title: Mutations at domain II, loop 3, of Bacillus thuringiensis CryIAa and CryIAb delta-endotoxins suggest loop 3 is involved in initial binding to lepidopteran midguts.
abstract: Alanine substitutions of loop 3 residues, 438SGFSNS443, of CryIAb toxin were constructed to study the functional role of these residues in receptor binding and toxicity to Manduca sexta and Heliothis virescens. Experiments with trypsin and insect gut juice enzyme digestions of mutant toxins showed that these mutations did not produce any gross structural changes to the toxin molecule. Bioassay data showed that mutant G439A (alanine substitution of residue Gly439) and F440A significantly reduced toxicity toward M. sexta and H. virescens. In contrast, mutants S438A, S441A, N442A, and S443A were similar or only marginally less toxic (2-3 times) to the insects compared to the wild-type toxin. Binding studies with brush border membrane vesicles prepared from M. sexta and H. virescens midgut membranes revealed that the loss of toxicity of mutants G439A and F440A was attributable to substantially reduced initial binding. Consistent with the initial binding, mutants G349A and F440A showed 3.5 times less binding to M. sexta and H. virescens brush border membrane vesicles, although the off-rate of bound toxins was not affected. The role of hydrophobic residue, Phe440, is distinctly different from our previous observation that alanine substitution of Phe371 at loop 2 of CryIAb did not affect initial binding but reduced irreversible association of the toxin to the receptor or membrane toward M. sexta (Rajamohan, F., Alcantara, E., Lee, M. K., Chen, X. J., and Dean, D. H. (1995) J. Bacteriol. 177, 2276-2282). Likewise, deletion of relatively hydrophobic CryIAa loop 3 residues, 440AAGA443 (D3a), resulted in reduced toxicity to Bombyx mori (>62 times less) and M. sexta (28 times less). The loss of toxicity was correlated with reduced initial binding to midgut vesicles prepared from these insects. However, alanine substitution of residues 437LSQ439 (A3a), contiguous to loop 3, altered neither toxicity nor receptor binding toward B. mori or M. sexta. These results suggest that the loop 3 residues of CryIAb and CryIAa toxins establish hydrophobic interactions with the receptor molecule, and mutations at these hydrophobic residues affect initial binding.
authors.affiliation: Department of Biochemistry, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: F
authors.initials: F
authors.lastname: Rajamohan
authors.forename: S R
authors.initials: SR
authors.lastname: Hussain
authors.forename: J A
authors.initials: JA
authors.lastname: Cotrill
authors.forename: F
authors.initials: F
authors.lastname: Gould
authors.forename: D H
authors.initials: DH
authors.lastname: Dean
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity*; D001427:Bacterial Toxins; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004064:Digestive System / Q000378:metabolism*; D004731:Endotoxins / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity*; D004926:Escherichia coli; D005260:Female; D006460:Hemolysin Proteins; D019476:Insect Proteins; D007700:Kinetics; D007814:Larva; D018402:Manduca; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010572:Pest Control, Biological; D010649:Phenylalanine; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C095416:Cry toxin receptors; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010649:Phenylalanine