Bacillus thuringiensis Cry4A and Cry4B mosquito-larvicidal proteins: homology-based 3D model and implications for toxin activity

Three-dimensional (3D) models for the 65-kDa activated Cry4A and Cry4B delta-endotoxins from Bacillus thuringiensis subsp. israelensis that are specifically toxic to mosquito-larvae were constructed by homology modeling, based on atomic coordinates of the Cry1Aa and Cry3Aa crystal structures. They were structurally similar to the known structures, both derived 3D models displayed a three-domain organization: the N-terminal domain (I) is a seven-helix bundle, while the middle and C-terminal domains are primarily comprise of anti-parallel beta-sheets. Circular dichroism spectroscopy confirmed the secondary structural contents of the two homology-based Cry4 structures. A structural analysis of both Cry4 models revealed the following: (a) Residues Arg-235 and Arg-203 are located in the interhelical 5/6 loop within the domain I of Cry4A and Cry4B, respectively. Both are solvent exposed. This suggests that they are susceptible to tryptic cleavage. (b) The unique disulphide bond, together with a proline-rich region within the long loop connecting alpha4 and alpha5 of Cry4A, were identified. This implies their functional significance for membrane insertion. (c) Significant structural differences between both models were found within domain II that may reflect their different activity spectra. Structural insights from this molecular modeling study would therefore increase our understanding of the mechanic aspects of these two closely related mosquito-larvicidal proteins.     

Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae. [BMB Reports 2014; 47(10): 546-551]     

The crystal structure of a major dust mite allergen Der p 2, and its biological implications

The crystal structure of the common house mite (Dermatophagoides sp.) Der p 2 allergen was solved at 2.15 A resolution using the MAD phasing technique, and refined to an R-factor of 0.209. The refined atomic model, which reveals an immunoglobulin-like tertiary fold, differs in important ways from the previously described NMR structure, because the two beta-sheets are significantly further apart and create an internal cavity, which is occupied by a hydrophobic ligand. This interaction is structurally reminiscent of the binding of a prenyl group by a regulatory protein, the Rho guanine nucleotide exchange inhibitor. The crystal structure suggests that binding of non-polar molecules may be essential to the physiological function of the Der p 2 protein.     

Insertion behavior of the Bacillus thuringiensis Cry4Ba insecticidal protein into lipid monolayers

Toxicity mechanisms of Bacillus thuringiensis Cry insecticidal proteins involve membrane insertion and lytic pore formation in lipid bilayers of the target larval midgut cell membranes. The B. thuringiensis Cry4Ba mosquito-larvicidal protein has been shown to be capable of permeabilizing liposome vesicles and of forming ion channels in planar lipid bilayers. Here, the membrane interaction of the 65-kDa activated Cry4Ba protein with the lipid monolayers, comprising dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, and cholesterol (Chol), was studied using Langmuir-Blodgett technique. The interactions of the Cry4Ba protein with the lipid monolayers were measured from the surface pressure versus area isotherms of the protein-lipid monolayers. The increase in the mean molecular area was demonstrated as an incorporation of the protein into lipid monolayers. The insertion of the Cry4Ba protein was monitored by measuring as an increase of the surface pressure at constant molecular area. For a given monolayer, the membrane insertion of the Cry4Ba reduced as the initial surface pressure increased. The Cry4Ba protein showed a strong preference of an insertion towards a Chol monolayer. In addition, the mixed monolayers of Chol showed an enhanced effect on the insertion kinetics of Cry4Ba into lipid films, suggesting its involvement in the modulation of the protein insertion. These findings provide the first evidence that the Cry4Ba protein is capable of inserting itself into lipid monolayers, depending on the packing density of the monolayers. Our results also indicate that only a limited part of the protein is likely to be involved in the insertion.     

Asn183 in alpha5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin

The proposed toxicity mechanism of the Bacillus thuringiensis Cry insecticidal proteins involves membrane penetration and lytic pore formation of the alpha4-alpha5 hairpins in the target larval midgut cell membranes. In this study, alanine substitutions of selected polar residues (Tyr(178), Gln(180), Asn(183), Asn(185), and Asn(195)) in the hydrophobic helix-alpha5 of the Cry4Ba mosquito-larvicidal protein were initially conducted via PCR-based directed mutagenesis. Upon IPTG induction, all the 130-kDa mutant protoxins were highly expressed in Escherichia coli as cytoplasmic inclusions, with yields similar to the wild-type protoxin. When E. coli cells expressing each mutant toxin were tested against Stegomyia aegypti mosquito larvae, the larvicidal activity of the N183A mutant was almost completely abolished whereas the four other mutants showed only a small reduction in toxicity. Additionally, replacements of this critical residue with various amino acids revealed that the uncharged polar residue at position 183 in alpha5 is crucial for larvicidal activity. Further characterisation of the N183K bio-inactive mutant revealed that the 65-kDa activated toxin was unable to form oligomers in lipid vesicles and its ability to induce the release of entrapped calcein from liposomes was much weaker than that of the wild-type toxin. These results suggest that the highly conserved Asn(183) located in the middle of the transmembrane alpha5 of Cry4Ba plays a crucial role in toxicity and toxin oligomerisation in the lipid membranes.     

Crystal structure of atypical cytoplasmic ABC-ATPase SufC from Thermus thermophilus HB8

SufC, a cytoplasmic ABC-ATPase, is one of the most conserved Suf proteins. SufC forms a stable complex with SufB and SufD, and the SufBCD complex interacts with other Suf proteins in the Fe-S cluster assembly. We have determined the crystal structure of SufC from Thermus thermophilus HB8 in nucleotide-free and ADP-Mg-bound states at 1.7A and 1.9A resolution, respectively. The overall architecture of the SufC structure is similar to other ABC ATPases structures, but there are several specific motifs in SufC. Three residues following the end of the Walker B motif form a novel 3(10) helix which is not observed in other ABC ATPases. Due to the novel 3(10) helix, a conserved glutamate residue involved in ATP hydrolysis is flipped out. Although this unusual conformation is unfavorable for ATP hydrolysis, salt-bridges formed by conserved residues and a strong hydrogen-bonding network around the novel 3(10) helix suggest that the novel 3(10) helix of SufC is a rigid conserved motif. Compared to other ABC-ATPase structures, a significant displacement occurs at a linker region between the ABC alpha/beta domain and the alpha-helical domain. The linker conformation is stabilized by a hydrophobic interaction between conserved residues around the Q loop. The molecular surfaces of SufC and the C-terminal helices of SufD (PDB code: 1VH4) suggest that the unusual linker conformation conserved among SufC proteins is probably suitable for interacting with SufB and SufD.     

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.     

Targeted mutagenesis of loop residues in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin affects larvicidal activity

Loop residues in domain II of Bacillus thuringiensis Cry delta-endotoxins have been demonstrated to be involved in insecticidal specificity. In this study, selected residues in loops beta6-beta7 (S(387)SPS(390)), beta8-beta9 (S(410), N(411), T(413), T(415), E(417) and G(418)) and beta10-beta11 (D(454)YNS(457)) in domain II of the Cry4Ba mosquito-larvicidal protein were changed individually to alanine by PCR-based directed mutagenesis. All mutant toxins were expressed in Escherichia coli JM109 cells as 130-kDa protoxins at levels comparable to the wild type. Only E. coli cells that express the P389A, S410A, E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type. In addition, E. coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild-type levels revealed a significantly higher loss in larvicidal activity of approximately 70%. Similar to the wild-type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These results indicate that S(410) and E(417) in the beta8-beta9 loop, and Y(455) and N(456) in the beta10-beta11 loop are involved in larvicidal activity of the Cry4Ba toxin.     

Crystal structure of human Edc3 and its functional implications

Edc3 is an enhancer of decapping and serves as a scaffold that aggregates mRNA ribonucleoproteins together for P-body formation. Edc3 forms a network of interactions with the components of the mRNA decapping machinery and has a modular domain architecture consisting of an N-terminal Lsm domain, a central FDF domain, and a C-terminal YjeF-N domain. We have determined the crystal structure of the N-terminally truncated human Edc3 at a resolution of 2.2 Å. The structure reveals that the YjeF-N domain of Edc3 possesses a divergent Rossmann fold topology that forms a dimer, which is supported by sedimentation velocity and sedimentation equilibrium analysis in solution. The dimerization interface of Edc3 is highly conserved in eukaryotes despite the overall low sequence homology across species. Structure-based site-directed mutagenesis revealed dimerization is required for efficient RNA binding, P-body formation, and likely for regulating the yeast Rps28B mRNA as well, suggesting that the dimeric form of Edc3 is a structural and functional unit in mRNA degradation.     

Binding characteristics to mosquito-larval midgut proteins of the cloned domain II-III fragment from the Bacillus thuringiensis Cry4Ba toxin

Receptor binding plays an important role in determining host specificity of the Bacillus thuringiensis Cry delta-endotoxins. Mutations in domains II and III have suggested the participation of certain residues in receptor recognition and insect specificity. In the present study, we expressed the cloned domain II-III fragment of Cry4Ba and examined its binding characteristics to mosquito-larval midgut proteins. The 43-kDa Cry4Ba-domain II-III protein over-expressed in Escherichia coli as inclusion bodies was only soluble when carbonate buffer, pH 10.0 was supplemented with 4 M urea. After renaturation via stepwise dialysis and subsequent purification, the refolded domain II-III protein, which specifically reacts with anti Cry4Ba-domain III monoclonal antibody, predominantly exists as a beta-sheet structure determined by circular dichroism spectroscopy. In vitro binding analysis to both histological midgut tissue sections and brush border membrane proteins prepared from susceptible Aedes aegypti mosquito-larvae revealed that the isolated Cry4Ba-domain II-III protein showed binding functionality comparable to the 65-kDa full-length active toxin. Altogether, the data present the 43-kDa Cry4Ba fragment comprising domains II and III that was produced in isolation was able to retain its receptor-binding characteristics to the target larval midgut proteins.     

Crystal structure of the BAFF-BAFF-R complex and its implications for receptor activation

B-cell activating factor (BAFF) is a key regulator of B-lymphocyte development. Its biological role is mediated by the specific receptors BCMA, TACI and BAFF-R. We have determined the crystal structure of the extracellular domain of BAFF-R bound to BAFF at a resolution of 3.3 A. The cysteine-rich domain (CRD) of the BAFF-R extracellular domain adopts a beta-hairpin structure and binds to the virus-like BAFF cage in a 1:1 molar ratio. The conserved DxL motif of BAFF-R is located on the tip of the beta-turn and is indispensable in the binding of BAFF. The crystal structure shows that a unique dimeric contact occurs between the BAFF-R monomers in the virus-like cage complex. The extracellular domain of TACI contains two CRDs, both of which contain the DxL motif. Modeling of TACI-BAFF complex suggests that both CDRs simultaneously interact with the BAFF dimer in the virus-like cage.     

Crystal structure of the jacalin-T-antigen complex and a comparative study of lectin-T-antigen complexes

Thomsen-Friedenreich antigen (Galbeta1-3GalNAc), generally known as T-antigen, is expressed in more than 85% of human carcinomas. Therefore, proteins which specifically bind T-antigen have potential diagnostic value. Jacalin, a lectin from jack fruit (Artocarpus integrifolia) seeds, is a tetramer of molecular mass 66kDa. It is one of the very few proteins which are known to bind T-antigen. The crystal structure of the jacalin-T-antigen complex has been determined at 1.62A resolution. The interactions of the disaccharide at the binding site are predominantly through the GalNAc moiety, with Gal interacting only through water molecules. They include a hydrogen bond between the anomeric oxygen of GalNAc and the pi electrons of an aromatic side-chain. Several intermolecular interactions involving the bound carbohydrate contribute to the stability of the crystal structure. The present structure, along with that of the Me-alpha-Gal complex, provides a reasonable qualitative explanation for the known affinities of jacalin to different carbohydrate ligands and a plausible model of the binding of the lectin to T-antigen O-linked to seryl or threonyl residues. Including the present one, the structures of five lectin-T-antigen complexes are available. GalNAc occupies the primary binding site in three of them, while Gal occupies the site in two. The choice appears to be related to the ability of the lectin to bind sialylated sugars. In either case, most of the lectin-disaccharide interactions are at the primary binding site. The conformation of T-antigen in the five complexes is nearly the same.     

Crystal structure of zinc-finger domain of Nanos and its functional implications

Nanos is an RNA-binding protein that is involved in the development and maintenance of germ cells. In combination with Pumilio, Nanos binds to the 3' untranslated region of a messenger RNA and represses its translation. Nanos has two conserved Cys-Cys-His-Cys zinc-finger motifs that are indispensable for its function. In this study, we have determined the crystal structure of the zinc-finger domain of zebrafish Nanos, for the first time revealing that Nanos adopts a novel zinc-finger structure. In addition, Nanos has a conserved basic surface that is directly involved in RNA binding. Our results provide the structural basis for further studies to clarify Nanos function.     

Crystal structure of CD14 and its implications for lipopolysaccharide signaling

Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. Here, we present the first crystal structure of CD14 to 2.5 angstroms resolution. A large hydrophobic pocket was found on the NH2-terminal side of the horseshoe-like structure. Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide-binding site. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding.     

Crystal structure of pyruvate dehydrogenase phosphatase 1 and its functional implications

Pyruvate dehydrogenase phosphatase 1 (PDP1) catalyzes dephosphorylation of pyruvate dehydrogenase (E1) in the mammalian pyruvate dehydrogenase complex (PDC), whose activity is regulated by the phosphorylation-dephosphorylation cycle by the corresponding protein kinases (PDHKs) and phosphatases. The activity of PDP1 is greatly enhanced through Ca2+ -dependent binding of the catalytic subunit (PDP1c) to the L2 (inner lipoyl) domain of dihydrolipoyl acetyltransferase (E2), which is also integrated in PDC. Here, we report the crystal structure of the rat PDP1c at 1.8 A resolution. The structure reveals that PDP1 belongs to the PPM family of protein serine/threonine phosphatases, which, in spite of a low level of sequence identity, share the structural core consisting of the central beta-sandwich flanked on both sides by loops and alpha-helices. Consistent with the previous studies, two well-fixed magnesium ions are coordinated by five active site residues and five water molecules in the PDP1c catalytic center. Structural analysis indicates that, while the central portion of the PDP1c molecule is highly conserved among the members of the PPM protein family, a number of structural insertions and deletions located at the periphery of PDP1c likely define its functional specificity towards the PDC. One notable feature of PDP1c is a long insertion (residues 98-151) forming a unique hydrophobic pocket on the surface that likely accommodates the lipoyl moiety of the E2 domain in a fashion similar to that of PDHKs. The cavity, however, appears more open than in PDHK, suggesting that its closure may be required to achieve tight, specific binding of the lipoic acid. We propose a mechanism in which the closure of the lipoic acid binding site is triggered by the formation of the intermolecular (PDP1c/L2) Ca2+ binding site in a manner reminiscent of the Ca2+ -induced closure of the regulatory domain of troponin C.     

Crystal structure of an asymmetric complex of pyruvate dehydrogenase kinase 3 with lipoyl domain 2 and its biological implications

A homodimer of pyruvate dehydrogenase kinase (PDHK) is an integral part of pyruvate dehydrogenase complex (PDC) to which it is anchored primarily through the inner lipoyl-bearing domains (L2) of transacetylase component. The catalytic cycle of PDHK and its translocation over the PDC surface is thought to be mediated by the "symmetric" and "asymmetric" modes, in which the PDHK dimer binds to two and one L2-domain(s), respectively. Whereas the structure of the symmetric PDHK/L2 complex was reported, the structural organization and functional role of the asymmetric complex remain obscure. Here, we report the crystal structure of the asymmetric PDHK3/L2 complex that reveals several functionally important features absent from the previous structures. First, the PDHK3 subunits have distinct conformations: one subunit exhibits "open" and the other "closed" configuration of the putative substrate-binding cleft. Second, access to the closed cleft is additionally restricted by local unwinding of the adjacent alpha-helix. Modeling indicates that the target peptide might gain access to the PDHK active center through the open but not through the closed cleft. Third, the ATP-binding loop in one PDHK3 subunit adopts an open conformation, implying that the nucleotide loading into the active site is mediated by the inactive "pre-insertion" binding mode. Altogether our data suggest that the asymmetric complex represents a physiological state in which binding of a single L2-domain activates one of the PDHK protomers while inactivating another. Thus, the L2-domains likely act not only as the structural anchors but also modulate the catalytic cycle of PDHK.     

Structural and functional relationships among the RTX toxin determinants of Gram-negative bacteria

Abstract The RTX (repeats in toxin) cytolytic toxins r represent a family of important virulence factors that have disseminated widely among Gram-negative bacteria. They are characterised by a series of glycine-rich repeat units at the C-terminal end of each protein. They also have other features in common. Secretion from the cell occurs without a periplasmic intermediate by a novel mechanism which involves recognition of a signal sequence at the C-terminus of the toxin by membrane-associated proteins that export the toxin directly to the outside of the cell. The structural gene for each protein encodes an inactive toxin which is modified post-translationally to an active cytotoxic form by another gene product before secretion. The genes for toxin synthesis, activation and secretion are for the most part grouped together on the chromosome and form an operon. The toxins all create pores in the cell membrane of target cells leading to eventual cell lysis and they appear to require Ca²⁺ for cytotoxic activity. Although the toxins have a similar mode of action, they vary in target cell specificity. Some are cytotoxic for a wide variety of eukaryotic cell types while others exhibit precise target cell specificity and are only active against leukocytes from certain host species. The characteristic glycine-rich repeat units have been identified in other exoproteins besides those with cytotoxic activity and it is likely that the novel secretory mechanism has been harnessed by a variety of pathogens to release important virulence-associated factors from the cell or to locate them on the cell surface.     

Crystal structure of archaeal toxin-antitoxin RelE-RelB complex with implications for toxin activity and antitoxin effects

The Escherichia coli chromosome encodes toxin-antitoxin pairs. The toxin RelE cleaves mRNA positioned at the A-site in ribosomes, whereas the antitoxin RelB relieves the effect of RelE. The hyperthermophilic archaeon Pyrococcus horikoshii OT3 has the archaeal homologs aRelE and aRelB. Here we report the crystal structure of aRelE in complex with aRelB determined at a resolution of 2.3 A. aRelE folds into an alpha/beta structure, whereas aRelB lacks a distinct hydrophobic core and extensively wraps around the molecular surface of aRelE. Neither component shows structural homology to known ribonucleases or their inhibitors. Site-directed mutagenesis suggests that Arg85, in the C-terminal region, is strongly involved in the functional activity of aRelE, whereas Arg40, Leu48, Arg58 and Arg65 play a modest role in the toxin's activity.     

Characterization of enterotoxin and soluble hemagglutinin from Vibrio mimicus: identity with V. cholerae O1 toxin and hemagglutinin

Abstract Eight strains of Vibrio mimicus isolated from patients with diarrhoea in Bangladesh were all found to produce an extracellular toxin identical to cholera toxin produced by Vibrio cholerae O1 bacteria, with regard to subunit structure and immunological properties. Like cholera toxin, but in contrast to heat-labile enterotoxin from Escherichia coli most of the toxin from V. mimicus was found extracellularly and was proteolytically 'nicked' in its A subunit. This may relate to the finding that V. mimicus also produced an extracellular hemagglutinin which was immunologically indistinguishable from the soluble hemagglutinin/nicking protease of V. cholerae O1.