Involvement of two amino acid residues in the loop region of Bacillus thuringiensis Cry1Ab toxin in toxicity and binding to Lymantria dispar.
Two amino acids, Gly and Ser, at positions 282 and 283 in the loop region of domain II of Cry1Ab2 toxin are substituted with Ala and Leu in the Cry1Ab9-033 toxin. Cry1Ab2 exhibited about a 10-fold increase in toxicity and a 9-fold increase in binding affinity to Lymantria dispar compared to Cry1Ab9-033. However, these toxins showed similar toxicity and binding affinity to Manduca sexta and Spodoptera exigua. Heterologous competition assays and brush border membrane vesicle (BBMV) ligand blotting experiments demonstrated that Cry1Ab2 and Cry1Ab9-033 toxins recognized the same 210-kDa L dispar BBMV protein. No measurable differences in dissociation binding assays were observed between these two toxins. Digestion of these toxins with L dispar gut enzymes and BBMV proteases indicated no differences in stability. Ala and Leu residues in Cry1Ab9-033 were substituted with Gly and Ser by site-directed mutagenesis to produce mutant Cry1Ab alpha 8. This toxin exhibited full recovery of toxicity and binding affinity for L dispar. These data suggested that the residues Gly and Ser in the loop region might be directly involved in receptor binding and toxicity in L dispar.
Lee
M K
MK
Department of Biochemistry, Ohio State University, Columbus 43210, USA.
You
T H
TH
Curtiss
A
A
Dean
D H
DH
D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D001667:Binding, Competitive; D004064:Digestive System / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D005998:Glycine / Q000235:genetics; D006460:Hemolysin Proteins; D018402:Manduca / Q000187:drug effects; D009036:Moths / Q000187:drug effects* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012694:Serine / Q000235:genetics; D013329:Structure-Activity Relationship
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D012694:Serine; D005998:Glycine