Bt Research 2024, Vol.15, No.1, 42-52 http://microbescipublisher.com/index.php/bt 45 4 Mechanisms of Toxin-Receptor Interactions 4.1. Binding affinity and specificity The binding affinity and specificity of Bt toxins to their receptors are critical for their insecticidal activity. The Cry1A toxins (Cry1Aa, Cry1Ab, and Cry1Ac) produced by Bacillus thuringiensis (Bt) bind specifically to the cadherin receptor BT-R1 in the midgut of Manduca sexta with high affinity (Kd = 1.1 nM) (Liu et al., 2022). This high-affinity binding is essential for the initiation of the insecticidal process. The specificity of the interaction is demonstrated by the fact that Cry1A toxins compete for the same binding site on BT-R1, and the inhibition patterns of insecticidal activity are similar for all three toxins (Liu et al., 2022). Additionally, the receptor BT-R1 has been shown to exhibit high-affinity binding to Cry1A toxins when expressed in heterologous cell cultures, further confirming its specificity. 4.2 Conformational changes upon binding Upon binding to their receptors, Bt toxins undergo conformational changes that are crucial for their function. The binding of Cry1Ab toxin to BT-R1 triggers a Mg2+ dependent signaling pathway, which involves the stimulation of the G-protein α-subunit and subsequent signaling cascades. This binding event is highly specific and involves the formation of a heterodimeric complex between Cry1Ab and the 12th ectodomain region (EC12) of BT-R1, with extremely high affinity (Kd = 19.5 ± 1.6 nM) (Liu et al., 2018). The interaction between Cry1Ab and specific epitopes on BT-R1, such as the 368RRPFNIGINNQQ379 region, is critical for the toxin's specificity and function. 4.3 Cellular responses and signal transduction The binding of Bt toxins to their receptors initiates a series of cellular responses and signal transduction pathways that lead to insect cell death. The Cry1Ab toxin, upon binding to BT-R1, activates a previously undescribed signaling pathway involving the stimulation of G protein (Gαs) and adenylyl cyclase, leading to increased cAMP levels and activation of protein kinase A (PKA). This signaling cascade results in cytological changes such as membrane blebbing, appearance of ghost nuclei, cell swelling, and lysis, ultimately causing cell death (Hu et al., 2018). Additionally, the binding of Cry1Ab monomer to BT-R1 correlates with cell death, whereas oligomeric forms of the toxin do not produce lytic pores or kill insect cells (Stevens et al., 2017). The involvement of cadherin receptors and other proteins, such as ATP-binding cassette transporters, further modulates the cytotoxic effects of Bt toxins. 5 Advances in Research Techniques 5.1. Structural biology methods Structural biology methods have significantly advanced our understanding of Bt toxin-receptor interactions. Techniques such as X-ray crystallography and homology modeling have been pivotal in elucidating the three-dimensional structures of both toxins and their receptors. For instance, the three-dimensional structure of the Cry1Ac toxin-binding region in Plutella xylostella cadherin-like receptor was constructed using homology modeling, which revealed the specific domains involved in binding (Rathinam et al., 2019). Additionally, X-ray crystallography has been used to resolve the structure of the toxin-binding site of the cadherin G-protein-coupled receptor BT-R1, providing insights into the molecular determinants of toxin binding (Liu et al., 2022). 5.2 Molecular docking and simulation Molecular docking and simulation techniques have been instrumental in predicting and validating the interactions between Bt toxins and their receptors. These computational methods allow for the exploration of binding affinities and the stability of toxin-receptor complexes. For example, molecular docking studies have shown that the Cry1Ac toxin interacts with specific regions of the cadherin-like receptor in Plutella xylostella, with hydrogen bonding and hydrophobic interactions playing crucial roles (Hu et al., 2018). Furthermore, molecular dynamics simulations have substantiated the stability of interactions between the chimeric Cry1AcF toxin and aminopeptidase1 receptors fromHelicoverpa armigera and Spodoptera litura, demonstrating the broad-spectrum efficacy of the engineered toxin (Rathinam et al., 2019).
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