Pore-forming Peptides and Protein Toxins
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To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. The two forms of the toxin were resolved to 2.
Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin pfam Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin.
Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated.
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A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. Cry6 proteins are members of the alpha helical pore-forming toxins — a structural class not previously recognized among the Cry toxins of B. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.
Bacillus thuringiensis strains produce a range of toxins active against invertebrates with enormous potential for use in the control of pests of importance in agriculture and health [ 1 ]. However, the Cry nomenclature is not limited to these proteins and includes several distinct and unrelated lineages. While much is known of the structure and function of the three-domain toxins [ 4 ] and the structure of a protoxin form has recently been published [ 5 ], very little is known of the non-three-domain Cry proteins.
One such Cry protein is Cry6Aa [ 13 ], a protein with activity against Coleoptera such as the Western Corn Rootworm Diabrotica virgifera virgifera [ 14 ] and a range of nematodes, including both free-living Caenorhabditis elegans and Panagrellus redivivus and plant pathogenic Heterodera glycines and Meloidogyne incognita species [ 15 — 18 ] that cause large-scale losses to agriculture [ 19 ]. Cry6B is a protein that is closely related to Cry6A but lacks 88 amino acids that are seen at the C-terminus of the latter protein.
Cry6B is reported to be active against the coleopteran lucerne weevil Hypera postica [ 20 ] but showed little or no activity against a range of nematode targets [ 15 ].
The absence of further data on the structure and function of these proteins has limited our ability to understand their activity against target invertebrates. As a result, development and exploitation of the toxins in the control of agricultural pest insects and nematodes pathogenic to plants and animals may be limited. This, in turn, inhibits their use to supplement the current chemotherapeutic approaches to nematicidal treatments that are very toxic and are being phased out [ 21 ]. In this study we applied both crystallographic techniques and state of the art ab initio modeling to probe the structure of Cry6Aa in protoxin and trypsin-cleaved forms.
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The structures obtained are novel among invertebrate-active toxins and are consistent with Cry6Aa acting as a pore-forming toxin. We demonstrated pore formation in vivo and used our predictions to construct a mutant expected to show compromised toxicity. Spores were harvested and crystals of Cry6Aa were isolated and purified by discontinuous sucrose density gradient ultracentrifugation according to previously described methods [ 6 ]. The cry6Aa gene was amplified from the above B. Following ligation of the PCR product and transformation of E. For recombinant production of Cry6Aa proteins in E.
For in vivo pore formation assays, a distinct clone of the cry6Aa gene in the Bam HI and Pst I sites of the vector pQE9 was produced adding an N-terminal His-tag and a seven amino-acid extension to the C-terminus, encoded by the vector. An equivalent clone in pQE9 containing the cry5Ba gene as a positive control for in vivo pore formation has been described previously [ 24 ].
Pore-forming Peptides and Protein Toxins
The supernatant was discarded. This step was repeated twice. The buffer exchanged Cry6Aa sample was then filtered through a 0.
The concentrated sample was further purified by size exclusion chromatography. For each run, 4. The fractions from peak 2 contained predominantly monomer and were pooled and used in crystallization experiments. The measured pH of 9. The sample was eluted using a 2. The programs gave results that were similar to each other and relatively independent of the reference protein set used. Initial crystals were obtained using Rigaku Reagents, Inc.
Data were indexed and processed with the HKL software suite [ 25 ]. The poly-alanine chain of the crystal structure of hemolysin B from Bacillus cereus [PDB: 2NRJ] consisting of residues 19— was used as a search model. The final structure of the complex was obtained by carrying out several cycles of refinement consisting of manual model building using COOT [ 28 ], followed by restrained refinement with REFMAC [ 29 ] using Translation, Libration and Screw-rotation TLS refinement and the reference and secondary structure restraints.
The trypsin-cleaved Cry6Aa structure, solved at higher resolution, was used as the reference structure in this refinement. SDS-PAGE analysis of protein samples obtained by dissolving the crystals in SDS-buffer did not reveal any degradation products and confirmed the presence of only full-length Cry6Aa protein in the crystals used for the data collection. Data were indexed and processed with HKL [ 25 ]. The crystals belonged to orthorhombic space group P2 1 2 1 2 and contained one molecule of full-length Cry6Aa per asymmetric unit.
The structure of full-length Cry6Aa was solved by molecular replacement using the structure of the truncated form of Cry6Aa as a search model. The Cry6Aa tertiary structure was modeled ab initio using the Rosetta software [ 30 ]. After entering the Cry6Aa sequence, the first stage of the web-based process returned a secondary structure prediction with options to continue full protein structure prediction either using de novo or database structure comparison methods. Both methods were followed to produce a selection of five possible structure outcomes from both de novo and database predictions 10 models in total.
Each sample was diluted to a concentration of 0. The mass was calculated using the Mass Hunter Qualitative Analysis software and the maximum entropy de-convolution algorithm.
Changes in the observed charge state distribution CSD of proteins analyzed by liquid chromatography—mass spectrometry LC-MS , as described above, were used as an indirect probe of the overall conformational flexibility of the protein under different conditions. Digestion of Cry6Aa with trypsin results in two fragment fractions, a large fragment and a small fraction containing two peptides.
The C-terminal peptide CTP sample was diluted with 0. The mass spectrum was collected and analyzed using flex analysis software. A standard mix of 20 phenylthiohydantoin-amino acids Shimadzu, catalog was run each time.
The amino acid residues from each Edman degradation cycle were determined based on their retention times from the C column compared to standards. To measure intoxication by Cry6Aa, 30—40 fourth-staged larvae of C. Three replica plates were used for each strain. Three to five worms were randomly picked from each of the different treatment conditions for image collection. Worm growth was assessed by measuring the area of clearly separated, individual worms worms overlapping on image collection were not measured , using Image J version 1.
The experiment was independently repeated three times. Data were analyzed with JMP v. Pore formation was assessed microscopically using the method described by Los et al. Worms were then washed off the OP50 plate, washed twice to remove bacteria, and pipetted onto agar plates spread with the transformed JM Worms were washed twice to remove excess dye and pipetted onto agar pads on microscope slides and imaged.
At least 30 worms were imaged for each condition, and the entire experiment was conducted three times. The Cry6Aa protein is active in the gut of nematodes such as C. The Cry6Aa crystals produced in B.
Serratia Type Pore Forming Toxins | Bentham Science
As a result, further analyses utilized recombinant Cry6Aa produced in P. The secondary structure content of the full-length Cry6Aa protein from this source was estimated through CD analysis. Overall, and considering the inherent margins of error in CD determinations, these results suggest that the protein has a stable secondary structure across a broad pH range of 3.
The structure of the trypsin-resistant core Fig. The Cry6Aa trypsin-resistant core structure is unlike any previously described Cry protein although it is similar to several known toxins, including HlyE and HblB see below. Crystal structure of Cry6a toxin. N- and C-termini and the putative transmembrane region are labeled. The CysCys disulfide bond is shown and, in the insert box, the final 2Fo-Fc electron density map calculated at 1. Side and main chains of the amino acid residues are presented as sticks and colored by the atoms.