Mutations in domain I interhelical loops affect the rate of pore formation by the Bacillus thuringiensis Cry1Aa toxin in insect midgut brush border membrane vesicles

Abstract

Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues, caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C, T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.

FIG. 1.

Positions of the mutated residues within domain I of Cry1Aa. The three-dimensional rendering of the activated toxin crystal structure (26) was obtained using SPDV software (27). Each of the indicated residues was replaced individually by a cysteine as described in Materials and Methods. The α2-α3, α4-α5, and α6-α7 loops on the lower side of the illustration are most likely to contact the membrane first since they are located on the same side of the toxin molecule as the domain II loops that have been shown to bind to the receptors (47, 51).

FIG. 2.

Effect of interhelical loop cysteine mutants of Cry1Aa on the osmotic swelling of M. sexta brush border membrane vesicles. Midgut membrane vesicles isolated from fifth-instar M. sexta larvae and equilibrated overnight in 10 mM HEPES-KOH (pH 7.5) were preincubated for 60 min with the indicated concentrations (in pmol toxin/mg membrane protein) of the T122C (A) or V150C (B) mutant. Vesicles were rapidly mixed with an equal volume of 150 mM KCl-10 mM HEPES-KOH (pH 7.5), directly in a cuvette, using a stopped-flow apparatus. Scattered-light intensity was monitored at 90° in a PTI spectrofluorometer. Each trace corresponds to the average data for five experiments performed with the same representative vesicle preparation.

FIG. 3.

Pore-forming ability of domain I interhelical loop mutants. Brush border membrane vesicles equilibrated overnight in 10 mM HEPES-KOH (pH 7.5) (▪) or Caps-KOH (pH 10.5) (•) were preincubated for 60 min with the indicated concentrations of Cry1Aa (A) or the F50C (B), I88C (C), P121C (D), T122C (E), N123C (F), V150C (G), Q151C (H), Y153C (I), W182C (J), W219C (K), or S252C (L) mutant. Their permeability to KCl was monitored following rapid mixing with 150 mM KCl and 10 mM HEPES-KOH (pH 7.5) (▪) or Caps-KOH (pH 10.5) (•). Percent volume recovery was calculated for the 3-s time point from traces like those shown in Fig. 2. Asterisks indicate a statistically significant difference, with the corresponding value measured at the same pH for wild-type Cry1Aa (*, P < 0.05; **, P < 0.01).

FIG. 4.

Effect of Cry1Aa domain I interhelical loop mutants on vesicle permeability to N-methyl-d-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose. Vesicles equilibrated in 10 mM HEPES-KOH (pH 7.5) (open bars) or Caps-KOH (pH 10.5) (hatched bars) were preincubated for 60 min with 150 pmol toxin/mg membrane protein. Their permeability was monitored following rapid mixing with either 150 mM N-methyl-d-glucamine hydrochloride (A) or potassium gluconate (B) or 300 mM sucrose (C) or raffinose (D) and with 10 mM HEPES-KOH (pH 7.5) (open bars) or Caps-KOH (pH 10.5) (hatched bars), as described in the legend of Fig. 2. Asterisks indicate statistically significant differences, with the corresponding values measured at the same pH for Cry1Aa (*, P < 0.05; **, P < 0.01).

FIG. 5.

Kinetics of pore formation. Brush border membrane vesicles equilibrated overnight in 10 mM HEPES-KOH (pH 7.5) or Caps-KOH (pH 10.5) were mixed with an equal volume of 150 mM KCl, 10 mM HEPES-KOH (pH 7.5) or Caps-KOH (pH 10.5), and 150 pmol of I88C/mg membrane protein. Percent volume recovery was calculated for each experimental point, and control values were subtracted from those obtained in the presence of toxin. For clarity, error bars are shown for only every 50th data point.