The Vip3Ag4 insecticidal protoxin from Bacillus thuringiensis adopts a tetrameric configuration that is maintained on proteolysis

The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus 27 thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that 28 may involve pore formation and apoptosis. These proteins are promising supplements to our 29 arsenal of insecticidal proteins but the molecular details of their activity are not understood. As a 30 first step in the structural characterisation of these proteins we have analysed their secondary 31 structure and resolved the surface topology of a tetrameric complex of the Vip3Ag4 protein by 32 transmission electron microscopy. Sites sensitive to proteolysis by trypsin are identified and the 33 trypsin-cleaved protein appears to retain a similar structure as an octomeric complex comprising 34 four copies each of the ~65 kDa and ~21 kDa products of proteolysis. This processed form of the 35 toxin may represent the active toxin. The quality and monodispersity of the protein produced in 36 this study make Vip3Ag4 a candidate for more detailed structural analysis using cryo-electron 37 microscopy