Complete structure elucidation of a functional form of the Bacillus thuringiensis Cry4Ba δ-endotoxin: Insights into toxin-induced transmembrane pore architecture

The insecticidal nature of Cry δ-endotoxins produced by Bacillus thuringiensis is generally attributed to their ability to form transmembrane pores, causing lysis of target insect cells. Previously, the truncated tertiary structure of the chymotrypsin-treated Cry4Ba toxin lacking the N-terminal helices-α1 and α2 was reported. To elucidate a more complete functional structure, a 65-kDa trypsin-activated form of the Cry4Ba-R203Q mutant toxin was thus generated for X-ray crystallography by eliminating the Arg²⁰³-tryptic cleavage site. The 2.0 Å crystal structure of Cry4Ba-R203Q with R-factor of 21.5% and R_free of 23.7.%, as subsequently improved with homology-based modeling and molecular dynamics (MD) simulations, revealed a wedge-shaped arrangement of three domains: a well-defined N-terminal domain of eight α-helices (α1, α2a, α2b, α3, α4, α5, α6 and α7) responsible for pore formation, a three-β-sheet prism displaying two functional motifs and a C-terminal β-sandwich domain. A full-atom structural model of the Cry4Ba pre-pore trimer constructed using a single-particle 3D-reconstructed template revealed that each toxin monomer forms the stable trimer by packing α3 and α4 together at the central interface. When MD simulations of a membrane-associated trimeric pore model comprising three α4-loop-α5 hairpins were performed, an stable open-pore structure at the membrane-water interface was clearly observed. Two conserved side-chains-Asn¹⁶⁶ and Tyr¹⁷⁰ in the α4-α5 loop were found to interact directly with phospholipid head groups, leading to pore opening and stability. Overall data provide the first complete view of the 3D structure of the Cry4Ba mosquito-active toxin and its trimeric pore architecture, underlining the importance of two critical loop residues-Asn¹⁶⁶ and Tyr¹⁷⁰.