Structural and functional studies of α-helix 5 region from Bacillus thuringiensis Cry1Ab δ-endotoxin

Abstract

The crystal insecticidal proteins from Bacillus thuringiensis are modular proteins comprised of three domains connected by single linkers. Domain I is a seven α-helix bundle, which has been involved in membrane insertion and pore formation activity. Site-directed mutagenesis has contributed to identify regions that might play an important role in the structure of the pore-forming domain within the membrane. There are several evidences that support that the hairpin α4-α5 inserts into the membrane in an antiparallel manner, while other helices lie on the membrane surface. We hypothesized that highly conserved residues of α5 could play an important role in toxin insertion, oligomerization and/or pore formation. A total of 15 Cry1Ab mutants located in six conserved residues of Cry1Ab, Y153, Y161, H168, R173, W182 and G183, were isolated. Eleven mutants were located within helix α5, one mutant was located in the loop α4-α5 and three mutants, W182P, W182I and G183C, were located in the loop α5-α6. Their effect on binding, K + permeability and toxicity against Manduca sexta larvae was analyzed and compared. The results provide direct evidence that some residues located within α5 have an important role in stability of the toxin within the insect gut, while some others also have an important role in pore formation. The results also provide evidence that conserved residues within helix α5 are not involved in oligomer formation since mutations in these residues are able to make pores in vitro.