ABS 045: Exploring Binding Between the Cry1Ab Mutant F328E and its Cognate Receptor EC12

Kareena Kamal ¹ , Jiahe Zhi ¹ , Stefanie D. Boyd ², Duane D. Winkler ²

¹ Winkler Lab, University of Texas at Dallas
² Department of Biological Sciences, University of Texas at Dallas

Van Wickle (2025) Volume 1, ABS045

Introduction: As the global need for nutritious food grows, more attention is spotlighted on the design of effective biopesticides. Cry1Ab is a protein toxin expressed in Bacillus thuringiensis (Bt) that targets tobacco hornworms (M. sexta). Cry1Ab binds to the Bt-R1 receptor in midgut epithelial cells and launches a signaling cascade that results in cell death. Past research demonstrates that the 12th ectodomain of Bt-R1 (EC12) is the toxin binding site (TBS). This work focuses on the interaction interface of Cry1Ab that binds to EC12, and specifically, the “Loop 3” region of Cry1Ab that has previously been shown to be important for binding and toxicity. After using co-evolutionary modeling and in vitro biochemical assays, we pinpointed six point mutations, including Cry1Ab F328E, which eliminates binding. This mutant’s lack of binding to the EC12 receptor is further supported by ELISA assays, where only the receptor should be attracted to the nickel beads, and Size Exclusion Chromatography (SEC), which is size-dependent molecular separation. Future experiments will involve testing the toxicity of this mutant in the hornworm by analyzing the hornworm’s response when Cry1Ab F328E is injected into its food. Determining this mutant’s efficiency will bring us closer to creating a new generation of smart biopesticides that will overcome costly and environmentally hazardous chemical pesticides.

Methods: Mutagenesis was performed to create a single point mutation in the toxin at amino acid residue F328, generating Cry1Ab F328E. The construct was then transformed into BL21 DE3 pLysS E. coli competent cells, followed by harvesting and purification. Purification included adding a histidine tag on the N-terminus of Cry1Ab F328E for cobalt binding. To test for Cry1Ab F328E’s toxicity, an Enzyme-Linked Immunosorbent Assay (ELISA) was performed on a 96-well plate coated with EC12; cell lysate was added and incubated. For detection, anti-his antibody and HRP-conjugated goat anti-mouse antibody were used, while TMB induced a color change. Through Size-Exclusion Chromatography (SEC), EC12 and Cry1Ab F328E were mixed in a 1:1 molar ratio to further test binding. After 15 minutes of incubation, the sample was separated on a size-exclusion column. WT Cry1Ab was our control and ran on the SEC column after the same incubation period to confirm ELISA’s binding result.

Results: Through ELISA and SEC results, we observed that replacing phenylalanine with glutamic acid at amino acid residue 328 leads to a lack of binding between the EC12 receptor and Cry1Ab toxin, and thus, no complex forms on the gel with the columns where both the receptor and mutated toxin are present. Therefore, we have proven that the Cry1Ab F328E mutation significantly disturbs the toxin-receptor interaction in the binding interface, implying that this is a critical binding site for Bt’s toxic effect on the hornworm’s midgut.

Discussion: Due to this mutated toxin’s significant impact on the binding interface, we now look forward to hatching hornworm eggs to inject the toxin into their food and studying them. Cry1Ab F328E is an exciting feat, as this is the first mutant that has disturbed the toxin-receptor interaction to this extent. From experimenting with possible mutants to now unveiling a successful mutant, we are inching closer to creating a new generation of biopesticides based on a critical peptide region that will overtake chemical pesticides. Moreover, this new biopesticide would be selective in targeting the EC12 receptor in hornworms attacking our crops.