Regulating inhibitory activity of potato I-type proteinase inhibitor from buckwheat by rutin and quercetin

Xiaodong Cui1 | Yifan Lv1 | Zhuanhua Wang2 | Jiao Li2 | Chen Li2

1 | INTRODUC TION

Plants are usually unavoidably faced with abiotic and biotic stresses. They respond to these stresses through a number of inducible de- fense mechanisms, including thickening of the cell wall, changing the lipid composition in the cellular membranes, increasing the concentration of phytohormones, accumulating secondary metab- olites, producing active forms of oxygen and nitrogen, activating the synthesis of defense proteins and peptides, and developing a hypersensitivity response and a systemic acquired resistance (Nurnberger et al., 2004). There are a large number of proteins or peptides with various properties in the plant immune response, some of which comprise classes of large family of proteins associ- ated with pathogenesis, namely pathogenesis-related (PR) proteins (Finkina et al., 2019). PR proteins comprise 17 classes of proteins/ peptides with different molecular masses, structures and func- tions. The expressions of these proteins increase at certain stages of ontogenesis, as well as when plants are under abiotic and biotic stresses (van Loon et al., 2006). The proteins in this family include: glucanases (PR-2), chitinases (PR-3,4,8,11) and endoproteases (PR-7), which are proteins hydrolyzing the fungal cell wall; protease inhib- itors (PIs, PR-6); lipid transfer proteins (LTPs, PR-14), which are the Bet v1 homologs (PR-10); and antifungal proteins (PR-1), thaumatin- like proteins(PR-5), thionins (PR-13), and defensins (PR-12), which are peptides and proteins with pronounced antimicrobial activity (Stintzi et al., 1993). Two of the proteins in the PR protein family, PR-10 and PR-14, are characterized by their ability to bind lipids and other hy- drophobic ligands (as well as secondary metabolites) and transfer them to the target sites (Shenkarev et al., 2017; Śliwiak et al., 2016). PR-14 (or LTPs) and PR-10 (or Bet v1 homologs) can form complexes with ligands in vitro, but the success of the complication can depend on the size of the hydrophobic cavity and the nature of amino acid residues in the proteins, the spatial structure of ligands, and the ex- perimental conditions (Melnikova et al., 2019).

PIs (or PR-6) are storage proteins that control the protein degradation by inhibiting endogenous enzymes. The expression levels of PIs in plants has been shown to increase in response to various abi- otic and biotic stresses, particularly in defending against insect pests (Jamal et al., 2012). In addition, PIs protect plants by suppressing ac- tivity of proteolytic enzymes of phytopathogens, insects, nematodes, and herbivorous animals. Since PIs can block intestinal protease, they were first thought to also cause malabsorption of amino acids in animals and can inhibit their growth (Wang et al., 2003). Standard plant serine proteinase inhibitors are categorized into different fam- ilies, including Kunitz, Bowman-Birk, Potato I and II and squash fam- ilies (Haq et al., 2004). These inhibitors are known as antinutritional factors in human diet as they can significantly reduce the digestion and absorption of essential nutrients and lead to adverse health ef- fects in human or animals. To ensure the safety and nutrition, it is necessary and important to reduce or inactivate the trypsin inhibitor activity (TIA) by proper methods. Some natural components from plants from plants have been found to deactivate the TIA. Some re- searchers have reported that tea polyphenols (TPs) can deactivate both Kunitz trypsin inhibitor (KTI) and Bowman-Birk trypsin inhib- itor (BBTI) (Chen et al., 2020; Huang et al., 2004; Liu et al., 2017). Stevioside can inactivate the trypsin inhibitor activity (TIA) of BBTI by forming a complex with BBTI, blocking it from binding to trypsin (Liu et al., 2019). Buckwheat trypsin inhibitor (BTI) is a member of the potato I-type family inhibitor that can be found in buckwheat seed; its molecular structure is different from those of BBTI and KTI (Wang et al., 2011, 2015). Rutin and quercetin are main components that constitute dietary flavonoids presented in buckwheat (Fabjan et al., 2003). Rutin has been used in pharmaceutical drugs because of its anti-oxidative, anti-bacterial, and anti-hyperglycaemic properties and quercetin is more efficient in oxidant removal and cell protection than rutin (Jia et al., 2019). The phenolic part (quercetin) of rutin is linked to a sugar molecule, a hydrophilic moiety of the molecule (Cui & Wang, 2012). Although this linkage slightly reduces the biological effect of the molecule, it makes the molecule become more soluble. Rutin in buckwheat can be cleaved into quercetin and rutinose by rutin-hydrolyzing enzyme (Cui & Wang, 2012). However, the inacti- vation effect and mechanism of these secondary metabolites (rutin and quercetin) from the buckwheat on rBTI remain unclear.

In this paper, we determined the effects of two flavonoids from buckwheat, rutin and quercetin, on the inhibitory activity of recom- binant buckwheat trypsin inhibitor (rBTI) and investigated the inter- actions between them, with an aim to understand whether rutin and quercetin can bind and regulate the trypsin inhibitory activity (TIA) of rBTI. We also report the molecular binding modes between these fla- vonoids and rBTI. The results can allow us to better understand the binding interactions between ligands and potato I-type inhibitor family.

2 | MATERIAL S AND METHODS

2.1 | Materials

Trypsin was purchased from Solarbio Life Science (Beijing, China). Nα-benzoyl-DL-arginine p-nitroanilide (MFCD00012846) was obtained from Sigma Aldrich (St Louis, USA). Mutan BEST kit was purchased from TaKaRa (Dalian, China). Rutin (>98% pure) and quercetin (>98% pure) were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Escherichia coli DH5α and E. coli BL21 (DE3) pLysS cells were purchased from TransGen Biotech Co., Ltd. (Beijing, China). All other chemicals used in this study were of ana- lytical grade.

2.2 | Sequence and hydrophobic core analysis

Amino acid sequence alignment was conducted using ClustalW (http://www.genome.jp/Tools-bin/clustalw) to identify the con- served regions among potato I-type family inhibitors from differ- ent plants. The sequence alignment was generated using program ESPript3.0 (Robert & Gouet, 2014), and the structural annotation was manually added. Disulfide pairs are indicated above the logo. Residue numbers and helices according to the rBTI structure are shown below the sequence. Tryptophan residues and hydrophobic core in the structure of rBTI (PDB ID: 3rdy) were also analyzed.

2.3 | Expression and purification of rBTI

Recombinant buckwheat trypsin inhibitor (rBTI) was produced by cloning the coding sequence of BTI into pExSec I expression vec- tor; the resulting DNA construct was named pExSec I-BTI. After confirming the sequence identity, the vector was transformed into E. coli BL21 (DE3) pLysS cells according to the instruction manual. To prepare a starter culture, single colonies were inocu- lated into LB medium and then incubated at 37°C for 16 hr under shaking at 250 rpm. After that, the starter culture was diluted 50 folds in fresh LB medium and was further incubated at the same conditions until the optical density at 600 nm reached 0.6–0.8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was then added to the culture to a final concentration of 1 mM to induce the protein production. The induction was carried out at 37°C for 4 hr under shaking at 250 rpm. The cells were then centrifuged at 8,000g for 20 min at 4°C, and the cell pellet was resuspended in 20 mM Tris-HCl buffer (pH 7.5) and was immediately lysed by ultrasonica- tion. The crude cell lysate was incubated at 80°C for 30 min and was then centrifuged at 12,000g for 10 min at 4°C. The superna- tant obtained was loaded onto a Resource Q column equilibrated with 20 mM Tris-HCl buffer (pH 7.5), and the target protein was eluted with the same buffer containing 0.5 M NaCl. The eluted protein that exhibited inhibitor activity was further separated on a Superdex 75 10/300 GL gel filtration column (GE Healthcare, Uppsala, Sweden). The purified rBTI was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 15.0% polyacrylamide gel. The gel was stained by Coomassie brilliant blue R-250 and then scanned by a densitometer to deter- mine the concentration of the target protein.

2.4 | Site-directed mutagenesis of rBTI

Based on the amino acid sequence alignment of rBTI and other plant proteinase inhibitors, two tryptophan (Trp, W) residues, Trp10 and Trp53, were mutated to alanine (Ala, A), and the previously con- structed pExSec I-BTI plasmid was used as a template. The PCR amplification of the target gene with the mutation site was carried out using TaKaRa Mutan BEST Kit and the following primers: for- ward primers 5ʹ- CAAGAAGCCCCAGAGGTC-3ʹ and reverse primer 5ʹ-TTTACCGGAGCACTGAC G-3ʹ for rBTI-W10A; and forward primer 5ʹ-GACCGTGTGGCCGTTTTCGTA-3ʹ and reverse primer 5ʹ- ACATCGGAGGTCTCTAGGCAC-3ʹ for rBTI-W53A. The resultant pExSec I-BTI-W10A and pExSec I-BTI-W53A plasmids were trans- formed into E. coli BL21 (DE3) cells. The expression and purification of the mutant proteins were conducted according to Section 2.3.

2.5 | Fluorescence spectroscopy

Measurement of fluorescence spectra of rBTI and mutants in the presence of rutin or quercetin was performed using a LS55 Fluorescence spectrometer (Perkin Elmer, USA) at 298 K and 310 K at an excitation wavelength (λex) of 280 nm and at emission wave- lengths (λem) of 290–450 nm. The excitation and emission slit widths were set at 5.0 nm. The fluorescence intensity at the maximum fluorescence emission wavelength (λmax) was used to calculate the quenching parameters and the binding constants.Synchronous fluorescence spectra of rBTI or mutants in the absence and presence of rutin or quercetin were recorded at λem = 275–400 nm and Δλ = 60 nm. The excitation and emission band- widths were set at 5.0 nm.

2.6 | Circular dichroism (CD) spectroscopy

CD spectroscopy of rBTI in the absence and presence of rutin/ quercetin were performed on a Chirascan spectropolarimeter (Applied Photophysics Ltd., England) using a quartz cuvette with a path length of 0.1 cm. rBTI was first incubated with rutin or querce- tin for 20 min; after that, its CD profiles were recorded at a wave- length range of 190 to 260 nm. The scan was carried out three times, each at a scan rate of 100 nm min–1 at a 2 nm bandwidth and 0.2 nm spectral resolution. The contents of the secondary structure of the protein samples were estimated using an online Circular Dichroism website: http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d3/ (Louis- Jeune et al., 2012).

2.7 | Determination of effects of rutin and quercetin on rBTI inhibitory activity

The inhibitory activity of rBTI against trypsin was determined ac- cording to a method reported by Cui et al. (2018). Briefly, rBTI was mixed with rutin or quercetin in 4.4 ml of reaction buffer (20 mM Tris- HCl buffer, pH 8.0 containing 10 mM CaCl2), incubated for 10 min at 25°C or 37°C, and then 20 μl of trypsin (1 mg/ml, dissolved in 1 mM HCl) was added. The final concentration of trypsin is 0.2 μmol/L. The mixture was incubated at the same temperatures for another 10 min. After that, 40 μl of BApNA (50 mg/ml, trypsin substrate) was then rapidly added, and the mixture was thereafter incubated for another 10 min at same temperature. The reaction was terminated by 0.5 ml of 33% acetic acid. Finally, the absorbance at 410 nm of the reaction solutions was measured on a UV-Vis spectrophotometer.

2.8 | Molecular docking

The molecular docking was performed using EADock available on the SwissDock server to determine the binding sites and evaluate the binding mode of rutin and quercetin to rBTI at the molecular level (Grosdidier et al., 2011a, 2011b). The structure of rutin and quercetin were retrieved from PubChem, and the 3D structure of rBTI (PDB ID: 3rdy) was derived from RCSB PDB (http://www.rcsb. org/pdb/home/home.do). The Swiss-Dock results were given as full fitness and ΔG (kcal/mol). The docking results were analyzed on PyMOL (version 2.1) and Discovery Studio 4.5 Visualizer.

2.9 | Statistical analysis

All measurements were conducted in triplicate, and the results were expressed as means of the three measurements ± standard deviations (SDs). The statistical analysis was carried out using SPSS version 20.0. One-way ANOVA test (Tukey’s test) was used to deter- mine the statistical significant differences, and the difference with a p value of .05 or .01 was considered significant.

3 | RESULTS AND DISCUSSION

3.1 | Sequence and structural analysis of rBTI

The sequence alignment results are shown in Figure 1a. The struc- ture of rBTI mainly comprises a single α-helix (α1, residues 18–28), a central parallel β-sheet consisting of two strands (β1, residues 30–38 and β2, residues 51–56), an inhibitory loop (residues 39–50) and two irregular structures at the N-terminus (residues 3–15) and the C-terminus (residues 61–69). Multiple sequence alignment of the homologous PI-I proteins revealed that two motifs, WPELVG and DRVWV, were conserved across the PI-I family. The amino acid sequences had high similarity, especially at the two conserved Cys residues. Additionally, the crystal structure of rBTI has been solved (Wang et al., 2011). From the structure of rBTI, two tryptophan residues were observed in the sequence of rBTI: one (Trp10) is in the hydrophobic core, and the other (Trp53) is in the inhibitory loop of rBTI. Trp10 was conservative in both BTI and other inhibitors,which suggests that Trp10 may play a key role in protein structure retention. However, in some inhibitors such as BGTI, MCTI, and CMTI, Trp53 was replaced with Arg (Figure 1b). Trp53 affects the binding of the inhibitor to trypsin because the rBTI-W53R/trypsin complex is highly unstable. Compared with wild-type rBTI, the mu- tant rBTI-W53R had a slightly lower association rate but a higher dissociation rate (Song et al., 2011). As shown in Figure 1c, the hy- drophobic core in rBTI is formed by α1, β1, β2 and two short loops (side chains of Trp10, Ile25, Val32, Val52 and Pro66).

FI G U R E 1 Sequence alignment and structural analysis of rBTI. (a) Sequence alignment of rBTI and other plant potato I-type inhibitors. Spirals indicate helices, and arrows indicate β-sheets. BTI: buckwheat trypsin inhibitor; AHTI: Amaranthushypochondriacus trypsin inhibitor; BGTI: bitter gourd trypsin inhibitor; CMTI: Cucurbita maxima trypsin inhibitor; JCPI: Jatropha curcas proteinase inhibitor; LUTI: Linum usitatissinum trypsin inhibitor; MCTI: Momordica charantia trypsin inhibitor; and ZEPI: Zinnia elegans protease inhibitor. (b) Tertiary structure of rBTI. R45 is the inhibitory activity site (Red). W10 and W53 are tryptophan residues in rBTI. (c) Hydrophobic core in rBTI.

3.2 | Expression and purification of rBTI and mutants

The wild-type rBTI and mutants were analyzed by SDS-PAGE. rBTI and rBTI-W53A had similar apparent molecular weights, however, the band of rBTI-W10A was higher than that of rBTI and rBTI-W53A (Figure 2a), indicating that the structure of rBTI-W10A may have undergone conformational change. In Cucurbita maxima trypsin inhibitor-V (CMTI-V), Trp9 (in rBTI is Trp10) is located in a hydro- phobic pocket and has van der Waals contacts with the α-helix resi- dues Ile24, Gln27, and Asn28 and the β-sheet residues Val51, Pro65, Arg66, and Ile67 (Cai et al., 2002). There is a hydrogen bond be- tween Trp9 and Ile23, which holds together the N-terminal segment and the α-helix (Cai et al., 1995). Mutation of Trp10 may make struc- ture rBTI-W10A looser than wild rBTI. The fluorescence emission spectra of rBTI and mutants were compared. As shown in Figure 2b, at an excitation wavelength of 280 nm, the maximal fluorescence emission wavelength of rBTI was 341 nm, while that of rBTI-W10A and rBTI-W53A were 355 nm and 348 nm, respectively. The fluores- cence intensities of the two mutants decreased dramatically, due to the mutations in rBTI-W10A and rBTI-W53A. These results suggest that the structure of the mutants may be significantly altered, and their tryptophan residues may be located in a relatively more hydro- philic position than those in the wild type rBTI.

CD can sensitively and directly monitor the structural changes of proteins or their bindings to ligands. Comparison of changes of the secondary structure contents (α-helix, β-sheet and ran- dom coils) of rBTI and mutants calculated by CD Pro software are listed in Table S1. According to the far UV-CD profile (190–240 nm; Figure 2c), rBTI exhibited a single negative peak at 208 nm, indicating that the dominant secondary structure in rBTI is β-sheet. Compared with wild-type rBTI, rBTI-W10A underwent more significant struc- tural changes, but rBTI-W53A underwent less significant structural changes. This result further confirms that Trp10 plays an important role in maintaining the integrity of the structure of rBTI.

3.3 | Intrinsic fluorescence spectroscopy

Fluorescence spectroscopy is a powerful technique that can be used to study the molecular interactions between proteins and quenchers.(Trp10 and Trp53) in the sequence of rBTI allow rBTI to exhibit a strong intrinsic fluorescence signal at near 341 nm. We observed that the fluorescence intensity of rBTI dramatically decreased in the presence of rutin and quercetin. This observation indicates that rutin and quercetin interact with rBTI and cause the florescence quenching. The fluorescence intensity of rBTI was lowest when the concentration of rutin and quercetin reached 10 μΜ (Figure 3a,d). Additionally, in the presence of rutin, the maximal fluorescence emission wavelength (λmax) of rBTI, which was at 341 nm, was slightly blue shifted to 339 nm, indicating that Trp in rBTI became more ex- posed to the hydrophilic environment upon the addition of rutin. The effects of rutin and quercetin on the two mutants, rBTI-W10A and rBTI-W53A, were also investigated. As shown in Figure 3b-e, the ef- fect on rBTI-W10A and rBTI-W53A was similar to that on wild type rBTI. At the same concentration, the quenching ability of rutin was higher than that of quercetin. The λmax of the two mutants was sig- nificantly blue shifted in the presence of rutin and quercetin.The fluorescence data were further analyzed by the Stern- Volmer equation as follows (Liu et al., 2018):

FI G U R E 2 Purification and spectral properties rBTI and mutants. (a) SDS-PAGE of wild-type rBTI and mutants. Mr: molecular mass markers; lane 1: wild-type rBTI; lane 2: rBTI-W10A; and lane 3: rBTI-W53A. (b) Fluorescence emission spectra of rBTI and mutants (λex = 280 nm, λem = 290–450 nm). (c) Circular dichroism spectra of rBTI and mutants where, F0 and F are the steady-state fluorescence intensities in the absence and presence of the quencher, respectively; Ksv is the Stern- Volmer dynamic quenching constant, which can be obtained from the slope of the curve of F0/F versus [Q]; [Q] is the concentration of the quencher; kq is the bimolecular quenching constant; and τ0 is the flu- orophore life time in the absence of the quencher, which is 10−8 s for a biopolymer.

The quenching parameters of rBTI and mutants by rutin and quercetin at 298 K and 310 K calculated by Equation (1) are tabu- lated in Table S2. The Ksv values of the complex between rBTI or mutants and rutin decreased with increasing temperature. This may be due to that at high temperatures, the stability of rutin/rBTI, rutin/ rBTI-W10A, and rutin/rBTI-W53A complexes are reduced, which are correspondence with the static quenching. By contrast, the Ksv values of the complex between rBTI or mutants and quercetin in- creased with increasing temperature. According to the literature, the maximum value of kq in the dynamic quenching is 2 × 1010 mol–1 s– 1; however, the kq value for the quercetin/rBTI complex was higher than this maximum value, suggesting that the quenching rBTI or mu- tants by quercetin is a static quenching process. That is, the complex is more stable at high temperatures. This may be that quercetin is more hydrophobic than rutin.

In a static quenching process, the binding constant (Ka) and the number of binding sites could be calculated by the double logarith- mic Stern-Volmer equation (Chen et al., 2020) as follows:It has been widely used to investigate the quenching mechanism, as well as the binding properties including binding sites, binding mode and binding constants (Czubinski & Dwiecki, 2017). Trp and tyrosine (Tyr) are the major residues that contribute to protein intrinsic fluo- rescence. At an excitation wavelength of 280 nm, two Trp residues where Ka and n represent the binding constant and the number of bind- ing sites, respectively. The Ka and n values are the intercept and the slope, respectively, of a plot of log(F0–F)/F versus log[Q]. The obtained where R is the universal gas constant and T is the temperature, and ΔH and ΔS can be determined from a plot between lnKa versus 1/T.

FI G U R E 3 Effects of rutin and quercetin on intrinsic fluorescence spectra of rBTI and mutants. (a) rBTI and rutin. (b) rBTI-W10A and rutin. (c) rBTI-W53A and rutin. (d) rBTI and quercetin. (e) rBTI-W10A and quercetin. (f) rBTI-W53A and quercetin. The concentration of rBTI or mutants was 1.0 μM. The molar concentrations of rutin and quercetin were 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 μM. The measurement was carried out at T = 298 K, pH 7.4, λex = 280 nm, and λem = 290–450 nm.

The value of free energy changes (ΔG) of complex formation was calculated using the following Gibb’s free energy equation (Shahabadi et al., 2019) as follows that there is only one quercetin binding site on rBTI (Table S3). On the other hand, the n value of rBTI/rutin complex was closer to 2, implying that there is more than one rutin binding site on rBTI (Table S3). These data suggest that quercetin binds to rBTI at a 1:1 ratio, whereas rutin binds at a 2:1 ratio.

3.4 | Thermodynamic analysis

The non-covalent interactions between proteins and small mole- cules include hydrophobic forces, van der Waals forces, electrostatic interactions, and hydrogen bonds. Thermodynamic parameters, such as the Gibb’s free energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS), can help to characterize these interactions.

Changes in ΔG, ΔH, and ΔS are provided in Table 1. The negative values of ΔG suggest that formation of rutin/rBTI and quercetin/rBTI com- plexes is a spontaneous process. According to Ross and Subramanian (2002): when ΔH > 0 and ΔS > 0, the binding is mainly through hydro- phobic interactions; when ΔH < 0 and ΔS < 0, the binding is mainly through van der Waals forces and hydrogen bonds; and when ΔH < 0 and ΔS > 0, the binding is mainly through electrostatic interactions. In this study, both the ΔH and ΔS of the rutin/rBTI and rutin/mutants complexes were negative(ΔH < 0 and ΔS < 0), indicating that the bind- ing between rutin and rBTI or mutants is mainly through hydrogen bonds and van der Waals forces (Table 1). On the other hand, the ΔH and ΔS of quercetin/rBTI and quercetin/mutants complexes were posi- tive (ΔH > 0 and ΔS > 0), demonstrating that the binding forces in these complexes are hydrophobic interactions (Table 1). This also reflects that when quercetin binds to its binding site on rBTI or mutants, there may be release of water molecules and ions as a result of the hydropho- bic interactions. These results show that rutin and quercetin bind to different binding sites on rBTI through different interactions.

3.5 | Synchronous fluorescence spectra

Synchronous fluorescence spectroscopy has been widely used for studying the changes in the microenvironment of the fluorophores of proteins. Because rBTI does not contain Tyr residues, we fixed the interval between the excitation (λex) and emission (λem) wavelengths at 60 nm (Δλ = 60 nm) to reflect only the microenvironment changes of Trp residues. At Δλ = 60 nm, the fluorescence intensity of rBTI or mutants significantly decreased upon the gradual addition of rutin or quercetin (Figure S1). This indicates that Trp residues cause the quenching of the intrinsic fluorescence of rBTI. At the same Δλ, the binding of rBTI and rutin or quercetin caused a slight blue shift from 298 to 297 nm (Figure S1a,d). Similarly, the binding between rBTI- W10A with and rutin or quercetin caused a slight blue shift from 297.5 to 295 nm (Figure S1b,e). Interestingly, the binding between rBTI-W53A and rutin or quercetin did not cause a blue or red shift (Figure S1c,f). This suggests that Trp53 in rBTI may become more exposed to the environment after rutin or quercetin binds to rBTI, causing the microenvironment to become more nonpolar. In con- trast, after rBTI binds to rutin and quercetin, the microenvironment of Trp10 was unchanged. Thus, it was evident that rutin or quercetin could induce the conformational change of rBTI by interacting with sites adjacent to Trp53 residues.

3.6 | Circular dichroism (CD) spectra of rBTI/ rutin and rBTI/quercetin complexes

The structure changes of rBTI, rBTI/rutin and rBTI/quercetin com- plexes are revealed by CD spectra. As shown in Figure 4a, in the presence of rutin or quercetin at the same concentration, the nega- tive ellipticities of rBTI were unchanged. But the positive ellipticity of rBTI/rutin was increased, suggesting that rutin has markly effects of the conformation of wild-type rBTI. The contents of α-helix and β-sheet of wild-type rBTI were 4.0% and 27.4%, respectively, those of rBTI/rutin complex were 4.7% and 29.14%, and those of rBTI/ quercetin complex were 4.03% and 27.3%, respectively (Table S1).

3.7 | Effects of rutin and quercetin on inhibitory activity of rBTI

The effects of rutin and quercetin on trypsin inhibitor activity (TIA) of rBTI were evaluated. Figure 4b shows the TIA of rBTI in the pres- ence of rutin or quercetin at various ratios. In the absence of rutin or quercetin, the TIAs of rBTI at 25°C and 37°C were 56.25% and 54.1%, respectively; by contrast, in the presence of rutin or querce- tin, the TIAs of rBTI decreased at both temperatures. The TIAs of rBTI decreased with the increase of rutin or quercetin concentration, suggesting that both rutin and quercetin can deactivate the inhibi- tory activity of rBTI. However, the ability of quercetin to inhibit rBTI was stronger than that of rutin. Comparing the effects at different temperatures, the TIA of rBTI in the presence of rutin at a 1:1 ratio increased with increasing temperature: the TIA was 38.98% at 25°C and was 42.6% at 37°C. The same trend was also observed at a 2:1 ratio. On the other hand, the TIA of rBTI in the presence of quercetin at the same ratios decreased as the temperature increased. These results demonstrate that both rutin and quercetin can inhibit the TIA of rBTI, and quercetin has stronger inhibitory ability than rutin. Some flavonoids including rutin and quercetin also inhibit serine proteases, such as thrombin, trypsin, and urokinase plasminogen activator (uPA) (Cuccioloni et al., 2009; Mozzicafreddo et al., 2008; Xue et al., 2017). Quercetin acted as competive inhibitors with serine proteases and forming the 1:1 reversible complexes. Trypsin and uPA activities were reported to be diminished by quercetin (IC50 = 15.4 μm and 7 μm, respectively) (Xue et al., 2017).

FI G U R E 4 Changes in structure and inhibitory activity. (a) Circular dichroism spectra of rBTI and rBTI/rutin and rBTI/quercetin complexes. (b) Inhibition of TIA of rBTI by rutin or quercetin. The final concentration of trypsin is 0.2 μmol/L in the reactive system.

FI G U R E 5 Molecular docking of rBTI in a complex with rutin or quercetin. (a), (d), (g) Three-dimensional docking conformation of rBTI/ rutin and rBTI/quercetin. (b), (e), (h) Interaction residues of rBTI with rutin or quercetin. (c), (f), (i) Two-dimensional docking conformation of rBTI-rutin/quercetin.

3.8 | Molecular docking studies of rBTI and rutin/ quercetin

In order to more clearly differentiate the interactions in the rBTI/ rutin and rBTI/quercetin complexes, molecular docking was em- ployed. The optimal poses of the two flavonoids with rBTI are shown in Figure 5, which shows that rBTI has two sites for rutin binding and one site for quercetin binding. Rutin was found to bind to the lateral side of the hydrophobic core (Figure 5a-c) and the inhibitory loop (Figure 5d-f) with the lowest binding energies of −6.963 kcal/mol and −6.139 kcal/mol, respectively. In binding site I, rutin forms two hydrogen bonds with Ser15 and Glu58 residues and three π-alkyl bonds with Val 36 and Val56 residues in rBTI. Additionally, the inter- action in this site mainly occurs between the C6-C3-C6 ring and rBTI amino acid residues (Figure 5b,c). In binding site II, rutin is located adjacent to the inhibitory activity site of rBTI and forms three hydro- gen bonds with the reactive residue Arg45 and π-σ interaction with Met69 residue (Figure 5e,f).

The molecular docking of rBTI with quercetin showed that the binding energy of the two molecules was −6.54 kcal/mol. Quercetin forms three conventional hydrogen bonds with Asp46, Thr65 and Val67 in rBTI. In addition to the two π-π T-Shape bands, three π-Alkyl bonds and one π-Sulfur bond were also observed (Figure 5g-i). In addition, van der Waals force was also observed in both the docked molecule between rBTI and rutin and that between rBTI and querce- tin, indicating that this force is an important for the binding between these molecules (Figure 5h,i). It is also apparent that the numbers of hydrogen bonds and hydrophobic interactions in the rBTI/quer- cetin complex were higher than those in the rBTI/rutin complexes. Furthermore, because hydrogen bonds and hydrophobic interactions are the main forces that stabilize flavonoid/protein complexes(Wani et al., 2021), it is unsurprising that quercetin can form a more stable complex with rBTI than rutin.

4 | CONCLUSION

In summary, the interaction mechanism of rBTI and rutin or querce- tin was first investigated. The results revealed that the interaction between rBTI and rutin or quercetin followed the static quenching mechanism. The results from synchronous fluorescence and CD spectroscopy further showed that upon binding to rutin or querce- tin, rBTI underwent conformational changes. Rutin was found to bind to rBTI via van der Waals forces and hydrogen bonds, while quercetin was found to bind to rBTI through hydrophobic forces. In addition, the results demonstrated that both rutin and quercetin can decrease the TIA of rBTI. The molecular docking further revealed the molecular binding mechanisms between the two flavonoids and rBTI, which can be helpful for the understanding of how flavonoid affects the inhibitory of rBTI.

ACKNOWLEDG MENTS

The research was supported by Applied Basic Research Programs of Shanxi Province (No.201801D121192) and Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (No. 201802020).

CONFLIC T OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper.

AUTHOR CONTRIBUTIONS

Xiaodong Cui: Conceptualization; Funding acquisition; Methodology; Writing-original draft; Writing-review & editing. Yifan Lv: Formal analysis; Methodology. Zhuanhua Wang: Funding acquisition; Resources. Jiao Li: Methodology. Chen Li: Data curation; Formal analysis.