RESUMO
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.
Assuntos
Toxina Adenilato Ciclase/metabolismo , Bordetella pertussis/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Toxina Adenilato Ciclase/genética , Alanina/genética , Alanina/metabolismo , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação , Calmodulina/genética , Dicroísmo Circular , Mutagênese Sítio-Dirigida , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
Adenylate cyclase toxin domain (CyaA-ACD) is a calmodulin (CaM)-dependent adenylate cyclase involved in Bordetella pertussis pathogenesis. Calcium (Ca2+) and magnesium (Mg2+) concentrations impact CaM-dependent CyaA-ACD activation, but the structural mechanisms remain unclear. In this study, NMR, dynamic light scattering, and native PAGE were used to probe Mg2+-induced transitions in CaM's conformation in the presence of CyaA-ACD. Mg2+ binding was localized to sites I and II, while sites III and IV remained Ca2+ loaded when CaM was bound to CyaA-ACD. 2Mg2+/2Ca2+-loaded CaM/CyaA-ACD was elongated, whereas mutation of site I altered global complex conformation. These data suggest that CyaA-ACD interaction moderates CaM's Ca2+- and Mg2+-binding capabilities, which may contribute to pathobiology.
