title: Calcium, acylation, and molecular confinement favor folding of Bordetella pertussis adenylate cyclase CyaA toxin into a monomeric and cytotoxic form.
abstract: The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.
authors.affiliation: Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Johanna C
authors.initials: JC
authors.lastname: Karst
authors.affiliation: Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: V Yvette
authors.initials: VY
authors.lastname: Ntsogo Enguéné
authors.affiliation: Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Sara E
authors.initials: SE
authors.lastname: Cannella
authors.affiliation: Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Orso
authors.initials: O
authors.lastname: Subrini
authors.affiliation: Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Hessel
authors.affiliation: Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Debard
authors.affiliation: Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France. Electronic address: daniel.ladant@pasteur.fr.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ladant
authors.affiliation: Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France. Electronic address: alexandre.chenal@pasteur.fr.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chenal
mesh_terms: D000215:Acylation; D037361:Adenylate Cyclase Toxin / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D001886:Bordetella pertussis / Q000201:enzymology*; D002118:Calcium / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology; D006461:Hemolysis; D011499:Protein Processing, Post-Translational; D058849:Protein Refolding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012756:Sheep; D014508:Urea / Q000737:chemistry
chemical_list: D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D014508:Urea; D002118:Calcium
keywords: Adenylate Cyclase (Adenylyl Cyclase); Bacterial Toxin; Bordetella pertussis; Calcium-binding Protein; Chromatography; Macromolecular Crowding; Molecular Confinement; Protein Acylation; Protein Aggregation; Protein Folding