Essential Role of Non-Conserved α4-His <sup>178</sup> in Stabilizing the α4-α5 Hairpin and Biotoxicity of the Cry4Aa Mosquitocidal Protein
Bibliographic record
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".