The role of β18–β19 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis

N546 in β18–β19 loop is important for binding and toxicity of the Bacillus thuringiensis Cry1Ac toxin

“…In our previous studies (Xia et al, 2008), N546 is located in the apex of b18-b19 loop of Cry1Ac5 crystal structure, and this loop is just located on the upper lip of a cavity mouth which recognized the GalNAc. Six mutants were generated using QuikChange site-directed mutagenesis kit, and in which, N546A showed almost two times increased activity than wild-type Cry1Ac toxin, mutant N546G had similar insecticidal activity to wild-type Cry1Ac, and mutant N546D, N546K and N546D showed a great loss in toxicity against H. armigera (Table 1).…”

“…Six mutants were generated using QuikChange site-directed mutagenesis kit, and in which, N546A showed almost two times increased activity than wild-type Cry1Ac toxin, mutant N546G had similar insecticidal activity to wild-type Cry1Ac, and mutant N546D, N546K and N546D showed a great loss in toxicity against H. armigera (Table 1). Since the side-chain of N546 is oriented towards the solvent, in the surface of the protein, we proposed this residue played a putative role in interaction to other macromolecules, but we cannot conclude well because additional experiments are needed to verify definitively the possible effects of these N546 mutations (Xia et al, 2008). In the current study, attempts were therefore made to determine the mechanism of toxicity change of these mutants.…”

“…N546 mutants were generated and expressed in our previous studies, the purification of crystals from Cry1Ac and its mutant toxins were performed as our early description (Xia et al, 2008). The protoxin was further purified by isoelectric precipitation, the crystals were solubilized in 50 mM Na 2 CO 3 /NaHCO 3 buffer containing 10 mM dithiothreitol, pH 9.5, at 37°C for 4 h, then adjusting pH to 5.5 with 2 M sodium acetate buffer solution pH (4.0), The purified protoxin was harvested by centrifugation for 10 min at 14,000g after stored at 4°C overnight.…”

“…But few groups paid attentions to the roles of asparagine residues in domain III. In our early studies (Xia et al, 2008), we produced a homology-based Cry1Ac5 model and analyzed the unique N546 by site-directed mutagenesis. We found the insecticidal activities of N546 mutants were severely affected, and which we explained as a conformational change or a consequence of differences in protein contacting to receptor macromolecules.…”

Evidence of the Involvement of E358, A498 and C571 of a New Cry1Ac δ-endotoxin of Bacillus thuringiensis in its High Insecticidal Activity Against Ephestia kuehniella

“…In fact, it was helpful to understand the mode of action of these toxins, sharing a high structural similarity and to predict the structure of other toxins. On the other hand, the protein modelling is very useful to study the structural effect of some mutations involved in the toxicity of Cry proteins [14] or in their binding to insect brush border membrane vesicles (BBMV) [15].…”