Comparative Biochemistry and Physiology, Part B 161 (2012) 86–92
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Structural insights regarding an insecticidal Talisia esculenta protein and its biotechnological potential for Diatraea saccharalis larval control Maria das Graças M. Freire a, Octávio L. Franco b, Carlos Eduardo G. Kubo c, Ludovico Migliolo b, Rodrigo H. Vargas b, Caio Fernando Ramalho de Oliveira c, José Roberto P. Parra d, Maria Ligia R. Macedo c,⁎ a
LAQUIBIO, ISECENSA, Campos dos Goytacazes-RJ, Brazil Universidade Católica de Brasília, Brasilia-DF, Brazil LPPFB, CCBS/UFMS, Campo Grande, MS, Brazil d Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, Escola Superior de Agricultura Luiz de Queiroz (ESALQ), USP, Piracicaba, SP, Brazil b c
a r t i c l e
i n f o
Article history: Received 9 August 2011 Received in revised form 22 September 2011 Accepted 23 September 2011 Available online 29 September 2011 Keywords: Reserve protein Insecticidal activity Binding sites Homology modeling Docking studies
a b s t r a c t Talisin is a seed-storage protein from Talisia esculenta that presents lectin-like activities, as well as proteinaseinhibitor properties. The present study aims to provide new in vitro and in silico biochemical information about this protein, shedding some light on its mechanistic inhibitory strategies. A theoretical three-dimensional structure of Talisin bound to trypsin was constructed in order to determine the relative interaction mode. Since the structure of non-competitive inhibition has not been elucidated, Talisin-trypsin docking was carried out using Hex v5.1, since the structure of non-competitive inhibition has not been elucidated. The predicted noncoincidence of the trypsin binding site is completely different from that previously proposed for Kunitz-type inhibitors, which demonstrate a substitution of an Arg64 for the Glu64 residue. Data, therefore, provide more information regarding the mechanisms of non-competitive plant proteinase inhibitors. Bioassays with Talisin also presented a strong insecticide effect on the larval development of Diatraea saccharalis, demonstrating LD50 and ED50 of ca. 2.0% and 1.5%, respectively. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Seed proteins play an important role in human and animal nutrition, providing a major contribution to dietary protein. These proteins may be classified as storage, structural and biologically active proteins (Fukusima, 1991). The major function of the storage proteins in plant tissues appear to be a nutritional resource for seed germination or tuber development (Yeh et al., 1997). Furthermore, storage proteins play a dual role in repository and defense mechanisms (Van Damme et al., 2002; Gaidamashvili et al., 2004). Vicilins, legume seed-storage proteins of globulin nature, from cowpeas (Sales et al., 1996) and other legumes, bind strongly to chitin and have a highly detrimental effect on the larval development of Callosobruchus maculatus (Yunes et al., 1998). Moreover, Kunitz-type proteinaceous inhibitors reversibly interact with enzyme targets, forming stable complexes influencing their catalytic activities in competitive and non-competitive ways (Prabhu and Pattabiraman, 1980; Bhattacharyya et al., 2006; Oliveira et al., 2007). These inhibitors have been widely isolated and characterized from plants (Macedo et al., 2000a, 2000b; Mello et al., 2001; Oliveira et al., 2002; Macedo et al., 2004a, 2004b; Ramos et al.,
⁎ Corresponding author. Tel.: + 55 67 33457612; fax: + 55 67 33457400. E-mail address:
[email protected] (M.L.R. Macedo). 1096-4959/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2011.09.010
2008) normally occurring as single polypeptide chains (Negreiros et al., 1991; Wu and Lin, 1993; Sattar et al., 2005). These proteinaceous compounds have been implicated in various physiological functions, such as the regulation of proteolytic cascades and safe storage of proteins, as well as defense molecules against plant pests and pathogens (Xavier-Filho, 1992). Kunitz-type inhibitors are characterized by molecular masses around 20 kDa, a low cysteine content forming two disulphide bonds and a common structural fold composed of a β-trefoil formed by 12 antiparallel β-strands with long interconnecting loops presenting one or two reactive sites for serine proteinases (Song and Suh, 1998; Krauchenco et al., 2003, 2004; Khamrui et al., 2005). Previous study (Freire et al., 2009) showed the biochemical characterization and cloning of the major protein from Talisia esculenta seeds (Talisin), a member of the Sapindaceae family. The deduced peptide presented high similarity with several storage proteins and all demonstrating amino acid sequences that were clearly related to the Kunitz family of the proteinase inhibitor (Freire et al., 2009). It has also been shown that Talisin could be a saccharide-binding protein characterized by a proven interaction with carbohydrates on neutrophil or mononuclear cells (Freire et al., 2003) and to with the chitin component of the peritrophic membrane (or equivalent structures) in the C. maculatus (Macedo et al., 2002). Dixon plot and negative staining against bovine trypsin indicate that Talisin presents a non-competitive inhibition of trypsin (Freire et al., 2009). Talisin
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also showed insecticidal activity against several insects (Macedo et al., 2004a, 2004b, 2011). Talisin's structural data are not available; in this report, we obtained the theoretical three-dimensional structure of Talisin by homology modeling. We used a molecular docking strategy in order to investigate the effects of this protein on the growth and development of Diatraea saccharalis, the major insect pest of sugarcane in Brazil and other South American countries, which is responsible for significant economic damage.
the larvae reached the fourth instar at standard conditions, the relationship between protein content and the weight and number of larvae were determined. Linear regression analysis was used to describe the response of D. saccharalis to various doses of Talisin. The effective dose for a 50% response (ED50) was defined as the concentration of Talisin that decreased the mass of the insect to 50% of that of control larvae. The lethal dose (LD50) corresponded to the concentration of Talisin that reduced the number of insects to 50% of the number found in control diet.
2. Materials and methods
2.7. Midgut preparation
2.1. Plant material and chemicals
Proteinases were obtained from the midguts of fourth-instar larvae, according to Macedo et al. (2007a). Fourth-instar larvae were cold-immobilized and the midgut, along with its contents, was removed in cold 150 mM NaCl and stored frozen (−20 °C). Guts from larvae of D. saccharalis were subsequently homogenized in 150 mM NaCl, centrifuged for 5 min at 6000 g at 4 °C, and the supernatants pooled and kept on ice for enzymatic assays.
T. esculenta (Sapindaceae) seeds were collected in the State of Ceará (Brazil). Unless indicated otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Amersham Biosciences (Uppsala, Sweden). 2.2. Insects
2.8. Enzyme assays D.saccharalis (Lepidoptera: Crambidae) larvae were from a laboratory colony and provided by Dr. J.R.P. Parra (Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, SP, Brazil). The colony was housed in standard conditions (25 ± 1 °C, 60 ± 10% relative humidity and L14:D10 photoperiod). 2.3. Extraction and purification of T. esculenta protein Defatted T. esculenta seeds were finely ground and extracted (meal to buffer ratio of 1:5) with 150 mM NaCl for 24 h at 4 °C and then centrifuged at 10,000 g for 30 min at the same temperature. The clear supernatant (crude extract or CE) was used to determine the protein content. The CE was diluted in 150 mM NaCl and applied to a Sephadex G-100 column (2.5 cm × 80 cm) equilibrated with the same solution. The protein-rich fraction was recovered and applied to a chitin column (1.5 cm × 10 cm) equilibrated with 50 mM phosphate buffer, pH 7.6, and eluted with 100 mM HCl. The purified protein was dialyzed and lyophilized. 2.4. Protein quantification Protein concentrations were determined by the dye-binding method of Bradford (1976), with bovine serum albumin as the standard. 2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE of protein was carried out according to Laemmli (1970), using 5% (w/v) stacking and 17% separating gels under reducing and non-reducing conditions. Proteins were stained with Coomassie Brilliant Blue R-250, and molecular mass was determined using molecular-mass references of phosphorylase B (94 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin (20.1 kDa), and R-lactalbumin (14.4 kDa). 2.6. Insect bioassays Effects of Talisin on D. saccharalis development were evaluated using the artificial medium system previously developed by Green et al. (1976). For the bioassays, neonate D. saccharalis larvae were selected and fed an artificial diet containing 1%, 1.5% and 2% of Talisin (w/w). Control larvae were fed with untreated diet. Each treatment was set up in glass containers (8.5 cm long × 2.5 cm diameter) and five larvae were transferred to each glass container (n = 75). After
Bovine pancreatic trypsin and midgut extracts were used for the enzymatic assays. Trypsin-like activities were assayed using N-benzoylDL-arginine-p-nitroanilide (BAPNA) as substrate (Erlanger et al., 1961). The substrate was used at a final concentration of 1 mM in 1% (v/v) dimethyl sulfoxide (DMSO) and pH 8.0 (0.1 M Tris-HCl buffer, pH 8.0). The ability of Talisin to inhibit the trypsin-like activities from insect midgut larvae, was determined by incubating a mixture of midgut extracts with different concentrations of Talisin. Incubation was performed in five repetitions. The inhibitory activity was measured by the difference in enzyme activity with and without Talisin. The effect of Talisin on the proteolytic activity of midgut extracts was measured using 1 mM BAPNA at pH 8.0 after incubation with Talisin at 37 °C for 20 min. The residual enzymatic activity was assayed as described above. The assays were run in triplicate with appropriate blanks. 2.9. Digestion of Talisin The digestion of Talisin by midgut extract was carried out as described by Macedo et al. (2002). The midguts of fourth-instar larvae were dissected, extracted in 1 mL of 0.1 M Tris buffer, pH 8.0, and processed as described above. Talisin was incubated with this homogenate in Tris buffer (final concentration, 2 mg/mL). The Talisin:midgut protein ratio was 1:1. Digestion was done for 1, 2, 4, 6, 12 and 24 h at 30 °C and was stopped by immersing the tubes in boiling water for 2 min. The degradation of bovine serum albumin (BSA) (2 mg/mL) was used as a positive control for serine proteinase activity. The digestion was stopped as described above. The relative molecular masses of the digestion products were estimated by 12.5% SDS-PAGE using protein markers of known molecular mass. 2.10. In silico analysis and molecular modeling An alignment of the Talisin sequence, using Bioinfo Meta-Server (http://meta.bioinfo.pl/submit_wizard.pl) and ClustalW (Thompson et al., 1994; Kumar et al., 2008) was performed for the proteins, in order to find a template with high identity and analyze primary sequence similarities, observing the characters with reactive sites and disulfide bonds. Homology modeling was performed using Modeller 9v6 (Berman et al., 2000), the template found with the highest identity was the protein under the Protein Data Bank (PDB) accession code 1R8N with 1.75 Å of resolution, a Kunitz-type inhibitor resolved by X-ray diffraction. Fifty models were built and the best one was chosen based on the DOPE scores (Laskowski et al., 1993) and overall
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2.11. Molecular docking HEX v.5.1 (Ritchie, 2008) program was used to examine possible modes of interaction of Talisin with trypsin (PDB ID: 1FN6) a hydrolase from the porcine pancreas (Deepthi et al., 2001). Briefly, this procedure performed global rotational and translational space scans by using Fourier transformations, which rank the output according to surface complementarity and electrostatic characteristics. A list of 500 complexes of trypsin inhibitor was ranked and biochemical data were available to filter out possible solutions. Previous knowledge of trypsin catalytic site location was used to filter binding models; enzyme–inhibitor complexes were discarded if they showed inhibitor atoms interacting with catalytic triad trypsin, according to the noncompetitive mechanism proposed. Furthermore, the best 50 postprocessing energy minimization model complexes were analyzed by Protein–Protein Interaction Server (http://www.bioinformatics. sussex.ac.uk/protorp/). 2.12. Database and phylogenetic tree From the National Center of Biotechnology Information (NCBI) portal, forty three sequences were mined out of the Entrez Protein Data Bank with the following criteria: 19 Kunitz-type inhibitor proteins, 16 lectin-like proteins and 7 storage proteins. Using these sequences an unrooted phylogenetic tree was built using maximum parsimony on MEGA software with 1000 bootstrap replicates (Kumar et al., 2008). The distribution of the representative's storage proteins, lectin-type proteins and Kunitz-type inhibitor was observed in a subclass level (Taxonomy: Rosids). 2.13. Statistical analysis
100
Tryptic Residual Activity (%)
resolution. Energy minimization was performed on GROMACS using the steepest descent method. The tridimensional model was validated utilizing PROCHECK (Wiederstein and Sippl, 2007) through Ramachandran Plot and g-factor of − 0.18 using PROSA II web server (Ritchie, 2008) observing an acceptable z-score of −4.85. The root main square deviation value RMSD: 0.367 Å was observed in order to find structural differences between template and target protein.
80
60
40
20
0 0.0
0.5
1.0
2.0
Fig. 1. Inhibition of D. saccharalis trypsin-likes by increasing amounts of Talisin.
effect of Talisin on larvae is shown in Fig. 2A and B. The weight of larvae fed on the control diet (represented by the Y-intercept value) was ca. 98 mg, while a diet containing 1.0%, 1.5% and 2% Talisin produced an approximately 34%, 47% and 60% decrease in weight, respectively. The insert of Fig. 2A shows the variation in the size of D. saccharalis larvae fed Talisin and the control diet (right and left, respectively). Regression analysis indicated that for each increase of 0.1% in Talisin content
A 110 100 90 80 70 60 50
All data were examined using one-way analysis of variance (ANOVA), and a p value b0.001 was considered to be significant. Means were compared by the Tukey test. The Statistica software was used for the analysis.
40
Y = 91.13 - 27.48X
2
R = 0.99
30 0.0
0.5
3. Results and discussion
1.0
1.5
2.0
Talisin (%, w/w)
B 100 90 80
Survival (%)
The phytophagous larvae of most lepidopteran species analyzed so far have alkaline midgut fluids, with serine proteinases and exopeptidases providing most of the midgut proteolytic activity. Talisin was assayed in vitro against proteinases from D. saccharalis (Fig. 1) in order to assess the general hypothesis that serine proteinase inhibitors can protect plants against herbivorous insects. The digestive enzymes extracted from larvae of D. saccharalis were trypsin-like enzymes (data not shown), and were inhibited to different degrees by Talisin. The substantial inhibition of gut proteinases of D. saccharalis in the in vitro assay suggested that Talisin may affect the growth and/or survival of this insect pest when incorporated into their diet. The strategy of interfering with digestion and, thus, affecting the nutritional status of the insect, is widely employed plants to defend themselves against pests and has been extensively reviewed (Carlini and Grossi-de-Sá, 2002; Ramos et al., 2009; Macedo et al., 2010). The effect of Talisin on larval development was monitored by feeding the larvae on an artificial diet (final protein concentrations ranged from 0.5% to 2.0% of total dietary protein) and then determining the number and mass of surviving fourth-instar larvae. The dose–response
1.5
Talisin (µg protein)
Weight (mg)
88
70 60 50 40
Y= 83.49 - 20.02X
2
R = 0.99
30 0.0
0.5
1.0
1.5
2.0
Talisin (%,w/w) Fig. 2. Effect of dietary Talisin on D. saccharalis larval weight (A) and survival (B). Insert: Variation in the size of fourth-instar D. saccharalis larvae fed with 1.5% Talisin (right) and control (left) diets. Bar = 1 cm.
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there was a 2.6 mg decrease in weight (R2 = 0.99). The mortality (Fig. 2B) of D. saccharalis larvae fed on control diet (represented by the y-intercept value) was about 10%, whereas diets containing 1.0%, 1.5% and 2% Talisin caused 30%, 40% and 52% mortality, respectively. Regression analysis showed that for each 0.1% increase in the Talisin dose, there was a 2.3% increase in mortality (R2 = 0.99). The concentration of Talisin used (0.5–2.0%, w/w) is markedly lower than that found in legume seeds and is similar to other insecticide proteins used by other workers (Carlini and Grossi-de-Sá, 2002; Macedo et al., 2007a, 2007b, 2007c). General proteinase activity in larvae reared on artificial diets containing 1.5% Talisin was observed for trypsin-like activity, utilizing BAPNA as a synthetic substrate. The enzyme assay showed that Talisin-fed larvae resulted in lower levels of trypsin activity per mg protein in the gut and no change in levels of trypsin activity as measured in feces (Fig. 3A and B). Tryptic activities in the midgut from fourth-instar D. saccharalis larvae reared on artificial diets containing 1.5% Talisin were altered by 27% (Fig. 3A). The lack of an increase in the trypsin activity of feces from Talisin-fed larvae suggests that Talisin did not cause rupture of the peritrophic membrane of D. saccharalis (Macedo et al., 2007a, 2007b, 2007c). After the incubation of Talisin with D. saccharalis midgut extracts, in vitro digestibility was monitored by 12.5% SDS-PAGE. The profiles of protein bands are presented in Fig. 4. Talisin was resistant to digestion during the first 24 h of incubation, in contrast to the control prepared with BSA where digestion of protein occurred during the first 3 h (data not shown). It is not known how these storage proteins become toxic to insects, but it is believed that they interfere with nutrient uptake by binding to chitin in the peritrophic membranes of the larval midgut (Chrispeels and Raikhel, 1991). Also, because of the proteolytic activity of the insect
A 22
[ ]BAPNA nmol/mgP/min
20
a
18 16
b
14 12 10 8 6 4 2 0
Control
Talisin
B 14
[ ]BAPNA nmol/mgP/min
a 12
a
10 8 6 4 2 0
Control
Talisin
Fig. 3. Trypsin-like activities in fourth-instar larvae feeding on a control artificial diet and a diet containing 1.5% Talisin. (A) Enzymatic activities of the midgut. (B) Enzymatic activities of the feces. Trypsin activity was evaluated using BAPNA as substrate.
89
94 66 43 30 Talisin
20.1 14.4 WM Talisin 1
2
4
6
12
24
Time (h) Fig. 4. SDS-PAGE patterns of Talisin digested by D. saccharalis midgut proteinases. (WM) weight marker.
midgut, these proteins must be insensitive to proteolysis. Therefore, resistance to gut proteolysis, specificity for carbohydrate receptors, the ability to bind to different parts of the small intestine, and functional and morphological changes caused would define the efficiency of the protein in terms of insecticidal activity (Pusztai et al., 1990). Several storage proteins with insecticidal activity are resistant to degradation by insect digestive enzymes, such as V. unguiculata vicilins (reserve protein-7S) (Macedo et al., 1993), maize zeatoxin (Macedo et al., 2000a) and coffee legumin-like proteins (Coelho et al., 2010). Talisin was insensitive to gut proteinases from D. saccharalis larvae, indicating that they may have a role in protecting plants against phytophagous insects. In order to investigate the distribution relation of Talisin and other plant proteins in taxonomic level Rosids, an unrooted phylogenetic tree was constructed based on the similar amino acid sequences of predicted Talisin and members of other plant species (Fig. 5). Forty two non redundant sequences were extracted from the Entrez Protein Data Bank, among these, nineteen Kunitz-type inhibitors, sixteen lectin proteins and seven storage proteins: these sequences were aligned on ClustalW and imported to MEGA software with the previously described parameters. The unrooted phylogenetic tree presented well-defined branches. It was possible to observe three clades; one represented by storage proteins, one by lectin-like proteins and other by Kunitz-type inhibitors. Talisin presented a high identity degree with a storage protein obtained from Litchi chinensis and similarity to a Kunitz-type inhibitor from Delonix regia, an ancestor of this type of inhibitors in Fabaceae (Norioka et al., 1988). Aiming to make a comparative analysis using protein sequences obtained through the Expasy Protein-Bank using the Bioinfo MetaServer, it was possible to observe 37% of identity to the D. regia Kunitz-type proteinase inhibitor (PDB ID: 1R8N) and 65% of identity with storage proteins, L. chinensis, which does not possess the tridimensional structure resolved by experimental methods (Fig. 6). The cysteine residues were conserved in Talisin and L. chinensis after the alignment, indicating a folding with disulfide bond formation, this characteristic was observed in several Kunitz-type inhibitors, such as Glycine max, Erythrina caffra and Copaifera langsdorffii (Onesti et al., 1991; Song and Suh, 1998; Krauchenco et al., 2004). After alignment analysis, atomic coordinates of the template 1r8n (PDB ID: 1R8N) were transferred into Talisin primary structure. The constructed model displays internal three-fold symmetry, as previously observed in SKTI (soybean Kunitz trypsin inhibitor), and one polypeptide chain linked by two disulfide bridge intrachains, similar to those of Kunitz-type inhibitors (Fig. 7). The amino acids were structurally organized into loops connecting consecutive β-strands, a pattern observed in Kunitz inhibitors. Overlap of these domains showed a high similarity among β-strands but not among the connecting loops presenting the classic barrel-shaped structure. The Procheck summary of Talisin showed that 98.3% of amino acid residues were in the favorable region and only three residues (Tyr 102,
90
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Fig. 5. Unrooted tree showing distribution of the following characters Kunitz-type Inhibitor (F), Storage protein (S) and Lectin protein (L). In circle is represented Talisin, a saccharidebinding protein from T. esculenta seeds with trypsin-inhibitory activity.
Asn 114 and Ser 135) were in the disallowed regions. These residues were present within loops and as such were not expected to affect the Talisin predicted structure. Structural differences between the template and predicted three-dimensional structure of the Talisin model were calculated by superimposing both structures. The RMSD values between the template and homology 12 model of Talisin calculated for Cα traces and main chain atom were 0.36. The RMSD values and small variability among experimental structures template and the structure modeled reflect the presence of strong restraints in most regions and emphasize a similar folding pattern among these
structures. Furthermore, the lower Z-score acquired for PROSA II and high score acquired for overall steric G-factor in the case of the Talisin were of −4.85 and − 0.18, respectively, indicating the high quality of the model. The result indicated that the constructed Talisin model presented its amino acid residues within the average of the observed parameters. On the other hand, the structure of lateral chains was considered well located, when compared to the experimental structures with the same resolution. The complex between the Talisin and trypsin structure (PDB ID: 1FN6) was used to study the enzyme–inhibitor interaction (Fig. 8).
Fig. 6. Multiple alignment between Talisin (TALI_ESCUL), a vegetative storage protein from Litchi chinensis (LICT_CHIN), a Kunitz-type inhibitor from Delonix regia (DELO_REGI) and a lectin from Dioclea lehmani (DIOC_LEHM).
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Fig. 7. The three-dimensional structure of Talisin and its surface is shown using cartoons as the display preference obtained trough homology modeling. Molecular model shows a RMSD of 0.367 Å when compared to template (1RN8).
The model of interaction showed a mechanism of inhibition of the non-competitive type identical to that observed in the Adenanthera pavonina inhibitor (Prabhu and Pattabiraman, 1980). The inhibitor reactive site is able to block substrate access due to five interactions with the most favorable enzyme region, forming a complex with trypsin, with a surface area of 1010 Å 2. These contacts prevent substrate access to the enzyme, although no direct reaction with the catalytic site was observed. The absence of the reactive site Arg 64, substituted by Glu 64 supports the inactivity of this loop. A similar blockade was observed by Migliolo et al. (2010) during in silico docking between ApTKI (Adenathera pavonina trypsin Kunitz inhibitor) and trypsin. A non-competitive type interaction was responsible for forming a surface area of 1.228 Å 2 with trypsin. These results are different for the Kunitz-type inhibitors, which interact with proteinases through this reactive loop (Major and Constabel, 2008). The OH− atom negative charge of Asp78 residue in Talisin interacts with the lateral chain of the Ser 147, which receives protons forming a hydrogen bound presenting 3.14 Å. The amino acid residue Ser94 in Talisin receives protons of the OH− from Tyr217 forming hydrogen bound
91
with 2.5 Å. The residue of Asn119 (donor protons) that forms a hydrogen bound with Asn97 (receiver protons) presented distance of 2.64 Å. An outer hydrogen bound was observed with a distance of 2.52 Å between the lateral chain Ser148 and lateral chain of Gln192. An electrostatic interaction was observed between the carboxyl group Glu151 (negative charge) and amine group of Lys60 (positive charge) with a 2.92 Å distance. The most favored HEX v5.1 result presented an interface surface area of 1010 Å 2 and a complementarity gap volume index score of 2.48. Planarity and circularity values (Gabb et al., 1997) of 2.7 and 0.44 are also typical. No other solutions had favorable combinations of large interface area and good complementarity; the mean values among the 50 analysis solutions were 860 ± 99 Å 2 and 3.4 ± 0.7 (mean ± SD). The values were also best when compared with interface area and complementarity in enzyme–inhibitor complexes with their upper limits at 785 Å 2 ± 75 and 2.2 ± 0.5, respectively. The evolutionary relationships between biologically inactive/ poorly active storage proteins and “normally active” enzymes/ bioactive proteins strongly suggest that (some) storage proteins may be derived from genes that originally encoded proteins with a well-defined enzymatic or other biological activity (Van Damme et al., 2000). In consequence, some carbohydrate-binding proteins work as multifunctional molecules, i.e. they may present NH2 terminal sequence homology to Kunitz-type inhibitors, no trypsin-inhibitory activity, but exhibit lectin-like activity such as Labramin, a protein from Labramia bojeri seeds (Macedo et al., 2007b, 2007c) or show sequence similarity to soybean trypsin inhibitor (SBTI), with no trypsin-inhibitory activity, but display hemagglutinating activity such as the lectin obtained from Pseudostellaria heterophylla roots (Van Damme et al., 2000). We suggest that the action of Talisin on D. saccharalis larvae may involve: (1) a strong resistance to digestion by midgut proteinases in D. saccharalis and (2) inhibition of endogenous trypsin-like activity. The strong negative effects of Talisin on D. saccharalis larvae, observed in this study, suggest that Talisin might be able to provide a viable alternative for designing insect-resistant transgenic crops or for use as a natural pesticide. 4. Conclusions The present study provided new in vitro and in silico biochemical information about Talisin, a storage protein with the structure of a non-competitive trypsin inhibitor. The trypsin binding site is completely different from that of previously proposed Kunitz-type inhibitors, making Talisin acts as a bifunctional protein. Acknowledgements This work was supported by the FUNDECT (Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FINEP (Financiamento de Estudos e Projetos/ Ministério da Ciência e Tecnologia). References
Fig. 8. Interaction between structural model from Talisin (black) against serine proteinase from porcine beta trypsin (grey) (PDB ID: 1FN6). In stick are represented amino acid residues involved in interaction and lines are represented catalytic triad (HIS57, ASP102 and SER195).
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