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.