Bordetella Pertussis Adenylate Cyclase Toxin: Potential Modulator of Calmodulin Metal-Binding Properties

Abstract

Calmodulin (CaM) is a potent activator of Bordetella pertussis adenylate cyclase toxin (CyaA) in the presence or absence of calcium (Ca2+). Physiological concentrations of magnesium (Mg2+) are sufficient to fully or partially saturate CaM at resting Ca2+ levels, which may facilitate CaM-dependent stimulation of CyaA, but it remains unclear what role metal-binding plays in toxin activation. In this study, multi-dimensional nuclear magnetic resonance (NMR), dynamic light scattering (DLS), and circular dichroism (CD) were used to examine the effects of Mg2+-binding on the structure and hydrodynamic properties of CaM/CyaA complexes. NMR structural investigations of partially (2Mg2+2Ca2+) and fully Ca2+-loaded (4Ca2+) CaM/CyaA complexes revealed that Mg2+-binding is largely localized to sites I and II of CaM. In the presence of CyaA, sites III and IV remained Ca2+-loaded, even when Mg2+ is in excess, indicating that CyaA prohibits metal exchange in the C-terminus of CaM. Moreover, interaction with CyaA stabilized Mg2+-binding at site II of CaM implying that CyaA modulates CaM's metal-sensing properties. DLS and CD analyses showed that differences exist in the global conformations of CaM/CyaA complexes in the 2Mg2+2Ca2+- and 4Ca2+-loaded states. The conformation and metal-binding properties of CaM's N-terminal domain were perturbed by mutations targeting the CaM/CyaA interface. However, these mutations had no detectable structural impact on sites III and IV of CaM, confirming CyaA interaction differentially modifies the conformation of each domain. These data suggest that CyaA alters the Ca2+- and Mg2+-binding properties of CaM, which would represent an alternative, novel mechanism of toxin function within the cell.