Food and Chemical Toxicology 74 (2014) 1–8

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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x

Binding of flavonoids to staphylococcal enterotoxin B ˇ rtomir Podlipnik b, Nataša Poklar Ulrih a,c,* Evgen Benedik a, Mihaela Skrt a, C a

Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškercˇeva 5, 1000 Ljubljana, Slovenia c Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CipKeBiP), Jamova 39, Ljubljana, Slovenia b



Article history: Received 17 April 2014 Accepted 21 August 2014 Available online 2 September 2014 Keywords: Flavones Catechins Molecular docking Fluorescence emission spectrometry Staphylococcal enterotoxin B


Staphylococcal enterotoxins are metabolic products of Staphylococcus aureus that are responsible for the second-most-commonly reported type of food poisoning. Polyphenols are known to interact with proteins to form complexes, the properties of which depend on the structures of both the polyphenols and the protein. In the present study, we investigated the binding of four flavonoid polyphenols to Staphylococcal enterotoxin B (SEB) at pH 7.5 and 25 °C: (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), kaempferol-3-glucoside (KAM-G) and kaempferol (KAM). Fluorescence emission spectrometry and molecular docking were applied to compare experimentally determined binding parameters with molecular modeling. EGCG showed an order of magnitude higher binding constant (1.4 × 105 M−1) than the other studied polyphenols. Our blind-docking results showed that EGCG and similar polyphenolic ligands is likely to bind to the channel at the surface of SEB that is responsible for the recognition of the T-cell beta chain fragment and influence the adhesion of SEB to T cells. © 2014 Published by Elsevier Ltd.

1. Introduction Intestinal infectious diseases are major contributors to morbidity and mortality in tropical countries, and they were estimated to have caused 1.5 million deaths in children in developing countries in 2002 (WHO, 2003). Staphylococcus enterotoxin B (SEB) is one of exotoxins produced by the ubiquitous Gram-positive facultative anaerobe Staphylococcus aureus which colonizes humans as well as domestic animals, and it is a common opportunistic pathogen. It is estimated that S. aureus persists in 20% of the general population, while another 60% are intermittent carriers (Kluytmans et al., 1997). The frequency of infections is higher in carriers than in noncarriers (von Eiff et al., 2001). Non-carriers commonly acquire infections through contaminated water or food, such as when food handlers who are carriers contaminate food during its preparation. In humans, the estimated 50% lethal dose (LD50) of SEB is 0.02 μg/ kg and the 50% effective dose (ED50) is 0.0004 μg/kg by aerosol exposure (Gill, 1982; Rusnak et al., 2004). There are no other data on LD50 and ED50 in humans by other routes of exposure. Symptoms of poisoning with SEB include high fever, nausea, vomiting, abdominal pain, cramps and hypotension, with or without diarrhea, toxic-shock syndrome, septicemia, and sometimes death. The

* Corresponding author. University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia. Tel.: +386 1 3230780; fax: +386 1 2566296. E-mail address: [email protected] (N.P. Ulrih). 0278-6915/© 2014 Published by Elsevier Ltd.

disease is usually self-resolving; it is rarely lethal, and the elderly are more susceptible (Pinchuk et al., 2010). Some studies have also considered enterotoxins to be a potential biological weapon (Mantis, 2005). There are 23 different enterotoxin types known, and they can be divided into five phylogenetic groups (Argudín et al., 2010; Vasconcelos and de Lourdes Ribeiro de Souza da Cunha, 2010). However, only a few of the enterotoxins have been studied in depth. The most common two of these are Staphylococcus enterotoxin A and SEB. Staphylococcal enterotoxins are similar to the gastrointestinal toxins, and they are responsible for the second-mostcommonly reported type of food poisoning through contaminated water and improperly prepared and stored food, with effects that can appear from a few minutes to 2 hours after ingestion (Argudín et al., 2010). These Staphylococcus enterotoxins have the remarkable ability to resist heat and acid. Therefore, they cannot be completely denaturated by mild cooking of contaminated food. They are pyrogenic and are resistant to inactivation by gastrointestinal proteases, including pepsin, trypsin, rennin and papain (Le Loir et al., 2003). SEB mediates immune activation on intestinal defensins (Dhaliwal et al., 2009). As well as being able to grow over a range of temperatures and pH, S. aureus can grow on a wide assortment of food. Therefore, if food that is contaminated with Staphylococcusenterotoxin-producing strains is left at temperatures that allow rapid growth of these bacteria (i.e., inadequate refrigeration), they become a common source of food poisoning (Pinchuk et al., 2010). Natural products from plants might be useful, particularly in developing countries where the availability of drugs is limited (Joung


E. Benedik et al./Food and Chemical Toxicology 74 (2014) 1–8

Table 1 Structures of the polyphenols used in this study. Catechins


Epigallocatechin-3-gallate (EGCG)

Kaempferol-3-glucoside (KAM-G)

Epigallocatechin (EGC)

Kaempferol (KAM)

et al., 2012). Plants contain high concentrations of polyphenols, which are considered to have antimicrobial, antiviral, antitoxic, antitumor, and anti-inflammatory properties (Daglia, 2012; Ferrazzano et al., 2011; Matsumoto et al., 2012), to reduce blood glucose and blood pressure (Ahn et al., 1999; Strobel et al., 2005), and have antiallergic activity (Maeda-Yamamoto et al., 2004). It has also been shown that the (-)-epigallocatechin-3-gallate (EGCG; the most abundant catechin in tea) has potential use as an adjunctive therapy in HIV-1 infections, because it binds to the T-cell receptor (Williamson et al., 2006). The known properties of such naturally occurring polyphenols mean that they are also of great interest to the pharmaceutical industry. The catechin polyphenols can act on different bacterial strains as well as on S. aureus, through several mechanisms. They can destabilize the cytoplasmic membrane, influence the permeabilization of the cell membrane, inhibit extracellular microbial enzymes, have direct actions on microbial metabolism, and sequester substrates required for microbial growth (Ferrazzano et al., 2011). The aim of our work is to investigate if some of the polyphenols, including two catechins, (-)-epigallocatechin-3-gallate (EGCG) and (-)-epigallocatechin (EGC), and two flavones, kaempferol-3glucoside (KAM-G) and kaempferol (KAM) (Table 1), can bind to SEB and to understand the structure–activity relationships between these selected polyphenols and their binding site on the SEB molecule. For this purpose the combination of fluorescence emission spectrophotometry and molecular docking was used to directly compare the predicted data with the experimental data, and to determine the potential modes of action of these polyphenols.

SEB was dissolved in 20 mM HEPES, pH 7.5, and further purified by dialysis against 20 mM HEPES, pH 7.5, for 36 h at 4 °C. The molar concentration of SEB in this aqueous HEPES solution was determined spectrophotometrically (model 8453 HewletPackard UV-VIS spectrophotometer) using ε277(SEB) = 39712.4 M−1 cm−1 (calculated with ProtParam from ExPASy) and MW = 28.37 kDa at 25 °C. The 1 mM stock solutions of each of the polyphenols were prepared in 96% ethanol. The working solutions of the polyphenols (c = 0.1 mM) were prepared by dilution of these stock solutions in 20 mM HEPES, pH 7.5. 2.2. Fluorescence emission spectrophotometry Fluorescence spectra were recorded with a Cary Eclipse spectrofluorimeter (Varian, Mulgrave, Australia) equipped with an electro-thermal temperature controller, using a 1-cm-path-length quartz cuvette. Slit widths with a nominal band-pass of 5 nm were used for both excitation and emission. The intrinsic fluorescence emission spectra of SEB (c = 0.012 ± 0.001 mg ml−1) were recorded from 290 nm to 450 nm, as a function of increasing concentrations of the polyphenols (titration). Excitation wavelengths of 275 nm and 295 nm were used to follow the SEB fluorescence. The emission spectra of SEB in the absence and presence of the polyphenols and after correction for the solvent blank were corrected for the dilution and for the photomultiplier-tube response. The wavelengths at maximum emission intensity, λmax, and the fluorescence intensity at 328 nm were determined. All of the experiments were conducted at 25 °C in 20 mM HEPES, pH 7.5, with two independent experiments, each carried out in triplicate. The data are expressed as means ± standard deviations (SD). 2.2.1. Fluorescence quenching parameters The fluorescence emission intensities for SEB measured at 328 nm as a function of increasing concentrations of the polyphenols were used for the construction of the binding profiles. Fluorescence quenching is the measure of the decrease in the quantum yield of the fluorescence induced by a variety of molecular interactions with a quencher molecule. Fluorescence quenching can be dynamic, resulting from collisional encounters between the fluorophore and the quencher, or static, resulting from the formation of a ground state between the fluorophore (protein) and the quencher (Lakowicz, 2006). Fluorescence quenching can be described by the linear Stern–Volmer equation:

2. Materials and methods 2.1. Reagents

Fo = 1 + K SV [Q ] F

Staphylococcal enterotoxin B (SEB; lyophilized powder) was from SigmaAldrich (Munich, Germany). EGCG, EGC, KAM-G and KAM (Table 1) were from Extrasynthese (Lyon, France). All of the chemicals used were of analytical grade.

In this equation, Fo and F are the fluorescence intensities in the absence and presence of the quencher, respectively, [Q] is the concentration of the quencher, and KSV is the Stern–Volmer quenching constant. A linear Stern–Volmer plot is generally in-


E. Benedik et al./Food and Chemical Toxicology 74 (2014) 1–8


dicative of a single class of fluorophore, with all of the molecules equally accessible to the quencher. If two fluorophore populations are present and one class is not accessible to the quencher, then the Stern–Volmer plots deviate from linearity. This is frequently seen for quenching of tryptophan (Trp) fluorescence in proteins by polar or charged quenchers. These molecules do not rapidly penetrate the hydrophobic interior of proteins, and only those Trp residues on the surface of the protein are quenched. SEB has one Trp (Trp197) that is located inside the folded protein, which makes it less accessible for interactions with polyphenols (Singh et al., 1988). The modified Stern–Volmer equation was applied here to analyze the fluorescence quenching:

⎡ (F − F ) ⎤ = log K A + n ⋅ log [Q ] log ⎢ o ⎣ F ⎥⎦


where, Fo and F are the fluorescence intensities in the absence and presence of the quencher, respectively, n is the number of polyphenols bound to the protein, [Q] is the concentration of the quencher, and KA is the modified Stern–Volmer quenching constant, which is very close to the binding constant (Lakowicz, 2006). The free energy of binding of phenolic compounds to SEB at 25 °C can be estimated from binding constants (KA) using the following equation:

ΔG o = − RT ln K A


2.3. Thermal stability of SEB in the absence and presence of polyphenols A Cary Eclipse spectrofluorimeter (Varian) was equipped with an electrothermal temperature controller that provided thermal programmability for the multiple cell unit, so that fluorescence emission intensity measurements can be performed directly as a function of temperature. Equilibrium thermal unfolding of SEB in the absence and presence of selected polyphenols was monitored as a function of temperature (range, 20 °C to 95 °C; heating rate, 1.0 °C min−1) by recording fluorescence emission intensity at 328 nm after excitation at 275 nm. The concentration of SEB was 1.12 μM. The molar ratio of polyphenols to SEB was 1:1. The samples were placed in a 1.0-cm-light-path quartz cell and sealed with a Teflon stopper to prevent evaporation. To check for the reversibility of the interaction with the protein, the samples were cooled to 20 °C and heated again in the same manner. The thermal denaturation curves were used to determine the temperature of denaturation, Td (the temperature at which half of the protein molecules are in the native state and half are in the denatured state (fD = fN = 0.5)), and the van’t Hoff enthalpy of unfolding, ΔH(Td). Based on the assumption that the temperature-induced SEB denaturation at pH 7.5 is a reversible two-state transition between the native, N, and the denatured, D, state, the van’t Hoff enthalpy can be calculated as described previously (Poklar and Vesnaver, 2000):

ΔH o (Td ) = 4RTd 2 × where, R (Jmol transition.


Fig. 1. Structure of the complex comprised of Staphylococcal enterotoxin B (red) and the human T-cell receptor beta chain (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

1 ΔT K−1)

16 docking experiments). In this case, the docking grids were constructed with an extension of 7.0 Å from any atom of the blind-docked EGCG, with the spacing of 0.375 Å between the grid points. All of the other settings remained the same as used for the blind-docking experiments. The resulting complexes between SEB and the ligands were refined using the YASARA energy minimization protocol as is described above. We have selected two resulting complexes of SEB (Site 1 and Site 2) and EGCG for further exploration with molecular dynamics simulation. All molecular dynamics simulations were run with YASARA Structure (Krieger et al., 2004), using the AMBER03 force field (Duan et al., 2003) under periodic-boundary conditions and with explicit water. A multiple time step of 1.25fs for intramolecular and 2.5 fs for intermolecular forces was used. Simulations were performed in cells set to be 15 Å larger than the protein along each axis. To ensure electroneutrality of the system, counter ions were added to a final concentration of 0.9% NaCl. A 7.86 Å cutoff was used for Lennard-Jones forces. The Particle Mesh Ewald method was used to treat electrostatics (Essmann et al., 1995). The system was at the beginning minimized by simulated annealing. After annealing, simulations were run at 298 K. Temperature was adjusted using a Berendsen thermostat based on time-averaged temperature (Berendsen et al., 1984). Ligands were parameterized using the AM1BCC protocol (Jakalian et al., 2002), and atomic charges were assigned by applying simple additive bond charge corrections (BCCs) to AM1 atomic charges. Protein side chain protonation patterns were assigned at pH 7.0 according to a protocol by Krieger et al. (2006).


3. Results is the gas constant, and ΔT is the temperature width of the

2.4. Molecular modeling and docking For the different molecular modeling and docking tasks, the Yet Another Scientific Artificial Reality Application (YASARA) Structure was used. YASARA is an easyto-use, reliable, universal package for molecular graphics, molecular modeling and molecular dynamics (Krieger and Vriend, 2002) ( The model of SEB that was used in our studies was extracted from the crystal structure (pdbid:1SBB) of the complex between T-cell receptor beta chain and superantigen SEB (Li et al., 1998). The structure of this complex is shown in Fig. 1. The Repair PDB utility of FoldX was run as a YASARA plugin (Van Durme et al., 2011) to prepare the complex for modeling. This utility minimizes the energy of a protein structure by rearranging the amino-acid side chains to obtain a better (lower) free energy of the protein. The native FoldX force field (Schymkowitz et al., 2005) is used for this purpose. The SEB structure soaked in water was optimized with the YASARA energy minimization macro (em_run.mcr) using the AMBER03 force field (Zhang et al., 2003). The resulting structure of SEB was used as a target for the blinddocking of EGCG, using Autodock 4.2 (Morris et al., 2009), to detect possible cavities that might serve as a binding site for EGCG and the other polyphenols in our study. All of the docking calculations were set using a YASARA macro (dock_run.mcr). The blind-docking exploration space was defined as a grid with an extension of 5.0 Å from all of the SEB protein atoms, resulting in a 106 × 126 × 82 grid of points equally distanced by 0.591 Å. The Lamarckian (Morris et al., 1998) genetic algorithm (population size = 150, maximum number of generations = 27000, maximum number of energy evaluations = 25 × 106) was used for searching for the optimal poses of the ligand in the active site of the receptor, by minimizing the free ligand–receptor binding energy. One hundred independent docking runs were performed, and the four best scored poses were selected for the binding site investigations. The docking of four ligands was finally performed to each of these four SEB protein binding sites (total,

3.1. Fluorescence emission spectrometry The interactions between the polyphenols and SEB were followed according to the changes in the fluorescence emission spectra of SEB in the absence and presence of the polyphenols. The changes observed in the emission spectra of SEB are likely to have occurred in response to polyphenol binding. The fluorescence emission intensity of SEB decreased with the successive addition of polyphenols, which indicated that these polyphenols can quench the intrinsic fluorescence of SEB (Supplementary Data 1 and 2). The emission maximum in the spectrum of SEB was at 328 nm, which suggests that the Trp residue is buried inside the molecule. No shifts in the maximum wavelengths (λmax) of the emission spectra were detected in SEB, regardless which λex was used (275 nm, 295 nm). The lack of a spectral shift is indicative that the Trp is not exposed to any change in polarity. 3.2. Fluorescence quenching The order of the Trp/Tyr quenching efficiencies of these polyphenols determined from the fluorescence emission intensity measurements at 328 nm was: EGC > EGCG ≅ KAM-G ≅ KAM (Supplementary Data 3). The modified Stern–Volmer equation (Eq. (2)) was applied here to obtain the KA (the modified Stern–Volmer quenching constant),


E. Benedik et al./Food and Chemical Toxicology 74 (2014) 1–8

Table 2 Binding characteristics of the polyphenols to SEB, including the Stern–Volmer quenching constant (KSV), the binding constant (KA), the number of polyphenols bound (n), and the Gibbs free energy of binding, ΔG°, at 25 °C and pH 7.5. Polyphenol

Ksv (×10−5) [M]−1

KA (×10−4) [M]−1


ΔG° (kJ/mol)


4.64 (0.9922)a 5.89 (0.9904)a 4.13 (0.9990)a 3.19 (0.9972)a

14.1 ± 1.1 3.0 ± 0.1 1.1 ± 0.1 0.1 ± 0.1

0.9 ± 0.1 0.8 ± 0.1 0.7 ± 0.1 0.6 ± 0.1

−29.4 ± 2.3 −25.6 ± 2.0 −23.1 ± 2.0 −17.3 ± 2.0


Table 3 Autodock scores for the polyphenols with SEB, according to the four sites, as described in the main text. Polyphenol


Autodock score (kJ/mol) Site 1

Site 2

Site 3

Site 4


−47.4 −41.3 −45.0 −34.2

−50.2 −40.9 −43.2 −38.0

−50.5 −45.8 −44.4 −36.7

−45.4 −37.6 −36.5 −33.1

−48.4 −41.4 −42.2 −35.5

R2 values.

which is very close to the binding constant, and the number of polyphenols bound to the protein. The data are presented in Table 2. The highest KA was obtained for EGCG, at 14.1 × 104 M−1, and there were 0.9 binding sites per SEB. The KA for EGC was 3.0 × 104 M−1, while for KAM-G and KAM the K A values were 1.1 × 10 4 M −1 and 0.1 × 104 M−1, respectively. 3.3. Thermal stability of SEB in the presence of flavonoids The impact of the interactions of these polyphenols on SEB was analyzed by thermal denaturation, which was monitored by fluorescence spectroscopy (Supplementary Data 4). The thermal denaturation was determined by heating of SEB together with the polyphenols at a molar ration of 1:1, with temperatures from 20 °C to 95 °C, and with the reversibility properties measured with cooling down to 20 °C and reheating to 95 °C (data not shown). When heated from 20 °C to 95 °C, the fluorescence intensity was monitored as a function of temperature at 328 nm. From the melting curves of SEB in the absence and presence of these polyphenols (Supplementary Data 4), it is clear that the denaturation temperatures do not change significantly. The temperature of denaturation of SEB in the absence of polyphenols at pH 7.5 was at 70.7 °C (Supplementary Data 5), which is higher than the previously determined 62.2 °C using UVcircular dichroism (Cavallin et al., 2003). No precipitation of SEB by polyphenols was observed during the denaturation process.

the results of this docking are collected in Table 3. From these data, we can conclude that EGCG has highest ‘overall’ binding affinity for SEB. The interaction maps of each of the four polyphenols with Site 2 of the SEB protein is presented in Fig. 3. Two complexes of SEB (Site 1 and Site 2) and EGCG were selected for further exploration with molecular dynamics simulation. The results of trajectory analysis were presented in Fig. 4. From Fig. 4 we may observe that after initial equilibration the total energy of both simulations are stable. From RMSD study of SEB protein we may observe a drift of RMSD to higher values that is more evident in the case of binding EGCG to Site 1 (final RMSD ~ 3.5 Å) than to the Site 2 (final RMSD ~ 2.5 Å). Analyses of SEB residues’ RMSF show evident fluctuations in SEB flexible loop region (Tyr94Thr113) that is important for the functional, physical and chemical properties of that protein (Yanaka et al., 2010). These fluctuations are more evident in case of binding EGCG to Site 1 that is in the loop region. The superposition of initial structure (black) and structure after 25 ns of molecular dynamics (gray) of EGCG to Site 1 and Site 2 are presented in Fig. 5. In Table 4 are presented results of MD analysis of important contacts between SEB protein and EGCG, and also some surface parameters relevant for binding. We may observe that EGCG has in average four HB contacts with Site 1 and the same number with Site 2. EGCG is in average shielded with four Tyr residues when it is bound to Site 1 and with one Trp and two Tyr residues in the case

3.4. Molecular modeling and docking The structure of SEB that we used for the docking studies was prepared according the protocol described in section 2.4. As we had no information where the SEB binding sites might be, we had to determine the potential binding sites for our ligands using blinddocking. The results of the blind-docking are represented visually (Fig. 2). We can observe that EGCG can fit in many of the numerous cavities at the surface of SEB (Fig. 2A). We selected the four bestscored binding sites that were detected by this blind-docking for the further analysis (Fig. 2B). Additional site-focused docking was performed with a denser grid (see section 2.4) for each of the four binding sites on SEB, and

Table 4 Analysis of hydrogen bond, Trp, Tyr contacts between SEB and EGCG, and analysis of lost solvent accessible surface. Data were sampled from MD trajectories of complexes between SEB and EGCG. Property

Site 1

Site 2

Avg. hydrogen bond contacts Max. hydrogen bond contacts Avg. Trp contacts Avg. Tyr contacts Lost surface of SEB/Å2 Lost surface of EGCG/Å2 Lost surface of SEB:EGCG complex/Å2

4.0 ± 0.7 6 0 3.9 ± 0.2 194 ± 17 138 ± 15 332 ± 32

4.1 ± 1.0 7 1 2.0 ± 0.2 193 ± 20 116 ± 11 309 ± 30

Fig. 2. Autodock blind-docking of EGCG to the SEB protein target. (A) 100 EGCG poses. (B) The four selected EGCG poses and the assignment of the binding sites.

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Fig. 3. Interaction maps of EGCG (A), EGC (B), KAM-G (C) and KAM (D) with SEB Site 2. Schrödinger Maestro 9.3 (Suite 2012: Maestro, version 9.3, Schrödinger, LLC, New York, NY, 2012) was used for the preparation of these interaction maps.

of interaction with Site 2. The average loss of total solvent accessible surface that is the consequence of EGCG binding to SEB is in the case of Site 1 (~330 Å2) slightly larger than for Site 2 (~300 Å2), regarding this information binding to Site 1 is a little bit more favored than to Site 2. 4. Discussion In the present study, we investigated the interactions of some naturally occurring polyphenols with SEB, using fluorescence emission spectrophotometry and molecular docking. We specifically included two catechins, (-)-epigallocatechin-3-gallate (EGCG) and (-)-epigallocatechin (EGC), and two flavones, kaempferol-3-glucoside (KAM-G) and kaempferol (KAM) (Table 1). Structurally, SEB is a single-chain polypeptide of 239 amino acids that contains one disulfide bond that is formed by two half cysteines that are located in the middle of the polypeptide chain, and which form the so-called cysteine loop (Papageorgiou et al., 1998). The SEB polypeptide contains one Trp residue (Trp197) that is located inside the folded protein, which makes it less accessible for interactions with polyphenols (Huseby et al., 2007; Singh et al., 1988). The changes observed in the fluorescence intensity of spectra of SEB upon increasing the polyphenol concentrations are likely to reflect interactions of these polyphenols with the 21 Tyr residues on the surface of the SEB molecule.

The modified Stern–Volmer equation was applied here to obtain the KA which is very close to the binding constant, and the number of polyphenols bound to the protein. The highest K A was obtained for EGCG, at 14.1 × 104 M−1, for other polyphenols the binding constant is an order of magnitude lower. The magnitude of these binding constants (~104 M−1) indicates that the binding of these compounds to SEB is relatively weak. Previously, we reported that the same phenolic compounds bind to bovine serum albumin with a binding constant of an order of magnitude higher (Skrt et al., 2012). The effect of studied polyphenol on SEB was analyzed by thermal denaturation monitored by fluorescence spectroscopy. The calculated thermodynamic values from the fluorescence emission spectrometry for SEB in the absence and presence of these polyphenolic compounds reveals that at the molar ratio of SEB to polyphenols of 1:1, the Td did not shift significantly (343.6 ± 0.5 K). Similarly, the ΔH(Td) values were all positive and in the range of 325 ± 12 kJ/mol for SEB in the absence and presence of polyphenols. These data confirm the previous data: these polyphenols do not significantly influence the thermal and enthalpic stability of SEB. If we take into account the average values of the Autodock scores for the best poses of all four of these binding sites, we can order these ligands by affinity as: EGCG > EGC ≅ KAM-G > KAM. The highest affinity observed in our docking experiments was for EGCG within Site 3 (−50.5 kJ/mol). For our study, Sites 1 and 2 are more relevant. Site 1 is part of a channel at the surface of SEB that is


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Fig. 4. Analysis of MD trajectory for complex between SEB Site 1 (Site 2) and EGCG.

responsible for the recognition of the T-cell beta chain fragment (see Supplementary Data 6). The blind-docking results showed that EGCG and similar polyphenolic ligands can bind to this SEB channel. Inspired by the promising docking result we may suggest that the binding of polyphenols like EGCG to SEB Site 1 has the influence to the adhesion of SEB to T cells, and thus prevents hyperactivation of the immune system (Krakauer, 2012; Watson et al., 2005). We have also revealed that the Trp197 quenched residue in the fluorescence quenching (section 3.1) is in Site 2, which thus provides a bridge between the experimental and docking data presented in the present study. The interaction maps of each of the four polyphenols with Site 2 of the SEB protein fit closely to Site 2 (Fig. 3). EGCG and EGC are connected within SEB Site 2 through five and six hydrogen bonds, respectively. Hydrogen bonding interactions of EGCG and EGC with the Trp197 ligand were observed, which indicate that EGCG and EGC are very closely positioned to Trp197 and that they might act as quenchers of Trp197 in the fluorescence emission spectroscopy (section 3.2). There were three hydrogen bonds predicted for KAM-G (Fig. 3C) and KAM (Fig. 3D), and for these two ligands, there was no evidence of very close interactions with Trp197. Based on our data for the observed very low binding constants and ΔG° of these polyphenols, and based on the lack of effects on the thermal stability of SEB, we suggest that the binding of polyphenols like EGCG to SEB is relatively weak, although it is likely that this binding will have an influence on the adhesion of SEB to T cells, and thus prevent hyperactivation of the immune system (Krakauer, 2012; Watson et al., 2005). SEB is commonly referred as a ‘bacterial superantigen’ because it is an extremely potent activator of T cells, with stimulation of the massive production and secretion of various proinflammatory cytokines and chemokines that mediate many of the toxic effects of SEB. SEB also inhibits the naturally occurring regulatory T-cell activity (Ahanotu et al., 2006; Brosnahan and Schlievert, 2011; Krakauer, 2013). Recently, it was demonstrated for the first time that beta toxin can kill proliferating human lymphocytes (Huseby et al., 2007). The SEB structure is similar to that of the sphingomyelinases of Listeria ivanovii and Bacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to the sphingomyelinases, the proteins most structurally related to beta toxin include human endonuclease HAP1, Escherichia coli endonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 from Bombyx mori (Huseby et al., 2007). These exotoxins bind to both the major histocompatibility complex (MHC) class II molecules on

Fig. 5. Superposition of MD snapshots for EGCG bound to Site 1 (A) and Site 2 (B). Legend: 0 ns – black color; 25 ns – gray color. From MD data we may observe that binding to Site 1 strongly disturbs SEB flexible loop region (Tyr94-Thr113). This effect is also observed for Site 2 but less evident.

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antigen-presenting cells and the specific Vβ regions of the T-cell antigen receptors (Choi et al., 1989; Mollick et al., 1991). MHC class II represents the major receptor-binding site for superantigens such as SEB, and therefore any cell expressing MHC class II is a potential target (Krakauer and Stiles, 2013). 5. Conclusions To summarize, our blind-docking results showed that EGCG and similar polyphenolic ligands can bind to the SEB channel, which is likely to influence the adhesion of SEB to T cells, and thus prevent hyperactivation of the immune system. Our model can explain the literature data showing that the EGCG in green tea can suppress the induction of proinflammatory cytokines and chemokines produced in vitro in response to superantigens by both T cells and monocytes (Krakauer, 2012; Ravindranath and Ravindranath, 2011). Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgment Evgen Benedik would like to thank Dr. Ajda Ota for her help with data processing. Financial support from the Slovenian Research Agency through the P4-0121 and P1-0201 Research program is gratefully acknowledged. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.fct.2014.08.012. References Ahanotu, E., Alvelo-Ceron, D., Ravita, T., et al., 2006. Staphylococcal enterotoxin B as a biological weapon: recognition, management, and surveillance of staphylococcal enterotoxin. Appl. Biosafety 11 (3), 120–126. Ahn, H.Y., Hadizadeh, K.R., Seul, C., et al., 1999. Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172). Mol. Biol. Cell 10 (4), 1093–1104. Argudín, M.Á., Mendoza, M.C., Rodicio, M.R., 2010. Food poisoning and Staphylococcus aureus enterotoxins. Toxins 2 (7), 1751–1773. Berendsen, H.J.C., Postma, J.P.M., van Gunsteren, W.F., et al., 1984. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81 (8), 3684. Brosnahan, A.J., Schlievert, P.M., 2011. Gram-positive bacterial superantigen outside-in signaling causes toxic shock syndrome. FEBS J. 278 (23), 4649–4667. Cavallin, A., Petersson, K., Forsberg, G., 2003. Spectrophotometric methods for the determination of superantigen structure stability. In: T. Krakauer. (Ed.), Superantigen Protocols. Humana Press, New Jersey, pp. 55–63. Choi, Y.W., Kotzin, B., Herron, L., et al., 1989. Interaction of Staphylococcus aureus toxin “superantigens” with human T cells. Proc. Natl Acad. Sci. U.S.A. 86 (22), 8941– 8945. Daglia, M., 2012. Polyphenols as antimicrobial agents. Curr. Opin. Biotechnol. 23 (2), 174–181. Dhaliwal, W., Kelly, P., Bajaj-Elliott, M., 2009. Differential effects of Staphylococcal enterotoxin B-mediated immune activation on intestinal defensins. Clin. Exp. Immunol. 156 (2), 263–270. Duan, Y., Wu, C., Chowdhury, S., et al., 2003. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J. Comput. Chem. 24 (16), 1999–2012. Essmann, U., Perera, L., Berkowitz, M.L., et al., 1995. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577–8593. Ferrazzano, G.F., Amato, I., Ingenito, A., et al., 2011. Plant polyphenols and their anti-cariogenic properties: a review. Molecules 16 (2), 1486–1507.


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Binding of flavonoids to staphylococcal enterotoxin B.

Staphylococcal enterotoxins are metabolic products of Staphylococcus aureus that are responsible for the second-most-commonly reported type of food po...
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