Cyt1Aa toxin: crystal structure reveals implications for its membrane-perforating function

During sporulation, Bacillus thuringiensis subsp. israelensis produces a mosquito larvicidal protein complex containing several crystalline and cytolytic (Cyt) toxins. Here, the activated monomeric form of Cyt1Aa, the most toxic Cyt family member, was isolated and crystallized, and its structure was determined for the first time at 2.2 Å resolution.Cyt1Aa adopts a typical cytolysin fold containing a β-sheet held by two surrounding α-helical layers. The absence of a β-strand (between residues V26 and I37) in the dimeric structure of Cyt2Aa led us to deduce that this is the only essential segment for dimer formation and that activation of the toxin occurs by proteolytic processing of its N-terminus. Based on the Cyt1Aa structure, we suggest that the toxicity of Cyt1Aa and other nonrelated proteins, all sharing a cytolysin fold, is correlated with their ability to undergo conformational changes that are necessary prior to their membrane insertion and perforation. This fold allows the α-helical layers to swing away, exposing the β-sheet to insert into the membrane. The identification of a putative lipid binding pocket between the β-sheet and the helical layer of Cyt1Aa supports this mechanism. Sequence-based structural analysis of Cyt1Aa revealed that the lack of activity of Cyt1Ca may be related to the latter's inability to undergo this conformational change due to its lack of flexibility. The pattern of the hemolytic activity of Cyt1Aa presented here (resembling that of pore-forming agents), while differing from that imposed by ionic and nonionic detergents, further supports the pore-forming model by which conformational changes occur prior to membrane insertion and perforation.     

Cloning and characterization of a cytolytic and mosquitocidal delta-endotoxin from Bacillus thuringiensis subsp. jegathesan.

A cytolytic toxin gene encoding a 30.1-kDa Cyt2Bb1 toxin protein from B. thuringiensis subsp. jegathasan was cloned employing a limited-growth PCR screening method with forward and reverse oligonucleotide primers designed from N-terminal amino acid sequences of native and trypsin-cleaved protein, respectively. The expressed protein showed little cross-reactivity to the antibody raised against the Cyt1Aa protein. Unlike Cyt1Aa and Cyt2Aa expression, there was little or no visible crystal inclusion formation under microscopic observation. The amino acid sequence alignment indicated 31 and 66% identity to Cyt1Aa and Cyt2Aa, respectively. The sequence alignment for five known cytolytic proteins indicated three highly conserved regions, two in the loop regions between alpha-helices and beta-sheets and one in the loop region between beta-sheets 5 and 6. beta-Blocks 4 to 7 are also conserved, not only structurally but also among the amino acids in the hydrophobic faces. Mosquitocidal activity assays indicated that the Cyt2Bb toxin had less toxicity than Cyt1Aa and had about 600-times-lower toxicity than the wild-type whole toxin crystal. However, both the Cyt2Bb and the Cyt1Aa toxin showed comparable levels of hemolytic activity.     

Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells

Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 Å. The protein is unusually elongated and mainly comprises long β-strands aligned with its long axis. It is similar to aerolysin-type β-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small β-sheet sandwiched by short α-helices, is probably the target-binding module. Two other domains are both β-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming β-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The β-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.     

An ADAM metalloprotease is a Cry3Aa Bacillus thuringiensis toxin receptor

Bacillus thuringiensis insecticidal proteins toxic action relies on the interaction with receptor molecules on insect midgut target cells. Here, we describe an ADAM metalloprotease as a novel type of B. thuringiensis toxin receptor on the basis of the following data: (i) by ligand blot and N-terminal analysis, we detected a Colorado potato beetle Cry3Aa toxin binding molecule that shares homology with an ADAM10 metalloprotease; (ii) Colorado potato beetle brush border membrane vesicles display ADAM activity since it cleaves an ADAM fluorogenic substrate; (iii) Cry3Aa acts as a competitor of the cleavage of the ADAM fluorogenic substrate; (iv) Cry3Aa sequence contains the recognition motif R(345)FQPGYYGND(354) present in ADAM10 substrates. Accordingly, a peptide representative of the recognition motif localized within loop 1 of Cry3Aa domain II (Ac-F(341)HTRFQPGYYGNDSFN(358)-NH(2)) effectively prevented Cry3Aa proteolytic processing and nearly abolished pore formation, evidencing the functional significance of the Cry3Aa-ADAM interaction in relation to this toxin mode of action.     

Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism

Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop β6-αE K198A, E204A and β7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop α-8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.     

Prediction of the structure of the replication initiator protein DnaA

The secondary structure of DnaA protein and its interaction with DNA and ribonucleotides has been predicted using biochemical, biophysical techniques, and prediction methods based on multiple-sequence alignment and neural networks. The core of all proteins from the DnaA family consists of an “open twisted α/β structure,” containing five α-helices alternating with five β-strands. In our proposed structural model the interior of the core is formed by a parallel β-sheet, whereas the α-helices are arranged on the surface of the core. The ATP-binding motif is located within the core, in a loop region following the first β-strand. The N-terminal domain (80 aa) is composed of two α-helices, the first of which contains a potential leucine zipper motif for mediating protein-protein interaction, followed by a β-strand and an additional α-helix. The N-terminal domain and the α/β core region of DnaA are connected by a variable loop (45–70 aa); major parts of the loop region can be deleted without loss of protein activity. The C-terminal DNA-binding domain (94 aa) is mostly α-helical and contains a potential helix-loop-helix motif. DnaA protein does not dimerize in solution; instead, the two longest C-terminal α-helices could interact with each other, forming an internal “coiled coil” and exposing highly basic residues of a small loop region on the surface, probably responsible for DNA backbone contacts. © 1997 Wiley-Liss Inc.     

Partial restoration of antibacterial activity of the protein encoded by a cryptic open reading frame (cyt1Ca) from Bacillus thuringiensis subsp. israelensis by site-directed mutagenesis

Insecticidal crystal proteins of Bacillus thuringiensis belong to two unrelated toxin families: receptor-specific Cry toxins against insects and Cyt toxins that lyse a broad range of cells, including bacteria, via direct binding to phospholipids. A new cyt-like open reading frame (cyt1Ca) encoding a 60-kDa protein, has recently been discovered (C. Berry et al., Appl. Environ. Microbiol. 68:5082-5095, 2002). Cyt1Ca displays the structure of a two-domain fusion protein: the N-terminal moiety resembles the full-length Cyt toxins, and the C-terminal moiety is similar to the receptor-binding domains of several ricin-like toxins, such as Mtx1. Neither the larvicidal activity of cyt1Ca expressed in Escherichia coli nor the hemolytic effect of His-tagged purified Cyt1Ca has been observed (R. Manasherob et al., unpublished). This was attributed to five amino acid differences between the sequences of its N-terminal moiety and Cyt1Aa. The 3' end of cyt1Ca was truncated (removing the ricin-binding domain of Cyt1Ca), and six single bases were appropriately changed by site-directed mutagenesis, sequentially replacing the non-charged amino acids by charged ones, according to Cyt1Aa, to form several versions. Expression of these mutated cyt1Ca versions caused loss of the colony-forming ability of the corresponding E. coli cells to different extents compared with the original gene. In some mutants this antibacterial effect was associated by significant distortion of cell morphology and in others by generation of multiple inclusion bodies spread along the cell envelope. The described deleterious effects of mutated cyt1Ca versions against E. coli may reflect an evolutionary relationship between Cyt1Aa and Cyt1Ca.     

Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane

Lysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon-water interface from the water subphase which suggested self-association of lysenin-His in solution. An assembly of premature oligomers by lysenin-His in solution was confirmed by blue native gel electrophoresis. When a monolayer composed of sphingomyelin and cholesterol was present at the interface, the rate of insertion of lysenin-His into the monolayer was considerably enhanced. Analysis of FTIR spectra of soluble lysenin-His demonstrated that the protein contained 27% β-sheet, 28% aggregated β-strands, 10% α-helix, 23% turns and loops and 12% different kinds of aggregated forms. In membrane-bound lysenin-His the total content of α-helices, turns and loops, and β-structures did not change, however, the 1636cm⁻¹ β-sheet band increased from 18% to 31% at the expense of the 1680cm⁻¹ β-sheet structure. Spectral analysis of the amide I band showed that the α-helical component was oriented with at 41° to the normal to the membrane, indicating that this protein segment could be anchored in the hydrophobic core of the membrane.     

Design,synthesis and evaluation of antimicrobial activity of N-terminal modified Leucocin A analogues

Class IIa bacteriocins are potent antimicrobial peptides produced by lactic acid bacteria to destroy competing microorganisms. The N-terminal domain of these peptides consists of a conserved YGNGV sequence and a disulphide bond. The YGNGV motif is essential for activity, whereas, the two cysteines involved in the disulphide bond can be replaced with hydrophobic residues. The C-terminal region has variable sequences, and folds into a conserved amphipathic α-helical structure. To elucidate the structure–activity relationship in the N-terminal domain of these peptides, three analogues (1–3) of a class IIa bacteriocin, Leucocin A (LeuA), were designed and synthesized by replacing the N-terminal β-sheet residues of the native peptide with shorter β-turn motifs. Such replacement abolished the antibacterial activity in the analogues, however, analogue 1 was able to competitively inhibit the activity of native LeuA. Native LeuA (37-mer) was synthesized using native chemical ligation method in high yield. Solution conformation study using circular dichroism spectroscopy and molecular dynamics simulations suggested that the C-terminal region of analogue 1 adopts helical folding as found in LeuA, while the N-terminal region did not fold into β-sheet conformation. These structure–activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins.     

High-resolution crystal structure of activated Cyt2Ba monomer from Bacillus thuringiensis subsp. israelensis

The Cyt family of proteins consists of δ-endotoxins expressed during sporulation of several subspecies of Bacillus thuringiensis. Its members possess insecticidal, hemolytic, and cytolytic activities through pore formation and attract attention due to their potential use as vehicles for targeted membrane destruction. The δ-endotoxins of subsp. israelensis include three Cyt species: a major Cyt1Aa and two minor proteins, Cyt2Ba and Cyt1Ca. A cleaved Cyt protein that lacks the N- and C-terminal segments forms a toxic monomer. Here, we describe the crystal structure of Cyt2Ba, cleaved at its amino and carboxy termini by bacterial endogenous protease(s). Overall, its fold resembles that of the previously described volvatoxin A2 and the nontoxic form of Cyt2Aa. The structural similarity between these three proteins may provide information regarding the mechanism(s) of membrane-perforating toxins.     

A membrane associated metalloprotease cleaves Cry3Aa Bacillus thuringiensis toxin reducing pore formation in Colorado potato beetle brush border membrane vesicles

Insect proteases are implicated in Bacillus thuringiensis insecticidal proteins mode of action determining toxin specificity and sensitivity. Few data are available on the involvement of proteases in the later steps of toxicity such as protease interaction with toxin-receptor complexes and the pore formation process. In this study, a Colorado potato beetle (CPB) midgut membrane metalloprotease was found to be involved in the proteolytic processing of Cry3Aa. Interaction of Cry3Aa with BBMV membrane proteases resulted in a distinct pattern of proteolysis. Cleavage was demonstrated to occur in protease accessible regions of domain III and was specifically inhibited by the metalloprotease inhibitors 1,10-phenanthroline and acetohydroxamic acid. Proteolytic inhibition by a peptide representing a segment of proteolysis in domain III and the metalloprotease inhibitor acetohydroxamic acid correlated with increased pore formation, evidencing that Cry3Aa is a specific target of a CPB membrane metalloprotease that degrades potentially active toxin.     

Binding of Cyt1Aa and Cry11Aa toxins of Bacillus thuringiensis serovar israelensis to brush border membrane vesicles of Tipula paludosa (Diptera: Nematocera) and subsequent pore formation

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.     

Alanine-162 of Bacillus thuringiensis Cyt2Aa2 toxin is essential for membrane binding and oligomerisation

Cyt2Aa2 is a cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. It is specifically toxic to dipteran larvae in vivo and is also active against several cell types, such as erythrocytes. The active toxin is proposed to bind to the cell membrane, and membrane pore formation by toxin oligomerisation leads to cell lysis. This study aimed to characterise the role of residues (I139, S159, L160, S161, A162, D209 and V215) potentially involved in the membrane binding of Cyt2Aa2. All mutants, except I139A and V215A, showed similar characteristics to the wild-type toxin after proteinase K cleavage. Three mutants, S159A, L160A and S161A, showed high haemolytic activity but low toxicity against Aedes aegypti. Membrane interaction assays showed that these mutants could bind to rat red blood cells (rRBCs) and oligomerise. The mutant D209N had no haemolytic activity but was still mildly toxic to A. aegypti. The mutant A162V could not lyse rRBCs, even at high concentrations, and showed no toxicity against A. aegypti. Our data suggest that alanine 162 of the Cyt2Aa2 toxin is involved in membrane binding and oligomerisation. Substitution of this amino acid altered the conformation of the toxin and affected its biological activity.     

A membrane associated metalloprotease cleaves Cry3Aa Bacillus thuringiensis toxin reducing pore formation in Colorado potato beetle brush border membrane vesicles

Insect proteases are implicated in Bacillus thuringiensis insecticidal proteins mode of action determining toxin specificity and sensitivity. Few data are available on the involvement of proteases in the later steps of toxicity such as protease interaction with toxin-receptor complexes and the pore formation process. In this study, a Colorado potato beetle (CPB) midgut membrane metalloprotease was found to be involved in the proteolytic processing of Cry3Aa. Interaction of Cry3Aa with BBMV membrane proteases resulted in a distinct pattern of proteolysis. Cleavage was demonstrated to occur in protease accessible regions of domain III and was specifically inhibited by the metalloprotease inhibitors 1,10-phenanthroline and acetohydroxamic acid. Proteolytic inhibition by a peptide representing a segment of proteolysis in domain III and the metalloprotease inhibitor acetohydroxamic acid correlated with increased pore formation, evidencing that Cry3Aa is a specific target of a CPB membrane metalloprotease that degrades potentially active toxin.     

Structure-function relationships of a membrane pore forming toxin revealed by reversion mutagenesis

Cyt2Aa1 is a haemolytic membrane pore forming toxin produced by Bacillus thuringiensis subsp. kyushuensis. To investigate membrane pore formation by this toxin, second-site revertants of an inactive mutant toxin Cyt2Aa1-I150A were generated by random mutagenesis using error-prone PCR. The decrease in side chain length caused by the replacement of isoleucine by alanine at position 150 in the αD-β4 loop results in the loss of important van der Waals contacts that exist in the native protein between I150 and K199 and L203 on αE. 28 independent revertants of I150A were obtained and their relative toxicity can be explained by the position of the residue in the structure and the effect of the mutation on side-chain interactions. Analysis of these revertants revealed that residues on αA, αB, αC, αD and the loops between αA and αB, αD and β5, β6 and β7 are important in pore formation. These residues are on the surface of the molecule suggesting that they may participate in membrane binding and toxin oligomerization. Changing the properties of the amino acid side-chains of these residues could affect the conformational changes required to transform the water-soluble toxin into the membrane insertion competent state.     

Proteolytic processing of the Cyt1Ab1 toxin produced by Bacillus thuringiensis subsp. medellin

Bacillus thuringiensis produces d-endotoxins that require proteolytic processing to become active. The activation of the B. thuringiensis subsp. medellin 28 kDa (Cyt1Ab1) cytolytic toxin by trypsin, chymotrypsin and gut extract from Culex quinquefasciatus larvae was analyzed. The Cyt1Ab1 toxin of B. thuringiensis subsp. medellin was processed by all proteases tested to fragments between 23 and 25 kDa, while processing of the Cyt1Aa1 toxin produce fragments between 22.5 and 24.5 kDa. The Cyt1Ab1 toxin was preferentially processed at the alkaline pH of 12. The in vitro proteolytic processing of the Cyt1Ab1 toxin by C. quinquefasciatus larvae midgut extract showed a 25 kDa fragment; a similar result was observed when the activation was performed in the in vivo experiments. The solubilized Cyt1Ab1 toxin and the protease resistant cores generated by in vitro processing showed hemolytic activity but not mosquitocidal activity. Amino terminal sequence of the C. quinquefasciatus gut extract resistant fragment indicated that the cutting site was located between Lys31 and Asp32, with a sequence DDPNEKNNHNS; while for the trypsin-resistant fragment the cutting site was determined between Leu29 and Arg30, and for the chymotrypsin-resistant fragment between Arg30 and Lys31.     

Binding of Cyt1Aa and Cry11Aa Toxins of Bacillus thuringiensis Serovar israelensis to Brush Border Membrane Vesicles of Tipula paludosa (Diptera: Nematocera) and Subsequent Pore Formation

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.     

Structure of the minor pseudopilin EpsH from the Type 2 secretion system of Vibrio cholerae

Many Gram-negative bacteria use the multi-protein type II secretion system (T2SS) to selectively translocate virulence factors from the periplasmic space into the extracellular environment. In Vibrio cholerae the T2SS is called the extracellular protein secretion (Eps) system,which translocates cholera toxin and several enzymes in their folded state across the outer membrane. Five proteins of the T2SS, the pseudopilins, are thought to assemble into a pseudopilus, which may control the outer membrane pore EpsD, and participate in the active export of proteins in a “piston-like” manner. We report here the 2.0 Å resolution crystal structure of an N-terminally truncated variant of EpsH, a minor pseudopilin from Vibrio cholerae. While EpsH maintains an N-terminal α-helix and C-terminal β-sheet consistent with the type 4a pilin fold, structural comparisons reveal major differences between the minor pseudopilin EpsH and the major pseudopilin GspG from Klebsiella oxytoca: EpsH contains a large β-sheet in the variable domain, where GspG contains an α-helix. Most importantly, EpsH contains at its surface a hydrophobic crevice between its variable and conserved β-sheets, wherein a majority of the conserved residues within the EpsH family are clustered. In a tentative model of a T2SS pseudopilus with EpsH at its tip, the conserved crevice faces away from the helix axis. This conserved surface region may be critical for interacting with other proteins from the T2SS machinery.     

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis. Structural implications

Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N- and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.     

Bacillus thuringiensis insecticidal Cry1Aa toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary success.

Bacillus thuringiensis insecticidal protein, Cry1Aa toxin, binds to a specific receptor in insect midguts and has insecticidal activity. Therefore, the structure of the receptor molecule is probably a key factor in determining the binding affinity of the toxin and insect susceptibility. The cDNA fragment (PX frg1) encoding the Cry1Aa toxin-binding region of an aminopeptidase N (APN) or an APN family protein from diamondback moth, Plutella xylostella midgut was cloned and sequenced. A comparison between the deduced amino acid sequence of PX frg1 and other insect APN sequences shows that Cry1Aa toxin binds to a highly conserved region of APN family protein. In this paper, we propose a model to explain the mechanism that causes B. thuringiensis evolutionary success and differing insect susceptibility to Cry1Aa toxin.