Abstract
The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) translocates its adenylate cyclase (AC) enzyme domain into target cells in a step that depends on membrane cholesterol content. We thus examined what role in toxin activities is played by the five putative cholesterol recognition amino acid consensus (CRAC) motifs predicted in CyaA hemolysin moiety. CRAC-disrupting phenylalanine substitutions had no impact on toxin activities and these were not inhibited by free cholesterol, showing that the putative CRAC motifs are not involved in cholesterol binding. However, helix-breaking proline substitutions in these segments uncovered a structural role of the Y632, Y658, Y725 and Y738 residues in AC domain delivery and pore formation by CyaA. Substitutions of Y940 of the fifth motif, conserved in the acylated domains of related RTX toxins, did not impact on fatty-acylation of CyaA by CyaC and the CyaA-Y940F mutant was intact for toxin activities on erythrocytes and myeloid cells. However, the Y940A or Y940P substitutions disrupted the capacity of CyaA to insert into artificial lipid bilayers or target cell membranes. The aromatic ring of tyrosine 940 side chain thus appears to play a key structural role in molecular interactions that initiate CyaA penetration into target membranes.
Highlights
The 1706 residue-long adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in virulence of pathogenic Bordetellae[1, 2]
Helix-preserving alanine substitutions of the same tyrosine residues had no impact on the activities of CyaA within target membranes (Supplementary Fig. S2). These results suggest that rather than being part of cholesterol recognition/interaction amino acid consensus (CRAC) motifs, the aromatic side chains of the tyrosine residues 632, 658, 725 and 738 play a structural role in α-helical segments that are involved in membrane translocation of the AC domain and in formation of CyaA pores
We show here that the conserved tyrosine 940 residue of the putative CRAC motif in the acylated domain of CyaA plays a central structural role in membrane insertion and penetration of the toxin
Summary
The 1706 residue-long adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in virulence of pathogenic Bordetellae[1, 2]. The Hly moiety oligomerizes into cation-selective pores that permeabilize cell membrane and allow efflux of potassium from cells[8,9,10,11] Both toxin activities depend on the posttranslational activation of proCyaA to CyaA by covalent fatty-acylation of the ε-amino groups of lysine residues 860 and 983 by a co-expressed protein acyltransferase, CyaC12–15. Two other RTX toxins, the Aggregatibacter actinomycetemcomitans leukotoxin LtxA and the Escherichia coli α-hemolysin HlyA, were previously shown to bind cholesterol[41,42,43] These toxins possess in their pore-forming domains the so called cholesterol recognition/interaction amino acid consensus (CRAC) motifs. Single residue substitutions in this CRAC motifs were found to markedly impair interaction of diverse proteins with target membranes[45, 46]
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