Limits...
In silico modeling and functional interpretations of Cry1Ab15 toxin from Bacillus thuringiensis BtB-Hm-16.

Kashyap S - Biomed Res Int (2013)

Bottom Line: The novel structural differences found are the presence of β0 and α3, and the absence of α7b, β1a, α10a, α10b, β12, and α11a while α9 is located spatially downstream.Validation by SUPERPOSE and with the use of PROCHECK program showed folding of 98% of modeled residues in a favourable and stable orientation with a total energy Z-score of -6.56; the constructed model has an RMSD of only 1.15 Å.These increments of 3D structure information will be helpful in the design of domain swapping experiments aimed at improving toxicity and will help in elucidating the common mechanism of toxin action.

View Article: PubMed Central - PubMed

Affiliation: National Bureau of Agriculturally Important Microorganisms (ICAR), Kusmaur, Kaithauli, Maunath Bhanjan, Uttar Pradesh 275101, India.

ABSTRACT
The theoretical homology based structural model of Cry1Ab15 δ-endotoxin produced by Bacillus thuringiensis BtB-Hm-16 was predicted using the Cry1Aa template (resolution 2.25 Å). The Cry1Ab15 resembles the template structure by sharing a common three-domain extending conformation structure responsible for pore-forming and specificity determination. The novel structural differences found are the presence of β0 and α3, and the absence of α7b, β1a, α10a, α10b, β12, and α11a while α9 is located spatially downstream. Validation by SUPERPOSE and with the use of PROCHECK program showed folding of 98% of modeled residues in a favourable and stable orientation with a total energy Z-score of -6.56; the constructed model has an RMSD of only 1.15 Å. These increments of 3D structure information will be helpful in the design of domain swapping experiments aimed at improving toxicity and will help in elucidating the common mechanism of toxin action.

Show MeSH

Related in: MedlinePlus

The comparative three-dimensional, three-domain structure of the Cry1Ab15 ((b), (d), (f)) and Cry1Aa ((a), (c), (e)) molecules.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3818814&req=5

fig3: The comparative three-dimensional, three-domain structure of the Cry1Ab15 ((b), (d), (f)) and Cry1Aa ((a), (c), (e)) molecules.

Mentions: Sequence alignment showed 88.3% identity (Smith Waterman Score-3356; Z-Score-3981.3; E Value-6.4e-215) between the Cry1Ab15 and Cry1Aa. It is observed that a model tends to be reliable if identity percentage between the template and target protein is above 40%. Low degree of reliability arises when identity decreases below 20% [26]. Identity difference in the present case is sufficiently high to carry out the theoretical modeling for the Cry1Ab15 toxin stretch of 84–661 residues (Figure 1). Sequence alignment of domain I, domain II, and domain III was straightforward within the possible limits of flanking domains. Domain III is quite well conserved both on the N-terminal and C-terminal sides. Domain I is composed of residues 86–341 and consists of 9 α-helices and too small β-strands. All the helices in the Cry1Ab15 model were slightly longer than those in Cry1Aa (Table 1). The amphiphilicity (Hoops and Woods) values indicated an exposed nature of a few of the helices of domain I (α1, α2a, α2b, α3, and α6). These values correspond well with the accessibility calculated with Swiss PDB, except for α1, which is packed against domain II (Figure 2). It is possible that this helix will have some mobility, with an emphasis that one of the cutting sites by gut proteases is located close to the middle of this helix [27]. On the other hand, membrane insertion and pore formation are thought to occur through elements of domain I, composed of a bundle of six amphipathic α-helices surrounding the highly hydrophobic helix α5 [7]. Spectroscopic studies with synthetic peptides corresponding to domain I helices revealed that α4 and α5 have the greatest propensity for insertion into artificial membranes, although insertion and pore formation were more efficient when α4 and α5 were connected by a segment analogous to the α4-α5 loop of the toxin [28, 29]. A particularly large number of single-site mutations with altered amino acids from these helices, which lead to a strong reduction in the toxicity and pore-forming ability of the toxin, have been characterized [30–33]. Also, a site-directed chemical modification study has provided strong evidence that α4 lines the lumens of the pores formed by the toxin [34]. Recent studies have established that toxin activity is especially sensitive to modifications not only in the charged residues of α4 [33] but also in most of its hydrophilic residue [30]. Furthermore, the loss of activity of most of these mutants did not result from an altered selectivity or the size of the pores, but from a reduced pore-forming capacity of the toxin [34]. The charge distribution pattern in the Cry1Ab15 theoretical model corresponds to a negatively charged patch along β4 and β13 (Figures 3 and 4) of domains II and III, respectively. The Cry1Ab15 domain I model relates well with the data from Gerber and Shai [29] who have suggested that α4 and α5 insert into the membrane in an antiparallel manner as a helical hairpin. It is possible that according to the surface electrostatic potential of helices 4 and 5 there was a neutral region in the middle of the helices which probably indicates, if we follow the umbrella model and consider it to be correct, that both helices cross the membrane with their polar sides exposed to the solvent as it has been suggested by the results of mutagenesis experiments done by Girard et al. [31] with the Cry1Ac toxin. This region is also the most conserved among the Cry toxins. Girard et al. [31] demonstrated that mutations in the base of helix 3 and the loop between α3 and α4 that cause alterations in the balance of negative charged residues may cause loss of toxicity. Mutations in helices α2 and α6 and the surface residues of α3 have no important effect on toxicity; meanwhile, helices α4 and α5 seem to be very sensitive to mutations. Helix α1 probably does not play an important part in toxin activity after the cleavage of the protoxin. It is possible that the mutations aimed to an increasing the amphiphilicity in these helices will improve the pore-forming activity of the Cry1Ab15 type toxins. The structure of domain I of the toxin, the effect of site-directed mutagenesis in this domain on toxin activity, and the studies with hybrid toxins [35–37] all suggest that domain I, or parts of it, inserts 125 into the membrane and forms a pore. This idea is further supported by studies that show that truncated proteins corresponding to domain I of CryIA(c) [38] δ-endotoxin form ion channels in model lipid membranes similar to those formed by the intact toxins. After receptor binding, the network of contacts between α7, the helix in the interface between the pore-forming domain and the receptor-binding domain, and α5, α6, and, presumably, α4 helices may assist at the insertion of the α4-α5 hairpin into the membrane by the unpacking of the helical bundle that exists in the nonmembrane-bound form of the toxin. This hypothesis might account for the observation that α7 mutants are susceptible to proteolysis by either trypsin or midgut juice [39]. Our model also supports the notion that the α4-α5 hairpin is the major structural component in the lining of the pores formed by δ-endotoxin. Therefore, it is possible to create toxin variants with better membrane permeability potential by stabilizing the hairpin antiparallel structure by cross-linking α4 with α5. This postulation is important because mutations within transmembrane segments of proteins usually decrease or have no effect on the biological activities of these proteins. Thus, it is conceivable that the introduction of several salt bridges or other bonds between α4-α5 helices or the stabilization of the α4-α5 hairpin by the creation of bridging interactions between the α3-α4 and α5-α6 loops may result in a significantly enhanced toxic activity. Other studies also support the umbrella-like model for domain I insertion into membranes [34, 40, 41]. As for other Cry toxins, domain II of the Cry1Ab15 toxin consists of three Greek key beta sheets arranged in a beta prism topology. It is comprised of residues 350–508, one helix (α8), and 11 β-strands (Table 1). In the case of the three domain Cry toxins, specificity is mostly attributed to their capacity to bind to certain proteins located on the surface of the intestinal membrane through specific segments of domains II and III, composed mainly of β sheets [42, 43]. Loop β4-β5 is mostly hydrophilic, and the charged residues located at the tip of the loop are probably important determinants of insect specificity. As in loop β2-β3, few glycine residues are also present before a negatively charged residue supporting the hypothesis that correct orientation of charged residues in the specificity loops could be important in receptor recognition. Mutations in defined regions of the Cry1Aa toxin have identified residues 365–371 (equivalent to residues in the Cry1Ab15 β6-β7 loop) as essential for binding to the membrane of midgut cells of Bombyx mori [35, 44]. In the Cry1Ab15 model, this region is shorter than their counterparts in Cry1Aa. Loop β2-β3 seems also to be able to modulate the toxicity and specificity of Cry1C [45]. The dual specificity of Cry2Aa for Lepidoptera and Diptera has been mapped to residues 307–382 that corresponds in the Cry1Ab15 theoretical model to sheet 1, strand β6, and loop β6-β7. Domain III comprised residues 471–608 and showed high conservation of residues and the only important modification is a 3-residue deletion between β16 and β17. Several studies indicate that site mutations in conserve blocks reduce toxicity and alter channel properties at least in Cry1Ac [7] and Cry1Aa [42, 46], and divergence in block 5 element [8, 41] postulates an alternative mechanism of membrane permeabilization.


In silico modeling and functional interpretations of Cry1Ab15 toxin from Bacillus thuringiensis BtB-Hm-16.

Kashyap S - Biomed Res Int (2013)

The comparative three-dimensional, three-domain structure of the Cry1Ab15 ((b), (d), (f)) and Cry1Aa ((a), (c), (e)) molecules.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818814&req=5

fig3: The comparative three-dimensional, three-domain structure of the Cry1Ab15 ((b), (d), (f)) and Cry1Aa ((a), (c), (e)) molecules.
Mentions: Sequence alignment showed 88.3% identity (Smith Waterman Score-3356; Z-Score-3981.3; E Value-6.4e-215) between the Cry1Ab15 and Cry1Aa. It is observed that a model tends to be reliable if identity percentage between the template and target protein is above 40%. Low degree of reliability arises when identity decreases below 20% [26]. Identity difference in the present case is sufficiently high to carry out the theoretical modeling for the Cry1Ab15 toxin stretch of 84–661 residues (Figure 1). Sequence alignment of domain I, domain II, and domain III was straightforward within the possible limits of flanking domains. Domain III is quite well conserved both on the N-terminal and C-terminal sides. Domain I is composed of residues 86–341 and consists of 9 α-helices and too small β-strands. All the helices in the Cry1Ab15 model were slightly longer than those in Cry1Aa (Table 1). The amphiphilicity (Hoops and Woods) values indicated an exposed nature of a few of the helices of domain I (α1, α2a, α2b, α3, and α6). These values correspond well with the accessibility calculated with Swiss PDB, except for α1, which is packed against domain II (Figure 2). It is possible that this helix will have some mobility, with an emphasis that one of the cutting sites by gut proteases is located close to the middle of this helix [27]. On the other hand, membrane insertion and pore formation are thought to occur through elements of domain I, composed of a bundle of six amphipathic α-helices surrounding the highly hydrophobic helix α5 [7]. Spectroscopic studies with synthetic peptides corresponding to domain I helices revealed that α4 and α5 have the greatest propensity for insertion into artificial membranes, although insertion and pore formation were more efficient when α4 and α5 were connected by a segment analogous to the α4-α5 loop of the toxin [28, 29]. A particularly large number of single-site mutations with altered amino acids from these helices, which lead to a strong reduction in the toxicity and pore-forming ability of the toxin, have been characterized [30–33]. Also, a site-directed chemical modification study has provided strong evidence that α4 lines the lumens of the pores formed by the toxin [34]. Recent studies have established that toxin activity is especially sensitive to modifications not only in the charged residues of α4 [33] but also in most of its hydrophilic residue [30]. Furthermore, the loss of activity of most of these mutants did not result from an altered selectivity or the size of the pores, but from a reduced pore-forming capacity of the toxin [34]. The charge distribution pattern in the Cry1Ab15 theoretical model corresponds to a negatively charged patch along β4 and β13 (Figures 3 and 4) of domains II and III, respectively. The Cry1Ab15 domain I model relates well with the data from Gerber and Shai [29] who have suggested that α4 and α5 insert into the membrane in an antiparallel manner as a helical hairpin. It is possible that according to the surface electrostatic potential of helices 4 and 5 there was a neutral region in the middle of the helices which probably indicates, if we follow the umbrella model and consider it to be correct, that both helices cross the membrane with their polar sides exposed to the solvent as it has been suggested by the results of mutagenesis experiments done by Girard et al. [31] with the Cry1Ac toxin. This region is also the most conserved among the Cry toxins. Girard et al. [31] demonstrated that mutations in the base of helix 3 and the loop between α3 and α4 that cause alterations in the balance of negative charged residues may cause loss of toxicity. Mutations in helices α2 and α6 and the surface residues of α3 have no important effect on toxicity; meanwhile, helices α4 and α5 seem to be very sensitive to mutations. Helix α1 probably does not play an important part in toxin activity after the cleavage of the protoxin. It is possible that the mutations aimed to an increasing the amphiphilicity in these helices will improve the pore-forming activity of the Cry1Ab15 type toxins. The structure of domain I of the toxin, the effect of site-directed mutagenesis in this domain on toxin activity, and the studies with hybrid toxins [35–37] all suggest that domain I, or parts of it, inserts 125 into the membrane and forms a pore. This idea is further supported by studies that show that truncated proteins corresponding to domain I of CryIA(c) [38] δ-endotoxin form ion channels in model lipid membranes similar to those formed by the intact toxins. After receptor binding, the network of contacts between α7, the helix in the interface between the pore-forming domain and the receptor-binding domain, and α5, α6, and, presumably, α4 helices may assist at the insertion of the α4-α5 hairpin into the membrane by the unpacking of the helical bundle that exists in the nonmembrane-bound form of the toxin. This hypothesis might account for the observation that α7 mutants are susceptible to proteolysis by either trypsin or midgut juice [39]. Our model also supports the notion that the α4-α5 hairpin is the major structural component in the lining of the pores formed by δ-endotoxin. Therefore, it is possible to create toxin variants with better membrane permeability potential by stabilizing the hairpin antiparallel structure by cross-linking α4 with α5. This postulation is important because mutations within transmembrane segments of proteins usually decrease or have no effect on the biological activities of these proteins. Thus, it is conceivable that the introduction of several salt bridges or other bonds between α4-α5 helices or the stabilization of the α4-α5 hairpin by the creation of bridging interactions between the α3-α4 and α5-α6 loops may result in a significantly enhanced toxic activity. Other studies also support the umbrella-like model for domain I insertion into membranes [34, 40, 41]. As for other Cry toxins, domain II of the Cry1Ab15 toxin consists of three Greek key beta sheets arranged in a beta prism topology. It is comprised of residues 350–508, one helix (α8), and 11 β-strands (Table 1). In the case of the three domain Cry toxins, specificity is mostly attributed to their capacity to bind to certain proteins located on the surface of the intestinal membrane through specific segments of domains II and III, composed mainly of β sheets [42, 43]. Loop β4-β5 is mostly hydrophilic, and the charged residues located at the tip of the loop are probably important determinants of insect specificity. As in loop β2-β3, few glycine residues are also present before a negatively charged residue supporting the hypothesis that correct orientation of charged residues in the specificity loops could be important in receptor recognition. Mutations in defined regions of the Cry1Aa toxin have identified residues 365–371 (equivalent to residues in the Cry1Ab15 β6-β7 loop) as essential for binding to the membrane of midgut cells of Bombyx mori [35, 44]. In the Cry1Ab15 model, this region is shorter than their counterparts in Cry1Aa. Loop β2-β3 seems also to be able to modulate the toxicity and specificity of Cry1C [45]. The dual specificity of Cry2Aa for Lepidoptera and Diptera has been mapped to residues 307–382 that corresponds in the Cry1Ab15 theoretical model to sheet 1, strand β6, and loop β6-β7. Domain III comprised residues 471–608 and showed high conservation of residues and the only important modification is a 3-residue deletion between β16 and β17. Several studies indicate that site mutations in conserve blocks reduce toxicity and alter channel properties at least in Cry1Ac [7] and Cry1Aa [42, 46], and divergence in block 5 element [8, 41] postulates an alternative mechanism of membrane permeabilization.

Bottom Line: The novel structural differences found are the presence of β0 and α3, and the absence of α7b, β1a, α10a, α10b, β12, and α11a while α9 is located spatially downstream.Validation by SUPERPOSE and with the use of PROCHECK program showed folding of 98% of modeled residues in a favourable and stable orientation with a total energy Z-score of -6.56; the constructed model has an RMSD of only 1.15 Å.These increments of 3D structure information will be helpful in the design of domain swapping experiments aimed at improving toxicity and will help in elucidating the common mechanism of toxin action.

View Article: PubMed Central - PubMed

Affiliation: National Bureau of Agriculturally Important Microorganisms (ICAR), Kusmaur, Kaithauli, Maunath Bhanjan, Uttar Pradesh 275101, India.

ABSTRACT
The theoretical homology based structural model of Cry1Ab15 δ-endotoxin produced by Bacillus thuringiensis BtB-Hm-16 was predicted using the Cry1Aa template (resolution 2.25 Å). The Cry1Ab15 resembles the template structure by sharing a common three-domain extending conformation structure responsible for pore-forming and specificity determination. The novel structural differences found are the presence of β0 and α3, and the absence of α7b, β1a, α10a, α10b, β12, and α11a while α9 is located spatially downstream. Validation by SUPERPOSE and with the use of PROCHECK program showed folding of 98% of modeled residues in a favourable and stable orientation with a total energy Z-score of -6.56; the constructed model has an RMSD of only 1.15 Å. These increments of 3D structure information will be helpful in the design of domain swapping experiments aimed at improving toxicity and will help in elucidating the common mechanism of toxin action.

Show MeSH
Related in: MedlinePlus