Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery: Bridging Experiments and Computations - September 10-14, 2014, Istanbul, Turkey

Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery Poster Session II

57-POS Board 10 Activation of Bordetella Pertussis Adenyl Cyclase by Calmodulin Therese Malliavin 1 , Edithe Selwa 2,1 , Marilyne Davi 1 , Alexandre Chenal 1 , Ana-Cristina Sotomayor-Perez 1 , Elodie Laine 2 , Daniel Ladant 1 . 1 CNRS/Institut Pasteur, Paris, France, 2 Université Pierre et Marie Curie, Paris, France. The adenyl cyclase (AC) is an essential toxin from Bordetella pertussis able to invade eukaryotic cells where it is activated upon binding to calmodulin. Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of calmodulin (C-CaM), we previously carried out molecular dynamics simulations (Selwa et al, 2012) which suggested that three residues, Arg 338 , Asn 347 and Asp 360 , might be important for the stabilization of the AC/CaM interaction. These residues belong to a loop-helix-loop motif, LHL, at the C-terminal end of AC, located at the interface between CaM and the AC catalytic loop. In the present work, we have characterized in silico and in vitro, three AC variants in which, one (Asn 347 ; ACm1A), two (Arg 338 and Asp 360 ; ACm2A), or three residues (Arg 338 , Asn 347 and Asp 360 ; ACm3A) were modified to Ala. Biochemical studies revealed that the affinity for CaM of ACm1A and ACm2A was not significantly affected, while that of ACm3A was drastically reduced. To understand the effect of the modifications, molecular dynamics simulations were performed on the modified proteins. The MD trajectories recorded on the ACm3A/C-CaM complex revealed that the calcium-binding loops of C-CaM display large fluctuations that could be related to the weakened interaction of ACm3A with its activator. All together, our results suggest that the LHL motif at the C-terminal end of AC is crucial to link CaM-binding to the stabilization of the AC catalytic loop in an active configuration. References: Selwa et al. Model of the activation of Bordetella pertussis adenyl cyclase by calmodulin: molecular dynamics and mutagenesis studies. In revision at J Biol Chem. Selwa et al. Differential role of Calmodulin and Calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 2012;80:1028-40.

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