The role played by ␣-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-D-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that ␣-helix 4 plays a crucial role in the mechanism of pore formation.Bacillus thuringiensis is the most extensively used commercial biopesticide worldwide and is presently the sole source of toxin genes for the development of insect-resistant transgenic plants (13,15,42). The insecticidal activity of B. thuringiensis is primarily associated with its ability to synthesize a crystalline parasporal inclusion body containing highly specific insecticidal proteins (22,36). The mode of action of these insecticides involves solubilization of the crystal in the highly alkaline lepidopteran midgut lumen, activation of the toxins by intestinal proteases, recognition of one or more binding sites on the midgut brush border membrane surface followed by pore formation, and cell lysis leading ultimately to insect death (36).The elucidation by X-ray diffraction analysis of the threedimensional structures of the activated Cry1Aa (21), Cry2Aa (31), Cry3Aa (28), and Cry3Bb (16) toxins has revealed a common three-domain folding pattern. Domain I is made of seven ␣-helices, and domains II and III are composed mostly of ␤-sheets. While domain I is considered to be responsible for pore formation (37, 43, 44), domains II and III are involved in receptor binding and host specificity (11,12,24,47). Domain III is also thought to play a role in protein stability (28). The domains of the activated toxins were shown to interact with each other to yield their overall toxic effect (33, 34). Exchanging domain I from different toxins can affect crystal formation, stability, pore formation, and membrane permeability as well as the size of the pores and toxicity.The toxin is thought to form pores in the cell membrane by first inserting a hairpin composed of the hydrophobic ␣5 and the amphipathic ␣4 h...

Helix 3 of the Cry1Aa toxin from Bacillus thuringiensis possesses eight charged amino acids. These residues, with the exception of those involved in intramolecular salt bridges (E90, R93, E112, and R115), were mutated individually either to a neutral or to an oppositely charged amino acid. The mutated genes were expressed, and the resultant, trypsin-activated toxins were assessed for their toxicity to Manduca sexta larvae and their ability to permeabilize M. sexta larval midgut brush border membrane vesicles to KCl, sucrose, raffinose, potassium gluconate, and N-methyl-D-glucamine hydrochloride with a light-scattering assay based on osmotic swelling. Most mutants were considerably less toxic than Cry1Aa. Replacing either E101, E116, E118, or D120 by cysteine, glutamine, or lysine residues had only minor effects on the properties of the pores formed by the modified toxins. However, half of these mutants (E101C, E101Q, E101K, E116K, E118C, and D120K) had a significantly slower rate of pore formation than Cry1Aa. Mutations at R99 (R99C, R99E, and R99Y) resulted in an almost complete loss of pore-forming ability. These results are consistent with a model in which alpha-helix 3 plays an important role in the mechanism of pore formation without being directly involved in determining the properties of the pores.

The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs, BinA formed voltage-dependent channels of approximately 100-200 pS with long open times and a high open probability. Larger channels (> or =400 pS) were also observed. BinB, which inserted less easily, formed smaller channels (< or =100 pS) with shorter mean open times. Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior. Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding component of the toxin, playing a role in promoting channel activity.

linking domains I and II in Cry1Aa were abolished individually in ␣-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp 242 -Arg 265 bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.During sporulation, Bacillus thuringiensis produces a parasporal crystal body composed of one or more proteins that are toxic to a number of insect larvae (1) or to other invertebrates (2). After solubilization in the insect midgut and activation by intestinal proteases, these proteins bind to specific receptors at the surface of the apical brush border membrane of epithelial columnar cells, insert into the membrane, and form pores that disrupt midgut cellular functions (3-5).Elucidation of the crystal structure of the coleopteranspecific Cry3A toxin (6) and the lepidopteran-specific Cry1Aa toxin (7) revealed a similar three-domain structure for both proteins. Domain I, composed of eight amphipathic ␣-helices, is thought to be involved in membrane insertion and pore formation (8 -15). Domain II, composed of three ␤-sheets and two short ␣-helices, is involved in the binding of the toxin to its receptor on the epithelial cell surface (16 -23). Domain III, composed of two ␤-sheets forming a face-to-face ␤-sandwich, appears to be involved in the stability (6), specificity (24 -26), and binding (27-34) of the toxin.These domains are closely packed together with the largest number of interdomain contacts found between domains I and II (6, 7). In Cry1Aa, domains I and II are linked by four salt bridges: Asp

Influence of domain I exchange on the stability and production of Bacillus thuringiensis Cry1 protoxins as well as on the shape of inclusion and toxicity to Spodoptera exigua and Plutella xylostella larvae was investigated. Chimeric genes were prepared by exchanging the regions coding for domain I between Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The AcCC chimera accumulated into bipyramidal inclusion bodies, whereas CEE produced round-shaped inclusion bodies, and ECC and AaEE protoxins produced small granules. AbEE and EAaAa did not produce any inclusion body and were visualized by immunodetection only. AcCC, CEE, ECC, and AaEE were stable to trypsin, whereas AbEE and EAaAa were not. Bioassays showed that the chimeras were not toxic in vivo. However, S. exigua larvae fed with the activated AcCC toxin displayed a lower growth rate.