Background: The molecular description of oligomeric pore formation by B. thuringiensis insecticidal toxins remains unclear.Results: Cry4Ba mosquito-active toxins assemble into a stable prepore trimer upon interaction with non-ionic micelles or lipid membranes. Conclusion: A membrane-bound state of monomers is required for facilitating a potential trimer assembly. Significance: This study reveals a requirement of membrane-bound monomers for forming a prepore trimer capable of perturbing target membranes.

Cry4Aa toxin is one of the highly specific mosquito-larvicidal proteins produced by the bacterium Bacillus thuringiensis subspecies israelensis. It is thought to form pores in the larval midgut membrane that cause membrane leakage and subsequent insect death. Therefore, Cry4Aa and other Cry toxins have been used as efficient and safe bacterial insecticides to control the disease-carrying mosquitoes such as Aedes, Anopheles, and Culex. However, we still do not clearly understand how Cry toxins kill mosquito-larvae at molecular details. Recent electron crystallographic images of Cry4Ba toxin, another toxin closely related to Cry4Aa toxin, have suggested that the protein forms trimer in aqueous solution and in lipid monolayer. Moreover, the unit cell of X-ray crystal structure of Cry4Ba toxin has been shown to be trimeric. In this study, we constructed the first full-atom structural model of Cry4Aa trimer using the trimeric unit cell structure of Cry4Ba toxin as a template and then used the methods of molecular dynamics (MD) and molecular mechanics combined with Poisson-Boltzmann and surface area (MM-PBSA) to show that the trimeric structure of Cry4Aa toxin is stable in 150 mM KCl solution on 10 ns timescale. The results reveal that Cry4Aa toxins use polar amino acid residues on α-helices 3, 4, and 6 to form trimer and suggest that the proteins form trimer to reduce their non-polar interactions with surrounding water. Based on the obtained trimeric structure of Cry4Aa toxins, we propose that pore formation of Cry toxins may involve a 90°-hairpin rotation during the insertion of their three α4-α5 hairpins into the membrane. This process may be mediated by water and ions.PACS Codes: 87.15.ap, 87.15.bk, 87.14.ep

Previously, the 126-kDa Bordetella pertussis CyaA pore-forming/hemolysin (CyaA-Hly) domain was shown to retain its hemolytic activity causing lysis of susceptible erythrocytes. Here, we have succeeded in producing, at large quantity and high purity, the His-tagged CyaA-Hly domain over-expressed in Escherichia coli as a soluble hemolytically-active form. Quantitative assays of hemolysis against sheep erythrocytes revealed that the purified CyaA-Hly domain could function cooperatively by forming an oligomeric pore in the target cell membrane with a Hill coefficient of ~3. When the CyaA-Hly toxin was incorporated into planar lipid bilayers (PLBs) under symmetrical conditions at 1.0 M KCl, 10 mM HEPES buffer (pH 7.4), it produced a clearly resolved single channel with a maximum conductance of ~35 pS. PLB results also revealed that the CyaA-Hly induced channel was unidirectional and opened more frequently at higher negative membrane potentials. Altogether, our results first provide more insights into pore-forming characteristics of the CyaA-Hly domain as being the major pore-forming determinant of which the ability to induce such ion channels in receptor-free membranes could account for its cooperative hemolytic action on the target erythrocytes.

Bacillus thuringiensis Cry4Ba toxin is lethal to mosquito-larvae by forming ion-permeable pores in the target midgut cell membrane. Previously, the polarity of Asn(166) located within the α4-α5 loop composing the Cry4Ba pore-forming domain was shown to be crucial for larvicidal activity. Here, structurally stable-mutant toxins of both larvicidal-active (N166D) and inactive (N166A and N166I) mutants were FPLC-purified and characterized for their relative activities in liposomal-membrane permeation and single-channel formation. Similar to the 65-kDa trypsin-activated wild-type toxin, the N166D bio-active mutant toxin was still capable of releasing entrapped calcein from lipid vesicles. Conversely, the two other bio-inactive mutants showed a dramatic decrease in causing membrane permeation. When the N166D mutant was incorporated into planar lipid bilayers (under symmetrical conditions at 150mM KCl, pH8.5), it produced single-channel currents with a maximum conductance of about 425pS comparable to the wild-type toxin. However, maximum conductances for single K(+)-channels formed by both bio-inactive mutants (N166I and N166A) were reduced to approximately 165-205pS. Structural dynamics of 60-ns simulations of a trimeric α4-α5 pore model in a fully hydrated-DMPC system revealed that an open-pore structure could be observed only for the simulated pores of the wild type and N166D. Additionally, the number of lipid molecules interacting with both wild-type and N166D pores is relatively higher than those of N166A and N166I pores. Altogether, our results further signify that the polarity at the α4-α5 loop residue-Asn(166) is directly involved in ion permeation through the Cry4Ba toxin-induced ionic pore and pore opening at the membrane-water interface.

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.