Specific mutations within the alpha4-alpha5 loop of the Bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170

The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry delta-endotoxins suggests that helices alpha4 and alpha5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (As...

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Bibliographic Details
Published in:Molecular biotechnology 2003-05, Vol.24 (1), p.11
Main Authors: Kanintronkul, Yodsoi, Sramala, Issara, Katzenmeier, Gerd, Panyim, Sakol, Angsuthanasombat, Chanan
Format: Article
Language:English
Subjects:
Aedes - drug effects
Amino Acid Sequence
Animals
Asparagine - chemistry
Asparagine - metabolism
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
Bacterial Proteins - pharmacology
Bacterial Toxins
Endotoxins - chemistry
Endotoxins - metabolism
Endotoxins - pharmacology
Hemolysin Proteins
Inclusion Bodies - metabolism
Insecticides - pharmacology
Larva - drug effects
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Protein Structure, Tertiary
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Recombinant Proteins - pharmacology
Structure-Activity Relationship
Tyrosine - chemistry
Tyrosine - metabolism
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Summary:The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry delta-endotoxins suggests that helices alpha4 and alpha5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the alpha4-alpha5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-beta-Dthiogalactopyranoside induction. When E. coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity. Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within the alpha4-alpha5 loop play a crucial role in the larvicidal activity of the Cry4B toxin.
ISSN:1073-6085