The conserved tyrosine residue 940 plays a key structural role in membrane interaction of Bordetella adenylate cyclase toxin

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.

Cytotoxic activity of Kingella kingae RtxA toxin depends on post-translational acylation of lysine residues and cholesterol binding

“…Previous studies of other RTX toxins, namely the B. pertussis toxin CyaA 22 – 24 , the E. coli α-hemolysin HlyA 25 , 26 and the A. actinomycetemcomitans leukotoxin LtxA 27 , demonstrated that they are post-translationally activated through amide-linked fatty acylation of the ε-amino groups of two conserved internal lysine residues. Sequence alignment analysis of these three RTX toxins with RtxA revealed lysine residues 558 and 689 of proRtxA to be putative acylation sites (Fig.…”

“…Although it is generally accepted that the RTX toxins are post-translationally acylated on the ε-amino groups of two conserved lysine residues, the type of acyl chains has only been experimentally identified for three RTX toxins, namely the B. pertussis CyaA 22 – 24 , the E. coli α-hemolysin HlyA 25 , 26 and the A. actinomycetemcomitans leukotoxin LtxA 27 . Analysis of the predicted acylated segment of RtxA and its alignment with the corresponding regions of CyaA, HlyA and LtxA revealed that the RtxA protein contains two conserved lysine residues, K558 and K689 (Fig.…”

“…The second most abundant acyl group linked to RtxA, a monohydroxylated form of C14:0 (Supplementary Table 3 ), was previously identified in LtxA 27 . Only a small proportion of the RtxA molecules (~8%) were covalently modified at the K689 residue by the monounsaturated C16:1 fatty acyl chain (Supplementary Table 3 ), which together with the saturated C16:0 acyl group are two major acyl modifications of the K860 and K983 residues of recombinant CyaA 23 , 24 . In contrast to the results of Lim et al for HlyA 26 , but in agreement with those of Fong et al for LtxA 27 , we were unable to identify odd-carbon fatty acyl groups, such as C15:0 and C17:0, attached to the RtxA molecule.…”

Structure–Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes

“…The hydrophobic pore-forming domain of CyaA, comprised between residues 500 to 700 ( Figure 1 ), consists of several predicted amphipathic and hydrophobic α-helical structures. These play a key role both in AC domain translocation, as well as in the formation of cation-selective oligomeric CyaA pores within target membranes [ 11 , 19 , 25 , 66 , 67 , 68 , 69 ]. By using the method of osmotic solute exclusion, the size of the pore formed by CyaA in the cell membrane was estimated to be between 0.6 to 0.8 nm in diameter [ 26 ].…”

Bordetella Pertussis Adenylate Cyclase Toxin Does Not Possess a Phospholipase A Activity; Serine 606 and Aspartate 1079 Residues Are Not Involved in Target Cell Delivery of the Adenylyl Cyclase Enzyme Domain

“…An “AC-to-Hly-linking” segment of CyaA was identified and its arginine residues appear to be involved in the disruption of the membrane bilayer, which enables insertion and translocation of the AC domain of CyaA across the plasma membrane of cells [ 5 , 6 , 7 ]. Furthermore, the structural integrity of the putative transmembrane helices I, III, and IV of the hydrophobic domain was shown to be essential for AC domain translocation across the plasma membrane of both CD11b + and CD11b − cells [ 32 , 33 , 34 , 35 ].…”