Ion channels formed in planar lipid bilayers by the dipteran-specific Cry4BBacillus thuringiensistoxin and its α1–α5 fragment

Abstract

Trypsin activation of Cry4B, a 130-kDa Bacillus thuringiensis (Bt) protein, produces a 65-kDa toxin active against mosquito larvae. The active toxin is made of two protease resistant-products of ca. 45 kDa and ca. 20 kDa. The cloned 21-kDa fragment consisting of the N-terminal region of the toxin was previously shown to be capable of permeabilizing liposomes. The present study was designed to test the following hypotheses: (1) Cry4B, like several other Bt toxins, is a channel-forming toxin in plannar lipid bilayers; and (2) the 21-kDa N-terminal region, which maps for the first five helices (alpha1-alpha5) of domain 1 in other Cry toxins, and which putatively shares a similar tri-dimensional structure, is sufficient to account for the ion channel activity of the whole toxin. Using circular dichroism spectroscopy and planar lipid bilayers, we showed that the 21-kDa polypeptide existed as an alpha-helical structure and that both Cry4B and its alpha1-alpha5 fragment formed ion channels of 248 +/- 44 pS and 207 +/- 23 pS, respectively. The channels were cation-selective with a potassium-to-chloride permeability ratio of 6.7 for Cry4B and 4.5 for its fragment. However, contrary to the full-length toxin, the alpha1-alpha5 region formed channels at low dose; they tended to remain locked in their open state and displayed flickering activity bouts. Thus, like the full-length toxin, the alpha1-alpha5 region is a functional channel former. A pH-dependent, yet undefined region of the toxin may be involved in regulating the channel properties.

Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: A critical review

“…In agreement with the suggested involvement of pre-pores in the mechanism of action of Cry toxins, the monomeric form of Cry1Ab was reported to cause only noisy fluctuations in the planar lipid bilayers, whereas well-resolved channel currents were observed with purified oligomers at a concentration at least 20-fold lower than that used for the Cry1Ab monomers (PardoLópez et al, 2006;Rausell et al, 2004b). The absence of well-defined current steps produced in planar lipid bilayers by the monomeric toxin is very surprising, however, in light of the fact that singlechannel currents induced by a variety of other activated Cry toxins, including Cry1Aa (Grochulski et al, 1995), Cry1Ac (Slatin et al, 1990), Cry1Ca (Lorence et al, 1995;Schwartz et al, 1993), Cry2Aa , Cry3Aa (Slatin et al, 1990), Cry4Ba (Puntheeranurak et al, 2004), and the Cry34Ab/Cry35Ab binary toxin , have been described. Actually, as shown in Fig.…”

Crystal Structure of the Mosquito-larvicidal Toxin Cry4Ba and Its Biological Implications

“…57 It is a functional channel-former like the full-length toxin, but there are subtle differences in the channel properties that may be regulated by an undefined region of the toxin. 57 In all the Cry toxin structures [12][13][14][15][16][17] including Cry4Ba, the helical domain I is connected to the b-sheet domain II by a single long linker extending from the C terminus of a7 to the first strand, b1 in sheet 3, of domain II. The unfolding of the protein around a hinge region linking domains I and II was shown, by engineered inter-domain disulphide bridges, to be a necessary step for the pore formation.…”

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

“…18 Like many other bacterial toxins, Cry toxins incorporate into lipid bilayer rafts as well as brush border membrane vesicles (BBMV). 19 Lipid bilayerand BBMV-associated Cry toxin molecules have been reported to appear in oligomeric (dimeric and tetrameric) and monomeric forms, 20,21 and these associations have been correlated with changes in membrane permeability. 22 The oligomeric forms of Cry toxin were presumed to be pores that cause cell death by osmotic lysis.…”