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 s elected N-terminal positions to study the function of domain I. Struct urally stable mutant proteins were tested for toxicity, receptor bindi ng kinetics, and pore function. Substitutions of tyrosine at position 153 with arginine (Y153R) or alanine (Y153A) did not affect toxicity a ppreciably, 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 bind ing 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 asso ciation and dissociation binding assays showed that irreversible binding of these two mutant t oxins to Manduca sexta brush border membrane vesicles was significantl y reduced. The decrease in irreversible binding was correlated with th e changes in toxicity and may reflect a severely disturbed membrane in sertion process in these two mutant toxins, leading to reduced pore fu nction and toxicity, The results support the model that domain I is in volved in membrane integration and pore formation.