Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin

Sriwimol, Wilaiwan; Aroonkesorn, Aratee; Sakdee, Somsri; Kanchanawarin, Chalermpol; Uchihashi, Takayuki; Ando, Toshio; Angsuthanasombat, Chanan

doi:10.1074/jbc.m114.627554

Cited by 30 publications

(19 citation statements)

References 46 publications

(63 reference statements)

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“…This similar finding could be observed in some other bacterial protein toxins from entirely different family, for instance, Bacillus thuringiensis Cry4Ba insecticidal protein, which was also demonstrated to form a trimeric pore structure [36,37]. These two pore-forming toxins potentially share a similar pore structure and mechanism of pore formation, although they have no sequence similarities outside of their pore-lining constituents.…”

Section: Resultssupporting

confidence: 75%

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

Kurehong

Kanchanawarin

Powthongchin

et al. 2017

Toxins

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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.

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Section: Resultssupporting

confidence: 75%

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

Kurehong

Kanchanawarin

Powthongchin

et al. 2017

Toxins

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“…In Vibrio cholerae cytolysin, variants that could not form oligomers lost the vast majority of their haemolytic activities against human erythrocytes (Rai & Chattopadhyay, 2015). It was also shown that some Cry toxins, such as Cry1A, Cry3, Cry4, and Cry11, could form oligomeric structures when activated in the presence of midgut receptors or brush border membrane vesicles (Gómez et al, 2014;Munoz-Garay et al, 2009;Rausell et al, 2004;Sriwimol et al, 2015). Here, we showed for the first time that the exposure of helices α4 and α5 could trigger the oligomerisation of Cry2Ab, resulting in the formation of a 250-kDa oligomeric structure (Figure 5a).…”

Section: Discussionmentioning

confidence: 99%

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure

Pan

Zhang

et al. 2018

Cellular Microbiology

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Cry2Ab, a pore-forming toxin derived from Bacillus thuringiensis, is widely used as a bio-insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250-kDa prepore oligomer. Site-directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.

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“…In addition, Cry1AbMod inserted in synthetic membranes showed a trimeric organization in 2D crystals observed by electron microscopy 29 . Similarly the Cry4Ba toxin showed a trimeric organization after incorporation into synthetic membrane observed by electron microscopy 28 . The trimeric 3D structure of Cry4Ba was used to construct a model of the trimeric structure of Cry4Aa and the authors hypothesized that this structure may resemble the oligomer structure involved in pore formation and toxicity of Cry4Aa 30 .…”

Section: Introductionmentioning

confidence: 89%

“…It was shown that similar to the Cry1A toxins, the Cry11Aa and Cry3Aa toxins also require binding to CAD to oligomerize 25 – 27 . In contrast the Cry4Ba toxin is an exception because it is able to oligomerize in vitro in the absence of CAD binding 27 , 28 .…”

Section: Introductionmentioning

confidence: 99%

Helix α-3 inter-molecular salt bridges and conformational changes are essential for toxicity of Bacillus thuringiensis 3D-Cry toxin family

Pacheco

Gómez

Sánchez

et al. 2018

Sci Rep

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Bacillus thuringiensis insecticidal Cry toxins break down larval midgut-cells after forming pores. The 3D-structures of Cry4Ba and Cry5Ba revealed a trimeric-oligomer after cleavage of helices α-1 and α-2a, where helix α-3 is extended and made contacts with adjacent monomers. Molecular dynamic simulations of Cry1Ab-oligomer model based on Cry4Ba-coordinates showed that E101 forms a salt-bridge with R99 from neighbor monomer. An additional salt bridge was identified in the trimeric-Cry5Ba, located at the extended helix α-3 in the region corresponding to the α-2b and α-3 loop. Both salt-bridges were analyzed by site directed mutagenesis. Single-point mutations in the Lepidoptera-specific Cry1Ab and Cry1Fa toxins were affected in toxicity, while reversed double-point mutant partially recovered the phenotype, consistent with a critical role of these salt-bridges. The single-point mutations in the salt-bridge at the extended helix α-3 of the nematicidal Cry5Ba were also non-toxic. The incorporation of this additional salt bridge into the nontoxic Cry1Ab-R99E mutant partially restored oligomerization and toxicity, supporting that the loop between α-2b and α-3 forms part of an extended helix α-3 upon oligomerization of Cry1 toxins. Overall, these results highlight the role in toxicity of salt-bridge formation between helices α-3 of adjacent monomers supporting a conformational change in helix α-3.

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Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin

Cited by 30 publications

References 46 publications

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

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

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure

Helix α-3 inter-molecular salt bridges and conformational changes are essential for toxicity of Bacillus thuringiensis 3D-Cry toxin family

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