Essential Role of Non-Conserved α4-His178in Stabilizing the α4-α5 Hairpin and Biotoxicity of the Cry4Aa Mosquitocidal Protein
8
Issued Date
2025-01-01
Resource Type
ISSN
09298665
eISSN
18755305
Scopus ID
2-s2.0-105023163006
Pubmed ID
40916407
Journal Title
Protein and Peptide Letters
Volume
32
Issue
8
Start Page
584
End Page
596
Rights Holder(s)
SCOPUS
Bibliographic Citation
Protein and Peptide Letters Vol.32 No.8 (2025) , 584-596
Suggested Citation
Imtong C., Bourchookarn W., Bourchookarn A., Sakdee S., Li H.C., Angsuthanasombat C. Essential Role of Non-Conserved α4-His178in Stabilizing the α4-α5 Hairpin and Biotoxicity of the Cry4Aa Mosquitocidal Protein. Protein and Peptide Letters Vol.32 No.8 (2025) , 584-596. 596. doi:10.2174/0109298665393672250715000125 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/113417
Title
Essential Role of Non-Conserved α4-His178in Stabilizing the α4-α5 Hairpin and Biotoxicity of the Cry4Aa Mosquitocidal Protein
Corresponding Author(s)
Other Contributor(s)
Abstract
Background: Bacillus thuringiensis Cry toxins are well known for their insecticidal properties, primarily through the formation of ion-leakage pores via α4-α5 hairpins. His<sup>178</sup>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-His<sup>178</sup>through experimental and computational analyses. Methods: Ten His<sup>178</sup>-substituted Cry4Aa mutants (H178D, H178E, H178K, H178R, H178G, H178F, H178Y, H178S, H178C, and H178Q) were generated via site-directed mutagenesis and expressed in Escherichia coli. Toxin solubility was assessed in carbonate buffer (pH 10.0), and biotoxicity was tested against Aedes aegypti 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 via MD simulations and sequence alignments with known Cry toxins. Results: All His<sup>178</sup>-substituted mutants were overexpressed as 130-kDa protoxin inclusions at levels comparable to the wild-type (WT). Replacing His<sup>178</sup>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 in vitro 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-His<sup>178</sup>likely forms a hydrogen bond with His<sup>217</sup>, 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: His<sup>178</sup>in Cry4Aa-α4 potentially forms a stabilizing hydrogen bond with α5-His<sup>217</sup>, which maintains the structural integrity of the α4-α5 hairpin. This structural stability is essential for efficient membrane insertion and optimal larvicidal activity.