Allosteric Activation of Bordetella pertussis Adenylyl Cyclase by Calmodulin: MOLECULAR DYNAMICS AND MUTAGENESIS STUDIES* |
| |
Authors: | Edithe Selwa Marilyne Davi Alexandre Chenal Ana-Cristina Sotomayor-Pérez Daniel Ladant Thérèse E Malliavin |
| |
Institution: | From the ‡Institut Pasteur and CNRS UMR 3528, Rue du Dr. Roux, Unité de Bioinformatique Structurale, 75015 Paris, France.;the ¶Institut Pasteur and CNRS UMR 3528, Rue du Dr. Roux, Unité de Biochimie des Interactions Macromoléculaires, 75015 Paris, France, and ;the §Université Pierre et Marie Curie, Cellule Pasteur UPMC, Rue du Dr. Roux, 75015 Paris, France |
| |
Abstract: | Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg338, Asn347, and Asp360) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn347; ACm1A), two (Arg338 and Asp360; ACm2A), or three residues (Arg338, Asn347, and Asp360; ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3A·C-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration. |
| |
Keywords: | Bioinformatics Calcium Molecular Modeling Mutagenesis Virulence Factor |
|
|