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Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis - PubMed

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Review

Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis

Sabino Pacheco et al. Front Insect Sci. 2023.

Abstract

Different Bacillus thuringiensis (Bt) strains produce a broad variety of pore-forming toxins (PFTs) that show toxicity against insects and other invertebrates. Some of these insecticidal PFT proteins have been used successfully worldwide to control diverse insect crop pests. There are several studies focused on describing the mechanism of action of these toxins that have helped to improve their performance and to cope with the resistance evolved by different insects against some of these proteins. However, crucial information that is still missing is the structure of pores formed by some of these PFTs, such as the three-domain crystal (Cry) proteins, which are the most commercially used Bt toxins in the biological control of insect pests. In recent years, progress has been made on the identification of the structural changes that certain Bt insecticidal PFT proteins undergo upon membrane insertion. In this review, we describe the models that have been proposed for the membrane insertion of Cry toxins. We also review the recently published structures of the vegetative insecticidal proteins (Vips; e.g. Vip3) and the insecticidal toxin complex (Tc) in the membrane-inserted state. Although different Bt PFTs show different primary sequences, there are some similarities in the three-dimensional structures of Vips and Cry proteins. In addition, all PFTs described here must undergo major structural rearrangements to pass from a soluble form to a membrane-inserted state. It is proposed that, despite their structural differences, all PFTs undergo major structural rearrangements producing an extended α-helix, which plays a fundamental role in perforating their target membrane, resulting in the formation of the membrane pore required for their insecticidal activity.

Keywords: Bacillus thuringiensis; Cry toxin; Tc toxin; Vip3 toxin; pore-forming activity.

Copyright © 2023 Pacheco, Gómez, Peláez-Aguilar, Verduzco-Rosas, García-Suárez, do Nascimento, Rivera-Nájera, Cantón, Soberón and Bravo.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1

Proteolytic activation of the Cry toxin. Deposited 3D structure of a long Cry1Ac protoxin (PDB: 4W8J), a short Cry2Aa protoxin (PDB: 1I5P), and the trypsin-activated Cry1Aa toxin (PDB: 1CIY) were used to generate this figure by using the PyMol program. The trypsin-like proteases present in the insect midgut cleaves the long protoxin of 130 kDa, removing domains IV–VII to form an activated protein of approximately 60 kDa composed of three structural domains resistant to proteolysis. Note that the N-terminal region (approximately 30 residues) of the Cry1Ac protoxin is not shown and is also removed upon activation. In the case of the short 70-kDa protoxin, the N-terminal region (shown in black) is removed by the action of trypsin protease, resulting in a similar activated toxin composed of three structural domains resistant to proteolysis.

Figure 2
Figure 2

“Umbrella” model for Cry toxins. The helical domain I of the Cry protein is reordered to insert the hairpin α-4/α-5 into the cell membrane (dotted line) to form the lytic pore. The hydrophilic face of helix α-4 is facing the pore lumen and the hydrophobic helix α-5 is oriented at the lipid membrane. Helices α-1, α-2, α-3, α-6, and α-7 are partially embedded on the membrane surface, whereas domains II and III remain solvent exposed. A tetrameric conformation was proposed. The cell membrane is indicated by the dotted line. The figure was generated by using the PyMol program.

Figure 3
Figure 3

“Buried dragon” model for Cry toxins. Domain I of the Cry toxin inserts into the cell membrane. Domain II is solvent exposed and domain III is buried. The fate of helix α-1 is unclear in this model and is shown in gray. The cell membrane is indicated by the dotted line. The figure was generated by using the PyMol program.

Figure 4
Figure 4

“Penknife” model. Helices α-1 to α-3 of the Cry3Aa toxin is a flexible region that swings away from the core protein and is proposed to be proteolyzed. The rest of the Cry toxin is inserted into the cell membrane (dotted line) forming a pore with domains II and III oriented to the lumen of the pore and the hydrophobic regions of helices α-4 to α-7 are facing the membrane lipids. The figure was generated by using the PyMol program.

Figure 5
Figure 5

Multistep “folding cane” model. The trypsin-activated Cry toxin binds to membrane receptors, triggering rearrangements at the N-terminal helices of domain I. Helices α-1 to α-3 form a single extended helix that exposes key residues to establish an intermolecular interaction for oligomerization to form a pre-pore. The extended helix requires further conformational changes to form a longer extended helix composed of helices α-1 to α-4 during insertion into the membrane. The region that is proposed to be embedded into the cell membrane (dotted line) corresponds to helices α-1 and α-2a. In agreement with the “umbrella” model, the upper region of the lumen pore is limited by helix α-4, and in this “folding cane” model the pore is along the structure of the extended-helix α-1 to α-4. The long distance between domains II and III and the membrane plane is potentially occupied by protein receptors. The proposed oligomer is composed of three or four Cry toxin subunits. The figure was generated by using the PyMol program.

Figure 6
Figure 6

Cryo-electron microscopy (cryo-EM) structures of pre-pore PDB: 6TFK (A) and pore PDB: 6TFJ (B) of the Vip3 toxin were used to generate this figure by using the PyMol program. The monomer and oligomer of the Vip3 toxin from each conformation are shown. The monomers contain five structural domains, indicated using different colors. Domains are defined according to Núñez-Ramírez et al. (12) and the trypsin cleavage site between helices α-4 and α-5 (A) is shown as a dotted loop. After activation, the N-terminal region forms a long four-helical coiled-coil helix needle at the base of the oligomeric complex that is needed for insertion into the membrane and pore formation. It was proposed that the tip of the extended long helix composed of helices α-1 and α-2 (residues 1–94) is inserted into the membrane to form the pore conformation, but these regions were not resolved in the cryo-EM 3D structure. A model of this region is shown in gray. The long distance between the core of the protein and membrane plane is proposed to be occupied by protein receptors.

Figure 7
Figure 7

Cryo-electron microscopy (cryo-EM) structures of the Tc holotoxin from Photorhabdus luminescens. The tripartite complex comprising TcA (TcdA1), TcB (TcdB2), and TcC (TccC3) from P. luminescens is shown before (pre-pore, (A) and after (pore, (B) membrane insertion. The Tc holotoxin contains five subunits of the TcA component, forming a bell-shaped oligomer, and the TcB–TcC cocoon complex is positioned on the upper region of the TcA oligomer. Note that the linker region is partially structured in the pore conformation (red). The cell membrane is indicated by a dotted line. Figures were prepared from deposited cryo-EM structures of the Tc complex from P. luminescens, PDB:6H6E and 6SUF, by using the PyMol program.

Figure 8
Figure 8

Comparison of the proposed structures of the Cry, Vip3, and Tc toxins when the oligomeric structures of these toxins are inserted into the membrane. The figure was generated by using the PyMol program.

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This work was supported by CONACyT Ciencia de Frontera CF-6693; and DGAPA PAPIIT-IN206721, PAPIIT-IN210722 and PAPIIT-IN202623.

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