The CyaA protein is a toxin produced by the bacteria responsible for whooping cough. When it intoxicates target cells, CyaA binds to a molecule known as calmodulin, inducing the folding of a disordered region of the toxin, which leads to CyaA activation. This mechanism, which combines structural folding and enzyme activation of CyaA, is vital both to activate the toxin when it reaches its target cells and also to prevent its toxicity within the bacteria that produce it. These findings, based on extensive structural biology research, were published by scientists from the Institut Pasteur and the CNRS in PLOS Biology.
"The results of our research have helped elucidate the relationship between the structure and activation of CyaA," sums up Alexandre Chenal, Head of the BiophysiCyaA Group in the Biochemistry of Macromolecular Interactions Unit at the Institut Pasteur. Adenylate cyclase toxin (CyaA) is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. This disease, which can affect people of all ages, is particularly severe and potentially fatal for infants and vulnerable individuals.
How does calmodulin activate CyaA?
Calmodulin is a highly conserved eukaryotic protein that interacts with a wide variety of other proteins and enzymes. It controls their activities in response to variations in concentrations of intracellular calcium.
The CyaA toxin contributes to the early stages of bacterial colonization of the lungs in people infected by B. pertussis, the causative agent for whooping cough. CyaA is synthesized and secreted by the bacterium in an inactive form. The CyaA toxin employs a unique mechanism to access the cytosol in our cells (the liquid phase inside cells): the catalytic domain is directly transported via the plasma membrane of target cells through a process known as membrane translocation.
"After invading eukaryotic target cells, the catalytic domain of CyaA is activated by calmodulin to synthesize huge quantities of cAMP," explains Alexandre Chenal. cAMP is a molecule used in low concentrations as an intracellular messenger. But the large quantities produced by CyaA impair cell physiology and ultimately lead to cell death. "We have known for some time about the activation of CyaA by calmodulin – an act of molecular piracy –, but the molecular activation mechanism remained a mystery," says the scientist.
75 amino acids serve as bait
"We used an integrative structural biology approach combining several biophysical techniques to characterize the structural changes in the CyaA catalytic domain and in calmodulin when they interact," continues Alexandre Chenal. The scientists' research enabled them to elucidate the relationship between the structure and activation of CyaA by using a variety of techniques available at the Institut Pasteur and the SOLEIL synchrotron facility: small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS) and synchrotron radiation circular dichroism (SR-CD).
Transition from structural disorder to order in CyaA
"We demonstrated that a disordered region of 75 amino acids in the catalytic domain of CyaA serves as bait to capture calmodulin," explains Alexandre Chenal. "Binding induces significant folding in this region, a prerequisite for CyaA activation." Beyond the region where interaction occurs between calmodulin and the catalytic domain, the formation of the complex also triggers allosteric changes and stabilizes the distant catalytic site. But a catalytic loop (the blue star in the figure below) is maintained in a highly flexible state, which is essential for effective enzyme catalysis (more than 1,000 reactions per second!), allowing rapid association/dissociation of substrates (ATP) and products (cAMP and pyrophosphate). The production of cAMP irreversibly impairs the physiology of the immune system cells in the lungs, giving B. pertussis bacteria free rein to colonize this region.
Left: structural model of the catalytic domain of isolated, inactive CyaA. The regions folded into helices and sheets are represented in green, while the regions characterized by structural disorder are represented as a black line. Structural disorder inhibits catalytic activity.
Right: the active enzyme complex, formed by the catalytic domain of CyaA (in color) and calmodulin (in grey). The stabilization of the catalytic domain induced by calmodulin binding is represented by the color gradient red, yellow, green and blue; the red and yellow regions are the most stable (observed with HDX-MS). The flexible catalytic loop is indicated by a blue star. The structural models were obtained using experimental SAXS, HDX-MS and SR-CD data.
Making use of structural disorder to defend against the toxin
"This research opens up new avenues for the identification of CyaA inhibitors. Our aim is now to identify molecules that are capable of binding to the flexible region in the catalytic domain instead of calmodulin, but without inducing the allosteric effects that structure and activate the catalytic site," concludes the scientist. This collaborative project should be carried out in conjunction with the Institut Pasteur Korea (find out more about the Institut Pasteur International Network) and several technological platforms at the Institut Pasteur in Paris. The research is part of a large-scale, long-term project designed to further our fundamental understanding of the virulence factors of B. pertussis and improve CyaA-based biotechnological applications developed in the laboratory.
Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis, PLOS Biology, December 29, 2017
O’Brien DP1, Durand D2, Voegele A1, Hourdel V3, Davi M1, Chamot-Rooke J3, Vachette P2, Brier S3, Ladant D1, Chenal A1.
1 Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, Paris, France.
2 Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Paris-Sud Univ., Paris-Saclay University, Gif-sur-Yvette cedex, France.
3 Institut Pasteur, USR CNRS 2000, Chemistry and Structural Biology Department, CITECH, Paris, France.
The project is funded by the Institut Pasteur (PTR and PasteurInnoV), the CNRS, the ANR (CACSICE Equipex), the French Foundation for Medical Research (FRM) and the Greater Paris Authority (DIM MalInf)