N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin

Binding of the insecticidal Bacillus thuringiensis Cry1Ac toxin to the putative receptor aminopeptidase N is specifically inhibited by N-acetyl-galactosamine (GalNAc), suggesting that this toxin recognises GalNAc on the receptor. A possible structural basis for involvement of domain III of the toxin in carbohydrate-mediated receptor recognition was noted in the similarity between the domain III fold of the related toxin Cry3A and a carbohydrate-binding domain in the 1,4-beta-glucanase from Cellullomonas fimi. This possibility was investigated by making selected mutations in domain III of the CrylAc delta-endotoxin. Mutagenesis of residues Asn506, Gln509 or Tyr513 resulted in toxins with reduced binding and a slower rate of pore formation in Manduca sexta midgut membrane vesicles compared to the wild-type CrylAc. These mutants also showed reduced binding to the 120 kDa CrylAc putative receptor aminopeptidase N. Unlike the wild-type toxin, binding of the triple mutant N506D,Q509E,Y513A (Tmut) to M, sexta midgut membrane vesicles could not be inhibited by GalNAc. These data indicate that GalNAc binding is located on domain III of CrylAc and therefore support a lectin-like role for this domain. A preliminary analysis of the CrylAc crystal structure locates Asn506, Gln509 and Tyr513 in a region on and adjacent to beta-16 in domain III, which has a unique conformation compared to the other known Cry structures. These residues are in a favourable position to interact with either soluble or protein-bound carbohydrate. (C) 1999 Academic Press.