Folding of the nonacylated acylation domain of the adenylate cyclase toxin is templated by its C-terminal RTX domain.

Adenylate cyclase toxin (CyaA) is a key virulence factor secreted by the whooping cough-causing bacterium Bordetella pertussis. Its acylation domain (AD) is a conserved functional domain amongst various pore-forming toxins. In CyaA, AD connects the C-terminal repeats-in-toxin (RTX) domain to the N-terminal functional domains, such as the hydrophobic domain and the adenylate cyclase domain, and its proper folding is important for the acquisition of the active conformation and full toxin activity of CyaA. However, the mechanism of AD folding remains elusive. Using single-molecule optical tweezers, here we investigated the folding pathway of nonacylated AD in the context of a truncated RTX scaffold. We showed that a truncated RTX construct, ΔRTX-i/v, in which the N-terminal part of RTX-i was connected to the C-terminal part of RTX-v, is folded upon binding of Ca2+ and can scaffold the folding of the AD. In the absence of Ca2+, the AD was unfolded. Upon Ca2+ binding, the AD folded sequentially, templated by the folding of its C-terminal RTX block, with the Ca2+-binding segment folding before its N-terminal segment. Selective perturbation of ΔRTX-i/v using a β-hairpin insertion slowed down the folding of ΔRTX-i/v, and subsequently gatekept the folding of AD. Our results indicated that the folding of nonacylated AD is templated by its C-terminal RTX domain. These findings suggested that the templated folding mechanism extends beyond the RTX domain into the adjacent functional AD domain and proceeds hierarchically from the RTX C-terminus to the AD, providing a molecular basis for how CyaA couples secretion to folding to ensure efficient toxin maturation.