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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
Suryanarayana, Srividya Wang, Jenna L. Richter, Mark Shen, Yuequan Tang, Wei-Jen Lushington, Gerald H. Seifert, Roland
Suryanarayana, Srividya
Wang, Jenna L.
Richter, Mark
Shen, Yuequan
Tang, Wei-Jen
Lushington, Gerald H.
Seifert, Roland
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Abstract
Anthrax disease is caused by the spore-forming bacterium, Bacillus anthracis. Bacillus anthracis produces a calmodulin-activated adenylyl cyclase (AC) toxin, edema factor (EF). Through excessive cAMP accumulation EF disrupts host defence. In a recent study 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′-MANT- 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.
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Date
2009-08-01
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Elsevier
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Keywords
Edema factor, Calmodulin, MANT-nucleotide, Fluorescence spectroscopy, Molecular modelling
Citation
Suryanarayana, Srividya, Jenna L. Wang, Mark Richter, Yuequan Shen, Wei-Jen Tang, Gerald H. Lushington, and Roland Seifert. "Distinct Interactions of 2â ²- and 3â ²-O-(N-methyl)anthraniloyl-isomers of ATP and GTP with the Adenylyl Cyclase Toxin of Bacillus Anthracis, Edema Factor." Biochemical Pharmacology 78.3 (2009): 224-30.