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Channel Formation in Cry Toxins: An Alphafold-2 Perspective - PubMed

  • ️Sun Jan 01 2023

Channel Formation in Cry Toxins: An Alphafold-2 Perspective

Jaume Torres et al. Int J Mol Sci. 2023.

Abstract

Bacillus thuringiensis (Bt) strains produce pore-forming toxins (PFTs) that attack insect pests. Information for pre-pore and pore structures of some of these Bt toxins is available. However, for the three-domain (I-III) crystal (Cry) toxins, the most used Bt toxins in pest control, this crucial information is still missing. In these Cry toxins, biochemical data have shown that 7-helix domain I is involved in insertion in membranes, oligomerization and formation of a channel lined mainly by helix α4, whereas helices α1 to α3 seem to have a dynamic role during insertion. In the case of Cry1Aa, toxic against Manduca sexta larvae, a tetrameric oligomer seems to precede membrane insertion. Given the experimental difficulty in the elucidation of the membrane insertion steps, we used Alphafold-2 (AF2) to shed light on possible oligomeric structural intermediates in the membrane insertion of this toxin. AF2 very accurately (<1 Å RMSD) predicted the crystal monomeric and trimeric structures of Cry1Aa and Cry4Ba. The prediction of a tetramer of Cry1Aa, but not Cry4Ba, produced an 'extended model' where domain I helices α3 and α2b form a continuous helix and where hydrophobic helices α1 and α2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes α4/α5 hairpin insertion, together with helices α6 and α7. Another Cry1Aa tetrameric model was predicted after deleting helices α1 to α3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix α4. We propose that this second model corresponds to the 'membrane-inserted' structure. AF2 also predicted larger α4/α5 hairpin n-mers (14 ≤n ≤ 17) with high confidence, which formed even larger (~5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms.

Keywords: Alphafold; Cry toxins; pore formation.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1

Comparison between experimental and AF2-predicted structures: (A) Comparison of experimental and AF2-predicted α-helical stretches (helices α1 to α7) in domain I for Cry1Aa and Cry4Ba. Each α-helical stretch has been color-coded in this and following figures for easy visualization. The sequence numbering shown at the bottom corresponds to the Cry4Ba sequence, whereas in Cry1Aa, the position of the α-helical segments were aligned with those of Cry4Ba, e.g., helix α3 spans residues 106–136 in Cry4Ba, but residues 90–120 in Cry1Aa; (B,C) overlay of experimental (cylinders) and AF2 structures (ribbon) of trimeric domain I in Cry4Ba (top and side views) (B) and for monomeric domain I of Cry1Aa (C). A trimeric model for domain I of Cry1Aa is shown in Figure 2A; (D) Cry4Ba monomer including domains I-III (cyan: experimental; red: AF2); (E) same as (D) without domain I, removed for clarity. The structures were fitted using Matchmaker in Chimera X [67].

Figure 2
Figure 2

AF2−predicted Cry1Aa conformational change in a transition trimer-to-tetramer: (A) AF2-predicted trimeric structure of Cry1Aa domain I, and proposed movement of helices α1 and α2 away from the bundle. Helices α6 and α7 have been removed for clarity. HOLE profile is shown above; (B) AF2−predicted tetrameric ‘extended’ structure, where α3 forms a continuous α-helix with α2b and where α1 and α2a converge at the N-terminal tip of the oligomer that targets the host membrane. The central cavity is lined by helix α4 residues (cyan) away from the membrane and by helix α3 (yellow) closer to the membrane with residues Gln95 and Arg99. A view from the membrane-facing end shows that residues form a hydrophobic patch around the pore (gold), whereas, from the other end, residues from α4 are polar (cobalt blue). Plots corresponding to pLDDT and PAE for this tetrameric model are shown in Supplementary Figure S5; (C) HOLE profile of the model shown in (B); (D,E) top view of the main salt bridges in the trimeric model in (A) (helical fragments not involved in salt bridges have been removed for clarity), with salt bridge Arg93(α3)−Glu81(α2b) (D), and critical α4 residues (Arg131 and Glu128) involved in intermonomer contacts (E). Helix α3 has salt bridges via Glu112 with helix α6; (F,G) same as (D,E) for the tetrameric model in (B); a continuous helix α3-α2b is stabilized by salt bridges between Glu81, Arg87 and Glu90 (F), whereas Glu128 and Arg131 in α4 still stabilize weaker intermonomer contacts (G); (H) detail of salt bridges in the crystal structure of the Cry1Aa domain I monomer (PDB: 1CIY) in the α2b/α3 region.

Figure 3
Figure 3

Tetrameric ‘membrane-inserted’ model of Cry1Aa domain I: (A–C) AF2-predicted tetrameric structure of domain I: side (A), top (B) and HOLE profile view (C); (A) helices 6 and 7 of one monomer were deleted for clarity; (D,E) helix α4 residues lining the channel in Cry1Aa (D) and Cry28Aa (E) in two opposed helices. Two monomers have been removed for clarity.

Figure 4
Figure 4

Structure of Cry1Aa AF2-predicted larger oligomer: (A–C) Predicted pore formed by 14 α4–α5 hairpins colored according to pLDDT in side (A), bottom (B) and top (C) views; (D–F) same as (A–C) for the 14-mer depicted as surface electric charge. The helix lining the pore is α4, and polar residues facing the pore are indicated. (F) Half of the pore was deleted for clarity. The pLDDT and PAE plots for the 14-mer are shown in Supplementary Figure S8; (G,H) predicted side view of pore formed by 16 α4–α5 hairpins colored according to pLDDT (G); top view (H) where helices α4 and α5 are labeled cyan and red, respectively.

Figure 5
Figure 5

Proposed mechanism of insertion of Cry toxins: (A–C) The toxin is in a stable trimeric state in solution (A), and it may form tetramers after binding a receptor or the membrane. An unknown trigger straightens helix α3 towards α2b, forming an ‘extended’ model where hydrophobic helices α1 and α2a bind the membrane (B); the helix between α3 and α2b reverses its straight conformation and bends again, and the hairpin between helices α4 and α5 inserts into the membrane together with α6 and α7 producing the ‘membrane-inserted’ tetramer (D). The latter two helices are less hydrophobic and ‘float’ towards the membrane surface (orange arrows); the α4–α5 hairpin forms a tetrameric channel (E), and it may be enlarged by the addition of further hairpins, allowing translocation of toxin monomers to the host cytoplasm. (E) Helices α3 to α1 were deleted for clarity. HOLE profiles (dark blue) and minimum radius (r) are shown for the trimeric form (A), tetrameric ‘extended’ model (B) and tetrameric ‘membrane-inserted’ model (D,E).

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References

    1. Pardo-López L., Soberón M., Bravo A. Bacillus thuringiensis insecticidal three-domain Cry toxins: Mode of action, insect resistance and consequences for crop protection. FEMS Microbiol. Rev. 2013;37:3–22. doi: 10.1111/j.1574-6976.2012.00341.x. - DOI - PubMed
    1. Sanahuja G., Banakar R., Twyman R.M., Capell T., Christou P. Bacillus thuringiensis: A century of research, development and commercial applications. Plant Biotechnol. J. 2011;9:283–300. doi: 10.1111/j.1467-7652.2011.00595.x. - DOI - PubMed
    1. Gould E., Solomon T. Pathogenic flaviviruses. Lancet. 2008;371:500–509. doi: 10.1016/S0140-6736(08)60238-X. - DOI - PubMed
    1. Pierson T.C., Diamond M.S. The continued threat of emerging flaviviruses. Nat. Microbiol. 2020;5:796–812. doi: 10.1038/s41564-020-0714-0. - DOI - PMC - PubMed
    1. Crickmore N., Berry C., Panneerselvam S., Mishra R., Connor T.R., Bonning B.C. A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins. J. Invertebr. Pathol. 2021;186:107438. doi: 10.1016/j.jip.2020.107438. - DOI - PubMed

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