Channel Formation in Cry Toxins: An Alphafold-2 Perspective

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 alpha 4, whereas helices alpha 1 to alpha 3 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 angstrom 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 alpha 3 and alpha 2b form a continuous helix and where hydrophobic helices alpha 1 and alpha 2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes alpha 4/alpha 5 hairpin insertion, together with helices alpha 6 and alpha 7. Another Cry1Aa tetrameric model was predicted after deleting helices alpha 1 to alpha 3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix alpha 4. We propose that this second model corresponds to the 'membrane-inserted' structure. AF2 also predicted larger alpha 4/alpha 5 hairpin n-mers (14 <= n <= 17) with high confidence, which formed even larger (similar to 5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms.

Automated summary

This study used Alphafold-2 to predict the structure of Bt toxins and proposed possible oligomeric intermediates in the membrane insertion process. The research provides important clues for revealing the mechanism of action of Bt toxins.