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.

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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.

Abstract:Myosin binding protein C (MyBP-C) is a multi-domain protein that participates in the regulation of muscle contraction through dynamic interactions with actin and myosin. Three primary isoforms of MyBP-C exist: cardiac (cMyBP-C), fast skeletal (fsMyBP-C), and slow skeletal (ssMyBP-C). The N-terminal region of cMyBP-C contains the M-motif, a three-helix bundle that binds Ca 2+ -loaded calmodulin (CaM), but less is known about N-terminal ssMyBP-C and fsMyBP-C. Here, we characterized the conformation of a recombinant N-terminal fragment of ssMyBP-C (ssC1C2) using differential scanning fluorimetry, nuclear magnetic resonance, and molecular modeling. Our studies revealed that ssC1C2 has altered thermal stability in the presence and absence of CaM. We observed that site-specific interaction between CaM and the M-motif of ssC1C2 occurs in a Ca 2+ -dependent manner. Molecular modeling supported that the M-motif of ssC1C2 likely adopts a three-helix bundle fold comparable to cMyBP-C. Our study provides evidence that ssMyBP-C has overlapping structural determinants, in common with the cardiac isoform, which are important in controlling protein-protein interactions. We shed light on the differential molecular regulation of contractility that exists between skeletal and cardiac muscle.

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