Novel calcium-binding sites in the acylated segment control the folding and membrane penetration of RTX toxins

ABSTRACT: The Repeats in ToXin (RTX) domain of RTX cytolysins harbors numerous hexacoordinated Ca2+-binding sites that facilitate cooperative and vectorial folding of the RTX β-rolls. Recent study identified Ca2+-binding sites in the acylated N-terminal extension of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA), where the side chains of residues D880, D918 and N936 coordinate two calcium ions. Substitution of N936 with a bulky leucine did not affect the essential acylation of internal lysines or the capacity of the CyaA N936L to bind the receptor CR3. However, this substitution impaired the calcium-dependent folding of the acylated segment and abolished the capacity of the toxin to penetrate cell membrane. Similarly, substitution of the homologous D639 residue strongly reduced the cytotoxicity of Escherichia coli α-hemolysin (HlyA). While the substitution of D918 with leucine had no effect on CyaA activity, the D880L variant enhanced cell binding, adenylate cyclase (AC) domain translocation to cell cytosol and both the specific hemolytic activity of the CyaA D880L on erythrocytes and the membrane activity in artificial lipid bilayers. Hydrogen/deuterium exchange revealed that the D880L substitution altered the folding of the acylated segment and reduced the deuteration accessibility of surafce residues in the two loops bearing the acyl chains. Additionally, substitution of the β-turn-forming glycine G934 in CyaA and G637 in HlyA impaired the folding of the acylated segment and decreased the cytotoxicity of both toxins. These results highlight the critical role of the acylated segment in the membrane penetration capacity of RTX toxins from Gram-negative pathogens.

INSTRUMENT(S):

ORGANISM(S): Bordetella Pertussis

SUBMITTER: Zuzana Kalaninova  

LAB HEAD: Petr Man

PROVIDER: PXD058094 | Pride | 2026-04-22

REPOSITORIES: Pride