E b D I M a ( b M B a A R R 2 A A K N F Q F M H 1 i w m i S 1 y ( f m f h 0 International Journal of Biological Macromolecules 85 (2016) 40–47 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac xperimental evidence and molecular modeling of the interaction etween hRSV-NS1 and quercetin eriane Elias Gomesa,b, Ícaro Putinhon Carusoa,b, Gabriela Campos de Araujoa,b, sabella Otenio de Lourenç oa,b, Fernando Alves de Meloa,b, Marinônio Lopes Cornélioa,b, arcelo Andrés Fosseya,b, Fátima Pereira de Souzaa,b,∗ Departamento de Física—Instituto de Biociências, Letras e Ciências Exatas (IBILCE) Universidade Estadual Paulista “Júlio de Mesquita Filho” UNESP)—Campus de São José do Rio Preto—SP. Rua Cristóvão Colombo, 2265, Jardim Nazareth, Cep: 15054-000 São José do Rio Preto, SP, Brazil Centro Multiusuário de Inovaç ão Biomolecular (CMIB)—Instituto de Biociências, Letras e Ciências Exatas (IBILCE) Universidade Estadual Paulista “Júlio de esquita Filho” (UNESP)—Campus de São José do Rio Preto—SP. Rua Cristóvão Colombo, 2265, Jardim Nazareth, Cep: 15054-000 São José do Rio Preto, SP, razil r t i c l e i n f o rticle history: eceived 14 November 2014 eceived in revised form 9 November 2015 ccepted 14 December 2015 vailable online 21 December 2015 eywords: on structural protein 1 lavonoids uercetin a b s t r a c t Human Respiratory Syncytial Virus is one of the major causes of acute respiratory infections in children, causing bronchiolitis and pneumonia. Non-Structural Protein 1 (NS1) is involved in immune system eva- sion, a process that contributes to the success of hRSV replication. This protein can act by inhibiting or neutralizing several steps of interferon pathway, as well as by silencing the hRSV ribonucleoproteic complex. There is evidence that quercetin can reduce the infection and/or replication of several viruses, including RSV. The aims of this study include the expression and purification of the NS1 protein besides experimental and computational assays of the NS1-quercetin interaction. CD analysis showed that NS1 secondary structure composition is 30% alpha-helix, 21% beta-sheet, 23% turn and 26% random coils. The melting temperature obtained through DSC analysis was around 56 ◦C. FRET analysis showed a dis- tance of approximately 19 Å between the NS1 and quercetin. Fluorescence titration results showed that −6 luorescence spectroscopy olecular modeling uman syncytial respiratory virus the dissociation constant of the NS1-quercetin interaction was around 10 M. In thermodynamic anal- ysis, the enthalpy and entropy balanced forces indicated that the NS1-quercetin interaction presented both hydrophobic and electrostatic contributions. The computational results from the molecular mod- eling for NS1 structure and molecular docking regarding its interaction with quercetin corroborate the experimental data. © 2015 Elsevier B.V. All rights reserved. . Introduction The participation of Human Respiratory Syncytial Virus (hRSV) n respiratory diseases during childhood can be life-threatening, ith severe consequences like pneumonia and bronchiolitis. The ajor strategy that contributes to the success of hRSV replication s its immune system evasion efficacy, a process provided by Non- tructural Proteins (NS1 and NS2). NS1 is a small protein that has 39 amino acids [1] with 15 kDa. Extensive studies have shown that ∗ Corresponding author. E-mail addresses: derianegomes1@gmail.com (D.E. Gomes), krocaruso@hotmail.com (Í.P. Caruso), bicamposaraujo@hotmail.com G.C.d. Araujo), isabella o.lourenco@hotmail.com (I.O.d. Lourenç o), ernanmello@gmail.com (F.A.d. Melo), mario@ibilce.com.br (M.L. Cornélio), arcelo@ibilce.unesp.br (M.A. Fossey), fatyssouza@gmail.com, atyssouza@yahoo.com.br (F.P.d. Souza). ttp://dx.doi.org/10.1016/j.ijbiomac.2015.12.051 141-8130/© 2015 Elsevier B.V. All rights reserved. NS1 plays an important role in the modulation of the host response to infection, antagonizing the interferon-mediated antiviral state [2]. It has been reported that NS1 (1) co-localizes with the mitochon- drial signaling protein (MAVS), inhibiting MAVS-RIG-I interaction and decreasing protein levels of members of the IFN induc- tion pathway, such as TRAF3 and IKKe [3,4]; (2) interacts with microtubule-associated protein 1B (MAP1B), which may be impor- tant for surface recognition of these proteins with other host structures [5]; and (3) interacts with M protein and the C-terminus of P protein [6]. Flavonoids have gained world interest due to their nutraceu- tical and therapeutic importance, exhibiting several biological activities such as antioxidant, anti-inflammatory, cardioprotective, antibacterial, antitumor, hepatoprotective, and antiviral activities [7]. Thus, because quercetin is the most studied flavonoid and it has antiviral properties [8–11], we decided to investigate how dx.doi.org/10.1016/j.ijbiomac.2015.12.051 http://www.sciencedirect.com/science/journal/01418130 http://www.elsevier.com/locate/ijbiomac http://crossmark.crossref.org/dialog/?doi=10.1016/j.ijbiomac.2015.12.051&domain=pdf mailto:derianegomes1@gmail.com mailto:ykrocaruso@hotmail.com mailto:bicamposaraujo@hotmail.com mailto:isabella_o.lourenco@hotmail.com mailto:fernanmello@gmail.com mailto:mario@ibilce.com.br mailto:marcelo@ibilce.unesp.br mailto:fatyssouza@gmail.com mailto:fatyssouza@yahoo.com.br dx.doi.org/10.1016/j.ijbiomac.2015.12.051 f Biol q s d s i ( [ d p o a p o 2 2 o o p 2 s I f b w 1 F d 7 s ( b p w l b i S H N t 9 i 2 t 1 o T c fi Q f o D.E. Gomes et al. / International Journal o uercetin interacts with NS1, and affects its immune-system eva- ion efficiency. Flavonoids are useful model molecules for spectroscopy studies ue to their interactions with relevant target proteins and some pectroscopic profiles of quercetin such as (1) the excited-state ntramolecular proton transfer, (2) dual fluorescence behavior and 3) the high intensity of the fluorescence signal in low polar solvents 12]. Biophysical binding properties have been explored in some etail using human serum albumin (HSA) and quercetin as binding artners [12]. Furthermore, the low toxicity and pharmacokinetics f quercetin are described in the literature [13], with reduced ther- peutic risk. In the present study, a combination of experimental rocedures with molecular docking has elucidated several aspects f the interaction between NS1 and quercetin. . Materials and methods .1. NS1 expression and purification Human Syncytial Respiratory Virus (type A) NS1 codon ptimization and pJexpress401-NS1 construction were devel- ped by DNA2.0 [14] BL-21(DE3) cells containing the plasmid Jexpress401-NS1 grown in 1.5 L of 2YT medium containing 5 �g/mL kanamycin and 34 �g/mL chloramphenicol with con- tant shaking at 37 ◦C until they reached the optical density of 0.8. nduction was performed with 0.5 mM IPTG (final concentration) or three hours at the same temperature. Cultures were harvested y centrifugation at 4 ◦C, 7000 rpm, for 30 min and the pellet cells ere stored at −80 ◦C until use. Cells were suspended in 40 mL lysis buffer (50 mM Tris–HCl; 00 mM NaCl; 5 mM �Me, pH 8.0) containing Animal Component ree Protease Inhibitor Cocktail (Sigma®, I3911-1BO) and were isrupted by sonication. The lysed cells were centrifuged at 4 ◦C, 000 rpm, for 30 min and the supernatant was loaded at a con- tant flow rate of 0.5 mL/min, in a Nickel Resin His60 Ni Superflow Clontech®), which was previously equilibrated with purification uffer (PB - 50 mM TRIS, 100 mM NaCl, 1 mM �Me, pH 8.0), cou- led to an AKTA Purifier (GE®). After that, the column was washed ith PB to remove non-binding proteins from lysed cells and fol- owed by PB containing 40 mM imidazole to remove non-specific ound contaminants. NS1 elution was performed using PB contain- ng 200 mM imidazole and the fractions collected were analyzed by DS-PAGE. Pure fractions were desalted and stored at 4 ◦C. NS1 desalting was performed on a PD-10 Desalting Column (GE ealthcare®) equilibrated with buffer (0.05 mM NaH2PO4, 300 mM aCl at pH 7.4) and kept on ice after that for up to 24 h before he interaction assays. Quercetin stock solution was prepared in 0% ethanol v/v and kept on ice after that for up to 24 h before the nteraction assays. .2. UV–Vis absorbance spectroscopy UV–Vis measurements were performed on a Cary-3E spec- rophotometer (Varian, Palo Alto, CA) equipped with a quartz cell of .0 cm path length, under the following conditions: scanning speed f 600 nm/min, 1.0 nm of interval and spectral bandwidth of 2.0 nm. he spectra were performed at room temperature. The stock oncentrations were obtained using the following extinction coef- cients: �375 nm = 21.880 M−1 cm−1 for quercetin (Sigma–Aldrich®, 4951) (at 375 nm) and �280 nm = 10.220−1 M−1 cm−1 (at 280 nm) or NS1 (Expasy-ProtParam [15]), obtained the final concentrations f 17.6 �M for NS1 and 7.1 mM for quercetin. ogical Macromolecules 85 (2016) 40–47 41 2.3. Circular dichroism spectroscopy (CD) Far-UV CD measurements were carried out on a Jasco J-810 spec- tropolarimeter (Jasco, USA) equipped with a quartz cell of 0.01 cm path length. The CD spectrum range 185–260 nm was recorded at a scan rate of 20 nm/min, a response time of 2.0 s, and spectral band- width of 1.0 nm. For the NS1 spectrum, six accumulations were performed. The measurement was performed at room temperature (with NS1 concentration of 17.6 �M). The CD spectrum was taken as millidegrees (�) and then expressed in terms of mean residue ellip- ticity (MRE or [�]) in deg cm2 dmol−1 using the following equation: [�] = �(mdeg) (10[P]ln) (1) where [P] is the molar concentration of the protein, n is the quantity of amino acid residue, and l is the path length (cm). The secondary structures of the protein NS1 were calculated with CONTINLL soft- ware from the CDPro package, using the reference set of proteins SMP37 [16]. 2.4. Differential scanning calorimetry Differential scanning calorimetry (DSC) measurements were recorded on a N-DSC III (TA Instruments, USA) in the temperature range of 20–80 C with a heating and cooling scan rate of 1 C/min. NS1 was diluted in phosphate buffer (50 mM NaH2PO4, 50 mM NaCl, pH 8.0) to a final concentration of 4.4 �M. Both calorimeter cells were loaded with the buffer solution, equilibrated at 20 ◦C for 10 min and scanned repeatedly as described above until the baseline was reproducible. Afterwards the sample cell was loaded with NS1 solution and scanned in the same way. Baseline correc- tion was conducted by subtracting the ‘buffer vs., buffer’ scan from the corresponding ‘protein vs., buffer’ scan. 2.5. Fluorescence spectroscopy The fluorescence spectroscopy measurements were performed using a Cary Eclipse Fluorescence Spectrophotometer (Varian, Palo Alto, CA) equipped with a quartz cell of 1.0 cm path length and a Peltier Single Cell Holder System. Both excitation and emission bandwidths were set at 4 nm. The excitation wavelength at 280 nm and emission spectrum were collected in the range 290–500 nm, which was corrected for the background fluorescence of the buffer and for the inner filter effect [17]. The fluorescence quenching experiments were carried out at the temperatures of 25 and 37 ◦C. The titrations were performed by adding small aliquots (≤10 �L) from quercetin stock solution to NS1 solution (2.0 mL) at a con- stant concentration of 1.1 �M. The final concentration of quercetin achieved in the protein solution was 25.6 �M. In every experiments, the final volume of ethanol in the buffer was 2.7%. 2.6. Molecular modeling of NS1 Since the NS1 model had not been proposed previously, protein modeling techniques were used to obtain a structural model. Due to low homology with other proteins with known atomic coordinates, the NS1 modeling method applied was the Rosetta Scoring func- tion and fragment insertion methodologies were developed for ab initio structure prediction [18]. First, a customized library of frag- ments for each three and nine residue in the protein sequence was selected from a database of known protein structure. Then these fragments were assembled using Monte Carlo simulated annealing search strategy in which fragments are randomly inserted into the protein sequence by replacing the backbone torsion angles in the protein chain with those in the fragment. For NS1 protein (145 aa), 56,475 structures were generated. Two structures with the lowest 42 D.E. Gomes et al. / International Journal of Biological Macromolecules 85 (2016) 40–47 Fig. 1. NS1 expression and Purification. (A) SDS-PAGE gel stained with Coomassie Brilliant Blue R-250 showing NS1 expression. Lane 1: PageRuler Prestained Protein Ladder; Lane 2: Before IPTG addition; Lanes 3 and 4: 1 and 3 h after IPTG addition (arrows indicates NS1), respectively. (B) SDS -PAGE gel stained with Coomassie Brilliant Blue R-250 s w thr p ck, abs o s w m s b m s 2 M c i m a t s t b t e t C t w s R o T w c w b 2 s q s t w howing NS1 purification. Lane 1: PageRuler Prestained Protein Ladder; Lane 2: flo urification in AKTA system (GE Healthcare Life Sciences) (wash and elution). In bla f pure protein. cores (as assessed by the number of the other 56,474 structures ithin 3 Å rmsd [19]) were visually inspected. The quality of the odel was validated using the PROQ [20] and the PROCHECK [21] ervers. The second model was showed to be like the first model ut with better estimated accuracy and agreement with experi- ental data. The Dali server was used to search for native-like ubstructures [22]. .7. Molecular dynamics simulation of NS1 in solution Molecular dynamics simulations were performed using a GRO- ACS molecular dynamics package (version 4.5.5) [23,24] to onfirm the stability of the NS1 protein model in water contain- ng 150 mM NaCl. We used the GROMOS53A6 force field [25] to odel the NS1 protein and SPC as a water model [26]. Temper- ture was kept constant at 310 K by means of a V-rescale [27] hermostat with coupling constant tau = 0.1. Pressure was held con- tant at 1 atm by a Berendsen barostat [28] with coupling constant au = 1. Integration of Newton’s equation of motion was performed y the leap-frog algorithm with a time step of 2 fs. Long-range elec- rostatic interaction was treated using fast particle-mesh Ewald lectrostatics (PME) [29]. A cutoff of 1.25 nm was implemented for he Lennard–Jones and the direct space part of the Ewald sum for oulombic interactions. The Fourier space part of the Ewald split- ing was computed by using the particle-mesh Ewald method [30], ith a grid length of 0.15 nm. The overall electric charge of the ystem was compensated by adding Cl− or Na+ ions, respectively. The simulation starting from the NS1 structure constructed by OSETTA, was equilibrated by 100 ps of MD with position restraints n the protein to allow for the relaxation of the solvent molecules. his first equilibration run was followed by another 50-ps run ithout position restraints on the protein. The production run at onstant temperature and pressure conditions, after equilibration, as 10 ns long. Stability of the NS1 model molecule was determined y means of the root mean square deviation (RMSD) using tools. .8. Docking calculation of the NS1-quercetin complex The g cluster tool from the GROMACS package [31] was used to elect the most likely NS1 model. The molecular structure of the uercetin was obtained from PRODRG [32]. AutoDockTools (ADT) oftware [33] of the MGL Tools 1.5.4 program was used to prepare he NS1 and quercetin. The protonation state of histidine side chains as set up according to the PropKa server [34], also using ADT ough; Lane 3: NS1 elution; Lane4: Pure NS1 (Arrow). (C) Chromatogram from NS1 orption in 280 nm, in red, concentration of imidazole. The arrow indicates the peak software. The grid maps were generated with 0.375 Å spacing and dimensions of 126 × 126 × 126 points by the AutoGrid 4.2 program [35]; these maps were centered in the protein. The AutoDock 4.2 program [35] was used to study the binding site between quercetin and NS1 by applying the Lamarckian Genetic Algorithm (LGA) for minimization using 50 million energy evaluations, a population size of 300 and root–square–mean deviation (RMSD) tolerance for cluster analysis of 2.0 Å. For each docking simulation, 100 differ- ent conformers were generated. The ADT software was used to visualize the docked conformations and to identify the interac- tions between amino acid residues of NS1 and quercetin. Chimera software [36] was used to generate the representation figures of the modeling and docking calculation. The map of the interactions involved in the binding site was calculated using LigPlot program [37]. 3. Results and discussion 3.1. NS1 expression, purification and characterization 3.1.1. NS1 expression and purification Fig. 1AA shows the results for NS1 expression in which the sam- ple collected before induction with IPTG, and at 1 hour and 3 h of growth after induction with IPTG can be seen in the second, third and fourth lanes, respectively. The most promising condition for protein expression was reached by growing cells for 3 h after induc- tion by using 0.5 mM IPTG, at 37 ◦C. The best condition for NS1 purification was determined washing two column volumes with purification buffer containing 40 mM of imidazole before eluting it against the purification buffer containing 200 mM imidazole, see Fig. 1C. The results can be seen in Fig. 1B in lanes 3 and 4, respec- tively. 3.1.2. Secondary structure analysis The structural characteristics of NS1in solution were studied using the CD technique. The far UV–CD spectrum of the NS1 pre- sented characteristics of a structured protein from its minimum at 209 nm and positive ellipticity at 190 nm (Fig. 2). The CD spec- trum of the NS1 presented the same spectral profile described in the literature [38]. The secondary structure analysis was performed using CONTINLL software [16] and indicating: 30% of �-helix, 21% of �-sheet, 23% of turn, and 26% of random coil. D.E. Gomes et al. / International Journal of Biol Fig. 2. Far UV–CD spectrum of NS1 (17.6 �M) which is represented by unfilled cir- cles. The black line represents the best result for curve fit performed by CONTINLL program. Fig. 3. DSC thermogram of the unfolding process of NS1. The apparent excess heat capacity curve was obtained for NS1 (4.4 �M) in phosphate buffer (50 mM NaH2PO4, 5 m p 3 t p t a a m l [ t u T 1 p t k w h 0 mM NaCl, pH 8.0) at a scan rate of 1 ◦C/min. The dotted lines (- - -) indicates the elting temperature (TM) of the protein. The insert corresponds to the Arrhenius lot of the unfolding process of NS1. .1.3. Thermal characterization The thermal stability of NS1 was investigated using the DSC echnique. Fig. 3 shows the DSC thermogram of the unfolding rocess (heating scan) of the protein. It can be seen from the hermogram that NS1 presents a melting temperature (TM) at pproximately 56 ◦C, with the endothermic transition starting at bout 50 ◦C and ending at 61 ◦C, This result corroborates the ther- al denaturation study of NS1 by using CD spectroscopy in the iterature, in which a melting temperature at 55 ◦C was obtained 38]. The cooling scan after the denaturation presented no transi- ions in the DSC thermogram of NS1, indicating that its thermal nfolding is irreversible as is also shown in the literature [38]. he calorimetric enthalpy change (�Hcal) was determined to be 2 kJ mol−1 from the total integral area under the thermogram eak. The rate constant (k) of the unfolding process at a given emperature can be obtained by using: = �CP (�Hcal − Q) (2) here v (1.0 ◦C min−1) stands for the scan rate, CP for the excess eat capacity, and Q for the heat evolved at a given temperature. ogical Macromolecules 85 (2016) 40–47 43 A value of 499 kJ mol−1 for the activation energy can be calculated from the Arrhenius plot in the insert of Fig. 3 [39]. 3.2. Characterization of the interaction between NS1 and quercetin 3.2.1. Fluorescence spectra and Förster resonance energy transfer Fig. 4aa shows the fluorescence emission spectra of the NS1 in the absence and presence of quercetin. The fluorescence spectrum of the NS1 excited at 280 nm presented an emission profile of its unique tryptophan residue (Trp) with an intensity maximum at 326 nm [38]. With the increment in the quercetin concentration in the NS1 solution, the fluorescence intensity of the protein at 326 nm was quenched with a concomitant increase in the inten- sity at 490 nm (see Fig. 4b), corresponding to quercetin emission [40–42]. Under the same condition, blank quercetin in the absence of NS1 does not show any detectable fluorescence [41]. Conse- quently, the fluorescence increment observed at 490 nm is due to the interaction between quercetin and NS1 and, subsequently, the resonance energy transfer of Trp residue to the flavonoid. In the literature, there are similar results demonstrating the resonance energy transfer between serum albumin and quercetin [41,42]. The energy transfer parameters and the average distance between the Trp residue in NS1 and quercetin was determined by applying Förster’s theory of resonance energy transfer (FRET) [43]. FRET is a photophysical process that involves the transfer of the excitation energy of an electronically excited donor to an acceptor molecule via non-radiative routes. According to Förster’s non-radiative energy transfer theory, it occurs under the follow- ing conditions: when the emission spectrum of the donor and the absorbance spectrum of the acceptor have a partial overlap; when orientation of the transition dipole of the donor and acceptor occurs; and when the distance between them is less than 8.0 nm. According to the theory, the distance r reflects the approximation between donor (NS1-Trp) and acceptor (Quercetin) which can be calculated by the equation: E = 1 − ( F F0 ) = R6 0 (R6 0 + r6) (3) where E is the efficiency of energy transfer, F0 and F are the flu- orescence intensity in the absence and presence of the acceptor (quercetin), respectively, and R0 is the critical distance when the transfer efficiency is 50%: R6 0 = 8.79 × 10−25K2n−4�J (4) In Eq. (4), K2 is the orientation factor related to the geometry of the donor (NS1-Trp) and acceptor (quercetin) and has been adopted to be 2/3 [44], which randomizes by rotational diffusion prior to energy transfer; n is the average refracted index of the medium in the wavelength range in which spectral overlap is significant and is taken as 1.36; � is the fluorescence quantum yield of the donor which was calculated taking the quantum yield of serum albumin a reference [45,46]; and J is the effect of the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor (see Fig. 5), which may be calculated by the following equation: ∞∫ 0 F (�) ε (�) �4d� J = ∞∫ 0 F (�) d� (5) 44 D.E. Gomes et al. / International Journal of Biological Macromolecules 85 (2016) 40–47 Fig. 4. (a) Normalized fluorescence spectra of NS1 in absence and presence of quercetin (�ex = 280 nm, pH = 7.4 and 25 ◦C). The inset corresponds to the detail of the emission region of quercetin. (b) Fluorescence intensity at 326 and 490 nm in different concentration of quercetin. [NS1] = 1.1 �M; (A–M) [Quercetin] = 0.0, 0.4, 0.8, 1.7, 2.5, 4.2, 5.9, 8.4, 10.9, 14.2, 17.5, 21.5 and 25.6 �M. F w t c q i a c p o 1 a w o 3 a a fi p w a c Fig. 6. Fluorescence quenching change of NS1 (�em = 326 nm) in function of quercetin concentration at (a) 25 and (b) 37 ◦C. The lines correspond to the adjust- ig. 5. Spectral overlaps of the NS1 fluorescence (A) with quercetin absorption (B). here F (�) is the corrected fluorescence intensity of the donor in he wavelength range from � to � + �� and ε (�) is the extinction oefficient of the acceptor at �. In order to obtain the average distance between NS1-Trp and uercetin, the F/F0 value in Eq. (3) was calculated by extrapolat- ng to zero at the linear of plot F/F0 vs., 1/[Quercetin], which gave value of 0.107 (Supplementary material, Fig. S1). Thus, the effi- iency of energy transfer (E) were determined at the saturation oint and its value was 0.893. According to Eqs. (3)–(5), the values f J = 3.776 × 10−15 cm3 L mol−1, R0 = 2.715 nm and r = 1.906 nm (or 9.06 Å) was calculated. The average distance between donor and cceptor fluorophore is in the range 0.5 R0 < r < 1.5 R0 [43–45], hich indicates that energy transfer from NS1-Trp and Quercetin ccurs with high probability. .2.2. Dissociation constant of the NS1-QCT complex The dissociation constants (Kd) for the interaction between NS1 nd quercetin were determined by fluorescence quenching change nalysis (Fig. 6). The fluorescence titration data at 25 and 37 ◦C were tted using the Eq. (6), which describes ligand binding to a single rotein site (1:1 complex) [47]: F0 − F F0 − FS = ([PT ] + [LT ] + Kd) − √ ([PT ] + [LT ] + Kd)2 − 4 [PT ] [LT ] 2 [PT ] (6) here F0 and F are the steady-state fluorescence intensities in the bsence and presence of quercetin, respectively. FS is the fluores- ence intensity of the fully complexed protein (saturated NS1 with ments of the experimental data made using Eq. (6). All the correlation coefficient are ≥0.99. quercetin), which was obtained by extrapolation at the plot of Fig. S1 (Supplementary material), [PT ] is the total concentration of NS1 and [LT ] is the total concentration of quercetin. Kd values for the NS1-quercetin interaction at 25 and 37 ◦C are shown in Table 1. It can be observed in Table 1 that, notwithstanding the fact that Kd values are similar at the two temperatures, a slight decrease of dissociation constant value occurs with the increase from 25 to 37 ◦C. 3.2.3. Thermodynamic analysis The protein-ligand interaction may involve the formation of hydrogen bonds, Van der Waals, electrostatic forces, and hydropho- bic interactions. In order to elucidate the interactions between NS1 and quercetin, the enthalpy change ( H) was calculated from the van’t Hoff equation: ln ( Kd1 Kd2 ) = ( −�H R )(1/ T2−1 T1 ) (7) where Kd1 and Kd2 are dissociation constants at the absolute tem- peratures T1 and T2, which were 298 K (25 ◦C) and 310 K (37 ◦C), respectively. R corresponds to the universal gas constant. The Gibbs D.E. Gomes et al. / International Journal of Biological Macromolecules 85 (2016) 40–47 45 Table 1 Dissociation constants (Kd) and thermodynamic parameters for NS1-quercetin interaction. T(◦C) Kd(�M) �H (kJ mol−1) −T�S (kJ mol−1) �G (kJ mol−1) �S (kJ mol−1 K−1) 25 2.86 5.1 – 36.7 – 31.6 0.123 37 2.64 – 38.2 – 33.1 T: temperature; �H: enthalpy change; �G: Gibbs free energy change; �S: entropy change. F H, −T. m ange ( t f c � N o t t a a p t t e N i b 3 d p ( s c a t A s R ig. 7. Thermodynamic profile of the binding of quercetin to NS1 at 25 and 37 ◦C. � ultiplied by the absolute temperature (T) (shown in green), and the free energy ch he reader is referred to the web version of this article.) ree energy ( G) and entropy changes ( S) for the NS1-quercetin omplex were calculated from the following equations: G = −RT ln( 1 Kd ) (8) S = (�H − �G) T (9) The thermodynamic parameters for the interaction between S1 and quercetin are shown in Table 1. The negative values btained for the Gibbs free energy indicated that the interac- ion process is spontaneous. H > 0 and S > 0 suggest that he binding reaction is endothermic and enthalpically unfavor- ble, and furthermore, that hydrophobic interactions could play n important role in the formation of the NS1-quercetin com- lex [48–50]. The analysis of the enthalpy–entropy balance shows hat the entropic term (−T S) makes a favorable contribution o Gibbs free energy ( G = H − T S) (Fig. 7), suggesting that lectrostatic interactions could be important in the stability of the S1-quercetin complex [50]. Thus, the thermodynamic parameters ndicated that the interaction between NS1 and quercetin presents oth hydrophobic and electrostatic contributions. .2.4. NS1 structure and its complex with quercetin The tridimensional structure of the protein NS1 has not been escribed in the literature. Therefore, a molecular model for the rotein NS1 was predicted using ab initio structure prediction Fig. 8). For the predicted model, a similar percentage of secondary tructure elements (�-helix: 32% and �-sheet: 12%) was found, ompared with data obtained by circular dichroism (�-helix: 30% nd �-sheet: 21%). Furthermore, the similarity of secondary struc- ures estimated by CD spectroscopy reinforces the NS1 modeling. n analysis of the model by the ProQ-SERVER resulted in a LG core of 2.302 and a Maxsub score of 0.011 [20]. Furthermore, the amachandran plot (Supplementary material, Fig. S2), calculated �S, and �G correspond to the enthalpy change (shown in red), the entropy change shown in blue). (For interpretation of the references to colour in this figure legend, by PROCHECK [21], showed 90.2% of the residues lying in the most favorable regions and 9.8% in additional allowed regions. Therefore, the local and global geometric properties indicate a good model and validate the NS1 model. The results of NS1 model MD simulation showed that the model is stable in solution, the RMSD value converged constantly, about 4 Å, from the model constructed by ROSETTA and the model clus- tered by GROMACS from the trajectories file, which retained the same structural characteristics (helix: 32%; �-sheet: 12%; turn and random coil: 56%) (Supplementary material, Fig. S3). Fig. 8 shows the docking result for the quercetin conformer of the lowest binding energy within the largest cluster. The bind- ing energy between NS1 and quercetin for the docking result is −29.7 kJ mol−1, which is in agreement with the values of Gibbs free energy present in Table 1. It can be observed from the hydrophobic- ity surface map in Fig. 8a that the interaction region of quercetin in NS1 presents a balance between hydrophilic and hydrophobic contributions. The theoretical distance determined between the single residue Trp of NS1 and quercetin was 20.12 Å (Fig. 8b), which is in good agreement with the distance found by FRET analysis (19.06 Å). The concordance present between the experimental and theoretical results for the binding energy and Trp-quercetin dis- tance, reinforces our assertion that both protein and complex were modeled property. Fig. 8c shows the analysis of interactions in the binding site of the NS1-quercetin complex performed by the LigPlot pro- gram [37]. His3, Tyr36, Asp70, Ile71, Cys72, Pro73, Ser105, and Leu143 residues. are involved in hydrophobic interactions with the flavonoid. His5, Ser69, Cys104, Gln106 and Asn108 residues form hydrogen bonds with the O5, O7, O4′, O3, and O5 oxygen atoms of quercetin, respectively. The NH group of the His5 side chain makes up one hydrogen bond with the studied ligand with a formation dis- tance of 2.86 Å. Each oxygen atom of Ser69, Cys104, and the Gln106 main chain forms one hydrogen bond with the Quercetin, having 46 D.E. Gomes et al. / International Journal of Biological Macromolecules 85 (2016) 40–47 F ockin o ich is n ing re b o t i q c o 4 s ( t I [ d o t r t H b e s r o [ r t ig. 8. Structural details of the interaction between NS1 and quercetin obtained by d f the quercetin molecule in the protein NS1 and its distance from the Trp residue, wh -terminal in blue and C-terminal in red. (c) Interaction region with quercetin show ond lengths of 2.81, 3.04, and 2.47 Å, respectively. The side chain xygen of the Asn108 residue builds up one hydrogen bond with he flavonoid with a formation distance of 3.26 Å. Docking calculation analysis demonstrates that electrostatic nteractions play an important role in the stability of the NS1- uercetin complex, as well as hydrophobic contacts. These results orroborate the description of the enthalpy–entropy balance btained from the thermodynamic analysis. . Conclusions It is known that NS1 is involved in immune system subver- ion, inhibiting or neutralizing several steps in the interferon IFN) pathway, as well as acting by silencing the ribonucleopro- eic complex of hRSV. NS1 is able to decrease cellular levels of KK� and TRAF3, which are components of the interferon cascade 4]. Furthermore, it interacts with many other cellular proteins, as escribed by Wu et al. [2]. Zandi et al. [8] tested the antiviral effects f flavonoids against type 2 Dengue Virus (DENV) in Vero cells; he results showed that quercetin exhibited significant anti-DENV eplication properties, affecting intracellular DENV viral replica- ion. In addition, Bachmetov et al. [9] have shown suppression of epatitis C Virus replication by quercetin, mediated by the inhi- ition of NS3 protease activity. Another study carried out by Choi t al. [10] showed the inhibition of viral replication in the initial tage of influenza A virus infection in the presence of quercetin 3- hamnoside. Finally, an antiviral activity assay using quercetin and ther flavonoids to investigate plaque-forming unit (pfu) reduction 11] showed that quercetin presents infectivity and intracellular eplication reduction properties against RSV. This scenario shows he importance of investigating interaction between viral proteins g method. (a) Surface representation of the NS1-quercetin complex. (b) Localization 20.12 Å. The cartoon schematic representation of NS1 structure colored in spectrum, sidues involved in the process and the type of interaction. and flavonoids, in particular quercetin and its derivatives, princi- pally regarding the description of the molecular mechanism in the interaction which leads to the prevention of viral infection. The combination of experimental and in silico approaches showed that the interaction between hRSV-NS1 and quercetin is stable in the studied conditions, with a dissociation constant of the order of 10−6 M. The enthalpic and entropic energetic bal- ance provided by thermodynamic analysis in addition to molecular modeling analysis indicates that the interactions involved in the formation and stabilization of the NS1-quercetin complex exhibits both hydrophobic and electrostatic contributions. From this per- spective, the present results describe hRSV-NS1 as a potential target for future translational research. Author contributions Performed the experiments: Deriane Elias Gomes, Ícaro Putin- hon Caruso and Isabella Otenio de Lourenç o; Performed theoretical studies: Gabriela Campos de Araujo and Ícaro Putinhon Caruso; Analyzed the data: Deriane Elias Gomes, Ícaro Putinhon Caruso, Gabriela Campos de Araujo, Fernando Alves de Melo, Marinônio Lopes Cornélio, Marcelo Andrés Fossey, Fátima Pereira de Souza; Wrote the paper: Deriane Elias Gomes, Ícaro Putinhon Caruso, Gabriela Campos de Araujo, Fernando Alves de Melo, Marinônio Lopes Cornélio, Marcelo Andrés Fossey, Fátima Pereira de Souza. Acknowledgments We thank Fundaç ão de Amparo à Pesquisa do Estado de São Paulo - Fapesp (Grants 2010/50211-0 and 2010/52266-6) for finan- f Biol c B s A i 1 R [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ mechanism of guaijaverin to human serum albumin: fluorescence spectroscopy and computational approach, Spectrochim. Acta A Mol. and Biomol. Spectrosc. 97 (2012) 449–455. 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