Molecular analysis of the interaction of Bordetella pertussis adenylyl cyclase with fluorescent nucleotides.
The calmodulin (CaM)-dependent adenylyl cyclase (AC) toxin from Bordetella pertussis (CyaA) substantially contributes to the pathogenesis of whooping cough. Thus, potent and selective CyaA inhibitors may be valuable drugs for prophylaxis of this disease. We examined the interactions of fluorescent 2',3'-N-methylanthraniloyl (MANT)-, anthraniloyl- and trinitrophenyl (TNP)-substituted nucleotides with CyaA. Compared with mammalian AC isoforms and Bacillus anthracis AC toxin edema factor, nucleotides inhibited catalysis by CyaA less potently. Introduction of the MANT substituent resulted in 5- to 170-fold increased potency of nucleotides. K(i) values of 3'MANT-2'd-ATP and 2'MANT-3'd-ATP in the AC activity assay using Mn(2+) were 220 and 340 nM, respectively. Natural nucleoside 5'-triphosphates, guanine-, hypoxanthine- and pyrimidine-MANT- and TNP nucleotides and di-MANT nucleotides inhibited CyaA, too. MANT nucleotide binding to CyaA generated fluorescence resonance energy transfer (FRET) from tryptophans Trp69 and Trp242 and multiple tyrosine residues, yielding K(d) values of 300 nM for 3'MANT-2'd-ATP and 400 nM for 2'MANT-3'd-ATP. Fluorescence experiments and docking approaches indicate that the MANT- and TNP groups interact with Phe306. Increases of FRET and direct fluorescence with MANT nucleotides were strictly CaM-dependent, whereas TNP nucleotide fluorescence upon binding to CyaA increased in the absence of CaM and was actually reduced by CaM. In contrast to low-affinity MANT nucleotides, even low-affinity TNP nucleotides generated strong fluorescence increases upon binding to CyaA. We conclude that the catalytic site of CyaA possesses substantial conformational freedom to accommodate structurally diverse ligands and that certain ligands bind to CyaA even in the absence of CaM, facilitating future inhibitor design.
Göttle
Martin
M
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Regensburg, Germany.
Dove
Stefan
S
Steindel
Phillip
P
Shen
Yuequan
Y
Tang
Wei-Jen
WJ
Geduhn
Jens
J
König
Burkhard
B
Seifert
Roland
R
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D001886:Bordetella pertussis / Q000201:enzymology*; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D009711:Nucleotides; D020033:Protein Isoforms; D000262:Adenylyl Cyclases