PROTEIN ENGINEERING OF BACILLUS-THURINGIENSIS DELTA-ENDOTOXIN - MUTATIONS AT DOMAIN-II OF CRYIAB ENHANCE RECEPTOR AFFINITY AND TOXICITY TOWARD GYPSY-MOTH LARVAE

Substitutions or deletions of domain II loop residues of Bacillus thur ingiensis delta-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in recept or binding and toxicity toward gypsy moth (Lymantria dispar). Substitu tion of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased t he toxicity against gypsy moth larvae 8-fold and enhanced binding affi nity to gypsy moth midgut brush border membrane vesicles (BBMV) approx imate to 4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (>21 times) as well as binding affinity suggesting that resi due N372 is involved in receptor binding, Interestingly, a triple muta nt, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced act ivity of DF-1 was correlated with higher binding affinity (18-fold) an d binding site concentrations, Dissociation binding assays suggested t hat the off-rate of the BBMV-bound mutant toxins was similar to that o f the wild type. However, DF-1 toxin bound 4 times more than the wild- type and N372A toxins. and it was directly correlated with binding aff inity and potency. Protein blots of gypsy moth BBMV probed with labele d N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused b y binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops ma). f it the toxin more efficiently to the binding pockets, These results of fer an excellent model system for engineering delta-endotoxins with hi gher potency and wider spectra of target pests by improving receptor b inding interactions.