Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin

Abstract

CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an a-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.

Use of allostery to identify inhibitors of calmodulin-induced activation of Bacillus anthracis edema factor

“…Yet strong experimental evidence in favor of this model was provided by the observation that the TUA compounds were also able to inhibit the related adenylyl cyclase from B. pertussis, CyaA. Catalytic domains of CyaA and EF only share 25% overall sequence identity, and the structural and thermodynamic features of their interaction with CaM differ significantly (32,43). Analysis of EF pockets (Table 1) revealed that the catalytic site and the SABC pocket and/or its SBC extension were the only ones to share significant homology with their CyaA counterparts.…”

“…CyaA is also activated by CaM and the structure of its catalytic site is highly similar to that of EF despite a low overall sequence homology (32). The compounds inhibited CyaA-CaM at 100 μM, but not at 10 μM, indicating that they were moderate inhibitors.…”

“…A number of thiophen ureidoacids thus selected were shown to inhibit EF activity in vitro with affinities in the low micromolecular range. Compounds from this series were also active against CyaA, the adenylyl cyclase toxin of Bordetella pertussis, the causative agent of whooping cough (32,33). (34).…”

“…Given the strong affinity of CaM for EF (32,43), we anticipated that molecules that could prevent CaM-induced activation of EF might not be able to inhibit the preformed EF-CaM complex. Indeed, the five weakest inhibitors were ineffective on preformed EF-CaM, but the strongest, TUAdiCl could partly inhibit the complex (Fig.…”

Sensor–response regulator interactions in a cross-regulated signal transduction network

“…For this purpose, we employed the BACTH method (bacterial adenylate cyclase two-hybrid) (Karimova et al, 1998), a widely used genetic approach for monitoring protein-protein interactions (Stynen et al, 2012). This assay reconstitutes Bordetella pertussis CyaA activity from trypsin-defined amino-terminal and carboxyl-terminal subdomains of 25 and 18 kDa, respectively (Guo et al, 2005). Interaction between T25 and T18 hybrid proteins generates cAMP, which activates lacZYA operon expression in an E. coli Dcya host strain lacking endogenous adenylate cyclase activity (Brickman et al, 1973).…”