Aromaticity of Tyr-202 in the alpha4-alpha5 loop is essential for toxicity of the Bacillus thuringiensis Cry4A toxin.
The current model for the mechanism of action of the Bacillus thuringiensis Cry delta-endotoxins involves the penetration of the alpha4-alpha5 hairpin into the target midgut epithelial cell membranes, followed by pore formation. In this study, PCR-based mutagenesis was employed to identify a critical residue within the alpha4-alpha5 loop of the 130kDa Cry4A mosquito-larvicidal protein. Alanine-substitutions of two charged (Asp-198 and Asp-200) and four polar (Asn-190, Asn-195, Tyr-201 and Tyr-202) residues in the alpha4-alpha5 loop were performed. Like the wild-type, all of the mutant toxins were over-expressed as inclusion bodies in Escherichia coli. When E. coli cells expressing each mutant toxin were bioassayed against Aedes aegypti larvae, larvicidal activity was completely abolished for the substitution of only Tyr-202, while replacements at the other positions still retained a high level of toxicity. Further replacement of Tyr-202 with an aromatic side chain, phenylalanine, did not affect the toxicity. These results revealed a crucial role in toxin activity for the conserved aromatic residue at the 202 position within the alpha4-alpha5 loop of the Cry4A toxin.
Laboratory of Molecular Biophysics, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
Walairat
W
Pornwiroon
Gerd
G
Katzenmeier
Sakol
S
Panyim
Chanan
C
Angsuthanasombat
D000330:Aedes; D000595:Amino Acid Sequence; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology / Q000633:toxicity*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology / Q000633:toxicity*; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology / Q000633:toxicity*; D006460:Hemolysin Proteins; D007814:Larva / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014443:Tyrosine