Structurally conserved aromaticity of Tyr249 and Phe264 in helix 7 is important for toxicity of the Bacillus thuringiensis Cry4Ba toxin.
Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry delta- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, Trp(243), Phe(246), Tyr(249) and Phe(264), in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at Tyr(249) and Phe(264) still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of Tyr(249) and Phe(264) within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.
Tiewsiri
Kasorn
K
Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. stcas@mahidol.ac.th
Angsuthanasombat
Chanan
C
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000633:toxicity*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000633:toxicity*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D009033:Culicidae / Q000187:drug effects; D004731:Endotoxins / Q000737:chemistry* / Q000633:toxicity*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000633:toxicity*; D006841:Hydrocarbons, Aromatic / Q000737:chemistry*; D007814:Larva / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000633:toxicity; D010649:Phenylalanine / Q000737:chemistry*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D006841:Hydrocarbons, Aromatic; D050505:Mutant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014443:Tyrosine; D010649:Phenylalanine