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Toxins 2017, 9(12), 389;

Site I Inactivation Impacts Calmodulin Calcium Binding and Activation of Bordetella pertussis Adenylate Cyclase Toxin

Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056, USA
Department of Microbiology, Miami University, Oxford, OH 45056, USA
Author to whom correspondence should be addressed.
Academic Editor: Alexandre Chenal
Received: 8 November 2017 / Revised: 26 November 2017 / Accepted: 27 November 2017 / Published: 30 November 2017
(This article belongs to the Special Issue Adenylate Cyclase (CyaA) Toxin)
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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. View Full-Text
Keywords: calcium; calmodulin; CyaA toxin; NMR calcium; calmodulin; CyaA toxin; NMR

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Johns, C.W.; Finley, N.L. Site I Inactivation Impacts Calmodulin Calcium Binding and Activation of Bordetella pertussis Adenylate Cyclase Toxin. Toxins 2017, 9, 389.

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