1.C.2 The Channel-forming δ-Endotoxin Insecticidal Crystal Protein (ICP) Family

The ICP family proteins (Cry proteins; δ-Endotoxins) are produced during sporulation by various strains of Bacillus thuringiensis as proteinaceous crystalline inclusions. They have been called ICPs. Each ICP is specific for a different group of related insects or other invertebrates. ICPs are widely used as biopesticides, particularly as insecticides. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with the cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion. Flexiblity is obtained by toxin oligomerization. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab, and this domain is inserted into the membrane (Pardo-Lopez et al., 2006).

Specificity against the following insect orders has been observed: Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Phthiraptera, Mallophaga and Acri. Other invertebrate orders susceptible to their action include Nemathelminthes, Platyhelminthes and Sarcomastigorphora. The ICPs were initially classified according to the host organism in which they act, but close homologues were subsequently found to act on many different hosts. A classification system based exclusively on sequence similarity has recently been proposed. More than 50 sequenced ICPs have been classified into 15 families. However, the phylogenetic tree of the channel-forming domains reveal three primary branches.

Native ICPs (pro-Cry proteins) are 50-140 kDa in size. They are proteolytically activated after ingestion by the host organism to give the toxic protein fragments. Each one probably binds to a glycoprotein receptor in the apical microvillar membranes of epithelial midgut cells. The toxin then undergoes a conformational change, inserting into the membrane to form an oligomeric pore that causes osmotic cell lysis.

The crystal structures of two homologous toxins (Cry3A; Coleopteran-specific) and (Cry1Aa; Lepidopteran-specific) have been reported. They contain three domains. Domain I (N-termini; 220 residues) is the channel-forming domain. It is a seven helix bundle in which a central helix (helix 5) is surrounded by the other six helices. The six helices are amphipathic and long enough to span the 30Å membrane bilayer. Domain II (central 200 residues) is the specificity-determining domain which binds the receptor proteins in the insect midgut membrane. Domain III (C-termini; ~150 residues) is functionally less well defined and may transduce information from domain II (receptor binding domain) to domain I (channel-forming domain). Domain III may also regulate channel activity, stabilize the toxin, and function together with Domain II in receptor binding. Toxicity has been explained by formation of transmembrane oligomeric pores or ion channels and by the ability of the monomeric toxin to subvert cellular signaling pathways. In vitro biophysical studies suggest that helices alpha4 and alpha5 in domain I insert into the lipid bilayer as an alpha-helical hairpin. A trimeric model for the ion conducting channel has been proposed (Torres et al., 2007).

The generalized transport reaction catalyzed by these toxins is:

Small molecules (in)  small molecules (out)