Receptor-mediated membrane insertion of Cry1Ac triggers cell lysis and oligomeric state remodeling.

The precise insecticidal mechanism of Cry toxins remains elusive, and the structural architecture of their transmembrane pores has yet to be elucidated. In this study, we constructed a C-terminally truncated Cry1Ac mutant, termed Cry1AcNC, which retains the N-terminal domain and toxin core. Using negative-stain electron microscopy, we visualized a tetrameric pore-ring structure formed by Cry1AcNC. To examine the functional role of helix flexibility in pore formation, we introduced disulfide bonds between the α-1 and α-2 helices to restrict their mobility. Cytotoxicity assays using Sf9 cells heterologously expressing BmABCC2, a key Cry toxin receptor, revealed that the double-site mutant Cry1AcNCLI (L42C/I63C) was completely inactive, despite retaining its ability to localize to the plasma membrane. Furthermore, electron microscopy analysis showed that Cry1AcNC formed extensive aggregates both before and after cellular infection. Collectively, these findings provide direct experimental evidence that the α-1 helix participates in ABC transporter-mediated membrane insertion and pore formation through its translocation, thereby advancing our understanding of the molecular mechanism underlying Cry toxin pore assembly.