title: Characterization of the regions involved in the calcium-induced folding of the intrinsically disordered RTX motifs from the bordetella pertussis adenylate cyclase toxin.
abstract: Repeat in toxin (RTX) motifs are nonapeptide sequences found among numerous virulence factors of Gram-negative bacteria. In the presence of calcium, these RTX motifs are able to fold into an idiosyncratic structure called the parallel beta-roll. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the best-characterized RTX cytolysins. CyaA contains a C-terminal receptor domain (RD) that mediates toxin binding to the eukaryotic cell receptor. The receptor-binding domain is composed of about forty RTX motifs organized in five successive blocks (I to V). The RTX blocks are separated by non-RTX flanking regions of variable lengths. It has been shown that block V with its N- and C-terminal flanking regions constitutes an autonomous subdomain required for the toxicity of CyaA. Here, we investigated the calcium-induced biophysical changes of this subdomain to identify the respective contributions of the flanking regions to the folding process of the RTX motifs. We showed that the RTX polypeptides, in the absence of calcium, exhibited the hallmarks of intrinsically disordered proteins and that the C-terminal flanking region was critical for the calcium-dependent folding of the RTX polypeptides, while the N-terminal flanking region was not involved. Furthermore, the secondary and tertiary structures were acquired concomitantly upon cooperative binding of several calcium ions. This suggests that the RTX polypeptide folding is a two-state reaction, from a calcium-free unfolded state to a folded and compact conformation, in which the calcium-bound RTX motifs adopt a beta-roll structure. The relevance of these results to the toxin physiology, in particular to its secretion, is discussed.
authors.affiliation: Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, CNRS URA 2185, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
authors.forename: Ana-Cristina
authors.initials: AC
authors.lastname: Sotomayor Pérez
authors.forename: Johanna C
authors.initials: JC
authors.lastname: Karst
authors.forename: Marilyne
authors.initials: M
authors.lastname: Davi
authors.forename: J Iñaki
authors.initials: JI
authors.lastname: Guijarro
authors.forename: Daniel
authors.initials: D
authors.lastname: Ladant
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chenal
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000737:chemistry* / Q000378:metabolism*; D020816:Amino Acid Motifs; D001886:Bordetella pertussis / Q000737:chemistry / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D013057:Spectrum Analysis
chemical_list: D037361:Adenylate Cyclase Toxin; D002118:Calcium