Functional significance of loops in the receptor binding domain of Bacillus thuringiensis CryIIIA delta-endotoxin.

Analysis of the three surface loops in domain II of Bacillus thuringiensis CryIIIA delta-endotoxin has been carried out to assess their role in receptor binding and toxicity. Site-directed mutagenesis was used to convert loop residues to alanine and the mutant proteins were analyzed for structural stability, toxicity to beetle larvae (Tenebrio molitor), binding to receptors on T. molitor brush border membrane vesicles (Tm-BBMV) and insertion into BBMV, as measured by irreversible membrane receptor binding. This study demonstrates the functional significance of loops for binding and insertion. Alanine replacements in loop I resulted in disruption of receptor binding or structural instability. The double mutation, Y350A, Y351A, could be suppressed by replacing a nearby R345 with alanine, and the resultant mutant protein also showed reduced receptor binding. Substitution of N353 and D354 in loop I with alanine residues caused the loss of binding ability and toxicity. A loop II double mutant, P412A, S413A, had no effect on binding or toxicity. A block mutation of loop III residues to alanine had the effect of reducing receptor binding while concomitantly increasing toxicity by 2.4-fold. We compared this up-mutant to wild-type toxin in each step of physiological processing of protoxin: solubility, proteolytic activation, and insertion into the Tm-BBMV. The loop III block mutant showed increased membrane insertion, but was similar to wild-type toxin in other parameters. These results reveal that loop I and loop III in domain II of CryIIIA delta-endotoxin are involved in receptor binding. In addition, the direct correlation between toxicity and irreversible binding of the loop III block mutant (despite the indirect relationship to reversible binding) suggests that loop III may play a role in membrane insertion.