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image of Essential Role of Non-Conserved α4-His178 in Stabilizing the α4-α5 Hairpin and Biotoxicity of the Cry4Aa Mosquitocidal Protein

Abstract

Background

Cry toxins are well known for their insecticidal properties, primarily through the formation of ion-leakage pores α4-α5 hairpins. His178 in helix 4 of the Cry4Aa mosquito-active toxin has been suggested to play a crucial role in its biotoxicity.

Objective

This study aimed to investigate the functional importance of Cry4Aa-His178 through experimental and computational analyses.

Methods

Ten His178-substituted Cry4Aa mutants (H178D, H178E, H178K, H178R, H178G, H178F, H178Y, H178S, H178C, and H178Q) were generated site-directed mutagenesis and expressed in . Toxin solubility was assessed in carbonate buffer (pH 10.0), and biotoxicity was tested against larvae. Trypsin-treated toxins were evaluated using fluorescent dye-release assays. Ion channel formation was studied in planar lipid bilayers (PLBs), and structural analysis was performed MD simulations and sequence alignments with known Cry toxins.

Results

All His178-substituted mutants were overexpressed as 130-kDa protoxin inclusions at levels comparable to the wild-type (WT). Replacing His178 with nonpolar or bulky polar residues reduced Cry4Aa biotoxicity to less than 10%, while substitutions with small, moderately polar, or negatively charged residues retained 50-85% activity, consistent with their solubility. Selected bioactive mutants, H178C and H178D, retained membrane-perturbing ability, like trypsin-activated WT, while the bioinactive H178Y mutant exhibited decreased membrane permeability. All tested mutants, including WT, induced cation-selective channels in PLBs with ~130-pS conductance. Sequence-structure analysis indicated that Cry4Aa-His178 likely forms a hydrogen bond with His217, a conserved His residue in helix 5.

Discussion

Specific physicochemical properties of residue 178 are critical for optimal larvicidal activity, making it a promising target for engineering more potent mosquito-control toxins.

Conclusion

His178 in Cry4Aa-α4 potentially forms a stabilizing hydrogen bond with α5-His217, which maintains the structural integrity of the α4-α5 hairpin. This structural stability is essential for efficient membrane insertion and optimal larvicidal activity.

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2025-09-05
2025-09-11

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Essential Role of Non-Conserved α4-His178 in Stabilizing the α4-α5 Hairpin and Biotoxicity of the Cry4Aa Mosquitocidal Protein
Bentham Science Publishers ; https://doi.org/10.2174/0109298665393672250715000125
/content/journals/ppl/10.2174/0109298665393672250715000125
/content/journals/ppl/10.2174/0109298665393672250715000125
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  • Article Type:     Research Article
Keywords: hydrogen bonding ; larval toxicity ; membrane perturbation ; structural integrity ; helical hairpin ; Cry insecticidal proteins

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