Bivalent sequential binding model of a Bacillus thuringiensis toxin to gypsy moth aminopeptidase N receptor

Specificity for target insects of Bacillus thuringiensis insecticidal Cry t oxins 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 realtime receptor binding analysi s using an optical biosensor (BIAcore), Wild-type Cry1Ac, a three-domain, l epidopteran-specific toxin, bound purified gypsy moth (Lymantria dispar) am inopeptidase N (APN) biphasically, Site 1 displayed fast association and di ssociation kinetics, while site 2 possessed slower kinetics, yet tighter af finity. We empirically determined that two Cry1Ac surface regions are invol ved in in vivo toxicity and APN binding. Mutations within domain III affect ed binding rates to APN site 1, whereas mutations in domain II affected bin ding rates to APN site 2, Furthermore, domain III contact is completely inh ibited 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 d ocking through recognition of an N-acetylgalactosamine moiety on L. dispar APN, Following primary docking, a higher affinity domain II binding mechani sm occurs, which is critical for insecticidal activity.