Structural Changes of the Cry1Ac Oligomeric Pre-Pore from Bacillus thuringiensis Induced by N-Acetylgalactosamine Facilitates Toxin Membrane Insertion

• Published in: Biochemistry (Easton) 2006-08, Vol.45 (34), p.10329-10336
• Main Authors: Pardo-López, Liliana, Gómez, Isabel, Rausell, Carolina, Sánchez, Jorge, Soberón, Mario, Bravo, Alejandra
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Animals; Bacillus thuringiensis; Bacillus thuringiensis - chemistry; Bacillus thuringiensis - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - pharmacology; Bacterial Toxins - chemistry; Bacterial Toxins - pharmacology; CD13 Antigens - chemistry; CD13 Antigens - metabolism; Endotoxins - chemistry; Endotoxins - pharmacology; Hemolysin Proteins; Insect Proteins - chemistry; Insect Proteins - metabolism; Insecticides - chemistry; Insecticides - pharmacology; Larva - metabolism; Larva - microbiology; Manduca - metabolism; Manduca - microbiology; Manduca sexta; Membranes, Artificial; Protein Binding; Protein Structure, Quaternary - drug effects
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi060297z

The primary action of Cry toxins produced by Bacillus thuringiensis is to lyse midgut epithelial cells in their target insect by forming lytic pores. The toxin−receptor interaction is a complex process, involving multiple interactions with different receptor and carbohydrate molecules. It has been proposed that Cry1A toxins sequentially interact with a cadherin receptor, leading to the formation of a pre-pore oligomer structure, and that the oligomeric structure binds to glycosylphosphatidyl-inositol-anchored aminopeptidase-N (APN) receptor. The Cry1Ac toxin specifically recognizes the N-acetylgalactosamine (GalNAc) carbohydrate present in the APN receptor from Manduca sexta larvae. In this work, we show that the Cry1Ac pre-pore oligomer has a higher binding affinity with APN than the monomeric toxin. The effects of GalNAc binding on the toxin structure were studied in the monomeric Cry1Ac, in the soluble pre-pore oligomeric structure, and in its membrane inserted state by recording the fluorescence status of the tryptophan (W) residues. Our results indicate that the W residues of Cry1Ac have a different exposure to the solvent when compared with that of the closely related Cry1Ab toxin. GalNAc binding specifically affects the exposure of W545 in the pre-pore oligomer in contrast to the monomer where GalNAc binding did not affect the fluorescence of the toxin. These results indicate a subtle conformational change in the GalNAc binding pocket in the pre-pore oligomer that could explain the increased binding affinity of the Cry1Ac pre-pore to APN. Although our analysis did not reveal major structural changes in the pore-forming domain I upon GalNAc binding, it showed that sugar interaction enhanced membrane insertion of soluble pre-pore oligomeric structure. Therefore, the data presented here permits to propose a model in which the interaction of Cry1Ac pre-pore oligomer with APN receptor facilitates membrane insertion and pore formation.