Protein engineering of Bacillus thuringiensis delta-endotoxin: mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae

Abstract

Substitutions or deletions of domain II loop residues of Bacillus thuringiensis delta-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) approximately 4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (> 21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of 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 affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering delta-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.

Figure 1

Comparison of the yields and stability of wild-type CryIAb and mutant δ-endotoxins on SDS/10% PAGE. (A) Protoxins, (B) trypsin-activated toxins, and (C) Western blot analysis of wild-type and mutant toxins after digestion with gypsy moth gut juice are shown. Masses of the protein markers (in kilodaltons) are shown on the left.

Figure 2

Comparison of CD spectra of thermally denatured wild-type and mutant toxins. Trypsin-activated toxins were subjected to thermal denaturation in the presence of 0.1 M guanidine hydrochloride and scanned on a Spex CD6 spectrophotometer.

Figure 3

Saturation binding of ¹²⁵I-labeled (1 nM) wild-type and mutant toxins. Toxins were labeled as described (8) and were incubated with increasing concentrations of gypsy moth BBMV (2.5 to 200 μg/ml).

Figure 4

Heterologous competition binding of ¹²⁵I-labeled wild-type (1 nM) in the presence of increasing concentrations on nonlabeled CryIAb, N372A, N372G, DF-1, and D3 toxins. Gypsy moth BBMV (200 μg/ml per tube) was used, and each point is an average value of three individual experiments. Binding is expressed as a percentage of the total amount bound upon incubation with labeled toxin.

Figure 5

Dissociation of membrane-bound ¹²⁵I-labeled wild-type and mutant toxins. Gypsy moth BBMV (20 μg per 100 μl) was incubated with 1 nM labeled toxins (association reaction) for 1 h. At the end of association reaction, 100 nM concentration of corresponding nonlabeled toxin was added to the test samples, and incubation (postbinding) was stopped at different time intervals. Nonspecific binding was obtained by incubating the labeled toxin with BBMV in the presence of a 1000-fold excessive corresponding nonlabeled toxin, and the value was subtracted from the total binding. (Inset) BBMV-bound labeled toxins at the end of association binding were resolved on SDS/10% PAGE and autoradiographed. The amounts of DF-1, N372A, CryIAb, and D3 toxins bound to the vesicles were 6050, 2200, 1390, and 321 cpm, respectively.

Figure 6

Binding of ¹²⁵I-labeled toxins to protein blots of gypsy moth BBMV proteins. A total of 40 μg of BBMV proteins was blotted and probed with 3 nM of labeled toxins.

Figure 7

Inhibition of I_sc across gypsy moth midgut by wild-type and mutant toxins. A total of 6.5 ng of toxin per ml was injected into the lumen side of the chamber in separate experiments, and the drop in I_sc was measured. The I_sc measured before the addition of the toxin was considered as 100%. Each point is an average of three independent experiments.