Chanan Angsuthanasombat

Structure of the Functional Form of the Mosquito Larvicidal Cry4Aa Toxin from Bacillus thuringiensis at a 2.8-Angstrom Resolution

The Cry4Aa delta-endotoxin from Bacillus thuringiensis is toxic to larvae of Culex, Anopheles, and Aedes mosquitoes, which are vectors of important human tropical diseases. With the objective of designing modified toxins with improved potency that could be used as biopesticides, we determined the structure of this toxin in its functional form at a resolution of 2.8 angstroms. Like other Cry delta-endotoxins, the activated Cry4Aa toxin consists of three globular domains, a seven-alpha-helix bundle responsible for pore formation (domain I) and the following two other domains having structural similarities with carbohydrate binding proteins: a beta-prism (domain II) and a plant lectin-like beta-sandwich (domain III). We also studied the effect on toxicity of amino acid substitutions and deletions in three loops located at the surface of the putative receptor binding domain II of Cry4Aa. Our results indicate that one loop is an important determinant of toxicity, presumably through attachment of Cry4Aa to the surface of mosquito cells. The availability of the Cry4Aa structure should guide further investigations aimed at the molecular basis of the target specificity and membrane insertion of Cry endotoxins.

Identification of Residues in the Dengue Virus Type 2 NS2B Cofactor That Are Critical for NS3 Protease Activation

ABSTRACT Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virus-encoded NS2B-NS3 two-component protease. The NS3 protease represents an attractive target for the development of antiviral inhibitors. The three-dimensional structure of the NS3 protease domain has been determined, but the structural determinants necessary for activation of the enzyme by the NS2B cofactor have been characterized only to a limited extent. To test a possible functional role of the recently proposed Φx3Φ motif in NS3 protease activation, we targeted six residues within the NS2B cofactor by site-specific mutagenesis. Residues Trp62, Ser71, Leu75, Ile77, Thr78, and Ile79 in NS2B were replaced with alanine, and in addition, an L75A/I79A double mutant was generated. The effects of these mutations on the activity of the NS2B(H)-NS3pro protease were analyzed in vitro by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of autoproteolytic cleavage at the NS2B/NS3 site and by assay of the enzyme with the fluorogenic peptide substrate GRR-AMC. Compared to the wild type, the L75A, I77A, and I79A mutants demonstrated inefficient autoproteolysis, whereas in the W62A and the L75A/I79A mutants self-cleavage appeared to be almost completely abolished. With exception of the S71A mutant, which had a kcat/Km value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced kcat values. These results indicate a pivotal function of conserved residues Trp62, Leu75, and Ile79 in the NS2B cofactor in the structural activation of the dengue virus NS3 serine protease.

Cloning and expression of 130-kd mosquito-larvicidal δ-endotoxin gene of Bacillus thuringiensis var. Israelensis in Escherichia coli

SummaryFive recombinant E. coli clones exhibiting toxicity to Aedes aegypti larvae were obtained from a library of 800 clones containing XbaI DNA fragments of 110 kb plasmid from B. thuringiensis var. israelensis. All the five clones (pMU 14/258/303/388/679) had the same 3.8-kb insert and encoded a major protein of 130 kDa which was highly toxic to A. aegypti larvae. Three clones (pMU 258/303/388) transcribed the 130 kD a gene in the same direction as that of lac Z promoter of pUC12 vector whereas the transcription of the other two (pMU 14/679) was in the opposite direction. A 1.9-kb fragment of the 3.8 kb insert coded for a protein of 65 kDa. Partial DNA sequence of the 3.8 kb insert, corresponding to the 5′-terminal of the 130 kDa gene, revealed a continuous reading frame, a Shine-Dalgarno sequence and a tentative 5′-regulatory region. These results demonstrated that the 3.8 kb insert is a minimal DNA fragment containing a regulatory region plus the coding sequence of the 130 kDa protein that is highly toxic to mosquito larvae.

Ion channels formed in planar lipid bilayers by the dipteran-specific Cry4B Bacillus thuringiensis toxin and its alpha1-alpha5 fragment

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 planar lipid bilayers; and (2) the 21-kDa N-terminal region, which maps for the first five helices (α1–α5) 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 α-helical structure and that both Cry4B and its α1–α5 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 α1–α5 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 α1–α5 region is a functional channel former. A pH-dependent, yet undefined region of the toxin may be involved in regulating the channel properties.

In vivo identification of Bacillus thuringiensis Cry4Ba toxin receptors by RNA interference knockdown of glycosylphosphatidylinositol-linked aminopeptidase N transcripts in Aedes aegypti larvae.

Bacillus thuringiensis Cry4Ba toxin selectively kills Aedes aegypti mosquito larvae as it is in part due to the presence of specific membrane-bound protein receptors. In this study, using data mining approach, we initially identified three potential glycosylphosphatidylinositol-linked aminopeptidase N (GPI-APN) isoforms, APN2778, APN2783 and APN5808, which are believed to act as Cry4Ba toxin receptors. These three isoforms that are functionally expressed in the larval midgut can be sequence-specific knocked down (ranging from ∼80 % to 95 %) by soaking the Aedes aegypti larvae in buffer of long double-stranded GPI-APN RNAs (∼300-680 bp). Finally, to see the physiological effect of APN knockdowns, the larvae were fed with Escherichia coli expressing Cry4Ba toxin. The results revealed that all the three identified GPI-APN isoforms may possibly function as a Cry4Ba receptor, particularly for APN2783 as those larvae with this transcript knockdown showed a dramatic increase in resistance to Cry4Ba toxicity.

Specific mutations within the α4–α5 loop of the bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170

The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry δ-endotoxins suggests that helices α4 and α5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the α4–α5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-β-d-thiogalactopyranoside induction. When E. coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity. Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within the α4–α5 loop play a crucial role in the larvicidal activity of the Cry4B toxin.

Effects on larvicidal activity of single proline substitutions in α3 or α4 of the Bacillus thuringiensis Cry4B toxin

The possible role of α‐helices 3 and 4 in toxicity of the dipteran‐active Bacillus thuringiensis Cry4B δ‐endotoxin was investigated by employing proline substitutions via site‐directed mutagenesis. Similar to the wild‐type Cry4B, the mutant toxins were over‐expressed in Escherichia coli as cytoplasmic inclusions and were structurally stable upon solubilization and trypsin activation. The substitution of glutamine 149 by proline in the center of helix 4 (Q149P) resulted in a nearly complete loss of toxicity against Aedes aegypti mosquito‐larvae. However, single proline replacements near the center of helix 3 (V119P) and at the N‐terminus of helix 4 (Q140P) did not decrease larvicidal activity. The toxicity of E. coli cells expressing the wild‐type toxin was significantly reduced by two‐hour preincubation with the non‐toxic mutant (Q149P), thus indicating that the primary binding step was not affected by the proline substitution in helix 4. The results therefore reveal a crucial role for helix 4 of the Cry4B toxin in toxicity, possibly in membrane insertion and pore formation rather than in receptor recognition.

Steady-state cleavage kinetics for dengue virus type 2 ns2b-ns3(pro) serine protease with synthetic peptides.

The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions. Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for K(1M), k(cat) and k(cat)/K(m) were obtained. The data presented here show that NS2B-NS3(pro) expressed in recombinant E. coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site. A marked difference in cleavage efficiency was found for the NS2B/NS3 substrate and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5 cleavage sites.

The mosquito larvicidal activity of 130 KDA delta-endotoxin of Bacillusthuringiensis var. israelensis resides in the 72 KDA amino-terminal fragment

Bacillus thuringiensis var. israelensis produces 130 kDa delta-endotoxin which is highly toxic to mosquito-larvae. The mosquito-larvicidal activity was delineated by sequential deletions from ends of the 1136 amino acids delta-endotoxin. A maximum of 459 amino acids could be removed from the carboxy-terminal of the toxin without a significant loss of the larvicidal activity. However, no more than 38 amino acids could be deleted from the amino-terminal without losing the toxicity. The truncated peptide of 72 kDa exhibited similar toxicity to the 130 kDa toxin and was between 39th and 677th amino acids.

Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3

BackgroundThe dengue virus two-component protease NS2B/NS3 mediates processing of the viral polyprotein precursor and is therefore an important determinant of virus replication. The enzyme is now intensively studied with a view to the structure-based development of antiviral inhibitors. Although 3-dimensional structures have now been elucidated for a number of flaviviral proteases, enzyme-substrate interactions are characterized only to a limited extend. The high selectivity of the dengue virus protease for the polyprotein precursor offers the distinct advantage of designing inhibitors with exquisite specificity for the viral enzyme. To identify important determinants of substrate binding and catalysis in the active site of the dengue virus NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine substitution mutagenesis and effects on enzyme activity were fluorometrically assayed.MethodsAlanine substitutions were introduced by site-directed mutagenesis at residues L115, D129, G133, T134, Y150, G151, N152, S163 and I165 and recombinant proteins were purified from overexpressing E. coli. Effects of these substitutions on enzymatic activity of the NS3 protease were assayed by fluorescence release from the synthetic model substrate GRR-amc and kinetic parameters Km, kcat and kcat/Km were determined.ResultsKinetic data for mutant derivatives in the active site of the dengue virus NS3 protease were essentially in agreement with a functional role of the selected residues for substrate binding and/or catalysis. Only the L115A mutant displayed activity comparable to the wild-type enzyme, whereas mutation of residues Y150 and G151 to alanine completely abrogated enzyme activity. A G133A mutant had an approximately 10-fold reduced catalytic efficiency thus suggesting a critical role for this residue seemingly as part of the oxyanion binding hole.ConclusionsKinetic data obtained for mutants in the NS3 protease have confirmed predictions for the conformation of the active site S1 and S2 pockets based on earlier observations. The data presented herein will be useful to further explore structure-activity relationships of the flaviviral proteases important for the structure-guided design of novel antiviral therapeutics.