Comparative Biochemistry and Physiology, Part B 161 (2012) 86–92

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Comparative Biochemistry and Physiology, Part B

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Structural insights regarding an insecticidal Talisia esculenta protein and itsbiotechnological 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, Brazilb Universidade Católica de Brasília, Brasilia-DF, Brazilc LPPFB, CCBS/UFMS, Campo Grande, MS, Brazild Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, Escola Superior de Agricultura Luiz de Queiroz (ESALQ), USP, Piracicaba, SP, Brazil

⁎ Corresponding author. Tel.: +55 67 33457612; fax:E-mail address: bioplant@terra.com.br (M.L.R. Mace

1096-4959/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.cbpb.2011.09.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 August 2011Received in revised form 22 September 2011Accepted 23 September 2011Available online 29 September 2011

Keywords:Reserve proteinInsecticidal activityBinding sitesHomology modelingDocking studies

Talisin is a seed-storage protein from Talisia esculenta that presents lectin-like activities, as well as proteinase-inhibitor properties. The present study aims to provide new in vitro and in silico biochemical informationabout this protein, shedding some light on its mechanistic inhibitory strategies. A theoretical three-dimensionalstructure of Talisin bound to trypsin was constructed in order to determine the relative interaction mode. Sincethe structure of non-competitive inhibition has not been elucidated, Talisin-trypsin docking was carried outusing Hex v5.1, since the structure of non-competitive inhibition has not been elucidated. The predicted non-coincidence of the trypsin binding site is completely different from that previously proposed for Kunitz-typeinhibitors, which demonstrate a substitution of an Arg64 for the Glu64 residue. Data, therefore, provide moreinformation regarding the mechanisms of non-competitive plant proteinase inhibitors. Bioassays with Talisinalso presented a strong insecticide effect on the larval development of Diatraea saccharalis, demonstratingLD50 and ED50 of ca. 2.0% and 1.5%, respectively.

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1. Introduction

Seed proteins play an important role in human and animal nutri-tion, providing a major contribution to dietary protein. These proteinsmay be classified as storage, structural and biologically active proteins(Fukusima, 1991). The major function of the storage proteins in planttissues appear to be a nutritional resource for seed germination ortuber development (Yeh et al., 1997). Furthermore, storage proteinsplay a dual role in repository and defense mechanisms (Van Dammeet al., 2002; Gaidamashvili et al., 2004). Vicilins, legume seed-storageproteins of globulin nature, from cowpeas (Sales et al., 1996) andother legumes, bind strongly to chitin and have a highly detrimentaleffect on the larval development of Callosobruchus maculatus (Yuneset al., 1998). Moreover, Kunitz-type proteinaceous inhibitors revers-ibly interact with enzyme targets, forming stable complexes influenc-ing their catalytic activities in competitive and non-competitive ways(Prabhu and Pattabiraman, 1980; Bhattacharyya et al., 2006; Oliveiraet al., 2007). These inhibitors have been widely isolated and charac-terized from plants (Macedo et al., 2000a, 2000b; Mello et al., 2001;Oliveira et al., 2002; Macedo et al., 2004a, 2004b; Ramos et al.,

2008) normally occurring as single polypeptide chains (Negreiroset al., 1991; Wu and Lin, 1993; Sattar et al., 2005). These proteina-ceous compounds have been implicated in various physiological func-tions, such as the regulation of proteolytic cascades and safe storageof proteins, as well as defense molecules against plant pests and path-ogens (Xavier-Filho, 1992). Kunitz-type inhibitors are characterizedby molecular masses around 20 kDa, a low cysteine content formingtwo disulphide bonds and a common structural fold composed of aβ-trefoil formed by 12 antiparallel β-strands with long interconnect-ing 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 char-acterization and cloning of the major protein from Talisia esculentaseeds (Talisin), a member of the Sapindaceae family. The deducedpeptide presented high similarity with several storage proteins andall demonstrating amino acid sequences that were clearly related tothe Kunitz family of the proteinase inhibitor (Freire et al., 2009). Ithas also been shown that Talisin could be a saccharide-binding pro-tein characterized by a proven interaction with carbohydrates onneutrophil or mononuclear cells (Freire et al., 2003) and to with thechitin component of the peritrophic membrane (or equivalent struc-tures) in the C. maculatus (Macedo et al., 2002). Dixon plot and nega-tive staining against bovine trypsin indicate that Talisin presents anon-competitive inhibition of trypsin (Freire et al., 2009). Talisin

87M.G.M. Freire et al. / Comparative Biochemistry and Physiology, Part B 161 (2012) 86–92

also showed insecticidal activity against several insects (Macedo etal., 2004a, 2004b, 2011).

Talisin's structural data are not available; in this report, weobtained the theoretical three-dimensional structure of Talisin by ho-mology modeling. We used a molecular docking strategy in order toinvestigate the effects of this protein on the growth and developmentof Diatraea saccharalis, the major insect pest of sugarcane in Braziland other South American countries, which is responsible for signifi-cant economic damage.

2. Materials and methods

2.1. Plant material and chemicals

T. esculenta (Sapindaceae) seeds were collected in the State of Ceará(Brazil). Unless indicated otherwise, all chemicals were purchased fromSigma-Aldrich (St. Louis, MO, USA) or Amersham Biosciences (Uppsala,Sweden).

2.2. Insects

D.saccharalis (Lepidoptera: Crambidae) larvaewere from a laborato-ry colony and provided by Dr. J.R.P. Parra (Departamento de Entomolo-gia, Fitopatologia e Zoologia Agrícola, Escola Superior de AgriculturaLuiz de Queiroz, Universidade de São Paulo, Piracicaba, SP, Brazil). Thecolony was housed in standard conditions (25±1 °C, 60±10% relativehumidity 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 andthen centrifuged at 10,000 g for 30 min at the same temperature.The clear supernatant (crude extract or CE) was used to determinethe protein content. The CE was diluted in 150 mM NaCl and appliedto a Sephadex G-100 column (2.5 cm×80 cm) equilibrated with thesame solution. The protein-rich fraction was recovered and appliedto a chitin column (1.5 cm×10 cm) equilibrated with 50 mM phos-phate buffer, pH 7.6, and eluted with 100 mM HCl. The purified pro-tein was dialyzed and lyophilized.

2.4. Protein quantification

Protein concentrationswere determined by the dye-bindingmethodof 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 reduc-ing and non-reducing conditions. Proteins were stained with Coo-massie Brilliant Blue R-250, and molecular mass was determinedusing molecular-mass references of phosphorylase B (94 kDa), bovineserum 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 evaluatedusing the artificial medium system previously developed by Greenet al. (1976). For the bioassays, neonate D. saccharalis larvae wereselected and fed an artificial diet containing 1%, 1.5% and 2% of Talisin(w/w). Control larvae were fed with untreated diet. Each treatmentwas set up in glass containers (8.5 cm long×2.5 cm diameter) andfive larvae were transferred to each glass container (n=75). After

the larvae reached the fourth instar at standard conditions, the rela-tionship between protein content and the weight and number of lar-vae were determined. Linear regression analysis was used to describethe response of D. saccharalis to various doses of Talisin. The effectivedose for a 50% response (ED50) was defined as the concentration ofTalisin that decreased the mass of the insect to 50% of that of controllarvae. The lethal dose (LD50) corresponded to the concentration ofTalisin that reduced the number of insects to 50% of the numberfound in control diet.

2.7. Midgut preparation

Proteinases were obtained from the midguts of fourth-instar lar-vae, according to Macedo et al. (2007a). Fourth-instar larvae werecold-immobilized and the midgut, along with its contents, was re-moved in cold 150 mM NaCl and stored frozen (−20 °C). Guts fromlarvae of D. saccharalis were subsequently homogenized in 150 mMNaCl, centrifuged for 5 min at 6000 g at 4 °C, and the supernatantspooled and kept on ice for enzymatic assays.

2.8. Enzyme assays

Bovine pancreatic trypsin andmidgut extracts were used for the en-zymatic assays. Trypsin-like activities were assayed using N-benzoyl-DL-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 insectmidgut larvae, was determined by incubating a mixture of midgut ex-tractswith different concentrations of Talisin. Incubationwas performedin five repetitions. The inhibitory activity was measured by the differ-ence in enzyme activity with and without Talisin.

The effect of Talisin on the proteolytic activity ofmidgut extracts wasmeasured using 1 mM BAPNA at pH 8.0 after incubation with Talisin at37 °C for 20 min. The residual enzymatic activity was assayed as de-scribed above. The assayswere run in triplicate with appropriate blanks.

2.9. Digestion of Talisin

The digestion of Talisin by midgut extract was carried out as de-scribed by Macedo et al. (2002). The midguts of fourth-instar larvaewere dissected, extracted in 1 mL of 0.1 M Tris buffer, pH 8.0, and pro-cessed as described above. Talisin was incubated with this homogenatein Tris buffer (final concentration, 2 mg/mL). The Talisin:midgut proteinratio was 1:1. Digestion was done for 1, 2, 4, 6, 12 and 24 h at 30 °C andwas stopped by immersing the tubes in boiling water for 2 min. Thedegradation of bovine serum albumin (BSA) (2 mg/mL) was used as apositive control for serine proteinase activity. The digestion wasstopped as described above. The relativemolecular masses of the diges-tion products were estimated by 12.5% SDS-PAGE using proteinmarkers 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 (Thompsonet al., 1994; Kumar et al., 2008) was performed for the proteins, inorder to find a template with high identity and analyze primary se-quence similarities, observing the characters with reactive sites anddisulfide bonds. Homology modeling was performed using Modeller9v6 (Berman et al., 2000), the template found with the highest id-entity was the protein under the Protein Data Bank (PDB) accessioncode 1R8N with 1.75 Å of resolution, a Kunitz-type inhibitor resolvedby X-ray diffraction. Fifty models were built and the best one waschosen based on the DOPE scores (Laskowski et al., 1993) and overall

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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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resolution. Energy minimization was performed on GROMACS usingthe steepest descent method. The tridimensional model was validat-ed utilizing PROCHECK (Wiederstein and Sippl, 2007) through Rama-chandran Plot and g-factor of −0.18 using PROSA II web server(Ritchie, 2008) observing an acceptable z-score of −4.85. The rootmain square deviation value RMSD: 0.367 Å was observed in orderto find structural differences between template and target protein.

2.11. Molecular docking

HEX v.5.1 (Ritchie, 2008) program was used to examine possiblemodes of interaction of Talisin with trypsin (PDB ID: 1FN6) a hydro-lase from the porcine pancreas (Deepthi et al., 2001). Briefly, this pro-cedure performed global rotational and translational space scans byusing Fourier transformations, which rank the output according tosurface complementarity and electrostatic characteristics. A list of500 complexes of trypsin inhibitor was ranked and biochemical datawere available to filter out possible solutions. Previous knowledgeof trypsin catalytic site location was used to filter binding models;enzyme–inhibitor complexes were discarded if they showed inhibitoratoms interacting with catalytic triad trypsin, according to the non-competitive mechanism proposed. Furthermore, the best 50 post-processing energy minimization model complexes were analyzedby 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) por-tal, forty three sequences were mined out of the Entrez Protein DataBank with the following criteria: 19 Kunitz-type inhibitor proteins, 16lectin-like proteins and 7 storage proteins. Using these sequences anunrooted phylogenetic tree was built using maximum parsimony onMEGA software with 1000 bootstrap replicates (Kumar et al., 2008).The distribution of the representative's storage proteins, lectin-typeproteins and Kunitz-type inhibitor was observed in a subclass level(Taxonomy: Rosids).

2.13. Statistical analysis

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 softwarewas used for the analysis.

3. Results and discussion

The phytophagous larvae of most lepidopteran species analyzed sofar have alkaline midgut fluids, with serine proteinases and exopepti-dases providing most of the midgut proteolytic activity. Talisin wasassayed in vitro against proteinases from D. saccharalis (Fig. 1) inorder to assess the general hypothesis that serine proteinase inhibi-tors can protect plants against herbivorous insects. The digestive en-zymes extracted from larvae of D. saccharalis were trypsin-likeenzymes (data not shown), and were inhibited to different degrees byTalisin. The substantial inhibition of gut proteinases of D. saccharalis inthe in vitro assay suggested that Talisin may affect the growth and/orsurvival of this insect pest when incorporated into their diet.

The strategy of interfering with digestion and, thus, affecting thenutritional status of the insect, is widely employed plants to defendthemselves against pests and has been extensively reviewed (Carliniand Grossi-de-Sá, 2002; Ramos et al., 2009; Macedo et al., 2010).

The effect of Talisin on larval development was monitored by feed-ing the larvae on an artificial diet (final protein concentrations rangedfrom 0.5% to 2.0% of total dietary protein) and then determining thenumber and mass of surviving fourth-instar larvae. The dose–response

effect of Talisin on larvae is shown in Fig. 2A and B. The weight of larvaefed 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 ap-proximately 34%, 47% and 60% decrease in weight, respectively. The in-sert of Fig. 2A shows the variation in the size of D. saccharalis larvae fedTalisin and the control diet (right and left, respectively). Regressionanalysis indicated that for each increase of 0.1% in Talisin content

Time (h)

9466

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WM Talisin 1 2 4 6 12 24

Talisin

Fig. 4. SDS-PAGE patterns of Talisin digested by D. saccharalis midgut proteinases.(WM) weight marker.

89M.G.M. Freire et al. / Comparative Biochemistry and Physiology, Part B 161 (2012) 86–92

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 bythe y-intercept value) was about 10%, whereas diets containing 1.0%,1.5% and 2% Talisin caused 30%, 40% and 52%mortality, respectively. Re-gression 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 markedlylower than that found in legume seeds and is similar to other insecti-cide 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 dietscontaining 1.5% Talisin was observed for trypsin-like activity, utiliz-ing BAPNA as a synthetic substrate. The enzyme assay showed thatTalisin-fed larvae resulted in lower levels of trypsin activity per mgprotein in the gut and no change in levels of trypsin activity as mea-sured in feces (Fig. 3A and B). Tryptic activities in the midgut fromfourth-instar D. saccharalis larvae reared on artificial diets containing1.5% Talisin were altered by 27% (Fig. 3A). The lack of an increase inthe trypsin activity of feces from Talisin-fed larvae suggests that Talisindid 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 profilesof protein bands are presented in Fig. 4. Talisin was resistant to diges-tion during the first 24 h of incubation, in contrast to the control pre-pared with BSA where digestion of protein occurred during the first3 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 tochitin in the peritrophic membranes of the larval midgut (Chrispeelsand Raikhel, 1991). Also, because of the proteolytic activity of the insect

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Fig. 3. Trypsin-like activities in fourth-instar larvae feeding on a control artificial dietand a diet containing 1.5% Talisin. (A) Enzymatic activities of the midgut. (B) Enzymaticactivities of the feces. Trypsin activity was evaluated using BAPNA as substrate.

midgut, these proteins must be insensitive to proteolysis. Therefore, re-sistance to gut proteolysis, specificity for carbohydrate receptors, theability to bind to different parts of the small intestine, and functionaland morphological changes caused would define the efficiency of theprotein in terms of insecticidal activity (Pusztai et al., 1990). Severalstorage proteins with insecticidal activity are resistant to degrada-tion by insect digestive enzymes, such as V. unguiculata vicilins (reserveprotein-7S) (Macedo et al., 1993), maize zeatoxin (Macedo et al., 2000a)and coffee legumin-like proteins (Coelho et al., 2010). Talisin was insen-sitive to gut proteinases from D. saccharalis larvae, indicating that theymay have a role in protecting plants against phytophagous insects.

In order to investigate the distribution relation of Talisin and otherplant proteins in taxonomic level Rosids, an unrooted phylogenetictree was constructed based on the similar amino acid sequences ofpredicted Talisin and members of other plant species (Fig. 5). Fortytwo non redundant sequences were extracted from the Entrez ProteinData Bank, among these, nineteen Kunitz-type inhibitors, sixteen lec-tin proteins and seven storage proteins: these sequences werealigned on ClustalW and imported to MEGA software with the previ-ously described parameters. The unrooted phylogenetic tree presentedwell-defined branches. It was possible to observe three clades; onerepresented by storage proteins, one by lectin-like proteins and otherby Kunitz-type inhibitors. Talisin presented a high identity degreewith a storage protein obtained from Litchi chinensis and similarity toa Kunitz-type inhibitor fromDelonix regia, an ancestor of this type of in-hibitors in Fabaceae (Norioka et al., 1988).

Aiming to make a comparative analysis using protein sequencesobtained through the Expasy Protein-Bank using the Bioinfo Meta-Server, it was possible to observe 37% of identity to the D. regiaKunitz-type proteinase inhibitor (PDB ID: 1R8N) and 65% of identitywith storage proteins, L. chinensis, which does not possess the tridi-mensional structure resolved by experimental methods (Fig. 6). Thecysteine residues were conserved in Talisin and L. chinensis after thealignment, indicating a folding with disulfide bond formation, thischaracteristic was observed in several Kunitz-type inhibitors, suchas Glycine max, Erythrina caffra and Copaifera langsdorffii (Onestiet 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. Theconstructed model displays internal three-fold symmetry, as previ-ously observed in SKTI (soybean Kunitz trypsin inhibitor), and onepolypeptide chain linked by two disulfide bridge intrachains, similarto those of Kunitz-type inhibitors (Fig. 7). The amino acids werestructurally organized into loops connecting consecutive β-strands,a pattern observed in Kunitz inhibitors. Overlap of these domainsshowed a high similarity among β-strands but not among the con-necting loops presenting the classic barrel-shaped structure.

The Procheck summary of Talisin showed that 98.3% of amino acidresidues were in the favorable region and only three residues (Tyr102,

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 saccharide-binding protein from T. esculenta seeds with trypsin-inhibitory activity.

90 M.G.M. Freire et al. / Comparative Biochemistry and Physiology, Part B 161 (2012) 86–92

Asn114 and Ser135) were in the disallowed regions. These residueswere present within loops and as such were not expected to affectthe Talisin predicted structure. Structural differences between thetemplate and predicted three-dimensional structure of the Talisinmodel were calculated by superimposing both structures. The RMSDvalues between the template and homology 12model of Talisin calcu-lated for Cα traces and main chain atom were 0.36. The RMSD valuesand small variability among experimental structures template andthe structure modeled reflect the presence of strong restraints inmost regions and emphasize a similar folding pattern among these

Fig. 6.Multiple alignment between Talisin (TALI_ESCUL), a vegetative storage protein from La lectin from Dioclea lehmani (DIOC_LEHM).

structures. Furthermore, the lower Z-score acquired for PROSA IIand high score acquired for overall steric G-factor in the case of theTalisin were of −4.85 and −0.18, respectively, indicating the highquality of the model. The result indicated that the constructed Talisinmodel presented its amino acid residues within the average of the ob-served parameters. On the other hand, the structure of lateral chainswas considered well located, when compared to the experimentalstructures 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).

itchi chinensis (LICT_CHIN), a Kunitz-type inhibitor from Delonix regia (DELO_REGI) and

Fig. 7. The three-dimensional structure of Talisin and its surface is shown using cartoonsas the display preference obtained trough homology modeling. Molecular model shows aRMSD of 0.367 Å when compared to template (1RN8).

91M.G.M. Freire et al. / Comparative Biochemistry and Physiology, Part B 161 (2012) 86–92

The model of interaction showed a mechanism of inhibition of thenon-competitive type identical to that observed in the Adenantherapavonina inhibitor (Prabhu and Pattabiraman, 1980).

The inhibitor reactive site is able to block substrate access due tofive interactions with the most favorable enzyme region, forming acomplex with trypsin, with a surface area of 1010 Å2. These contactsprevent substrate access to the enzyme, although no direct reactionwith the catalytic site was observed. The absence of the reactive siteArg64, substituted by Glu64 supports the inactivity of this loop. A sim-ilar blockade was observed by Migliolo et al. (2010) during in silicodocking between ApTKI (Adenathera pavonina trypsin Kunitz inhibi-tor) and trypsin. A non-competitive type interaction was responsiblefor forming a surface area of 1.228 Å2 with trypsin. These results aredifferent for the Kunitz-type inhibitors, which interact with protein-ases through this reactive loop (Major and Constabel, 2008). TheOH− atom negative charge of Asp78 residue in Talisin interacts withthe lateral chain of the Ser147, which receives protons forming a hydro-gen bound presenting 3.14 Å. The amino acid residue Ser94 in Talisinreceives protons of the OH− from Tyr217 forming hydrogen bound

Fig. 8. Interaction between structural model from Talisin (black) against serine pro-teinase from porcine beta trypsin (grey) (PDB ID: 1FN6). In stick are representedamino acid residues involved in interaction and lines are represented catalytic triad(HIS57, ASP102 and SER195).

with 2.5 Å. The residue of Asn119 (donor protons) that forms a hydrogenbound with Asn97 (receiver protons) presented distance of 2.64 Å. Anouter hydrogen bound was observed with a distance of 2.52 Å betweenthe lateral chain Ser148 and lateral chain of Gln192. An electrostatic inter-action was observed between the carboxyl group Glu151 (negativecharge) and amine group of Lys60 (positive charge) with a 2.92 Ådistance.

The most favored HEX v5.1 result presented an interface surfacearea of 1010 Å2 and a complementarity gap volume index score of2.48. Planarity and circularity values (Gabb et al., 1997) of 2.7 and0.44 are also typical. No other solutions had favorable combinationsof large interface area and good complementarity; the mean valuesamong the 50 analysis solutions were 860±99 Å2 and 3.4±0.7(mean±SD). The values were also best when compared with inter-face area and complementarity in enzyme–inhibitor complexes withtheir upper limits at 785 Å2±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 proteinsmay be derived from genes that originally encoded proteins with awell-defined enzymatic or other biological activity (Van Damme et al.,2000). In consequence, some carbohydrate-binding proteins work asmultifunctionalmolecules, i.e. theymay presentNH2 terminal sequencehomology to Kunitz-type inhibitors, no trypsin-inhibitory activity, butexhibit lectin-like activity such as Labramin, a protein from Labramiabojeri seeds (Macedo et al., 2007b, 2007c) or show sequence similarityto soybean trypsin inhibitor (SBTI), with no trypsin-inhibitory activity,but display hemagglutinating activity such as the lectin obtained fromPseudostellaria heterophylla roots (Van Damme et al., 2000).

We suggest that the action of Talisin on D. saccharalis larvae mayinvolve: (1) a strong resistance to digestion by midgut proteinasesin D. saccharalis and (2) inhibition of endogenous trypsin-like activity.The strong negative effects of Talisin on D. saccharalis larvae, observedin this study, suggest that Talisin might be able to provide a viable al-ternative for designing insect-resistant transgenic crops or for use as anatural pesticide.

4. Conclusions

The present study provided new in vitro and in silico biochemicalinformation about Talisin, a storage protein with the structure of anon-competitive trypsin inhibitor. The trypsin binding site is completelydifferent from that of previously proposedKunitz-type inhibitors,makingTalisin acts as a bifunctional protein.

Acknowledgements

This work was supported by the FUNDECT (Fundação de Apoio aoDesenvolvimento do Ensino, Ciência e Tecnologia do Estado de MatoGrosso do Sul), CNPq (Conselho Nacional de Desenvolvimento Científicoe Tecnológico) and FINEP (Financiamento de Estudos e Projetos/Ministério da Ciência e Tecnologia).

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