Imtong C.Bourchookarn W.Bourchookarn A.Sakdee S.Li H.C.Angsuthanasombat C.Mahidol University2025-12-072025-12-072025-01-01Protein and Peptide Letters Vol.32 No.8 (2025) , 584-59609298665https://repository.li.mahidol.ac.th/handle/123456789/113417Background: Bacillus thuringiensis Cry toxins are well known for their insecticidal properties, primarily through the formation of ion-leakage pores via α4-α5 hairpins. His¹⁷⁸in 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-His¹⁷⁸through experimental and computational analyses. Methods: Ten His¹⁷⁸-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 His¹⁷⁸-substituted mutants were overexpressed as 130-kDa protoxin inclusions at levels comparable to the wild-type (WT). Replacing His¹⁷⁸with 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-His¹⁷⁸likely 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: His¹⁷⁸in 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.Biochemistry, Genetics and Molecular BiologyArticleSCOPUS10.2174/01092986653936722507150001252-s2.0-1050231630061875530540916407