Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R 1 ) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R 1 promotes cleavage of the amino-terminal end, including helix ␣-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R 1 fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.Bacillus thuringiensis is a bacterium that produces crystalline inclusions formed by insecticidal proteins, called Cry toxins, during the sporulation phase of growth. Cry toxins are toxic to different insect orders as well as to other invertebrates, such as nematodes, mites, and protozoa (1). Cry toxins have been used worldwide in the control of insect pests in agriculture, either as transgenic crops or as spray formulations.The molecular mechanism proposed to describe the action of Cry1A toxins, which are active against different lepidopteran insect species, involves several steps. After larval ingestion of the crystalline inclusions, these are solubilized in midgut lumen and activated by proteases releasing a toxic 65-kDa fragment that binds, in a sequential manner, with at least two receptors located in midgut microvilli. The first interaction occurs with cadherin protein (Bt-R 1 2 in the case of Manduca sexta). This interaction promotes further proteolytic processing of the N-terminal end, including helix ␣-1 of the toxin, resulting in the formation of a prepore oligomeric structure (2). The oligomer has higher affinity to secondary receptors, which are anchored by glycosylphosphatidylinositol, such as aminopeptidase-N (APN) or alkaline phosphatase in the case of M. sexta or Heliothis virescens, respectively (3, 4). Glycosylphosphatidylinositol-anchored receptors are located in specific membrane regions called lipid rafts, where the oligomer inserts into the membrane-forming pores, disrupting the osmotic equilibrium and leading to cell death (1, 5). Although this mechanism of action is g...

Pore-forming toxins are biological weapons produced by a variety of living organisms, particularly bacteria but also by insects, reptiles, and invertebrates. These proteins affect the cell membrane of their target, disrupting permeability and leading eventually to cell death. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. The Cry toxins produced by Bacillus thuringiensis are widely used as insecticides. These proteins have been recognized as pore-forming toxins, and their primary action is to lyse midgut epithelial cells in their target insect. To exert their toxic effect, a prepore oligomeric intermediate is formed leading finally to membrane-inserted oligomeric pores. To understand the role of Cry oligomeric pre-pore formation in the insecticidal activity we isolated point mutations that affected toxin oligomerization but not their binding with the cadherin-like, Bt-R 1 receptor. We show the helix ␣-3 in domain I contains sequences that could form coiled-coil structures important for oligomerization. Some single point mutants in this helix bound Bt-R 1 receptors with similar affinity as the wild-type toxin, but were affected in oligomerization and were severally impaired in pore formation and toxicity against Manduca sexta larvae. These data indicate the pre-pore oligomer and the toxin pore formation play a major role in the intoxication process of Cry1Ab toxin in insect larvae. Bacillus thuringiensis (Bt)2 is a Gram-positive bacterium that produces insecticidal Cry toxins. Cry proteins are widely used for insect control in agriculture and forestry and against mosquitoes, due to their high specificity and safety for humans and for the environment (1, 2).Although Bt Cry toxins are widely used as insecticides their mode of action is still not completely understood. These Cry toxins are pore-forming toxins that induce cell death by forming ionic pores following insertion into the membrane, causing osmotic lysis of larvae midgut cells (1-3). However, recently an alternative model proposed that these toxins activate a signal pathway through Bt-R 1 receptor interaction, which results in insect cell death without the participation of lytic pores into the membrane (4). It is important to note that this alternative model was proposed based on the effect of Cry1Ab toxin to cultured Trichoplusia ni H5 insect cells expressing the Manduca sexta toxin receptor, Bt-R 1 .Nevertheless, in both models, receptor interaction with exposed regions in domains II and III of Cry1A toxins (1-4) is a key step that determines insect toxicity. In the case of Cry1A toxins, two receptors have been characterized in several lepidopteran species: cadherin-like proteins, known as Bt-R receptors (5) (Bt-R 1 in the case of M. sexta), and glycosylphosphatidylinositol-anchored proteins, as aminopeptidase-N or alkaline phosphatase (6, 7).In the pore-forming model, it is proposed that both receptors are important and participate in a sequential manner (3,8,9). After proteoly...

In all genome-sequencing projects completed to date, a considerable number of 'gaps' have been found in the biochemical pathways of the respective species. In many instances, missing enzymes are displaced by analogs, functionally equivalent proteins that have evolved independently and lack sequence and structural similarity. Here we fill such gaps by analyzing anticorrelating occurrences of genes across species. Our approach, applied to the thiamin biosynthesis pathway comprising approximately 15 catalytic steps, predicts seven instances in which known enzymes have been displaced by analogous proteins. So far we have verified four predictions by genetic complementation, including three proteins for which there was no previous experimental evidence of a role in the thiamin biosynthesis pathway. For one hypothetical protein, biochemical characterization confirmed the predicted thiamin phosphate synthase (ThiE) activity. The results demonstrate the ability of our computational approach to predict specific functions without taking into account sequence similarity.

Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into ␣-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high ␣-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into ␣-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics.

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