title: Stability, structural and functional properties of a monomeric, calcium-loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis.
abstract: Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins.
authors.affiliation: Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, 75724 PARIS cedex 15, France.
authors.forename: Sara E
authors.initials: SE
authors.lastname: Cannella
authors.affiliation: Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, 75724 PARIS cedex 15, France.
authors.forename: Véronique Yvette
authors.initials: VY
authors.lastname: Ntsogo Enguéné
authors.affiliation: Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, 75724 PARIS cedex 15, France.
authors.forename: Marilyne
authors.initials: M
authors.lastname: Davi
authors.affiliation: Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, 75724 PARIS cedex 15, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Malosse
authors.affiliation: Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, 75724 PARIS cedex 15, France.
authors.forename: Ana Cristina
authors.initials: AC
authors.lastname: Sotomayor Pérez
authors.affiliation: Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, 75724 PARIS cedex 15, France.
authors.forename: Julia
authors.initials: J
authors.lastname: Chamot-Rooke
authors.affiliation: Institut de Biologie Intégrative de la Cellule, UMR 9198, Université Paris-Sud, F-91405 ORSAY Cedex, France.
authors.forename: Patrice
authors.initials: P
authors.lastname: Vachette
authors.affiliation: Institut de Biologie Intégrative de la Cellule, UMR 9198, Université Paris-Sud, F-91405 ORSAY Cedex, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Durand
authors.affiliation: Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, 75724 PARIS cedex 15, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ladant
authors.affiliation: Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, 75724 PARIS cedex 15, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chenal
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000737:chemistry* / Q000378:metabolism*; D001886:Bordetella pertussis / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism*; D057446:Hydrodynamics; D013058:Mass Spectrometry; D021381:Protein Transport; D053838:Scattering, Small Angle
chemical_list: D037361:Adenylate Cyclase Toxin; D003067:Coenzymes; D002118:Calcium