38th Annual Meeting of the Society for Invertebrate Pathology

It was initially demonstrated that helices 4 and 5 of the Cry4Ba mosquito-larvicidal protein from Bacillus thuringiensis subsp. israelensis (Bti) are toxic determinants against Aedes aegypti larvae, likely being involved in membrane-pore formation. Further analysis revealed a crucial role in toxicity for the positively charged side-chain of Arg-158 in helix 4, conceivably involved in the passage of ions through the pore. The highly conserved Asn-183 in helix 5 was also found to be important for Cry4Ba toxin function. Directed mutations within the alpha4-alpha5 loop of Cry4Ba revealed that polarity of Asn-166 and highly conserved aromaticity of Tyr-170 are critically involved in larvicidal activity. A crucial role in toxicity was also revealed for the conserved aromatic residue at position 202 within the alpha4-alpha5 loop of the closely related Bti mosquito-larvicidal protein, Cry4Aa. Interestingly, both the proline-rich motif (P193PNP196) and the unique disulphide bond (C192-C199) in the alpha4-alpha5 loop were found to be structurally essential for Cry4Aa toxicity, possibly required for efficient penetration of the alpha4-alpha5 transmembrane hairpin into the lipid membrane. HPLC purified Cry4Ba alpha4-alpha5 hairpins were shown to be highly capable of inducing liposome permeability, constituting the region responsible for pore formation within the toxin molecule. Unrestrained molecular dynamics (MD) simulations performed with a modelled pore comprising six copies of the Cry4Ba alpha4-alpha5 hairpin or its derivatives placed in solvated lipid membrane bilayers (POPC/water) suggested that mutations at the critical Arg-158 residue affect structural integrity of the toxin-induced pore. Consistent with the reduced conductances observed for single channels formed by biological inactive N166 mutant toxins, MD simulations of the Cry4Ba-pore model also revealed a significant decrease in the extent of hydrogen bonding at position-166 with water molecules when Asn-166 was substituted with Ile, suggesting that Asn-166 is conceivably involved in ion conduction of the Cry4Ba toxin.