Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough

O’Brien, Darragh P.; Pérez, Ana Cristina Sotomayor; Karst, Johanna C.; Cannella, Sara; Enguéné, Véronique Yvette Ntsogo; Hessel, Audrey; Raoux-Barbot, Dorothée; Voegele, Alexis; Subrini, Orso; Davi, Marilyne; Guijarro, J. Iñaki; Raynal, Bertrand; Baron, Bruno; England, Patrick; Hernández, Belén; Ghomi, Mahmoud; Hourdel, Véronique; Malosse, Christian; Chamot‐Rooke, Julia; Vachette, Patrice; Durand, Dominique; Brier, Sébastien; Ladant, Daniel; Chenal, Alexandre

doi:10.1016/j.toxicon.2018.01.007

Cited by 22 publications

(20 citation statements)

References 71 publications

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“…These data have direct implications for CyaA secretion. We previously suggested that in the low calcium environment of the bacterial cytosol, RD adopts disordered conformations favorable for transport through the type 1 secretion machinery and folds upon binding calcium in the extracellular, calcium-rich environment (4,32,34,39,41,42). Our present data now extend this model to the full-length CyaA toxin.…”

Section: Relativesupporting

confidence: 73%

“…Finally, the C-terminal 701 residues (1006-1706) correspond to the cell receptor-binding domain, RTX domain (RD), which harbors ;40 copies of a glycine-and aspartate-rich nona-peptide repeat, characteristic of the RTX bacterial cytolysins. In the absence of calcium, these RTX motifs are intrinsically disordered but undergo a disorder-to-order transition upon calcium binding (4,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). The RTX motifs themselves constitute the primary sites of calcium binding within the protein (3,31,43,44).…”

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confidence: 99%

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Post‐translational acylation controls the folding and functions of the CyaA RTX toxin

et al. 2019

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The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro‐toxin, pro‐CyaA, and converted into its cytotoxic form upon acylation of two lysines. After secretion, CyaA invades eukaryotic cells and produces cAMP, leading to host defense subversion. To gain further insights into the effect of acylation, we compared the functional and structural properties of pro‐CyaA and CyaA proteins. HDX‐MS results show that the refolding process of both proteins upon progressive urea removal is initiated by calcium binding to the C‐terminal RTX domain. We further identified a critical hydrophobic segment, distal from the acylation region, that folds at higher urea concentration in CyaA than in pro‐CyaA. Once refolded into monomers, CyaA is more compact and stable than pro‐CyaA, due to a complex set of interactions between domains. Our HDX‐MS data provide direct evidence that the presence of acyl chains in CyaA induces a significant stabilization of the apolar segments of the hydrophobic domain and of most of the acylation region. We propose a refolding model dependent on calcium and driven by local and distal acylation‐dependent interactions within CyaA. Therefore, CyaA acylation is not only critical for cell intoxication, but also for protein refolding into its active conformation. Our data shed light on the complex relationship between post‐translational modifications, structural disorder and protein folding. Coupling calcium‐binding and acylation‐driven folding is likely pertinent for other repeat‐in‐toxin cytolysins produced by many Gram‐negative bacterial pathogens.—O'Brien, D. P., Cannella, S. E., Voegele, A., Raoux‐Barbot, D., Davi, M., Douché, T., Matondo, M., Brier, S., Ladant, D., Chenal, A. Post‐translational acylation controls the folding and functions of the CyaA RTX toxin. FASEB J. 33, 10065–10076 (2019). http://www.fasebj.org

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Section: Relativesupporting

confidence: 73%

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confidence: 99%

Post‐translational acylation controls the folding and functions of the CyaA RTX toxin

et al. 2019

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“…In contrast, in the absence of Ca 2+ ion, the RTX parallel β-roll motif repeat structure appeared to be disordered [27,28]. A previous study showed that the RTX parallel β-roll motif repeat structure became disordered and adequately unfolded in the absence of Ca 2+ ion [6][7][8][27][28][29]. In agreement with our previous laboratory work (CD spectra), in the absence of Ca 2+ ion (0 mM CaCl 2 ), the AMS8 lipase secondary structure, especially β-sheet content was decreased [14].…”

Section: Destabilization Of Rtx Parallel β-Roll Motif Repeat the Strumentioning

confidence: 94%

The Influence of Calcium toward Order/Disorder Conformation of Repeat-in-Toxin (RTX) Structure of Family I.3 Lipase from Pseudomonas fluorescens AMS8

Ali

Salleh

Leow

et al. 2020

Toxins

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Calcium-binding plays a decisive role in the folding and stabilization of many RTX proteins, especially for the RTX domain. Although many studies have been conducted to prove the contribution of Ca2+ ion toward the folding and stabilization of RTX proteins, its functional dynamics and conformational structural changes remain elusive. Here, molecular docking and molecular dynamics (MD) simulations were performed to analyze the contribution of Ca2+ ion toward the folding and stabilization of the RTX lipase (AMS8 lipase) structure. AMS8 lipase contains six Ca2+ ions (Ca1–Ca6). Three Ca2+ ions (Ca3, Ca4, and Ca5) were bound to the RTX parallel β-roll motif repeat structure (RTX domain). The metal ion (Ca2+) docking analysis gives a high binding energy, especially for Ca4 and Ca5 which are tightly bound to the RTX domain. The function of each Ca2+ ion is further analyzed using the MD simulation. The removal of Ca3, Ca4, and Ca5 caused the AMS8 lipase structure to become unstable and unfolded. The results suggested that Ca3, Ca4, and Ca5 stabilized the RTX domain. In conclusion, Ca3, Ca4, and Ca5 play a crucial role in the folding and stabilization of the RTX domain, which sustain the integrity of the overall AMS8 lipase structure.

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“…The parts of the consecutive turns and β-strands form a right-hand parallel β-helix that is stabilized by the binding of Ca 2+ ions. Meanwhile, without Ca 2+ ions, these RTX parallel β-roll motif repeat structures appear to be mostly disordered [3,21]. [7].…”

Section: Calcium-binding Site and Rtx β-Roll Motif Repeat Structure Omentioning

confidence: 99%

Calcium-Induced Activity and Folding of a Repeat in Toxin Lipase from Antarctic Pseudomonas fluorescens Strain AMS8

Ali

Salleh

Rahman

et al. 2020

Toxins

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It is hypothesized that the Ca2+ ions were involved in the activity, folding and stabilization of many protein structures. Many of these proteins contain repeat in toxin (RTX) motifs. AMS8 lipase from Antarctic Pseudomonas fluorescens strain AMS8 was found to have three RTX motifs. So, this research aimed to examine the influence of Ca2+ ion towards the activity and folding of AMS8 lipase through various biophysical characterizations. The results showed that CaCl2 increased lipase activity. The far-UV circular dichroism (CD) and Fourier-transform infrared (FTIR) analysis suggested that the secondary structure content was improved with the addition of CaCl2. Fluorescence spectroscopy analysis showed that the presence of CaCl2 increased protein folding and compactness. Dynamic light scattering (DLS) analysis suggested that AMS8 lipase became aggregated at a high concentration of CaCl2.The binding constant (Kd) value from the isothermal titration calorimetry (ITC) analysis proved that the Ca2+ ion was tightly bound to the AMS8 lipase. In conclusion, Ca2+ ions play crucial roles in the activity and folding of the AMS8 lipase. Calcium binding to RTX nonapeptide repeats sequences will induced the formation and folding of the RTX parallel β-roll motif repeat structure.

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Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough

Cited by 22 publications

References 71 publications

Post‐translational acylation controls the folding and functions of the CyaA RTX toxin

Post‐translational acylation controls the folding and functions of the CyaA RTX toxin

The Influence of Calcium toward Order/Disorder Conformation of Repeat-in-Toxin (RTX) Structure of Family I.3 Lipase from Pseudomonas fluorescens AMS8

Calcium-Induced Activity and Folding of a Repeat in Toxin Lipase from Antarctic Pseudomonas fluorescens Strain AMS8

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