Toxins, Vol. 14, Pages 823: Insights from the Structure of an Active Form of Bacillus thuringiensis Cry5B

Conceptualization, M.K.C. and M.M.L.; methodology, M.K.C., J.L., L.W. and M.K.; validation, M.K.C. and J.L.; formal analysis, M.K.C., L.W. and J.L.; investigation, M.K.C., J.L. and L.W.; resources, M.K.C.; writing—original draft preparation, M.K.C. and J.L.; writing—review and editing, M.K.C., J.L. and M.M.L.; supervision, M.K.C. and M.M.L.; project administration, M.K.C. and M.M.L.; funding acquisition, M.K.C. and M.M.L. All authors have read and agreed to the published version of the manuscript.

Figure 1. Structures of Cry5B(27–698) and Cry5B(112–698). Structure of (A) single subunit and tetramer in (B) side view and (C) top view of Cry5B(27–698). N-terminal functional extension (residues 27–107) in red, residues 112–140 in blue and the three-domain core (residues 141–698) colored in yellow. Structure of (D) single subunit and trimer in (E) side view and (F) bottom view of Cry5B(112–698) (PDB ID: 4D8M). Residues 112–140 in cyan and the three-domain core (residues 141–698) colored in orange. The black boxes in (B,E) showing the interaction between neighboring subunits. Hydrogen bond shown as black dashed lines.

Figure 1. Structures of Cry5B(27–698) and Cry5B(112–698). Structure of (A) single subunit and tetramer in (B) side view and (C) top view of Cry5B(27–698). N-terminal functional extension (residues 27–107) in red, residues 112–140 in blue and the three-domain core (residues 141–698) colored in yellow. Structure of (D) single subunit and trimer in (E) side view and (F) bottom view of Cry5B(112–698) (PDB ID: 4D8M). Residues 112–140 in cyan and the three-domain core (residues 141–698) colored in orange. The black boxes in (B,E) showing the interaction between neighboring subunits. Hydrogen bond shown as black dashed lines.

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Figure 2. CDH-8 CR7/8 can trigger the in vitro oligomerization of Cry5B(1–772). The blue and red arrows indicate the Cry5B(1–772) monomer and oligomer in native condition, respectively.

Figure 2. CDH-8 CR7/8 can trigger the in vitro oligomerization of Cry5B(1–772). The blue and red arrows indicate the Cry5B(1–772) monomer and oligomer in native condition, respectively.

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Figure 3. Toxicity test of Cry5B N-terminal truncations against C. elegans. (A) Relative length of worms indicated the toxicity of Cry5B constructs. (B) Each panel shows a typical C. elegans grown on E. coli BL21 expressing different constructs after three days. Scale bar represents 1 mm. Worms grown on empty vector and Cry5B(112–698) are obviously large and healthy, while those grown on Cry5B(1–698), Cry5B(12–698), Cry5B(21–698) and Cry5B(27–698) are much smaller, indicating intoxication. Two-way ANOVA, N = 15–25, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05; ns, not significant.

Figure 3. Toxicity test of Cry5B N-terminal truncations against C. elegans. (A) Relative length of worms indicated the toxicity of Cry5B constructs. (B) Each panel shows a typical C. elegans grown on E. coli BL21 expressing different constructs after three days. Scale bar represents 1 mm. Worms grown on empty vector and Cry5B(112–698) are obviously large and healthy, while those grown on Cry5B(1–698), Cry5B(12–698), Cry5B(21–698) and Cry5B(27–698) are much smaller, indicating intoxication. Two-way ANOVA, N = 15–25, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05; ns, not significant.

Toxins 14 00823 g003 Figure 4. A ring of hydrogen bonds at the apex of the tetramer Cry5B(27–698). Four subunits colored as in Figure 1B. Hydrogen bonds shown as black dashed lines. Figure 4. A ring of hydrogen bonds at the apex of the tetramer Cry5B(27–698). Four subunits colored as in Figure 1B. Hydrogen bonds shown as black dashed lines. Toxins 14 00823 g004

Figure 5. Toxicity test of Cry5B(1–772) Y495 mutants against C. elegans. (A) Relative length of worms indicated the toxicity of Cry5B constructs. (B) Each panel shows a typical C. elegans grown on E. coli BL21 expressing different constructs after three days. Scale bar represents 1 mm. Worms grown on empty vector and Cry5B(1–772) Y495A are obviously larger, while those grown on wild-type Cry5B(1–772), Cry5B(1–772) Y495F, Cry5B(1–772) Y495W and Cry5B(1–772) Y495R are much smaller. Two-way ANOVA, N = 10, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05; ns, not significant.

Figure 5. Toxicity test of Cry5B(1–772) Y495 mutants against C. elegans. (A) Relative length of worms indicated the toxicity of Cry5B constructs. (B) Each panel shows a typical C. elegans grown on E. coli BL21 expressing different constructs after three days. Scale bar represents 1 mm. Worms grown on empty vector and Cry5B(1–772) Y495A are obviously larger, while those grown on wild-type Cry5B(1–772), Cry5B(1–772) Y495F, Cry5B(1–772) Y495W and Cry5B(1–772) Y495R are much smaller. Two-way ANOVA, N = 10, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05; ns, not significant.

Toxins 14 00823 g005 Figure 6. Computational model of the Cry5B(27–698) with extended helix. (A) Proposed reorientation of N-terminal region. (B) Modeled Cry5B(27–698) tetramer structure when N-terminal region rearranged to form an extended helix (residues 112–162). Colored as in Figure 1A. Black boxes showing the close distance (~7 Å) between the proposed glycan-binding motif (GG and GPIEE shown as grey spheres) and the possible position of N-terminal region (red). Figure 6. Computational model of the Cry5B(27–698) with extended helix. (A) Proposed reorientation of N-terminal region. (B) Modeled Cry5B(27–698) tetramer structure when N-terminal region rearranged to form an extended helix (residues 112–162). Colored as in Figure 1A. Black boxes showing the close distance (~7 Å) between the proposed glycan-binding motif (GG and GPIEE shown as grey spheres) and the possible position of N-terminal region (red). Toxins 14 00823 g006

Table 1. Data Collection and Refinement Statistic.

Table 1. Data Collection and Refinement Statistic.

Cry5B(27–698)Data collection Wavelength (Å)0.99984Space groupP 4 21 2Cell dimensions: a, b, c (Å)114.4 114.4 263.4                              α, β, γ (°)90.0 90.0 90.0Resolution (Å)20 (4.5)Rmerge0.274 (0.559)I/σI2.2 (1.3)Completeness (%)99.7 (100)Redundancy5.3 (5.4)Refinement No. reflections10,836Rwork/Rfree23.76/28.85No. atoms: Protein10,254                    Ligand/ion0                    Water0B-factors(Å2): Protein107.08R.m.s. deviations: Bond lengths (Å)0.002                                Bond angles (°)0.54Range of residues27–83, 88–107, 113–165, 173–214, 224–698

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