Involvement of Two Amino Acid Residues in the Loop Region ofBacillus thuringiensisCry1Ab Toxin in Toxicity and Binding toLymantria dispar

Abstract

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 toLymantria disparcompared to Cry1Ab9-033. However, these toxins showed similar toxicity and binding affinity toManduca sextaandSpodoptera exigua.Heterologous competition assays and brush border membrane vesicle (BBMV) ligand blotting experiments demonstrated that Cry1Ab2 and Cry1Ab9-033 toxins recognized the same 210-kDaL. disparBBMV protein. No measurable differences in dissociation binding assays were observed between these two toxins. Digestion of these toxins withL. dispargut 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 α8. This toxin exhibited full recovery of toxicity and binding affinity forL. dispar.These data suggested that the residues Gly and Ser in the loop region might be directly involved in receptor binding and toxicity inL. dispar.