Importance of Cry1 δ-Endotoxin Domain II Loops for Binding Specificity in Heliothis virescens (L.)

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Abstract
We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens . Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with 125 I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens . Cry1Ac competed with high affinity (competition constant [ K com ] = 1.1 nM) for 125 I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for 125 I-Cry1Ab binding sites, though the K com values ranged from 179 to 304 nM. Cry1Ab competed for 125 I-Cry1Ac binding sites ( K com = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the 125 I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens . Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens . The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.

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