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 (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.
Laboratory of Molecular Biophysics, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
Yodsoi
Y
Kanintronkul
Issara
I
Sramala
Gerd
G
Katzenmeier
Sakol
S
Panyim
Chanan
C
Angsuthanasombat
D000330:Aedes / Q000187:drug effects*; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D001427:Bacterial Toxins; D004731:Endotoxins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D006460:Hemolysin Proteins; D002479:Inclusion Bodies / Q000378:metabolism; D007306:Insecticides / Q000494:pharmacology; D007814:Larva / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014443:Tyrosine; D001216:Asparagine