Delta Endotoxin
By Matthew Armentrout
The bacteria Bacillus thuringiensis produce the protein Delta Endotoxin, PDB ID 3eb7. Delta Endotoxin, also referred to as Cry Toxin, is a member of the Endotoxin Superfamily and acts as a pore forming toxin that crystallizes in the midgut epithelial cells of insects to form a transmembrane ionic pore1. This ionic pore is lethal to the insect host and is the reason for its common use as an insecticide in the agricultural industry. The Delta Endotoxin gene of B. thuringiensis is inserted into the genome of the target crop, most often maize, giving the crop the ability to produce the toxin. As a result insects that ingest the crop are poisoned and killed by the toxin2.
This protein exists initially as a protoxin in a monomeric state that is ingested by the insect. Proteases in the midgut cleave the protein activating it thus allowing the toxin to bind cadherin receptors that do an additional proteolytic cleavage. The cadherin receptor cleaves the α1-helix of the first domain and induces oligomerization. The ogliomeric structure binds a secondary receptor, either aminopeptidase N or alkaline phosphatase, which drives the protein into the membrane to form the pore. The ionic pore causes osmotic shock for the epithelial cell which leads to cell death and ultimately results in organismal death3.
Delta Endotoxin consists of three identical Cry8Ea1 protein monomers that have 589 amino acids each1. Each monomer consists of a series of α-helices and ß-sheets that aggregate together to form three domains.
Domain 1 is at the N-terminus of the protein and consists primarily of α helices. This domain belongs to the Endotoxin N Superfamily and functions as the pore-forming domain of the Delta Endotoxin5. Before cleavage, Domain 1 consists of seven α-helices which are numbered from one to seven starting at the N-terminus. Upon the binding of a single Cry8Ea1 protein to a cadherin receptor the α1-helix is cleaved. Oligomerization then takes place by the principle role of the α3-helix to form a 2.2Å crystal structure1. The α3-helix experiences a slight movement in position which allows more of its hydrophobic surface area to be exposed and as a result allows α3-helix to bind to helices of other Cry monomers1. As shown by Figure 1, the N-terminus of the α3-helix on three separate proteins associates with one another via van der Waals force. The middle of the α3-helix contacts the C-terminus of the neighboring molecule's α4-helix. The C-terminus of the α3-helix contacts the N-terminus of the neighboring molecule's α6-helix4.
Domain II is located in the middle of the protein and is composed of anti-parallel β-sheets5 that span from Asp-304 to His-497. Domain II associates and binds a receptor that causes a conformational change of the ß-sheet prism. This conformational change causes a change in the structural interface between Domain I and Domain II which results the disassembly of the protein’s Domain I. When Domain I disassembles the α5-helix, which is originally surrounded and stabilized by α3, α4, α6 and α7 helices, adopts an exposed confirmation in which it extends away from the protein along with the α4-helix, as shown by Figure 2, to form what is called the α4-α5 hairpin1. The α4-α5 hairpin contains a high concentration of hydrophobic residues which allows it to insert into the host’s lipid membrane and initiate pore formation4. Substitution of any residue in the α4 or α5 helix with a proline results in complete loss of toxicity. When the hairpin inserts into the lipid membrane it causes the remaining helices to rearrange and follow the “umbrella” model of membrane penetration1.
Domain III is located at the C-terminus of the protein and consists of two layers of anti-parallel beta sheets forming a beta-sheet sandwich. It’s primary function is believed to stabilize the protein structure5 and to bind carbohydrates which may contribute to the proteins binding specificity6. The ß20-ß21 loop of Domain III is a key structure and substitutions of select residues, which vary based upon each Cry toxin homolog, can result in major loss of toxicity5.
Delta Endotoxin has many different homologous molecules that target different organisms. For example, Cry2Aa targets Diptera7, while Cry4Ba is a mosquito-larvicidal protein8, and Cry1Ab targets Manduca sexta larvae9. By studying point mutations and substitutions in these homologs researchers are able to determine the function of the secondary structures and domains of Delta Endotoxin. As shown in the Figure 34, substitution of Lys-115, Arg-158, Asn-166, or Tyr-179 on the Cry4Ba toxin, for alanine will result in a loss of function of Domain 1. In these substituions there is toxin-receptor binding but no pore formation. Substitution of Q149 on α4-helix of the same toxin, colored magenta, with proline abolishes toxicity because a stable α4-α5 helical hairpin is unable to form. Additionally, There is major loss of toxicity when Ser-580, Leu-582, Gly-583, Asn-584, or Val-586 of the ß20-ß21 loop is substituted with alanine in the Cry1Ac toxin which lead researchers to confirm that Domain III plays a principle role in protein stability5.
Delta Endotoxin plays an important role in today's agricultural industry which means that it effects every single one of us. The only threat that the use of Delta Endotoxin has is that it strongly selects for insect resistant to the toxin. Already resistant strains of insects have emerged that are resistant to specific Cry toxins by having modified receptors in thier midgut that have a lower binding affinity for the toxin. The methodology behind how resistant strains of insects are developed is still in need of further investigation10. By developing a better understanding of how the toxin works researchers can attempt to make a more effective toxin that is less likely to produce strains of resistant insects.