Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: Comparison with membranous adenylyl cyclase I

Abstract

The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2′-(N-methylanthraniloyl)-3′-deoxy-adenosine 5′-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions.

Effects of 39 Compounds on Calmodulin-Regulated Adenylyl Cyclases AC1 and Bacillus anthracis Edema Factor

“…The catalytic activity of AC1 was essentially determined as previously described [ 21 , 54 ]. Sf9 membranes expressing AC1 were resuspended using syringes at 4°C and diluted with binding buffer (75 mM Tris-HCl, 12.5 mM MgCl 2 and 1 mM EDTA) to a final protein concentration of 1 μg/μl and a final pH of 7.4.…”

“…The three dimensional structure of CaM is modified by binding four Ca 2+ -ions. Ca 2+ -saturated CaM possesses a flexible linker region, connecting a C-terminal and an N-terminal globular region [ 16 ], which affords the interaction with numerous target proteins like myosin light-chain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE) and Bordetella pertussis AC toxin CyaA (CyaA) and the associated regulation of diverse physiological processes [ 16 – 21 ].…”

“…CyaA and sarcoplasmatic/endoplasmatic reticulum Ca 2+ -ATPase (SERCA)), where CDZ acts as a potent inhibitor, it was suggested that the potency of inhibition is correlated with the hydrophobicity and the size of the inhibitor [ 21 , 49 ]. In contrast to most of above CaM-inhibitors, CDZ inhibits many CaM-targets in a CaM-independent manner [ 18 , 21 , 49 – 52 ]. Table 1 summarizes the effects of CaM-inhibitors on CaM-target interactions.…”

Bordetella protein toxins

“…The crystal structure of the AC enzyme-calmodulin complex with adefovir diphosphate, thereby mimicking the binding of ATP, revealed the mechanism of catalytic activity of CyaA. Both the calmodulin-binding and the catalytic site of the AC enzyme have emerged as targets of inhibitors that can be used to inhibit the catalytic activity of CyaA and may be valuable drugs for the prophylaxis of B. pertussis infection [142][143][144][145][146][147][148][149][150][151].…”

The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant

“…Although ExoY shares significant homology in the catalytic domain with CyaA and edema factor, the previously characterized calmodulin-binding domain was absent from ExoY. Calmodulin is required to measure edema factor catalytic activity, and it stimulates CyaA activity over 500-fold (Karst et al 2010; Schuler et al 2012; Selwa et al 2012, 2014). ExoY, however, was not stimulated or activated by calmodulin in the Yahr studies (Yahr et al 1998).…”