Bivalent sequential binding model of a Bacillus thuringiensis toxin to gypsy moth aminopeptidase N receptor.
Specificity for target insects of Bacillus thuringiensis insecticidal Cry toxins is largely determined by toxin affinity for insect midgut receptors. The mode of binding for one such toxin-receptor complex was investigated by extensive toxin mutagenesis, followed by real-time receptor binding analysis using an optical biosensor (BIAcore). Wild-type Cry1Ac, a three-domain, lepidopteran-specific toxin, bound purified gypsy moth (Lymantria dispar) aminopeptidase N (APN) biphasically. Site 1 displayed fast association and dissociation kinetics, while site 2 possessed slower kinetics, yet tighter affinity. We empirically determined that two Cry1Ac surface regions are involved in in vivo toxicity and APN binding. Mutations within domain III affected binding rates to APN site 1, whereas mutations in domain II affected binding rates to APN site 2. Furthermore, domain III contact is completely inhibited in the presence of N-acetylgalactosamine, indicating loss of domain III binding eliminates all APN binding. Based upon these observations, the following model is proposed. A cavity in lectin-like domain III initiates docking through recognition of an N-acetylgalactosamine moiety on L. dispar APN. Following primary docking, a higher affinity domain II binding mechanism occurs, which is critical for insecticidal activity.
Jenkins
J L
JL
Department of Molecular Genetics and Department of Biochemistry, Ohio State University, Columbus, Ohio 43210, USA.
Lee
M K
MK
Valaitis
A P
AP
Curtiss
A
A
Dean
D H
DH
D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins; D001665:Binding Sites; D018826:CD13 Antigens / Q000378:metabolism*; D003198:Computer Simulation; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D009036:Moths / Q000378:metabolism*; D011485:Protein Binding
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens