Vegetative insecticidal proteins (Vip) from the entomopathogenic bacterium Bacillus thuringiensis are employed in pest management strategies due to their high specificity against lepidopteran insects. Once ingested by larvae, these proteins are processed by midgut proteases and reach the midgut epithelium, where they bind specifically and exert toxicity through pore formation.
Among the Vip proteins, Vip3Aa stands out as one of the most potent and commercially significant variants. CryoEM structural analysis revealed it forms a tetramer, with each monomer comprising five distinct structural domains. Furthermore, two conformational states were identified: the protoxin, with a pyramidal shape, and the activated form, in which the N-terminal Domain I, after proteolysis, rearranges into a coiled-coil that inserts into lipid membranes.
This presentation examines Vip3Aa’s mode of action from a structure-function perspective, highlighting how its conformational states and structural domains contribute to different steps of its toxic mechanism. We investigated the effects of point mutations and truncations on structure using NS-EM, assessed in vivo stability via proteolytic assays, analysed specific binding to midgut cells using radiolabelled protein binding assays, and evaluated functional binding through toxicity assays in the lepidopteran species Spodoptera exigua.
Key structural features were found to be critical for Vip3Aa's function: at the N-terminus, Domain I is vital for structural integrity, stability, and toxicity. Its conformational change is essential for the insecticidal activity, while the helix α1 within this domain contributes to protoxin state stabilization and facilitates membrane insertion and toxicity in the activated state. Both structural conformations can bind to functional receptors, with Domain III mediating specific interactions. While at the C-terminus, Domains IV and V are not necessary for specific binding but are required for effective functional receptor recognition and toxicity.
This study deepens our understanding of Vip3Aa’s structure-function relationship and provides useful insights for its improved biotechnological application.