Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

Springer, Tzvia I.; Goebel, Erich J; Hariraju, Dinesh; Finley, Natosha L.

doi:10.1016/j.bbrc.2014.09.048

Cited by 9 publications

(42 citation statements)

References 32 publications

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“…29 that CaM/CyaA‐ACD is elongated and that the conformation of free CyaA‐ACD differs from that in the complex. We have demonstrated that CyaA‐ACD interaction with both domains of CaM is necessary to maintain the complex in an extended global state 19. In that same study, we found that disruption of intermolecular contacts between the β‐hairpin of CyaA and CaM decreases the hydrodynamic radius of the complex.…”

Section: Resultsmentioning

confidence: 54%

“…We have previously shown that these tools are useful for probing CyaA‐ACD‐induced conformational transitions in 4Ca 2+ ‐CaM 19. In the present study, stable isotope labeled CaM was used to generate complexes with unlabeled CyaA‐ACD in the presence of Mg 2+ .…”

Section: Resultsmentioning

confidence: 89%

“…Recombinant CyaA‐ACD was overproduced and purified as previously described 19. Cell pellets containing insoluble CyaA‐ACD were resuspended on ice in buffer containing 8 m Urea, 500 m m NaCl, 40 m m imidazole, 20 m m Tris‐HCl, and 1 m m PMSF at pH 8.0 and lysed by sonication.…”

Section: Methodsmentioning

confidence: 99%

“…N‐terminal CaM is required for full activation even though CaM mutants defective in N‐terminal domain opening also activate CyaA‐ACD 6. The N‐terminal domain of CaM has a unique conformational state upon CyaA‐ACD association that does not resemble ‘closed’ CaM 19. Furthermore, we showed that N‐terminal CaM directly contacts CyaA‐ACD through interactions involving the β‐hairpin of CyaA‐ACD.…”

mentioning

confidence: 85%

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

et al. 2016

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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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Section: Resultsmentioning

confidence: 54%

Section: Resultsmentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 85%

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

et al. 2016

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“…CaM was produced and purified as previously described [28]. Following purification, protein samples were visualized by SDS-PAGE and determined to be homogenous.…”

Section: The Thermal Stability Of Ssmybp-c Is Impacted By Ca 2+ -Cammentioning

confidence: 99%

Calcium-Dependent Interaction Occurs between Slow Skeletal Myosin Binding Protein C and Calmodulin

et al. 2017

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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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Temperature Accelerated Molecular Dynamics with Soft-Ratcheting Criterion Orients Enhanced Sampling by Low-Resolution Information

Cortés-Ciriano

Bouvier

Nilges

et al. 2015

J. Chem. Theory Comput.

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Many proteins exhibit an equilibrium between multiple conformations, some of them being characterized only by low-resolution information. Visiting all conformations is a demanding task for computational techniques performing enhanced but unfocused exploration of collective variable (CV) space. Otherwise, pulling a structure toward a target condition biases the exploration in a way difficult to assess. To address this problem, we introduce here the soft-ratcheting temperature-accelerated molecular dynamics (sr-TAMD), where the exploration of CV space by TAMD is coupled to a soft-ratcheting algorithm that filters the evolving CV values according to a predefined criterion. Any low resolution or even qualitative information can be used to orient the exploration. We validate this technique by exploring the conformational space of the inactive state of the catalytic domain of the adenyl cyclase AC from Bordetella pertussis. The domain AC gets activated by association with calmodulin (CaM), and the available crystal structure shows that in the complex the protein has an elongated shape. High-resolution data are not available for the inactive, CaM-free protein state, but hydrodynamic measurements have shown that the inactive AC displays a more globular conformation. Here, using as CVs several geometric centers, we use sr-TAMD to enhance CV space sampling while filtering for CV values that correspond to centers moving close to each other, and we thus rapidly visit regions of conformational space that correspond to globular structures. The set of conformations sampled using sr-TAMD provides the most extensive description of the inactive state of AC up to now, consistent with available experimental information.

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Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

Cited by 9 publications

References 32 publications

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

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

Calcium-Dependent Interaction Occurs between Slow Skeletal Myosin Binding Protein C and Calmodulin

Temperature Accelerated Molecular Dynamics with Soft-Ratcheting Criterion Orients Enhanced Sampling by Low-Resolution Information

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