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 (K(i) values in the 0.1-5 microM 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. K(i) values of MANT-
nucleotides for inhibition of catalysis correlated with K(d) 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.