Evidence for intermolecular interaction as a necessary step for pore-formation activity and toxicity of Bacillus thuringiensis Cry1Ab toxin.
Based on the observation of large conductance states formed by Bacillus thuringiensis Cry toxins in synthetic planar lipid bilayers and the estimation of a pore size of 10-20 A, it has been proposed that the pore could be formed by an oligomer containing four to six Cry toxin monomers. However, there is a lack of information regarding the insertion of Cry toxins into the membrane and oligomer formation. Here we provide direct evidence showing that the intermolecular interaction between Cry1Ab toxin monomers is a necessary step for pore formation and toxicity. Two Cry1Ab mutant proteins affected in different steps of their mode of action (F371A in receptor binding and H168F in pore formation) were affected in toxicity against Manduca sexta larvae. Binding analysis showed that F371A protein bound more efficiently to M. sexta brush border membrane vesicles when mixed with H168F in a one to one ratio. These mutant proteins also recovered pore-formation activity, measured with a fluorescent dye with isolated brush border membrane vesicles, and toxicity against M. sexta larvae when mixed, showing that monomers affected in different steps of their mode of action can form functional hetero-oligomers.
Soberón
M
M
Instituto de Biotecnología, UNAM, Cuernavaca, Morelos, Mexico. mario@ibt.unam.mx
Pérez
R V
RV
Nuñez-Valdéz
M E
ME
Lorence
A
A
Gómez
I
I
Sánchez
J
J
Bravo
A
A
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D001427:Bacterial Toxins; D021961:Cell Membrane Structures / Q000378:metabolism*; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D006460:Hemolysin Proteins; D007473:Ion Channels / Q000502:physiology; D007814:Larva / Q000187:drug effects; D018402:Manduca / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008871:Microvilli; D009154:Mutation
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007473:Ion Channels; C030956:insecticidal crystal protein, Bacillus Thuringiensis