Received for publication January 24, 2007.
Revised June 6, 2007.
Accepted for publication June 6, 2007.

Molecular analysis of the interaction of Bordetella pertussis adenylyl cyclase with fluorescent nucleotides

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 to 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 nM 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 W69 and W242 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 F306. 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.

Key words: Adenylyl cyclases, cAMP, Structure-activity relationships and modeling, Fluorescence techniques, Protein targets, Antibiotic mechanisms