The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis d-endotoxin are consistent with an ‘‘umbrella-like’’ structure of the pore

The aim of this study was to elucidate the mechanism of membrane insertion and the structural organization of pores formed by Bacillus thuringiensis d-endotoxin. We determined the relative affinities for membranes of peptides corresponding to the seven helices that compose the toxin pore-forming domain, their modes of membrane interaction, their structures within membranes, and their orientations relative to the membrane normal. In addition, we used resonance energy transfer measurements of all possible combinatorial pairs of membrane-bound helices to map the network of interactions between helices in their membrane-bound state. The interaction of the helices with the bilayer membrane was also probed by a Monte Carlo simulation protocol to determine lowest-energy orientations. Our results are consistent with a situation in which helices a4 and a5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like the ribs of an umbrella (the ‘‘umbrella model’’). Our results also support the suggestion that a7 may serve as a binding sensor to initiate the structural rearrangement of the pore-forming domain. Growing public concern regarding the use of chemical insecticides has led to the extensive use of environment-friendly alternatives, of which the d-endotoxins are the preferred choice of the insect biocontrol market (1). The d-endotoxins are highly potent insecticidal toxins produced by Bacillus thuringiensis bacteria. The use of d-endotoxins rather than conventional chemical pesticides is preferable, both because of their high specificity and efficiency and because of their environmental safety and lack of harmful side effects. Research over the past three decades has attempted to elucidate the structure and activity of d-endotoxins. Although the crystal structure of one of the d-endotoxins was determined more than 6 years ago (2), the conformation of the pores formed by the toxin and the molecular mechanism of toxin interaction with and insertion into membranes are still not clear. Insights regarding the structure of the pore-forming domain within the membrane could significantly advance understanding of the mode of action of these toxins and advance the design of more potent toxins. In addition, structural studies of d-endotoxins within a membrane environment are highly important as a paradigm for the mechanism of insertion and organization and specific interactions within membranes of other membranepermeating toxins and integral membrane proteins. To date, elucidation of the function of membrane proteins has been very limited because of the difficulty in obtaining structural information within a membrane environment by crystallography or NMR spectroscopy (3). The toxins are released as protoxins, which are solubilized in the midgut of insects and activated by gut proteases. It is assumed that the trigger for the insertion of the pore-forming domain of the toxins into the epithelial cell membrane is a conformational change in the toxin, which occurs when another domain of the toxin binds to a receptor present on brush-border membranes (4, 5). The pore-forming properties of the toxins have been demonstrated by studies in which activated d-endotoxins form single ion channels in planar lipid bilayers and cultured insect cells (6, 7). The crystal structures of two d-endotoxins have been determined (2, 8). The toxins are composed of three distinct domains (Fig. 1). Domain I, the pore-forming domain, is composed of a bundle of six a-helices surrounding a5, the central helix. Domain II, the receptor-binding domain, is composed of three b-sheets with loops at the apex of the b-hairpin extensions, and domain III has a two antiparallel b-sheet sandwich structure. The toxins exhibit a remarkably high degree of similarity in their structures, particularly in the pore-forming domain. The structure of domain I of the toxin, the effect of site-directed mutagenesis in this domain on toxin activity, and studies with hybrid toxins (5, 9, 10) all suggest that domain I, or parts of it, inserts into the membrane and forms a pore. This idea is further supported by studies that show that truncated proteins corresponding to domain I of CryIA(c) (11) d-endotoxin form ion channels in model lipid membranes similar to those formed by the intact toxins. Extensive mutagenesis studies indicate that mutations in a5, but not a2 or a6, result in a substantial number of inactive or low-activity toxins (9, 12). Studies with synthetic peptides corresponding to a5 and a7, the most conserved helices of the pore-forming domain, from CryIIIA (13–15) and a5 of CryIA(c) (16) suggest that a5, but not a7, aggregates within lipid membranes, permeates phospholipid vesicles, and forms ion channels within planar lipid bilayers. Similar to the results obtained with model membranes, it was found that a5 binds insect midgut membranes, is protected from enzymatic proteolysis upon binding, and is cytotoxic to Sf-9 insect cells (14). In this study we compare membrane interactions, the structure within membranes, the orientation relative to the membrane plane, and the structural organization in the membrane-bound state of the seven helices composing the pore-forming domain of the CryIIIA d-endotoxin. Taken together, our results are consistent with an ‘‘umbrella’’ model for the structure of the pores formed by the toxin. These results also further support our previous suggestions regarding the role of the a7 helix as the binding sensor of the pore-forming domain (15). MATERIALS AND METHODS Peptide Synthesis, Fluorescent Labeling, and Purification. The peptides were synthesized by the solid-phase method (17) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9512289-6$2.00y0 PNAS is available online at www.pnas.org. Abbreviations: NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; Rho, rhodamine; SUV, small unilamellar vesicle; ATR, attenuated total reflectance; MC, Monte Carlo. §To whom reprint requests should be addressed. e-mail: bmshai@ weizmann.weizmann.ac.il.