Interaction of Fluorescently Labeled Cadherin G Protein-coupled Receptor with the Cry1Ab Toxin of Bacillus thuringiensis

Abstract

The Cry1Ab toxin produced by Bacillus thuringiensis binds to a conserved structural motif in the 12th ectodomain module (EC12) of BT-R1, a cadherin G protein-coupled receptor (GPCR) contained in the membrane of midgut epithelial cells of the tobacco hornworm Manduca sexta. Toxin binding transmits a signal into the cells and turns on a multi-step signal transduction pathway, culminating in cell death. Using chromatographically purified Cry1Ab and EC12 proteins, we demonstrated the direct formation of a stable complex between these two proteins in solution and visualized it on a native polyacrylamide gel. Moreover, we generated a fluorescent EC12 probe by converting the 36th residue to cysteine to enable maleimide-mediated conjugation of Alexa-488 fluorescent dye to EC12 by site-directed mutagenesis. In addition, we changed the 44th residue of EC12 to tryptophan, which greatly improved accuracy of protein quantification and traceability. Using the fluorescently labeled EC12 probe for direct and competitive binding assays, we were able to determine binding specificity in solution. These accomplishments will facilitate identification and characterization of the interface sequences for both the Cry1Ab toxin and BT-R1.

“The Defined Toxin-binding Region of the Cadherin G-protein Coupled Receptor, BT-R1, for the Active Cry1Ab Toxin of Bacillus thuringiensis”

“…All deletional and substitutional mutations were introduced into EC12 using the Phusion Site-Directed Mutagenesis Kit (ThermoFisher Scientific). As described in a recently published report [31], we substituted the 36 th residue in EC12 with a cysteine residue combined with a change of the 44 th tyrosine residue to tryptophan (EC12 A36C/Y44W ). This change enabled conjugating Alexa-488 fluorescent dye at the cysteine residue through a C-maleimide-mediated reaction.…”

“…Cells were harvested by centrifugation at 8000 rpm for 15 minutes at 4°C and re-suspended in equilibration buffer containing 20 mM Tris buffer (pH 8.0), 300 mM NaCl, 2 mM dithiothreitol (DTT) and 1 mM phenylmethanesulfonyl fluoride (PMSF), followed by sonication. The resulting soluble proteins were purified by nickel affinity chromatography as described [31] and quantified by Bradford assay (Bio-Rad, CA) using bovine serum albumin as control.…”

“…Protein samples were heated at 95°C for 5 min in SDS-PAGE sample buffer and subjected to SDS-PAGE (Mini-Protean Gel Electrophoresis System, Bio-Rad) using gels containing 2,2,2-tricloroethanol (TCE, Sigma- Aldrich) as previously described [31]. Crosslinking (covalent binding) of tryptophan residues in the proteins to TCE was activated by ultraviolet light [38].…”

“…To better understand the biochemical and biophysical properties that define binding of toxin to BT-R 1 in the context of specificity, selectivity and affinity, we purified both the Cry1Ab toxin and EC12 in their active states and examined the interaction of the two molecules directly in solution by size exclusion chromatography. Moreover, we applied size exclusion chromatography-multi-angle laser scattering (SEC-MALS) analysis to confirm the complex formation between Cry1Ab toxin and EC12, and fluorescence gel imaging to determine specificity of the interaction [31]. Here, we show that active Cry1Ab forms a stable heterodimeric complex with EC12 with very high affinity.…”

“…Here, we have refined our analysis of the toxin-binding region (TBR) and demonstrate that the TBR is a finite structural area within EC12 exclusively. EC12 contains a 112-amino acid sequence whose N- and C-terminal regions are highly conserved among homologous cadherin receptors in a number of moth species [30,31] but divergent from similar receptors present in other insects such as beetles and mosquitoes [1]. To better understand the biochemical and biophysical properties that define binding of toxin to BT-R 1 in the context of specificity, selectivity and affinity, we purified both the Cry1Ab toxin and EC12 in their active states and examined the interaction of the two molecules directly in solution by size exclusion chromatography.…”