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

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

Volume: 32 Issue: 8

Author(s): Chompounoot Imtong, Walairat Bourchookarn, Apichai Bourchookarn*, Somsri Sakdee, Hui-Chun Li and Chanan Angsuthanasombat*

Affiliation:

• Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand

• Center for Advanced Therapeutics, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand
• Department of Biochemistry, School of Medicine, Tzu Chi University, Hualien 97004, Taiwan
• Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand

Keywords: Cry insecticidal proteins, helical hairpin, hydrogen bonding, larval toxicity, membrane perturbation, structural integrity.

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. 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 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 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 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-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.

Cite this article as:

Imtong Chompounoot, Bourchookarn Walairat, Bourchookarn Apichai*, Sakdee Somsri, Li Hui-Chun and Angsuthanasombat Chanan*, Essential Role of Non-Conserved α4-His¹⁷⁸ in Stabilizing the α4-α5 Hairpin and Biotoxicity of the Cry4Aa Mosquitocidal Protein, Protein & Peptide Letters 2025; 32 (8) . https://dx.doi.org/10.2174/0109298665393672250715000125