Molecular Analysis of the Interaction ofBordetella pertussisAdenylyl 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 2Ј,3Ј-Nmethylanthraniloyl (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 3ЈMANT-2Јd-ATP and 2ЈMANT-3Јd-ATP in the AC activity assay using Mn 2ϩ were 220 and 340 nM, respectively. Natural nucleoside 5Ј-triphosphates, guanine-, hypoxanthine-and pyrimidine-MANTand 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 3ЈMANT-2Јd-ATP and 400 nM for 2ЈMANT-3Јd-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 lowaffinity 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.

Amidate Prodrugs of 9-[2-(Phosphonomethoxy)Ethyl]Adenine as Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis

“…Adefovir diphosphate (PMEApp) has been shown to inhibit adenylate cyclase activity of edema factor (EF) and adenylate cyclase toxin (ACT), the key virulence factors of Bacillus anthracis and Bordetella pertussis, respectively (10,11). Although the prodrug bis(POM)PMEA (compound 2, Fig.…”

“…Adefovir is currently used for the treatment of chronic hepatitis B virus infection (9). In addition to that, its active metabolite diphosphorylated form PMEA (PMEApp) has been found to inhibit activity of bacterial AC-based toxins, including ACT and edema factor (EF) of Bacillus anthracis (10,11). The crystal structures of ACT and EF in complex with calmodulin and PMEApp have been reported (12).…”

Bis-Halogen-Anthraniloyl-Substituted Nucleoside 5′-Triphosphates as Potent and Selective Inhibitors ofBordetella pertussisAdenylyl Cyclase Toxin

“…In other studies we have shown that 2Ј(3Ј)-O-MANT-substituted NTPs are moderately potent competitive CyaA inhibitors with K i values in the range of 0.2 to 10 M (Gille et al, 2004;Göttle et al, 2007). CyaA exhibits a broad base specificity for purine and pyrimidine nucleotides (Göttle et al, 2007(Göttle et al, , 2010, indicating that the catalytic site possesses substantial conformational flexibility and is spacious.…”

“…CyaA exhibits a broad base specificity for purine and pyrimidine nucleotides (Göttle et al, 2007(Göttle et al, , 2010, indicating that the catalytic site possesses substantial conformational flexibility and is spacious. Figure 1 provides an overview of structures of MANT nucleotides.…”

“…MANT nucleotides are fluorescent and can be used to monitor the binding of nucleotides to CyaA and conformational changes in CyaA upon binding of CaM. Interaction of the MANT group with Phe306 results in a CaM-dependent increase in fluorescence (Göttle et al, 2007).…”