Interaction with adenylate cyclase toxin from Bordetella pertussis affects the metal binding properties of calmodulin

Although CyaA has been studied for over three decades and revealed itself to be a very good prototype for developing various biotechnological applications, only a little is known about its functional dynamics and about the conformational landscape of this protein. Molecular dynamics simulations helped to clarify the view on these points in the following way. First, the model of interaction between AC and calmodulin (CaM) has evolved from an interaction centered on the surface between C-CaM hydrophobic patch and the α helix H of AC, to a more balanced view, in which the C-terminal tail of AC along with the C-CaM Calcium loops play an important role. This role has been confirmed by the reduction of the affinity of AC for calmodulin in the presence of R338, D360 and N347 mutations. In addition, enhanced sampling studies have permitted to propose a representation of the conformational space for the isolated AC. It remains to refine this representation using structural low resolution information measured on the inactive state of AC. Finally, due to a virtual screening study on another adenyl cyclase from Bacillus anthracis, weak inhibitors of AC have been discovered.

“…Independent experimental studies of calmodulin [ 31 ] reveal that the interaction of calmodulin with AC increases the apparent Ca

binding in C-CaM. This is a close relationship to the observations of Selwa et al [ 21 ], as the involvement of R90 in hydrogen bond with AC and the displacement of CaM

helices due to opening and closure of EF-hands [ 24 , 25 ] are directly connected.…”

Section: Interaction Between Calmodulin and Acsupporting

confidence: 66%

Molecular Modeling of the Catalytic Domain of CyaA Deepened the Knowledge of Its Functional Dynamics

Malliavin

2017

“…Site-specific mutations in the β-hairpin resulted in conformational perturbations in metal binding sites I and II of N-CaM, while no significant structural modifications were observed in C-CaM [ 56 ]. Intriguingly, when the AC domain interacts with intact CaM, the metal-binding sites I and II of CaM bind Mg 2+ ions, while the sites III and IV of CaM are loaded with Ca 2+ ions [ 57 ]. Mg 2+ ions are present in millimolar concentrations in the cytosol of cells and the action of CyaA and cAMP signaling induces entry of Ca 2+ ions into cells [ 28 , 58 , 59 ].…”

Section: Cyaa Structurementioning

confidence: 99%

Structure–Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes

Novák

Černý

Osičková

et al. 2017

Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin–hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αMβ2) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3′,5′-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.

“…The Ca 2+ -binding was monitored using 2D NMR to determine the amino proton-nitrogen chemical shifts for each amino acid residue mapping to metal binding sites I–IV in D22A. NMR chemical shift assignments were achieved by direct spectral comparison to previously reported values [ 20 , 21 ]. In the current study, we found that site-specific metal binding to D22A was evidenced by the direction and magnitude of amide proton-nitrogen chemical shift values observed in the presence of Ca 2+ -saturation ( Figure 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…It is known that maximal enzymatic activity of CyaA is stimulated through interaction with both the N -terminal and C -terminal lobes of CaM [ 18 ]. Intermolecular association has been reported between N -terminal CaM and CyaA-ACD [ 19 ], and mutation in CyaA-ACD disrupts this low-affinity protein-protein binding interface [ 20 , 21 ]; however, it remains to be determined how activation of CyaA-ACD is achieved through association with both domains of CaM.…”

Section: Introductionmentioning

confidence: 99%

“…While C -terminal CaM association involving bound Ca 2+ accounts for the high-affinity interaction with CyaA-ACD [ 26 ], studies have shown that inter-domain communication exists in CaM in the presence of CyaA-ACD and ligated metals [ 21 ], revealing the importance of understanding how intact CaM activates CyaA-ACD. Previously, it was reported that several residues spanning from amino acid 22 to 38 in CaM, including site I and the linker between helices 2 and 3, are involved in direct protein-protein contacts with CyaA-ACD [ 20 , 21 ]; but it is not understood how intermolecular binding and metal ligation in this region of CaM impact its ability to fully activate CyaA-ACD. The potential involvement of site I at position 3 in CyaA-ACD binding was particularly interesting, as one carboxylate oxygen of D22 directly coordinates metal, while the remaining one is solvent exposed in free CaM [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

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Site I Inactivation Impacts Calmodulin Calcium Binding and Activation of Bordetella pertussis Adenylate Cyclase Toxin

Johns

Finley

2017

Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable α-helical content to CaMWt, and only modest differences in α-helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C-terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N-terminal CaM and CyaA-ACD.