Structural basis for the activation of the Vip3Aa toxin

1 p.Bacillus thuringiensis (Bt) synthetizes potent insecticidal proteins commonly used in biotech and conventional agriculture. The members of the Vip3 family are synthesized in an inactive state, as protoxins, and they require to be activated by proteases present in the midgut tract of the insect. In particular, trypsin-like enzymes cleave the molecule at residue 198, generating two fragments of around 19 and 65 kDa that remain strongly associated. The activated toxin then recognizes specific receptors in the brush border of columnar cells and triggers cell death. Notably, although the first member of the Vip3 family was discovered more than 20 years ago,numerous questions persist regarding the three-dimensional organization, activation and mode of action of these proteins at molecular level. To shed light on some of these questions, we have determined the 2.9 Å resolution structures of Vip3Aa in the protoxin and toxin states by cryoelectron microscopy. The reconstruction of the uncleaved protein shows that it assembles into a highly stable tetrameric dimer of dimers that contains three putative sugar-binding domains poised for receptor engagement. Interestingly, moreover, the trypsin-activated structure reveals that, upon protease digestion, the N-terminal region of the toxin undergoes a large conformational change that leads to the formation of an extended four-helix coiled coil. Collectively, these results provide a high-resolution view of a Vip3 member in the protoxin and toxin conformation that serve as a strong foundation for the development of more efficient insecticidal proteins, and allows us to propose an activation mechanism for this family of entomopathogenic proteins akin to that of other toxins and viral fusion proteins.Peer reviewe