title: Single-reversal charge in the β10-β11 receptor-binding loop of Bacillus thuringiensis Cry4Aa and Cry4Ba toxins reflects their different toxicity against Culex spp. larvae.
abstract: Bacillus thuringiensis Cry4Aa toxin was previously shown to be much more toxic to Culex mosquito-larvae than its closely related toxin - Cry4Ba, conceivably due to their sequence differences within the β10-β11 receptor-binding loop. Here, single-Ala substitutions of five residues (Pro(510), Thr(512), Tyr(513), Lys(514) and Thr(515)) within the Cry4Aa β10-β11 loop revealed that only Lys(514) corresponding to the relative position of Cry4Ba-Asp(454) is crucial for toxicity against Culex quinquefasciatus larvae. Interestingly, charge-reversal mutations at Cry4Ba-Asp(454) (D454R and D454K) revealed a marked increase in toxicity against such less-susceptible larvae. In situ binding analyses revealed that both Cry4Ba-D454R and D454K mutants exhibited a significant increase in binding to apical microvilli of Culex larval midguts, albeit at lower-binding activity when compared with Cry4Aa. Altogether, our present data suggest that a positively charged side-chain near the tip of the β10-β11 loop plays a critical role in determining target specificity of Cry4Aa against Culex spp., and hence a great increase in the Culex larval toxicity of Cry4Ba was obtained toward an opposite-charge conversion of the corresponding Asp(454).
authors: Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Nakornpathom 73170, Thailand; Bacterial Protein Toxin Research Unit, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand.
authors: Sarinporn
authors: S
authors: Visitsattapongse
authors: Bacterial Protein Toxin Research Unit, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand.
authors: Somsri
authors: S
authors: Sakdee
authors: Bacterial Protein Toxin Research Unit, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand.
authors: Somphob
authors: S
authors: Leetacheewa
authors: Bacterial Protein Toxin Research Unit, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand; Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development (BIRD), Bangkok 10150, Thailand. Electronic address: chanan.ang@mahidol.ac.th.
authors: Chanan
authors: C
authors: Angsuthanasombat
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D003465:Culex / Q000187:drug effects / Q000382:microbiology*; D004731:Endotoxins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D007814:Larva / Q000187:drug effects / Q000382:microbiology; D008969:Molecular Sequence Data; D009032:Mosquito Control; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Bacillus thuringiensis; Charge-reversal mutation; Cry4 toxins; Culex mosquito-larvae; Receptor-binding loop