Domain III β4-β5 Loop and β14-β15 Loop of Bacillus thuringiensis Vip3Aa Are Involved in Receptor Binding and Toxicity.

Abstract

Vip3Aa, secreted by Bacillus thuringiensis, is effective at controlling major agricultural pests such as Spodoptera frugiperda. However, to control Vip3Aa resistance evolved in the field by different lepidoptera species, an in-depth study of sequence--structure--activity relationships is necessary to design new Vip3Aa variants. In this study, the four specific loops (β4-β5 loop, β9-β10 loop, β12-β13 loop, and β14-β15 loop) in domain III were selected and four loop mutants were constructed by replacing all residues in each specific loop with alanine. We obtained soluble proteins for three of the loop mutants, excluding the β9-β10 loop. These loop mutants have been characterized by toxicity bioassays against S. frugiperda, proteolytic processing, and receptor binding. These results indicate that the β4-β5 loop and β14-β15 loop are involved in receptor binding and Vip3Aa toxicity. Based on this, we constructed numerous mutants and obtained three single mutants (Vip3Aa-S366T, Vip3Aa-S366L, and Vip3Aa-R501A) that exhibited significantly increased toxicity of 2.61-fold, 3.39-fold, and 2.51-fold, respectively. Compared to Vip3Aa, the receptor affinity of Vip3Aa-S366T and Vip3Aa-S366L was significantly enhanced. Furthermore, we also analyzed and aligned the three-dimensional structures of the mutants and Vip3Aa. In summary, these results indicate that the loops in domain III have the potential to be targeted to enhance the insecticidal toxicity of the Vip3Aa protein.