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.
Highlights
Bacillus thuringiensis (Bt) is a group of spore-forming Grampositive bacteria that produce entomocidal parasporal crystalline proteins during the sporulation phase of their life cycle [1]
Based on our cytological and biochemical investigations, it is evident that univalent binding of the Cry1Ab toxin affects the structural and functional properties of BT-R1 to bring about cellular damage and total loss of function in midgut epithelial cells of tobacco hornworm larvae exposed to the toxin
Previous studies have shown that Cry toxin binding is located in a finite region within EC12 of BT-R1, the cadherin G protein-coupled receptor (GPCR) for Cry1Ab [3–7,15]
Summary
Bacillus thuringiensis (Bt) is a group of spore-forming Grampositive bacteria that produce entomocidal parasporal crystalline proteins during the sporulation phase of their life cycle [1]. The lethal action of Cry toxins is initiated by binding of the toxins to specific receptors in the midgut epithelium of susceptible insect larvae These receptors, represented by BT-R1 of the moth Manduca sexta (tobacco hornworm) [2,3], constitute a family of homologous cadherin G protein-coupled receptors (GPCRs) that are essential to the binding and activity of the various Cry toxins [1,3–7]. The 65-kDa Cry1Ab toxin produced by Bt binds to a conserved structural motif within the 12th ectodomain (EC12) of BT-R1 [8] This incident is essential for toxicity of Cry1Ab to the insect. We constructed a fluorescent EC12 probe for determining the specificity and affinity of EC12 binding to Cry1Ab by converting residue 36 (serine) in EC12 to cysteine This change permitted C-maleimide mediated conjugation of Alexa Fluor® 488 to the free thiol group on EC12. By monitoring the interaction of fluorescently labeled EC12 and Cry1Ab toxin in a non-denaturing polyacrylamide gel system, we verified formation of a highly stable, soluble complex that most likely typifies such structures formed in a variety of related moth species
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