Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment.
The Bacillus thuringiensis Cry1Ac delta-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane. Surface plasmon resonance analysis allowed the quantification of toxin binding to M. sexta APN in a supported lipid monolayer. The initial binding was rapid and reversible, with an affinity constant of 110 nM. The second phase was slower and resulted in an overall affinity constant of 3.0 nM. Reagents used to disrupt protein-protein interactions did not dissociate the toxin after high-affinity binding was attained. The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation. The results suggest that after binding to M. sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state. A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase. This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M. sexta larvae and its deficient membrane-permeabilizing activity on M. sexta midgut brush border membrane vesicles.
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
M A
MA
Cooper
J
J
Carroll
E R
ER
Travis
D H
DH
Williams
D J
DJ
Ellar
D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism* / Q000633:toxicity*; D001427:Bacterial Toxins / Q000378:metabolism* / Q000633:toxicity*; D015374:Biosensing Techniques; D018826:CD13 Antigens / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004347:Drug Interactions; D004731:Endotoxins / Q000378:metabolism* / Q000633:toxicity*; D006460:Hemolysin Proteins; D007700:Kinetics; D018402:Manduca / Q000201:enzymology*; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens