title: 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.
abstract: 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.
authors.affiliation: Department of Molecular Biosciences, The University of Kansas, Lawrence, KS 66045, USA.
authors.forename: Srividya
authors.initials: S
authors.lastname: Suryanarayana
authors.forename: Jenna L
authors.initials: JL
authors.lastname: Wang
authors.forename: Mark
authors.initials: M
authors.lastname: Richter
authors.forename: Yuequan
authors.initials: Y
authors.lastname: Shen
authors.forename: Wei-Jen
authors.initials: WJ
authors.lastname: Tang
authors.forename: Gerald H
authors.initials: GH
authors.lastname: Lushington
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000262:Adenylyl Cyclases / Q000737:chemistry*; D000942:Antigens, Bacterial / Q000737:chemistry*; D001408:Bacillus anthracis / Q000201:enzymology*; D001427:Bacterial Toxins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006160:Guanosine Triphosphate / Q000737:chemistry*; D007536:Isomerism; D008958:Models, Molecular; D062367:ortho-Aminobenzoates / Q000737:chemistry*
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; D062367:ortho-Aminobenzoates; C045473:3'-O-(N-methylanthraniloyl) ATP; C063981:2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases