The Vip3Ag4 Insecticidal Protoxin from Bacillus thuringiensis Adopts A Tetrameric Configuration That Is Maintained on Proteolysis

Abstract

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

Structural insights into the insecticidal Vip3A toxin ofBacillus thuringiensis

“…There are over 100 known proteins of the Vip3 family. Based on their high degree of sequence conservation and previous studies 9, 20, 26, 27 , they are very likely to share similar overall structures and domain compositions.…”

“…AY489126.1) in this study. Full-length Vip3Aa11 consists of 789 amino acids, which has been demonstrated to be digested between residues K198 and D199 by insect midgut juice 20, 26, 27 . Of the two resulting fragments, the C-terminal fragment (D199 to end) is considered to be the toxic core of Vip3A.…”

“…One of the main reasons for this is the lack of a high-resolution three-dimensional structure, which significantly impedes detailed molecular level functional and mechanistic studies and thus limits the development of their insecticidal potential. Accordingly, many groups have made great efforts to obtain or predict the atomic structure of Vip3A 21, 27, 28, 32, 33 . However, high-resolution crystal structure of Vip3 toxin family is still missing.…”

“…The scavenger receptor class C like protein (Sf-SR-C) and the fibroblast growth factor receptor (Fgfr) have been reported as potential receptors for Vip3A 13, 14 . However, although some in silico modeling efforts and low resolution cryo-EM structures have attempted to obtain structural insight for these toxins, the atomic structure of Vip3A is still not available, which makes it difficult to reveal the relationship between their structure and function 27, 28, 32, 33 .…”

Development of a sensitive monoclonal antibody-based sandwich ELISA to detect Vip3Aa in genetically modified crops

“…The vip3Aa20 gene was amplified from GM maize MIR162 genomic DNA and subcloned into the pET28a plasmid. pET28a-vip3Aa1/19 and pET28a-vip3Aa7/10 were generated by site-directed mutagenesis (Palma et al 2017). The vip3Aa14 and vip3Aa8 genes were chemically synthesized and subcloned into the pET28a plasmid.…”