Structural insights into Bacillus thuringiensis Cry, Cyt and parasporin toxins

Abstract

Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively.

Figure 1

The pore formation model of three-domain Cry1A toxin in the midgut lipid rafts. (A) Ribbon diagram of Cry1Aa structure. Three domains are colored in pale green, lemon and bright orange, respectively; (B) Sequential steps of the pore formation model. 1. Solubilized Cry1A is digested by the protease in the alkaline insect midgut; 2. Cry1A binds to the abundant GPI-anchored APN and ALP receptors in the lipid rafts with low affinity. This binding promotes the localization and concentration of the activated toxins; 3. Binding to the cadherin receptor facilitates the proteolytic cleavage of the helix α1 at N-terminal end; 4. and 5. N-terminal cleavage induces the formation of pre-pore oligomer and increases the oligomer binding affinity to GPI-anchored APN and ALP receptors; 6. Oligomer inserts into the membrane, leading to pore-formation and cell lysis.

Figure 2

Structural comparison and sequence alignment of domain I of all structure solved three-domain Cry toxins and the pore-forming domain of colicin A (PDB ID 1COL). (A) Ribbon diagram of domain I of Cry4Aa (top) and the pore-forming domain of colicin A (bottom); (B) Multiple sequence alignment of Cry toxins and colicin A. Secondary structure elements α-helices, 3₁₀-helices and β-strands are denoted in blue as α, η and β, respectively. Figure was prepared by ESPript [33].

Figure 3

Structural comparison and sequence alignment between domain II of three-domain Cry toxins and ZG16p protein (PDB ID 3APA). (A) Ribbon diagram of domain II of Cry4Aa (left) and ZG16p (right); (B) multiple sequence alignment of Cry toxins and ZG16p protein. Secondary structure elements α-helices, 3₁₀-helices and β-strands are denoted in blue and present as α, η and β, respectively. In ZG16p protein, putative sugar-binding involved GG loop, recognition loop, and binding loop are marked in orange, green, and purple, respectively.

Figure 4

Structural comparison and sequence alignment of domain III of all structural known three-domain Cry toxins and CBM6 family member Aga16B-CBM6-2 binding with neoaragohexaose (PDB ID 2CDP). (A) Ribbon diagram of domain III of Cry4Aa (left) and Aga16B-CBM6-2 (right); (B) multiple sequence alignment of Cry toxins and Aga16B-CBM6-2. Secondary structure elements α-helices, 3₁₀-helices and β-strands are denoted in blue as α, η and β, respectively. Strictly conserved and semi-conserved residues of CBM6 family are marked in green and orange triangle, respectively.

Figure 5

Structural comparison and sequence alignment of Cyt toxins and VVA2 toxin (PDB ID IVCY). (A) Ribbon diagram of Cyt1Aa and VVA2 toxins. The overall structure has a single domain of α/β architecture with a central β-sheet surrounded by two α-helical layers. The central β-sheet consists of six antiparallel β-strands with two α-helices on one side and three on the other; (B) multiple sequence alignment of Cyt toxins and VVA2 toxin. Secondary structure elements α-helices, 3₁₀-helices and β-strands are colored in blue and marked as α, η and β, respectively. The identical or semi-conserved hydrophobic residues in all sequences of the alignment are marked at the bottom with black solid circle and open circle, respectively.

Figure 6

Structural comparison among parasporin-2, the nontoxic 26 kDa protein and the aerolysin-type β-PFTs. The membrane binding related N-domain is colored in pale green. Membrane insertion and pore formation regions are colored in lemon and bright orange, respectively [142]. The pink colored amphipathic β-hairpin is suggested to be responsible for pore formation. Parasporin-4 is modeled using the nontoxic 26 kDa protein as template.

Figure 7

Transmembrane model of aerolysin in lipid bilayer (modified according to Ref [159]). Domain I and domain II of aerolysin are colored in orange; domain III in lemon; domain IV in pale green; and the amphipathic loop in pink. According to the aerolysin swirling model, receptor binding and transmembrane steps are as follows: 1. Solubilized aerolysin binds to the GPI-anchored receptors in the lipid rafts at N-terminus. This process facilitates aerolysin monomer a 180° rotation to make its N-terminal end facing the membrane. 2. After C-terminal proteolytic digestion, the activated aerolysin monomers assemble into a heptamer, as well as the N terminus of aerolysin remains binding to receptors during oligomerization. 3. The amphipathic loop of domain II swirls and rearranges into a transmembrane β-barrel.