title: The role of β20-β21 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis.
abstract: 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.
authors.affiliation: Key Lab of Microbial Molecular Biology of Hunan Province, College of Life Science, Hunan Normal University, Changsha, 410081, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Lv
authors.forename: Ying
authors.initials: Y
authors.lastname: Tang
authors.forename: Yunlei
authors.initials: Y
authors.lastname: Zhang
authors.forename: Liqiu
authors.initials: L
authors.lastname: Xia
authors.forename: Faxiang
authors.initials: F
authors.lastname: Wang
authors.forename: Xuezhi
authors.initials: X
authors.lastname: Ding
authors.forename: Siming
authors.initials: S
authors.lastname: Yi
authors.forename: Wenping
authors.initials: W
authors.lastname: Li
authors.forename: Jia
authors.initials: J
authors.lastname: Yin
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001413:Bacillus thuringiensis / Q000472:pathogenicity*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D003460:Crystallization; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D007306:Insecticides / Q000737:chemistry* / Q000633:toxicity*; D007814:Larva / Q000187:drug effects; D007915:Lepidoptera / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D016019:Survival Analysis
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D050505:Mutant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis