Cry5Ba is a delta-endotoxin produced by Bacillus thuringiensis PS86A1 NRRL B-18900. It is active against nematodes and has great potential for nematode control. Here, we predict the first theoretical model of the three-dimensional (3D) structure of a Cry5Ba toxin by homology modeling on the structure of the Cry1Aa toxin, which is specific to Lepidopteran insects. Cry5Ba resembles the previously reported Cry1Aa toxin structure in that they share a common 3D structure with three domains, but there are some distinctions, with the main differences being located in the loops of domain I. Cry5Ba exhibits a changeable extending conformation structure, and this special structure may also be involved in pore-forming and specificity determination. A fuller understanding of the 3D structure will be helpful in the design of mutagenesis experiments aimed at improving toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.
The β20-β21 loop is a unique structure in the domain III of Bacillus thuringiensis Cry proteins. In this study, the role of β20-β21 loop on insecticidal activity of Cry1Ac toxin was investigated. 10 residues in β20-β21 loop were substituted with alanine using PCR-based site-directed mutagenesis. All mutants were capable of producing diamond-shaped crystal and expressing a protein sized 130 kDa. The mutants S581A and I585A enhanced toxicity against Helicoverpa armigera larvae dramatically, while most of the rest mutants possess a reduced toxicity at different degrees. Indoor bioassay result revealed that mutants S581A and I585A had a 1.72- and 1.89-fold increasing in toxicity against Helicoverpa armigera larvae compared with the wild-type strain, respectively; On the contrary, G583A experienced a significant reduced insecticidal activity. Three-dimensional analysis of Cry1Ac5 protein demonstrated that the side chain of residues T579, S580, L582, and I585 extended to the surface of the protein, and might participate in the interaction between the protein and its receptor, whereas side chain of residues N576, F578, S581, N584, and V586 preferred the inside of the protein, and which might be critical to the stability of the protein structure. Our study for the first time clarified the special properties and the functions of the β20-β21 loop in domain III of Cry1Ac5. These findings also provided the latest biological evidence for the recognition and binding mechanism of the domain III in Cry1Ac, and its role in maintaining the structure stability of Cry1Ac.
Cry5Aa is a crystal protein produced by Bacillus thuringiensis serovar. damstadiensis during its stationary phase, this delta-endotoxin is active against nematodes and has great potential for nematodes control. The theoretical model of the three-dimensional structure of Cry5Aa was predicted by homology modeling on the structures of the Cry1Aa which is specific to Lepidopteran insects. The structure of the Cry5Aa resembles previously reported Cry toxin structures but shows the following distinctions. Cry5Aa has a long insertion in alpha2 of domain I. Some loops in the domain II and III of Cry5Aa are exposed to the solvent. In this work we give a brief description of our model and hypothesize the residues of the Cry5Aa that could be important in receptor recognition and pore formation. This model will be helpful for the design of mutagenesis experiments aimed to the improvement of toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.
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