Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins

Abstract

Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.

Structural basis for non-canonical integrin engagement by Bordetella adenylate cyclase toxin

“…A recent study also assessed the effect of alanine substitutions at this position in other pore-forming RTX toxins. Given the importance of this residue in ACT, it is striking that its mutation did not significantly affect the hemolytic activities of E. coli HlyA, and only modestly affected Kingella kingae RtxA, or Actinobacillus pleuropneumonia ApxIA (Lepesheva et al, 2021). This suggests that the pore-forming RTX toxins more similar to E. coli HlyA may have more stable acylation domains than ACT that can maintain the structure of the loop harboring the acylation site homologous to ACT Lys983 despite local unfolding of L0.…”

A conserved tryptophan in the acylated segment of RTX toxins controls their β2 integrin–independent cell penetration

“…To simplify the interpretation of the CD measurements, we used a truncated RTX719 protein comprising the acylated segment fused to a minimal RTX domain with C-terminal capping structure that supports the calcium-dependent vectorial folding of the RTX domain ( Fig. 1 D ) ( 28 , 37 ). As shown in Figure 1 E , the far-UV CD spectra of the RTX719 W876L protein refolded in the presence of 2 mM calcium ions were indistinguishable from those of the intact RTX719 protein and exhibited a negative peak at 218 nm, which is characteristic for the RTX β-roll structure.…”

“…Nonetheless, the structure-function relationships underlying the AC-translocating activity of the membrane-penetrating domain of CyaA (residues 400–700) remain poorly understood. Despite inferences from the functional characterization of mutant toxin variants, a mechanistic understanding of CyaA intoxication remains to be defined ( 9 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ). To partially address this gap, we report that the highly conserved tryptophan residues of CyaA (W876) and HlyA (W579) fine-tune the structural configuration of acylated segments of RTX leukotoxins to enable their integrin receptor-independent (nonassisted) membrane penetration.…”

Kingella kingae RtxA Cytotoxin in the Context of Other RTX Toxins

“…In addition, each RTX block-linking fragment of CyaA contains a conserved aromatic residue. These aromatic residues are thought to be crucial for the proper folding of the adjacent block of RTX repeats [139,184,252]. In CyaA, the binding of extracellular calcium ions accelerates the secretion process by triggering the formation of intramolecular Brownian ratchets.…”