Investigating the properties of Bacillus thuringiensis Cry proteins with novel loop replacements created using combinatorial molecular biology

Abstract

Cry proteins are a large family of crystalline toxins produced by Bacillus thuringiensis. Individually, the family members are highly specific, but collectively, they target a diverse range of insects and nematodes. Domain II of the toxins is important for target specificity, and three loops at its apex have been studied extensively. There is considerable interest in determining whether modifications in this region may lead to toxins with novel specificity or potency. In this work, we studied the effect of loop substitution on toxin stability and specificity. For this purpose, sequences derived from antibody complementarity-determining regions (CDR) were used to replace native domain II apical loops to create "Crybodies." Each apical loop was substituted either individually or in combination with a library of third heavy-chain CDR (CDR-H3) sequences to create seven distinct Crybody types. An analysis of variants from each library indicated that the Cry1Aa framework can tolerate considerable sequence diversity at all loop positions but that some sequence combinations negatively affect structural stability and protease sensitivity. CDR-H3 substitution showed that loop position was an important determinant of insect toxicity: loop 2 was essential for activity, whereas the effects of substitutions at loop 1 and loop 3 were sequence dependent. Unexpectedly, differences in toxicity did not correlate with binding to cadherins--a major class of toxin receptors--since all Crybodies retained binding specificity. Collectively, these results serve to better define the role of the domain II apical loops as determinants of specificity and establish guidelines for their modification.

FIG. 1.

The structure of Cry1Aa (30) (PDB code 1CIY) highlighting the three domains of the active toxin (a) and the three apical loops of domain II (b). The molecular graphic images were produced using the UCSF Chimera package (60) from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081).

FIG. 2.

Crybody libraries and their construction. (a) Schematic representation of the seven Crybody libraries derived from cry1Aa13 and a human scFv antibody library. Sequences encoding loop 1, loop 2, and loop 3 of cry1Aa13 are shown in blue, green, and yellow, respectively. CDR-H3 sequences are shown in red. The three domains encoded by cry1Aa13 are labeled I, II, and III. V_L and V_H indicate the antibody domains. (b) Sequences of Crybodies with CDR-H3 insertions at site 1, 2, or 3. Adaptor codons are shown in bold, and the general CDR-H3 sequence is in red. The block arrows show secondary structure as defined by Grochulski et al. (30). (c) Overview of the three-step assembly strategy used to construct the cc123 library, involving (1) the recovery of CDR-H3 sequences from the scFv antibody library and their ligation to synthetic double-stranded DNA adaptor molecules corresponding to sequences adjacent to cry1Aa13 domain II loops, (2) PCR amplification of cry1Aa13 sequences encoding the toxin framework adjacent to the domain II loops, and (3) a series of overlap extension PCRs to join the recovered CDR-H3 sequences and the toxin framework sequences. Primers, restriction sites, and DNA fragments relevant to the assembly are labeled.

FIG. 3.

Comparison of the lengths (a) and amino acid compositions (b) of Crybody-derived CDR-H3 sequences to those of antibody CDR-H3 sequences from a large database (84).

FIG. 4.

The average levels of hydropathy of (a) individual CDR-H3 sequences or (b) the combination of CDR-H3 sequences and natural toxin loops found in Crybodies with uninterrupted open reading frames. Crybodies that formed or failed to form crystalline inclusions when expressed in sporulating B. thuringiensis IPS 78/11 are shown in green and red, respectively. Correctly assembled Crybodies or those with framework mutations are represented by diamonds and plus signs, respectively. The average levels of hydropathy of the natural toxin loops are labeled and shown by gray horizontal lines. From left to right, the identity of each construct is as follows: for the cc1 library, 8, 11, 13, 14, 15, 16, 21, and 25; for the cc2 library, 2, 4, 5, 6, 9, 20, 22, 26, and 30; for the cc3 library, 2, 6, 9, 10, 16, 17, 20, and 24; for the cc12 library, 4, 11, 12, 13, 15, 17, 24, 25, 26, and 28; for the cc13 library, 3, 4, 9, 11, 12, 15, 16, 20, and 22; for the cc23 library, 1, 2, 3, 8, 14, 21, 23, and 24; and for the cc123 library, 1, 2, 5, 7, 8, 9, and 10.

FIG. 5.

The length of (a) individual CDR-H3 sequences or (b) the combination of CDR-H3 sequences and natural toxin loops found in Crybodies with uninterrupted open reading frames that formed (green) or failed to form (red) crystalline inclusion when expressed in sporulating B. thuringiensis IPS 78/11. Correctly assembled Crybodies are represented by diamonds, whereas Crybodies with framework mutations are shown by plus signs. The lengths of the natural toxin loops are labeled and shown by gray horizontal lines. The identity of each construct is as reported in the legend to Fig. 4.

FIG. 6.

Coomassie blue-stained SDS-PAGE gels showing solubilized (upper gels) or trypsin-treated (lower gels) Crybodies expressed in sporulating B. thuringiensis IPS 78/11. + and − indicate lanes containing Cry1Aa13 or cc2.2 (crystal-negative control), respectively. Constructs with framework mutations are indicated by an asterisk.

FIG. 7.

The relationship between the presence of arginine or lysine residues in CDR-H3 inserts and Crybody sensitivity to internal trypsin cleavage. (a) The number of arginine or lysine residues present in CDR-H3 inserts of Crybodies susceptible (red) or resistant (green) to internal trypsin cleavage. Correctly assembled Crybodies are represented by diamonds, whereas Crybodies with framework mutations are shown by plus signs. (b) The percentages of Crybodies resistant to internal trypsin cleavage, grouped by the number of arginine or lysine residues present in their CDR-H3 sequence. The ratio of resistant to total Crybodies is shown in parentheses at each data point. Only correctly assembled Crybodies were considered in this analysis.

FIG. 8.

Crybody toxicity toward M. sexta neonates. Strains of B. thuringiensis IPS 78/11 expressing wild-type Cry1Aa13 (+) or a Crybody (as indicated) were cultured in CCY medium supplemented with 6 μg/ml chloramphenicol until complete sporulation was observed by phase contrast microscopy (48 to 72 h). Twenty microliters of each culture was applied to the surface of M. sexta diet in 48-well plates. Control wells (−) contained only diet. Neonates were added and plates incubated for 5 days, at which point mortality was assessed. Crybodies from libraries cc1, cc2, and cc3 were tested in three independent trials, six insects per trial. Crybodies from libraries cc12 and cc23 were tested twice, six insects per trial. Asterisks indicate Crybodies with framework mutations.

FIG. 9.

Cry toxin receptor binding. (a) Crybody binding to the cadherin receptors CAD3 and M1429⁻, as determined by ELISA. Solubilized Crybodies, Cry1Aa13 (+), and a control without toxin (−) were used to coat the wells of microtiter plates. Wells were probed with CAD3, M1429⁻, or buffer alone and bound receptor detected with Ni-nitrilotriacetic acid horseradish peroxidase. Absorbance values were corrected by subtracting the absorbance of corresponding controls incubated without receptors. The means and standard deviations of the results from four replicates are shown. Asterisks indicate Crybodies with mutations in the toxin framework. (b) Analysis of Cry1Aa13 and Cry1Aa13.2.4D5 binding to M. sexta BBMV receptors by ligand blot assay. On duplicate gels, 230, 46, 9.2, 1.8, 0.37, or 0.070 μg of total BBMV protein was resolved by SDS-PAGE and then electrotransferred to nitrocellulose membranes. The blots were then blocked and probed with either activated Cry1Aa13 or Cry1Aa13.2.4D5. Toxin binding was detected by incubation with anti-Cry1Aa polyclonal antibodies followed by anti-rabbit IgG antibodies conjugated to horseradish peroxidase. (c) Analysis of Cry1Aa13, CryMK.123, and Cry3Aa binding to CAD3. Various concentrations of Cry1Aa13, CryMK.123, and Cry3Aa were mixed with a constant amount of CAD3 (4 nM) and allowed to reach equilibrium. A portion of each sample was then transferred to an ELISA plate previously coated with Cry1Aa13, and the amount of free CAD3 was detected as described for panel a. The means and standard deviations of the results from three independent experiments are shown.