Calcium-loaded acylated segment controls membrane penetration capacity of repeats-in-toxin cytolysins.

Loading of calcium ions into the numerous carboxy-proximal binding sites in the repeats-in-toxin (RTX) domains drives the cooperative and vectorial folding of RTX β-roll structures involved in cell binding and membrane penetration of RTX cytolysins. Two additional binding sites for calcium ions, coordinated by the side chains of residues D880, D918, and N936, were identified in the structure of the acylated cap of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA). We show that this calcium-binding structure plays a key role in membrane insertion of the toxin. An N936L residue substitution did not impact toxin acylation or CR3 receptor binding but disrupted the calcium-driven folding of the acylated segment and ablated the membrane penetration capacity of the toxin. Similarly, substitution of the corresponding D639 residue of Escherichia coli α-hemolysin abolished its cytolytic capacity. Moreover, disruption of the β-turn structures in the calcium-binding sites of the acylated segment of CyaA (G934L) and α-hemolysin (G637L) strongly impaired the cytotoxic capacities of both toxins. On the contrary, a D880L substitution yielded a CyaA toxin with an enhanced CR3-independent cell penetration and pore-forming capacity. Hydrogen/deuterium exchange probing revealed that the D880L substitution altered the fold of the acylated segment and the interaction of its two acylated β-hairpins. Hence, loading the calcium-binding sites in the acylated segment controls the structure and rules the interaction and the functional cooperation of the two acylated β-hairpins that facilitate penetration of the CyaA polypeptide into the cell membrane.