NMR Characterization of Adenylyl Cyclase Toxin-Calmodulin Complexes

Abstract

Bordetella pertussis, etiological agent of whooping cough, secrets a calmodulin (CaM) - activated adenylate cyclase toxin (CyaA) virulence factor which assists in respiratory colonization. The worldwide increase in pertussis cases emphasizes the necessity of understanding CyaA's role in disease progression. Intact CaM consists of tethered N- and C-terminal domains both of which can separately bind to and activate CyaA. Moreover, interaction with intact calmodulin substantially increases the binding affinity between CyaA and CaM by unknown structural mechanisms. A crystal structure determined in the absence of N-terminal CaM elucidates the molecular mechanisms by which C-terminal CaM activates CyaA. While it is reported that the second N-terminal calcium-binding site in CaM is likely involved in stabilizing interactions with the catalytic pocket of CyaA, no high-resolution structure detailing this protein-protein interface is currently available. In this study, nuclear magnetic resonance (NMR) chemical shift mapping and paramagnetic relaxation enhancement (PRE) techniques have been used to probe molecular interactions between intact CaM and CyaA. Chemical shift perturbations induced in labeled CaM by CyaA binding were localized primarily to helices IV and V, VI and VII in the N- and C-terminal domains of CaM, respectively. Binding of CyaA resulted in the structural modification of intact CaM as evidence by the protection of specific resonances in helices IV, V, and VI of labeled CaM from spin labeled induced broadening. These data suggest that conformational modulation in both the N- and C-terminal domains of CaM upon CyaA binding contributes to the maximal activation of the toxin.