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.

The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies

“…Standard protocols, including dilution and dialysis from denaturing buffers containing 8 M urea, recover high molecular weight species with variable activity levels. Recently, refolding on a size exclusion column was performed to prevent aggregation of partially folded species and resulted in purification of monomers with very high activity, which depended on the presence of calcium, acylation and molecular confinement (43). Although promising, the yields and scalability of this process are currently unclear.…”

“…The different monomer yields may reflect a time-dependent aggregation process or the presence of folding intermediates with different aggregation propensities in the two refolding procedures. A recent report reiterated the challenges of refolding ACT (43).…”

“…Structural data for RTX-containing proteins suggest the repeats fold into parallel ␤-helix structures. In the presence of calcium ions, the protein converts from an intrinsically disordered domain into a compact ␤-roll structure with an altered CD spectrum and a reduced hydrodynamic radius that appears to be further stabilized by acylation (43,49,50). RTX 985 exhibited a shift from largely monomer in the presence of calcium (78 kDa) to a mixture of oligomers (ϳ380 kDa) upon the addition of EGTA to chelate calcium ions.…”

Structure and function of RTX toxins

“…As suggested previously, the high calcium concentration in the extracellular medium should favor the folding of the RTX domain, which may serve as a nucleation site for the folding of the remaining regions of the secreted protein [78,87,88]. It is thought that the C-terminal extremity is released first into the extracellular medium, but this has not been proven yet.…”

“…Calcium ions likely balance the repulsive charges of the aspartate residues in the RTX repeats, allowing the polypeptide chain to collapse and fold into a compact and stable β-roll conformation [79] (Figure 23.3). Importantly, calcium binding to the RTX motifs also induces conformational changes in the other functional domains of the toxins [47,81,84,85] and affects the overall conformation of the full-length proteins [7,[86][87][88].…”