Functional Contributions of Positive Charges in the Pore-Lining Helix 3 of the Bordetella pertussis CyaA-Hemolysin to Hemolytic Activity and Ion-Channel Opening

Abstract

The Bordetella pertussis CyaA-hemolysin (CyaA-Hly) domain was previously demonstrated to be an important determinant for hemolysis against target erythrocytes and ion-channel formation in planar lipid bilayers (PLBs). Here, net-charge variations in the pore-lining helix of thirteen related RTX cytolysins including CyaA-Hly were revealed by amino acid sequence alignments, reflecting their different degrees of hemolytic activity. To analyze possible functional effects of net-charge alterations on hemolytic activity and channel formation of CyaA-Hly, specific mutations were made at Gln⁵⁷⁴ or Glu⁵⁸¹ in its pore-lining α3 of which both residues are highly conserved Lys in the three highly active RTX cytolysins (i.e., Escherichia coli α-hemolysin, Actinobacillus pleuropneumoniae toxin, and Aggregatibacter actinomycetemcomitans leukotoxin). All six constructed CyaA-Hly mutants that were over-expressed in E. coli as 126 kDa His-tagged soluble proteins were successfully purified via immobilized Ni²⁺-affinity chromatography. Both positive-charge substitutions (Q574K, Q574R, E581K, E581R) and negative-charge elimination (E581Q) appeared to increase the kinetics of toxin-induced hemolysis while the substitution with a negatively-charged side-chain (Q574E) completely abolished its hemolytic activity. When incorporated into PLBs under symmetrical conditions (1.0 M KCl, pH 7.4), all five mutant toxins with the increased hemolytic activity produced clearly-resolved single channels with higher open probability and longer lifetime than the wild-type toxin, albeit with a half decrease in their maximum conductance. Molecular dynamics simulations for 50 ns of a trimeric CyaA-Hly pore model comprising three α2-loop-α3 transmembrane hairpins revealed a significant role of the positive charge at both target positions in the structural stability and enlarged diameter of the simulated pore. Altogether, our present data have disclosed functional contributions of positively-charged side-chains substituted at positions Gln⁵⁷⁴ and Glu⁵⁸¹ in the pore-lining α3 to the enhanced hemolytic activity and ion-channel opening of CyaA-Hly that actually mimics the highly-active RTX (repeat-in-toxin) cytolysins.

Keywords: Bordetella pertussis; CyaA-hemolysin; MD simulations; RTX cytolysin; channel-open lifetime; pore-lining helix; trimeric pore.

Figure 1

(A) (Above) Schematic representation of CyaA showing adenylate cyclase (AC) domain and hemolysin (Hly) domain which contains the hydrophobic (HP) region, palmitoylation site (Lys⁹⁸³) and the RTX region. Five putative helices in the HP region are indicated by cylinders where α2 and α3, putative membrane-inserting constituents were marked in blue and red, respectively. Each line in the RTX region represents each repeat of (X-U-X-Gly-Gly-X-Gly-X-Asp, X for any amino acid and U for large hydrophobic residues); (B) Left panel: The homology-based model of the CyaA-Hly α2–α3 hairpin. Side chains at pore-lining region of α3 are shown as van der Waals (vdW) spheres and colored according to atoms: cyan, white, red, and blue for C, H, O, and N, respectively. Right panel: Conserved amino acids at pore-lining regions of thirteen related RTX cytolysins, as mentioned earlier in [25]. The side-chains are highlighted in bold letter and the charged residues are colored in red for negative and blue for positive; and (C) Side view of the hairpins of CyaA-Hly and three highly-active RTX cytolysins, i.e., HlyA from E. coli, ApxIA from Actinobacillus pleuropneumoniae and LtxA from Aggregatibacter actinomycetemcomitans, showing three key side chains at Glu⁵⁷⁰/Gln⁵⁷⁴/Glu⁵⁸¹ (for CyaA-Hly) and Glu/Lys/Lys (for HlyA, ApxIA, and LtxA) patches. Inset: Top view of individual hairpins. Amino acids are colored according to their charged/polar properties (red is negatively-charged, blue is positively-charged, and light-blue is N-containing polar uncharged) with H atoms omitted.

Figure 2

Time-course of hemolytic activity against sheep erythrocytes of individual purified toxins (10 μg/mL) as indicated. The control for 100% hemolysis is susceptible sheep erythrocytes with respect to a complete lysis when treated with 0.1% Triton X-100. Subset: SDS-PAGE (10% gel) of the Ni²⁺-NTA purified CyaA-Hly toxins, both wild-type (WT) and mutants (Q574K, Q574R, Q574E, E581K, E581R, and E581Q). Bands of CyaA-Hly toxins 126 kDa in length are marked by an arrow.

Figure 3

Ion-channel properties of CyaA-Hly wild-type (WT) and its mutant toxins (1 μg/mL). (A) Representative current traces were recorded at 100 mV holding potential. The closed stage level of channels is denoted by the letter c. Vertical and horizontal bars indicate measured current-time scales, respectively; and (B) current-voltage relations of all single channels formed by WT and its mutant toxins were recorded under symmetric conditions.

Figure 4

(A) MD simulations (50 ns) of trimeric CyaA-Hly channel models in membrane of the wild-type (WT, left) and two representative mutants; Q574K (middle) and E581K (right). Total net charges (z) inside the pores are shown; (B) C_α-RMSF profiles (Arg⁵²⁸–Ala⁵⁹³) obtained from MD trajectories of 50-ns simulations of individual CyaA-Hly pore structures; and (C) the average separation distance (C_α to C_α) between three Glu⁵⁷⁰ residues on the trimeric CyaA-Hly pore models versus time during 50-ns MD simulations. Left and right panels represent the systems of Gln⁵⁷⁴ and Glu⁵⁸¹ mutants compared to the wild-type, respectively.