Single amino acid changes in domain II of Bacillus thuringiensis CryIAb delta-endotoxin affect irreversible binding to Manduca sexta midgut membrane vesicles.
Deletion of amino acid residues 370 to 375 (D2) and single alanine substitutions between residues 371 and 375 (FNIGI) of lepidopteran-active Bacillus thuringiensis CryIAb delta-endotoxin were constructed by site-directed mutagenesis techniques. All mutants, except that with the I-to-A change at position 373 (I373A), produced delta-endotoxin as CryIAb and were stable upon activation either by Manduca sexta gut enzymes or by trypsin. Mutants D2, F371A, and G374A lost most of the toxicity (400 times less) for M. sexta larvae, whereas N372A and I375A were only 2 times less toxic than CryIAb. The results of homologous and heterologous competition binding assays to M. sexta midgut brush border membrane vesicles (BBMV) revealed that the binding curves for all mutant toxins were similar to those for the wild-type toxin. However, a significant difference in irreversible binding was observed between the toxic (CryIAb, N372A, and I375A) and less-toxic (D2, F371A, and G374A) proteins. Only 20 to 25% of bound, radiolabeled CryIAb, N372A, and I375A toxins was dissociated from BBMV, whereas about 50 to 55% of the less-toxic mutants, D2, F371A, and G374A, was dissociated from their binding sites by the addition of excess nonlabeled ligand. Voltage clamping experiments provided further evidence that the insecticidal property (inhibition of short-circuit current across the M. sexta midgut) was directly correlated to irreversible interaction of the toxin with the BBMV. We have also shown that CryIAb and mutant toxins recognize 210- and 120-kDa peptides in ligand blotting. Our results imply that mutations in residues 370 to 375 of domain II of CrylAb do not affect overall binding but do affect the irreversible association of the toxin to the midgut columnar epithelial cells of M. sexta.
Rajamohan
F
F
Department of Biochemistry, Ohio State University, Columbus 43210, USA.
Alcantara
E
E
Lee
M K
MK
Chen
X J
XJ
Curtiss
A
A
Dean
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D004252:DNA Mutational Analysis; D004064:Digestive System / Q000187:drug effects / Q000378:metabolism*; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006460:Hemolysin Proteins; D007814:Larva / Q000378:metabolism; D018402:Manduca / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010572:Pest Control, Biological; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis