Mode of Action of Bt Toxins

To date, the three-dimensional structures of eight different members of the 3d-Cry toxins with different insect specificity have been solved. Figure 8.2 shows a representative structure of one member of 3d-Cry group, the Cry8Ea, that was the most recent 3d-Cry structure to be identified.17 Domain I, a seven a-helix

Figure 8.2 Three-dimensional structure of Cry8Ea (PDB 3ebA) insecticidal toxin produced by Bacillus thuringiensis. Domain I (DI) is an a-helix bundle, domain II (DII) consists of three anti parallel b-sheets with exposed loop regions, and domain III (DIII) is a b-sandwich.

bundle, is implicated in membrane insertion, toxin oligomerization and channel formation.5 Domain II consists of a beta-prism of three anti-parallel b-sheets packed around a hydrophobic core, and domain III is a b-sandwich of two antiparallel b-sheets. Domains II and III are implicated in insect specificity by mediating specific interactions with several receptor proteins.5 The structure of other 3d-Cry members (Cry1Aa, Cry2Aa, Cry3Aa, Cry3Ba, Cry4Aa and Cry4Ba) is highly similar to Cry8Ea, despite the low amino acid sequence similarity, and share conserved residues located in the internal regions of domain I and III, as well as in interdomain contacts. This suggests that all members of the 3d-Cry family may share a similar mechanism of action.5 It is widely recognized that 3d-Cry toxins exert their toxic effect by forming pores in the insect midgut epithelium cells, resulting in cell lysis and disruption of the midgut epithelium.2,5 The mode of action of these group of proteins has been mostly studied in lepidopteran insects but more recently also in coleopteran and dipteran insects.18 The 3d-Cry toxins are produced as protoxins that need to be dissolved and processed proteolytically by insect proteases to release the active toxic fragment. Two groups of protoxins are produced by different Bt strains: some of them are large protoxins (such as Cry1Aa of 130 kDa) and some are short protoxins of 70 kDa (such as Cry2Aa). However, in both cases the activated toxins have a similar size of 60 kDa. Large protoxins lose half of the C-terminal end and 20 to 50 amino acids of the N-terminal end, while short protoxins are processed only at the N-terminal end. The activated toxin is composed of the three structural domains mentioned above. The activated toxin goes through complex sequential binding events with different insect gut proteins leading to membrane insertion and pore formation. In the case of the lepidopteran insect, Manduca sexta, it has been proposed that the first binding interaction of the activated Cry1Ab toxin occurs through exposed amino acid regions of domain II (specifically through loop 3) and domain III (through strand b-16) of the toxin, with at least two glycosylphosphatidylinositol (GPI)-anchored proteins identified as alkaline phosphatase (ALP) and ami-nopeptidase N (APN), which are highly abundant low affinity binding sites for the toxin (Kd 100 nM).5,19,20 These binding interactions help to concentrate the activated toxin in the microvilli membrane of the midgut cells where the toxin binds with high affinity through exposed domain II loops 2, 3 and alpha-8 to a low abundant transmembrane protein identified as a cadherin receptor (Kd

I nM).5,19,20 This high affinity binding interaction with cadherin facilitates further proteolytic cleavage of the toxin of the N-terminal end including helix a-1 of domain I. This proteolytic cleavage induces the formation of a toxin pre-pore oligomer.5,19,20 The oligomeric structure of the toxin shows an important increase of 200 fold in its affinity to GPI-anchored receptors ALP and APN involving domain II loop 2 region (Kd=0.5 nM).19 The binding of the pre-pore to the GPI-anchored proteins leads finally to insertion into the membrane causing pore-formation and cell lysis.5 Figure 8.3 depicts the molecular events that lead to Cry toxin membrane insertion and pore formation.

As mentioned previously, Cry toxins are highly specific and only kill a narrow spectrum of insect species. Insect specificity of these proteins is determined mainly by the specific recognition of insect midgut proteins, although in some cases proteolytic activation and solubilization of Cry protoxins could affect susceptibility in certain insect species.21 As mentioned previously, domain

II and domain III of Cry toxins are the structural determinants of specificity of Cry toxins. Example hybrid toxins that involve domain III or domain II loops exchange between different Cry toxins with a correlative change on insect

Figure 8.3 Mode of action of Bacillus thuringiensis Cry toxins. Below the toxin receptor interaction are the apparent binding affinities (Kd) and the toxin regions involved in each toxin interaction with the midgut receptors.

specificity have been reported.22 25 In addition, the analysis of the phylogenetic relationships of the isolated domains of members of the 3-Domain Cry family revealed that domain III swapping occurred during evolution of these proteins, suggesting that in vivo recombination has been a strategy for increasing novel specificities.26

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