3′-deoxy-GTP compared to the corresponding 3′-MANT-2′-deoxy-isomers and the 2′(3′)-racemates. Ki values of MANT-nucleotides for inhibition of catalysis correlated with Kd values of MANT-nucleotides in FRET studies. Molecular modelling indicated different positioning of the MANT-group in 2′-MANT-3′-deoxy-ATP/GTP and 3′-MANT-2′-deoxy-ATP/GTP bound to EF. Collectively, EF interacts differentially with 2′- and 3′-MANT-isomers of ATP and GTP, indicative for conformational flexibility of the catalytic site and offering a novel approach for the development of potent and selective EF inhibitors. Moreover, our present study may serve as a general model of how to use MANT-nucleotide isomers for the analysis of the molecular mechanisms of nucleotide/protein interactions.

By using enzymatic, fluorescence spectroscopy and molecular modelling approaches, this paper shows distinct interaction defined isomers of MANT-ATP and MANT-GTP with edema factor from Bacillus anthracis.Figure optionsDownload as PowerPoint slide