Structural and functional studies of alpha-helix 5 region from Bacillus thuringiensis Cry1Ab delta-endotoxin

The crystal insecticidal proteins from Bacillus thuringiensis are modular p roteins comprised of three domains connected by single linkers. Domain I is a seven a-helix bundle, which has been involved in membrane insertion and pore formation activity. Site-directed mutagenesis has contributed to ident ify regions that might play an important role in the structure of the pore- forming domain within the membrane. There are several evidences that suppor t that the hairpin alpha4-alpha5 inserts into the membrane in an antiparall el manner, while other helices lie on the membrane surface. We hypothesized that highly conserved residues of alpha5 could play an important role in t oxin insertion, oligomerization and/or pore formation. A total of 15 Cry1Ab mutants located in six conserved residues of Cry1Ab, Y153, Y161, H168, R17 3, W182 and G183, were isolated. Eleven mutants were located within helix a 5, one mutant was located in the loop a4-a5 and three mutants, W182P, W182I and Gl83C, were located in the loop alpha5-alpha6. Their effect on binding , K+ permeability and toxicity against Manduca sexta larvae was analyzed an d compared. The results provide direct evidence that some residues located within a5 have an important role in stability of the toxin within the insec t gut, while some others also have an important role in pore formation. The results also provide evidence that conserved residues within helix a5 are not involved in oligomer formation since mutations in these residues are ab le to make pores in vitro. (C) 2001 Published by Elsevier Science B.V.