Abstract

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 Mn²⁺ 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.