Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis

O’Brien, Darragh P.; Durand, Dominique; Voegele, Alexis; Hourdel, Véronique; Davi, Marilyne; Chamot‐Rooke, Julia; Vachette, Patrice; Brier, Sébastien; Ladant, Daniel; Chenal, Alexandre

doi:10.1371/journal.pbio.2004486

Cited by 35 publications

(63 citation statements)

References 60 publications

(75 reference statements)

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“…Finally, TR and AC are secreted, and, interestingly, their folding appears minimally affected by the acylation status. This is in agreement with the fact that the isolated AC and AC-TR polypeptides can autonomously fold in solution (15,16,24). Taken together, the calcium-induced folding, coupled with hydrophobic effects between distal regions containing apolar segments and acyl chains, is likely shared with other multidomain proteins.…”

Section: Relativesupporting

confidence: 86%

“…In addition to its pore-forming activity, CyaA possesses an extra domain endowed with adenylate cyclase activity. The N-terminal CyaA catalytic domain (AC) is delivered into the cytoplasm of target cells (13,14), where it is activated by calmodulin (15,16) to produce high levels of cAMP, subverting host immunity (10,17,18). CyaA is a large, multidomain protein of 1706 amino-acid residues, with AC (residues 1-364) (16), whereas the C-terminal hemolysin region (residues 365-1706) contains several domains responsible for AC membrane translocation and pore-forming activity (19)(20)(21)(22)(23)(24).…”

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confidence: 99%

“…The N-terminal CyaA catalytic domain (AC) is delivered into the cytoplasm of target cells (13,14), where it is activated by calmodulin (15,16) to produce high levels of cAMP, subverting host immunity (10,17,18). CyaA is a large, multidomain protein of 1706 amino-acid residues, with AC (residues 1-364) (16), whereas the C-terminal hemolysin region (residues 365-1706) contains several domains responsible for AC membrane translocation and pore-forming activity (19)(20)(21)(22)(23)(24). The translocation region (TR; residues 365-527) is involved in AC translocation across target cell membranes (24) and membrane permeabilization (25) and exhibits features similar to membrane-active peptides (24,26,27).…”

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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: 86%

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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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“…Interactions with IDPs are often accompanied by a disorder-to-order transition, which can be directly monitored by HDX-MS. 50,51 Like many proteins, CyaA, an adenylate cyclase toxin from bacteria, contains a large intrinsically disordered region (IDR). 52 Binding of calmodulin (CaM) to CyaA activates its enzymatic activity, generating toxic levels of cAMP. HDX-MS was used by O'Brien et al to elucidate the mechanism of CyaA activation through ligand binding.…”

Section: Monitoring Transiently Preferred Conformational States In mentioning

confidence: 99%

“…They surmise the rigidity in the crystal structure may be an artifact of symmetric crystal packing and not representative of the native environment. 52 HDX-MS can be used to characterize structural transitions that often define protein function and control protein-protein interactions. Though HDX data do not provide a three-F I G U R E 3 Ligand induced disorder to order transition is critical for catalytic function in CyaA.…”

Section: Monitoring Transiently Preferred Conformational States In mentioning

confidence: 99%

Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

2019

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Much of our understanding of protein structure and mechanistic function has been derived from static high‐resolution structures. As structural biology has continued to evolve it has become clear that high‐resolution structures alone are unable to fully capture the mechanistic basis for protein structure and function in solution. Recently Hydrogen/Deuterium‐exchange Mass Spectrometry (HDX‐MS) has developed into a powerful and versatile tool for structural biologists that provides novel insights into protein structure and function. HDX‐MS enables direct monitoring of a protein's structural fluctuations and conformational changes under native conditions in solution even as it is carrying out its functions. In this review, we focus on the use of HDX‐MS to monitor these dynamic changes in proteins. We examine how HDX‐MS has been applied to study protein structure and function in systems ranging from large, complex assemblies to intrinsically disordered proteins, and we discuss its use in probing conformational changes during protein folding and catalytic function. Statement for a Broad Audience The biophysical and structural characterization of proteins provides novel insight into their functionalities. Protein motions, ranging from small scale local fluctuations to larger concerted structural rearrangements, often determine protein function. Hydrogen/Deuterium‐exchange Mass Spectrometry (HDX‐MS) has proven a powerful biophysical tool capable of probing changes in protein structure and dynamic protein motions that are often invisible to most other techniques.

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A High‐Affinity Calmodulin‐Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells

et al. 2021

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The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells.

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Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis

Cited by 35 publications

References 60 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

Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

A High‐Affinity Calmodulin‐Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells

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