Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling

Abstract

The Cry11 family belongs to a large group of δ-endotoxins that share three distinct structural domains. Among the dipteran-active toxins referred to as three-domain Cry11 toxins, the Cry11Aa protein from Bacillus thuringiensis subsp. israelensis (Bti) has been the most extensively studied. Despite the potential of Bti as an effective biological control agent, the understanding of Cry11 toxins remains incomplete. In this study, five Cry11 variants obtained via DNA shuffling displayed toxic activity against Aedes aegypti and Culex quinquefasciatus. Three of these Cry11 variants (8, 23, and 79) were characterized via 3D modeling and analysis of docking with ALP1. The relevant mutations in these variants, such as deletions, insertions and point mutations, are discussed in relation to their structural domains, toxic activities and toxin-receptor interactions. Importantly, deletion of the N-terminal segment in domain I was not associated with any change in toxic activity, and domain III exhibited higher sequence variability than domains I and II. Variant 8 exhibited up to 3.78- and 6.09-fold higher toxicity to A. aegypti than Cry11Bb and Cry11Aa, respectively. Importantly, variant 79 showed an α-helix conformation at the C-terminus and formed crystals retaining toxic activity. These findings indicate that five Cry11 variants were preferentially reassembled from the cry11Aa gene during DNA shuffling. The mutations described in loop 2 and loop 3 of domain II provide valuable information regarding the activity of Cry11 toxins against A. aegypti and C. quinquefasciatus larvae and reveal new insights into the application of directed evolution strategies to study the genetic variability of specific domains in cry11 family genes.

Keywords: Aedes aegypti; Bacillus thuringiensis; Cry11; Culex quinquefasciatus; DNA shuffling; docking.

FIGURE 1

Structure of the Cry toxins, domains, and their mode of action. (A) Ribbon diagram of Cry deduced 3D structure. Three domains are colored in red blue and green, respectively. (B) Sequential binding mechanism. 1. The toxin binds to GPI-anchored APN and ALP receptors in the lipid rafts; 2. Binding to cadherin receptor 3. Proteolytic cleavage of the helix α1 at N-terminal end; 4. N-terminal cleavage induces the formation of pre-pore oligomer 5. Increasing of the oligomer binding affinity to GPI-anchored APN and ALP receptors; 6. Oligomer inserts into the membrane, leading to pore-formation and cell lysis; and 7. Cellular death.

FIGURE 2

Half lethal concentrations of Cry11 variants obtained via DNA shuffling in Aedes aegypti and Culex quinquefasciatus larvae. The values are expressed as ng/ml of spore-crystal mixtures, 95% confidence limit (CL).

FIGURE 3

Prediction of the 3D Structures of Cry11Aa and Variants 8, 23, and 79. (A) Conserved region of variant 8 in light blue and Cry11Aa in beige, RMSD: 1,084 with 247 aa. (B) Non-conserved region of variant 8 in light blue and Cry11Aa in beige. (C) Conserved region of variant 23 in light blue and Cry11Aa in beige, RMSD: 1,132 with 488 aa. (D) Non-conserved region of variant 23 in light blue and Cry11Aa in beige. (E) Conserved region of variant 79 in light blue and Cry11Aa in beige, RMSD: 1,084 with 247 aa. (F) Non-conserved region of variant 79 in light blue and Cry11Aa in beige. (G) Ribbon representation of the non-conserved region of variant 79 generated using the Robetta server.

FIGURE 4

Molecular docking of the interactions of domain II of Cry11Aa and its variants with ALP1. (A,B) Interactions formed by 11-amino acid peptides within domain II of Cry11Aa. (A) Variant 8 (B) Cry11Aa. (C–F) Interactions formed by 5-amino acid peptides within domain II of Cry11Aa (C) variant 8 (D) Cry11Aa (E) Variant 23 (F) Variant 79.

FIGURE 5

Interactions visualized in LigPlot of ALP1 against peptides of Cry11Aa and its variants 8 and 23. (A) Cry11Aa, (B) Variant 23, and (C) Variant 8.