Mutations in domain I of Bacillus thuringiensis delta-endotoxin CryIAb reduce the irreversible binding of toxin to manduca sexta brush border membrane vesicles.
Site-directed mutagenesis was used to generate CryIAb mutants at the selected N-terminal positions to study the function of domain I. Structurally stable mutant proteins were tested for toxicity, receptor binding kinetics, and pore function. Substitutions of tyrosine at position 153 with arginine (Y153R) or alanine (Y153A) did not affect toxicity appreciably, whereas replacing this tyrosine with aspartic acid (Y153D) resulted in a great loss of toxicity. Mutation of alanine at position 92 to glutamic acid (A92E) almost completely abolished toxicity. The initial receptor binding was unchanged as measured by competition binding assays among all mutant proteins. Reduced pore function, however, was observed for mutants A92E and Y153D as tested by voltage clamping. Further studies with specially designed association and dissociation binding assays showed that irreversible binding of these two mutant toxins to Manduca sexta brush border membrane vesicles was significantly reduced. The decrease in irreversible binding was correlated with the changes in toxicity and may reflect a severely disturbed membrane insertion process in these two mutant toxins, leading to reduced pore function and toxicity. The results support the model that domain I is involved in membrane integration and pore formation.
Chen
X J
XJ
Department of Biochemistry, Ohio State University, Columbus 43210.
Curtiss
A
A
Alcantara
E
E
Dean
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000737:chemistry; D001483:Base Sequence; D004064:Digestive System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006460:Hemolysin Proteins; D015151:Immunoblotting; D007700:Kinetics; D007814:Larva; D018402:Manduca; D008871:Microvilli / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011485:Protein Binding; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000083722:Bacillus thuringiensis Toxins; C029103:Bacillus thuringiensis protoxin; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis