| Bacillus thuringiensis is a common soil bacterium of agricultural significance due to its production of crystal (Cry) proteins with insecticidal activity against a range of pests. Cry toxins must bind to brush border membrane receptors in the insect midgut to exert their toxic effect. The binding mechanism of Cry1Aa, Cry1Ab, and Cry1Ac toxins to various lepidopteran larval receptors was examined by a combined approach of modeling, mutagenesis, and kinetic analysis. Additionally, the correlation between receptor affinity and toxicity was explored.; A three-dimensional structure of Cry1Ac was modeled from the crystal structure of Cry1Aa, and residues were selected for mutation based on surface exposure and previous mutagenesis results. To analyze mutant toxin binding, several receptors were biochemically purified, including aminopeptidase N (APN) from Lymantria dispar and Manduca sexta, and APN and a cadherin-like receptor from Bombyx mori. Real-time surface plasmon resonance (SPR) analysis of mutant kinetics indicated residues in both domains II and III of Cry1A toxins contribute to receptor binding. SPR studies with domain-switched toxins, carbohydrate solution competition, and variations in injection time provided substantiating evidence that Cry1Ac binds L. dispar APN in two steps. Sequential binding involves docking of an N-acetylgalactosamine (GalNAc) moiety on APN in a cavity on Cry1Ac domain III, followed by higher affinity domain II residue contact. In contrast to L. dispar APN kinetics, Cry1Aa and Cry1Ab bound one site on M. sexta APN, and Cry1Ac bound two sites heterogeneously. However, correlation of toxicity loss with loss of binding for domain II residues, but not domain III residues, was a conserved finding.; Using a computationally-based docking program, the GalNAc-binding cavity in Cry1Ac identified by mutagenesis was shown to specifically favor GalNAc docking over other sugar epimers. Simulated design of a similar cavity in the homologous position of Cry1Aa predicted favorable GalNAc binding as well.; Despite the prevalence of APN as a Bt toxin receptor, higher affinity binding was observed to two other receptors, a glycoreceptor from L. dispar, and cadherin-like glycoproteins from M. sexta and B. mori. Taken together, these studies advance our understanding of the molecular mechanisms of Cry1A toxin binding to their target receptors. |
