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 thu ringiensis Cry toxins in synthetic planar lipid bilayers and the estimation of a pore size of 10-20 Angstrom, it has been proposed that the pore could be formed by an oligomer containing four to six Cry toxin monomers. Howeve r, there is a lack of information regarding the insertion of Cry toxins int o the membrane and oligomer formation. Here we provide direct evidence show ing 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 bind ing and H168F in pore formation) were affected in toxicity against Manduca sexta larvae. Binding analysis showed that F371A protein bound more efficie ntly to M. sexta brush border membrane vesicles when mixed with H168F in a one to one ratio. These mutant proteins also recovered pore-formation activ ity, measured with a fluorescent dye with isolated brush border membrane ve sicles, and toxicity against M. sexta larvae when mixed, showing that monom ers affected in different steps of their mode of action can form functional hetero-oligomers. (C) 2000 Federation of European Microbiological Societie s. Published by Elsevier Science B.V. All rights reserved.