Binding Modes of Flavones to Human Serum Albumin - American

Sep 16, 2010 - (BLI) and its aglycon, baicalein (BLE)sto HSA using a combination of experimental and computational approaches. The association propert...
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J. Phys. Chem. B 2010, 114, 12938–12947

Binding Modes of Flavones to Human Serum Albumin: Insights from Experimental and Computational Studies Hui Liu, Wei Bao, Hanjing Ding, Jongchol Jang, and Guolin Zou* State Key Laboratory of Virology, College of Life Sciences, Center of Nanoscience and Nanotechnology, Wuhan UniVersity, Wuhan 430072, China ReceiVed: March 7, 2010; ReVised Manuscript ReceiVed: August 9, 2010

Pharmaceutical interactions with human serum albumin (HSA) are of great interest, because HSA is a pharmacokinetic determinant and a good model for exploring the protein-ligand interactions. Due to their hydrophobic nature, naturally occurring flavones, which possess various pharmacological activities, bind to HSA in human plasma. Here, we have identified the binding modes of two representative flavonessbaicalin (BLI) and its aglycon, baicalein (BLE)sto HSA using a combination of experimental and computational approaches. The association properties were measured by applying spectroscopic methods, and a higher affinity was found for BLE. As evidenced by displacement and chemical unfolding assays, both ligands bind at Sudlow site I. Furthermore, molecular docking was utilized to characterize the models of HSA-flavone complexes, and molecular dynamics (MD) simulations as well as free energy calculations were undertaken to examine the energy contributions and the roles of various amino acid residues of HSA in flavones binding; the mechanism whereby glycosylation affects the association was also discussed. The present work provides reasonable binding models for both flavones to HSA. Introduction Human serum albumin (HSA), the most abundant carrier protein in the human circulatory system, plays a crucial role in the transport, distribution, and metabolism of a wide variety of endogenous and exogenous ligands.1 It is a 585-residue monomer and consists of three homologous domains (I-III), each of which comprises two subdomains (A and B).2 In 1975, Sudlow et al.3 first proposed the existence of two primary drugbinding sites that are hydrophobic cavities in HSA. The hypothesis was confirmed nearly 2 decades later, and the sites were found to be located at subdomains IIA and IIIA, respectively.2,4 To date, a consensus has been reached that small insoluble drugs bind selectively at one of these two sites, termed Sudlow sites I and II.1,3,5 To get the characteristics of the HSA-ligand interactions, Ghuman et al.5 crystallographically determined the structures of HSA with 12 diverse drugs and toxins. On the basis of the results, they found a general preference of HSA for the ligands with small-sized, waterinsoluble, and acidic or electronegative features. The binding selectivities of Sudlow sites I and II originate from their shapes, sizes, and particular distributions of basic residues on the mostly hydrophobic interior cavities. However, so far there is not a general mechanism for elucidating the binding modes of HSA with its various classes of ligands. Knowledge of the mechanism of pharmaceutical interactions with HSA provides a molecular basis for understanding the pharmacokinetic properties of druglike ligands. Moreover, it leads to new approaches for drug design and therapy.6 Also, HSA is an excellent model for investigating the protein-ligand interactions, because of its availability, stability, and extraordinary binding capacity.7 In the context, this field of research is regarded as the second step in rational drug design,1 and much effort has been made.8-13 * To whom correspondence should be addressed. Tel: 86-27-87645674. Fax: 86-27-68752560. E-mail: [email protected].

Figure 1. Chemical structures of flavones and site markers used in this work.

Flavones, belonging to the flavonoid family, are a group of naturally occurring polyphenolic compounds (Figure 1). They are widely distributed in angiosperms and daily ingested by humans (mainly from fruit and vegetables).14 Intriguingly and importantly, as major components of many medicinal plants, members of flavones are found to have various biological and pharmacological activities, thus being of considerable therapeutic interest.15 Of these compounds, baicalin (BLI) and baicalein (BLE) are two representatives (Figure 1). They are highly accumulated in the roots of Scutellaria baicalensis, a traditional herbal medicine. After administration, BLI is partly converted to BLE in vivo via the cleavage of the glycoside moiety.16 They exhibit strong antioxidant and free radical scavenging activities.17,18 Moreover, their abilities to inhibit viral enzymes have been reported (e.g., HIV reverse transcriptase).19 They also show antitumor activities.20 HSA represents a major transport medium for flavones in plasma,15 and a recent study reported that a flavone derivative

10.1021/jp102053x  2010 American Chemical Society Published on Web 09/16/2010

Binding Modes of Flavones to HSA exhibited antioxidative activity in the HSA-bound form,21 thus indicating that not only the pharmacokinetic but also the pharmacodynamic properties of flavones are related to HSA. Therefore, insight into the detailed binding modes of flavones to HSA is of great importance from a pharmacological point of view. In fact, interactions of HSA with the flavonoid family have attracted much attention.8-11 Although a schematic representation of flavone binding on HSA has been postulated,10 the residue-specific information of the binding mode is not yet well-defined. Previous investigations of the HSA-ligand interactions were undertaken routinely by means of spectroscopic methods, owing in part to its specific fluorescent properties.8-13 Advances in computational methods, including molecular dynamics (MD) and free energy calculation, facilitate annotating the physical basis of the structure-function relationship of biomolecules.22,23 More recently, such methods have been applied in studies of HSA, exemplified by the identification of binding sites of fatty acids24 and AZT derivatives.25 In the present work, we have employed a combination of experimental and computational approaches, in an attempt to determine where and how flavones bind to HSA. Insight gained from the designed assays has mapped the locations of the flavones’ binding sites, and more explicit models have been constructed by computational approaches. The results obtained by these two methods have been found to be correlated well with each other. Additionally, we have discussed the mechanism whereby the structural diversity of flavones, especially the glycosylation, affects the association. Our findings thus provide a framework for elucidating the mechanisms of HSA-flavones binding and may throw light on future studies about HSA-drug interactions. Experimental Section Materials. HSA (fatty acid free), BLE, BLI, warfarin, 8-anilino-1-naphthalenesulfonic acid (ANS), and guanidine hydrochloride (GdnHCl) were purchased from Sigma-Aldrich (St. Louis, MO). Other chemicals were of analytic grade. Ultrapure water (Millipore, Milli-Q Lab) was used for solution preparation. HSA, warfarin, and ANS were dissolved in 0.1 M phosphate-buffered saline (PBS), pH 7.4, as stock solutions, whereas BLE and BLI were dissolved in ethanol before being diluted into the assay buffer to obtain the required concentrations. The final concentrations of the organic solvent were below 0.5% (v/v) throughout the measurements. The concentrations of HSA and ANS were determined using the extinction coefficients of ε280nm ) 36 600 M-1 cm-1 and ε350nm ) 5000 M-1 cm-1, respectively, with a Cary-5000 UV-vis spectrometer (Varian). Fluorescence Titration Assays. In each assay, a 2.0 mL sample containing 3.0 µM HSA was added into a 1 cm quartz cuvette and then was titrated by stepwise addition of small aliquots (1 µL) of a 1.5 mM BLE or BLI stock solution at intervals of 5 min. Steady-state fluorescence emission spectra of HSA were recorded from 300 to 400 nm on an F-4500 fluorescence spectrometer (Hitachi, Tokyo, Japan) at a λex of 295 nm with slit widths of 5 nm and a scan speed of 240 nm/ min. Synchronous fluorescence spectra were collected in the synchronous scan mode with an offset of 15 or 60 nm (∆λ ) λem - λex ) 15 or 60 nm). All the spectra were recorded at 298 K. Displacement of ANS and Warfarin. Before displacement, ANS (9.0 µM) or warfarin (3.0 µM) were incubated with HSA (3.0 µM) in 0.1 M PBS, pH 7.4, at 298 K for 30 min. Then a

J. Phys. Chem. B, Vol. 114, No. 40, 2010 12939 2 mL sample was added into a 1 cm quartz cuvette, followed by titration of aliquots of BLE (1.5 mM) or BLI (1.5 mM). The emission intensity of ANS was monitored and recorded from 400 to 600 nm at a λex of 375 nm, and that of warfarin was collected from 330 to 500 nm at a λex of 320 nm. Chemical Unfolding of HSA. Samples of HSA were denatured in 0.1 M PBS, pH 7.4, containing various concentrations of GdnHCl and kept at 298 K for 2 h. The concentration of HSA was maintained at 3.0 µM, and the concentrations of GdnHCl were 2.0, 4.0, and 6.0 M. The denatured protein sample was used for subsequent fluorescence assays as described above. Circular Dichroism (CD) Spectroscopy. Before the measurements, HSA (10.0 µM) was incubated with each drug in 0.1 M PBS, pH 7.4. CD spectra were collected on a J-810 spectropolarimeter (JASCO, Tokyo, Japan) with a 0.1 cm quartz cuvette. Data were recorded from 200 to 250 nm with a scan speed of 100 nm/min at 298 K. Secondary structures were determined by using SELCON3, CDSSTR, and CONTINLL methods.26 Molecular Dockings. BLE, BLI, and ANS were docked into the three-dimensional structure of HSA with the AutoDock software package (version 4.0.1).27,28 Crystallographic coordinates of BLE and ANS were obtained from the Cambridge Structural Database (CSD), whereas that of BLI was derived from the structure of an analogue in CSD by removing unnecessary methyl groups (Supporting Information, Figure S1). The crystal structure of defatted HSA (PDB code 1AO6)29 was obtained from the Protein Data Bank (PDB) and all water molecules were removed. Preparations of all ligands and the protein were performed with AutoDockTools (ADT). A docking cube with an edge of 33.75 Å (a grid spacing of 0.375 Å), which encompassed the whole subdomain IIA, was used throughout docking. On the basis of the Lamarckian genetic algorithm (LGA),27 250 runs were performed for each ligand with 150 individuals in the population; the maximum numbers of energy evaluations and of generations were 2.5 × 107 and 2.7 × 104, respectively; other parameters were set as default.28 The resulting docking solutions were subsequently clustered with a root-meansquare deviation (rmsd) tolerance of 2.0 Å and were ranked by binding energy values. MD Simulations. The final docking results of HSA-BLE and HSA-BLI were used as the initial structures for MD simulations. All the simulations were performed with the AMBER software package (version 10),30 employing the AMBER ff99SB force field.31 The AM1-BCC32 semiempirical partial charges and topology parameters were generated for BLE and BLI with the general AMBER force field33 (GAFF) by ANTECHAMBER. Hydrogen atoms were added by LEaP. The systems were then solvated with the TIP3P water model in a cubic periodic unit cell with each side at least 9 Å from the nearest solute atom. Sodium ions were added to neutralize the net charges of the entire systems. Each solvated system was fully minimized in 10 000 steps and then gradually heated from 0 to 298 K in 20 ps with the canonical (NVT) ensemble, followed by a 400 ps equilibration with slowly releasing the weak restraints on the heavy atoms of the complex. Finally, the production simulations of 8 ns were carried out with the isothermal-isobaric (NPT) ensemble at 298 K, 1 atm, without any restraints. Constant temperature and pressure were maintained using the Langevin thermostat and the Berendsen barostat, respectively. All bonds containing hydrogen atoms were constrained by employing the SHAKE34 algorithm, allowing the use of a 2 fs time step for MD simulations. Long-range electrostatic interactions were accounted for by the particle-

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mesh Ewald (PME) method35 with an 8 Å nonbonded cutoff. Trajectories were analyzed by PTRAJ and in-house scripts. Free Energy Calculations. The binding free energies (∆Gbind) of HSA-BLE and HSA-BLI complexes were calculated using the molecular mechanics and generalized Born surface area (MM-GBSA) method23,36 with the AMBER package. Before the calculations, counterions and water molecules were stripped out and then 500 snapshots (every 2 ps) were extracted from the last 1 ns trajectory for each complex. The binding free energy, which was computed for each snapshot and averaged, can be evaluated by a combination of the molecular mechanics energy in the gas phase (∆Egas), the solvation free energy (∆Gsol), and the configurational entropic contribution of the solute (-T∆Sconf):

∆Gbind ) 〈Gcomplex - (GHSA + Gflavone)〉

∆Gbind ) ∆Hgas + ∆Gsol - T∆S ≈ ∆Egas + ∆Gsol T∆Sconf

(1)

(2)

The molecular mechanics energy in the gas phase is composed of electrostatic (∆Emm,ele) and van der Waals (∆Emm,vdW) energies:

∆Egas ) ∆Emm,vdW + ∆Emm,ele

(3)

Figure 2. Fluorescence emission spectra of HSA at different concentrations of (A) BLE and (B) BLI. The concentration of HSA was 3.0 µM. The molar ratios of flavones versus HSA were 0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, and 2.5. The arrow indicates the direction of increasing flavones concentration. The spectra were collected at an excitation wavelength of 295 nm, pH 7.4, 298 K. (C) Stern-Volmer plot of HSA quenched by flavones. (D) Plot of log [(F0 - F)/F] versus log [Q].

Results and Discussion whereas the solvation free energy consists of the polar (∆Gsol,pol) and nonpolar (∆Gsol,npol) contributions:

∆Gsol ) ∆Gsol,pol + ∆Gsol,npol

(4)

In this work, the polar term was calculated from the reaction field energy using a modified generalized Born (GB) model,37 whereas the nonpolar one was estimated by

∆Gsol,npol ) σSASA

(5)

A value of 0.0072 kcal/(mol Å2) was used for the surface tension proportionality constant (σ).38 Then the linear combination of pairwise overlaps (LCPO) method39 was used to determine the solvent-accessible surface area (SASA). Subsequently, normal-mode analysis was applied to estimate the entropy term (-T∆Sconf) of eq 2 using NMODE. Since the calculation is extremely time-consuming and hardware-unavailable for such large systems (involving more than 9000 atoms without water molecules and counterions), only residues 1-300 (subdomains IA, IB, and IIA) as well as ligands were used and only 10 snapshots (every 100 ps) were extracted from the last 1 ns trajectory of each complex.24 Each snapshot was energyminimized with a cutoff of 0.0001 kcal/(mol Å) and the distancedependent dielectric model (ε ) 4r) was employed to account for solvent screening at 298 K. Moreover, the decomposition of binding free energy was executed on each residue for a further investigation of the HSA-flavone interactions, in which all the energy terms of ∆Gbind, except for the entropic term, were taken into account.

Determination of Binding Properties by Fluorescence Titration Assays. In proteins, the fluorescence property of the indole ring of Trp is sensitive to changes in the local environment (local electric field direction and strength).40 Therefore, Trp is often used as a reporter for examining conformational events and ligand-binding properties of proteins through monitoring variations in its fluorescence.41 We took advantage of the fact that HSA possesses only one Trp residue (Trp214) located in the hydrophobic cavity of subdomain IIA2 to perform fluorescence titration assays and detect the bindings of flavones to HSA. The fluorescence emission of HSA at 339 nm, when excited at 295 nm, was primarily due to Trp214. Upon addition of BLE into HSA sample, as shown in Figure 2A, the fluorescence intensity was quenched (i.e., the emission intensity decreased) by 56% at 339 nm in a concentration-dependent manner. Similarly, the interaction of BLI with HSA was also accompanied by a fluorescence quenching of 44% at 339 nm (Figure 2B). No fluorescence change was observed in the presence of ethanol instead of flavones (data not shown). Alternatively, the observation of fluorescence quenching was confirmed by synchronous fluorescence measurements, whereby the emission peaks of the Trp and Tyr were separated out. When the value of ∆λ was set to 60 nm, the synchronous fluorescence, a contribution from the Trp residue, decreased instantaneously upon addition of either BLE or BLI (Figure 3A,B), in agreement with the steady-state fluorescence results. In addition, the intensity of Tyr synchronous fluorescence with a ∆λ of 15 nm also significantly decreased (Figure 3C,D). The results show a change of the surrounding environment of the fluorophore Trp214, induced by the quenchers (BLE and BLI). It suggests that the binding regions of both ligands are in the vicinity of the Trp214 residue, because a distant event cannot

Binding Modes of Flavones to HSA

J. Phys. Chem. B, Vol. 114, No. 40, 2010 12941 TABLE 1: Experimentally Determined Quenching and Binding Parameters for HSA-BLE and HSA-BLI Complexesa parameter -1

kq (M s) Ksv (M-1) Kd (µM) n I50 (µM) Ki (µM)

HSA-BLE

HSA-BLI

3.62 × 10 1.81 × 105 2.58 1.07 1.79 1.19

2.12 × 1013 1.06 × 105 20.31 0.93 24.61 16.41

13

a kq and Ksv were obtained from fluorescence titration assays based on eq 6; Kd and n were also obtained from fluorescence titration assays based on eq 7; I50 and Ki were determined by displacement of warfarin based on eqs 8 and 9, respectively. All data were obtained at pH 7.4, 298 K.

Figure 3. Synchronous fluorescence spectra of HSA at different concentrations of BLE and BLI: (A) HSA-BLE with a ∆λ ) 60 nm, (B) HSA-BLI with a ∆λ ) 60 nm, (C) HSA-BLE with a ∆λ ) 15 nm, and (D) HSA-BLI with a ∆λ ) 15 nm. The concentration of HSA was 3.0 µM. The molar ratios of flavones versus HSA were 0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, and 2.5. The arrow indicates the direction of increasing flavones concentration. The spectra were collected at pH 7.4, 298 K.

cause its fluorescence quenching. It can also be deduced that some Tyr residues are adjacent to the binding sites of flavones. In this context, Sudlow site II seemed not to be a good choice, because it is far away from Trp214; Sudlow site I was a likelier choice, given that Trp214 is just located in subdomain IIA. However, more supports were required for such a hypothesis. The fluorescence quenching of Trp214 could be quantitatively elaborated by the Stern-Volmer equation,41 which expects a linear relationship between the fluorescence intensity in the absence of the quencher (F0), when compared to that in the presence of the quencher (F), with the quencher concentration ([Q]):

F0 ) 1 + kqτ0[Q] ) 1 + Ksv[Q] F

(6)

where Ksv is the Stern-Volmer constant and equals the product of the quenching rate constant (kq) and the fluorescence lifetime of HSA without a quencher (τ0). As shown in Figure 2C, the regression curves of F0/F versus [Q] were plotted with good linearity. The values of Ksv and kq were determined from the slopes of the curves. As listed in Table 1, both of the calculated values of kq are greater than the maximum dynamic quenching constant of various kinds of quenchers with biopolymers,42 thus indicating that a static quenching mechanism dominates in the present complexes.43 Furthermore, the dissociation constants (Kd) and stoichiometries (n) of BLE and BLI binding to HSA can be obtained by measuring the fluorescence change of Trp214, based on the following equation:12

log

F0 - F ) n log [Q] - log Kd F

(7)

where the intercept and slope of the plot (Figure 2D) give Kd and n, respectively. Calculated parameters were summarized in Table 1. A 1:1 binding stoichiometry could be concluded for both complexes. The dissociation constants Kd for BLE and BLI were calculated to be 2.58 and 20.31 µM, respectively. From the results, we noted that, compared to the case of its aglycon BLE, the affinity of BLI to HSA decreases by approximately 1 order of magnitude, which stems from the glycosylation of the A-ring (Figure 1). The findings are in agreement with an earlier report by Dufour and Dangles.10 The affinities are moderate and reasonable, because the documented Kd values of noncovalent association of HSA with drugs mostly range from 1 to 100 µM,1 and a higher affinity to carrier proteins may result in an ineffectiveness of potent drugs. Additionally, since the concentration of HSA under physiological conditions (∼600 µM) is far higher than the concentrations of common ligands, it is likely that interactions with even those with such affinities could be biologically significant. Identification of Binding Sites by Displacement of Site Markers. The emission spectra and intensities of extrinsic fluorescent probes are often used to determine a ligand’s location on a macromolecule. ANS (Figure 1) is such a widely used fluorescent probe. It has an inverse relationship between its fluorescent quantum yield and solvent polarity, thus being a very sensitive marker for hydrophobic binding sites. Figure 4A,B shows the binding of ANS to HSA under varying the flavones’ concentrations. As is shown, either BLE or BLI titration reduced the fluorescence intensity of the bound ANS at 466 nm (Figure 4A,B). The decrease of the bound ANS fluorescence in the presence of BLE or BLI could be due to a dissociation of ANS and HSA. The results along with quenching assays data suggest that flavones bind at a certain hydrophobic cavity in the proximity of Trp214, most likely subdomain IIA. However, on the basis of this assay, it is not possible to tell exactly where the location is, since ANS binds to HSA at more than one site. Next, we detected the effect of BLE and BLI on the association of HSA with warfarin to get a more explicit knowledge of the flavones’ binding sites. Warfarin is an anticoagulant drug and specifically binds at Sudlow site I, as determined by X-ray crystallography.44 Also, it is a good fluorescent probe. When binding to HSA in the hydrophobic cavity, its fluorescence increases dramatically, compared to that of the free form in an aqueous environment. The emission intensity at 390 nm was directly related to the bound fraction of warfarin. The specificities of HSA for BLE and BLI were thus examined by employing fluorescence assays that detected whether the flavones could result in a displacement of warfarin from its binding site. Similar to the ANS assays, addition of either flavone caused a decrease of the HSA-warfarin fluores-

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Liu et al. where I50 is the midpoint of competition and is defined as the concentration of the competitor at which the maximum fluorescence intensity of the HSA-warfarin complex is halfreduced. Fmin represents the maximum warfarin displacement produced by the competitor; [L] represents the concentration of the competitor. The calculated values of I50 were 1.79 and 24.61 µM for BLE and BLI, respectively (Table 1). Furthermore, the apparent inhibition constants (Ki) can be derived from

Ki )

Figure 4. Displacement of site markers from HSA by flavones. Fluorescence emission spectra of HSA-ANS in the presence of increasing concentrations of (A) BLE and (B) BLI; fluorescence emission spectra of HSA-warfarin in the presence of increasing concentrations of (C) BLE and (D) BLI. The arrow indicates the direction of increasing flavones concentration.

Figure 5. Displacement of warfarin from HSA by flavones. HSA (3.0 µM) incubated with warfarin (3.0 µM) was titrated with BLE or BLI. Solid lines represent the optimal curve fitting of the data (obtained from Figure 4C,D) to a sigmoidal dose-response function by nonlinear regression.

cence emission (Figure 4C,D). These data thus reflect the fact that warfarin was displaced from its binding cavity located at subdomain IIA. BLE showed more significant displacement of warfarin when titrated over the same concentration range, compared to BLI, thus suggesting that BLE was a stronger competitor for warfarin than BLI. The data support the idea that both the ligands, BLE and BLI, bind to HSA in the hydrophobic cavity of Sudlow site I (subdomain IIA). The measured dependence of the fluorescence changes of warfarin on the concentrations of added flavones (Figure 4C,D) also allowed the description of the binding process. Quantitatively, the data were used to determine binding affinities of BLE and BLI by nonlinearly fitting the dose-response curve (Figure 5):45

F ) Fmin +

Fmax - Fmin 1 + 10(log I50-log [L])p

(8)

I50 [warfarin] 1+ Kd,warfarin

(9)

in which a Kd for warfarin (Kd,warfarin) of 4 µM was used.3 The results are presented in Table 1 and are in close agreement with the quenching data, also suggesting a difference of roughly 1 order of magnitude in binding affinity. This conformity demonstrates the accuracy and specificity of the warfarin competitive binding assay. Chemical Unfolding Study. In order to characterize the binding properties in greater detail, we undertook a GdnHClinduced unfolding study of HSA. On the basis of previous reports, the denaturant GdnHCl is known to cause partial unfolding of HSA, exposing the interdomain cleft region to the surrounding aqueous environment. There was an initial domain separation resulting in the expanded form of the protein followed by complete unfolding of the domains at a high denaturant concentration. However, Sudlow site I is still capable of binding ligands throughout the denaturation.43 Thus, we separated the domains of HSA by using different concentrations of GdnHCl. The fluorescence measurements were used to monitor the protein unfolding in the studied GdnHCl concentration range. The spectra of the single Trp214, located in the linker segment between the two halves of domain II, suggest that in the native protein the residue is well-protected from the solvent, since incubation with GdnHCl led to a red shift from 339 to 351 nm and a decrease in the emission intensity (Supporting Information, Figure S2). The fluorescence of Trp214 was quenched by the binding of flavones (Supporting Information, Figures S2 and S3), similar to the native protein results. Additionally, a decrease of Tyr emission was also detected by monitoring its synchronous fluorescence (Supporting Information, Figure S4). According to the crystal structure of HSA, there are 18 Tyr residues in total, but only Tyr263 is located in subdomain IIA. Thus, Tyr263 is the very Tyr residue of which the emission was quenched by flavones binding, since other Tyr residues were away from the binding sites in the unfolding form. On the basis of the results above, we come to the conclusion that both flavones, BLE and BLI, bind specifically at Sudlow site I, near Trp214 and Tyr263. This binding framework shed light on the computational study in the following sections. Effects of BLE and BLI Binding on the Secondary Structure of HSA. We performed CD spectroscopy to examine the influence on HSA caused by association of flavones. CD is an efficient tool for determining the structure of soluble proteins. Far-UV CD measurements allow one to monitor changes in the secondary structure. In a control assay, the CD spectra of HSA with or without 0.5% ethanol (v/v) were virtually the same, suggesting that ethanol in experimental concentration did not result in formation of rigid secondary structure (data not shown). Figure 6 shows the CD spectra in the far-UV region (200-250 nm) of HSA recorded at pH 7.4 in the absence or presence of BLE and BLI. The spectrum of HSA was character-

Binding Modes of Flavones to HSA

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Figure 6. CD spectra of HSA in the absence and presence of flavones. The spectra were background-corrected. Each measurement was repeated at least three times and averaged out.

TABLE 2: Secondary Structure Percentages of HSA in Absence or Presence of Flavonesa structureb

apo-HSA

HSA-BLE

HSA-BLI

H(r) H(d) S(r) S(d) T Unrd

37.4 19.8 2.8 3.5 13.2 24.1

39.1 20.3 2.3 3.1 12.3 23.0

38.1 19.9 2.8 3.3 12.8 24.2

a All data were obtained from analysis of CD spectra by three methods (SELCON3, CDSSTR, and CONTNLL), and averaged values are shown. b Abbreviations used are as follows: H, helices; S, strands; T, turns; Unrd, unordered; r, regular; d, distorted.

ized by two negative bands at 208 and 222 nm, which is indicative of a high content of typical R-helix structures. In the presence of flavones, the spectra of binding complexes overlapped well with that of apo-HSA in the shape, except that there was a ∼6% increase in the amplitude of the negative 222 and 208 nm bands for BLE binding and ∼2% increase of each band amplitude for BLI binding. It is likely that, in the presence of either of the two flavones, HSA acquired R-helical secondary structure. The propensity for helix formation emerged more evident by systematically comparing secondary structure decomposition using three analysis methods26 (Table 2). Compared to the apo form, the binding of BLE or BLI resulted in an increase of the apparent R-helical content (ordered and distorted helixes) of HSA by 2.2 or 0.8%, respectively. The data reflect that slight conformational changes of HSA observed were induced by binding of these flavones. Compared to BLI, a stronger interaction between HSA and BLE was also indicated. Construction of Binding Models. Given the lack of tangible X-ray- or NMR-based structures of HSA-flavone complex, we applied the molecular docking approach to construct the binding complexes of the ligands to HSA. According to the aforementioned experimental results, it was revealed that binding sites of BLE and BLI were both located at Sudlow site I with a binding stoichiometry of 1:1. Therefore, the whole region of subdomain IIA was extracted for docking with AutoDock. With an rmsd tolerance of 2.0 Å, 250 distinct structures were obtained for each ligand. The structures with the lowest energy were chosen as the models for HSA-flavones complexes (Figure 7). As shown, docking data of HSA complexed with BLE and BLI were in both cases interpreted as the flavone moieties being buried in Sudlow site I. In the case of BLI, its glycoside group pointed out of the protein, where it can interact with the bulk solvent.

Figure 7. Overview of HSA structure (PDB code 1AO6) and comparison of the binding models for the ligands used in this work. The bottom panel is a close-up view of the binding sites in the box of the top panel. The ligands are shown in stick representation and colored as follows: BLI, green; BLE, orange; ANS, white; warfarin, gray.

In an additional docking, we investigated the possible binding mode for the fluorescence probe ANS in Sudlow site I. The result (Figure 7) indicates an overlap between ANS and BLE as well as BLI binding site; it clarifies that the results of ANS fluorescence decay are due to the dissociation from HSA caused by a competitive association of the flavones which share a similar site. Furthermore, the crystal structure of warfarin binding site (PDB code 1H9Z)44 was fitted to the docking results. As shown in Figure 7, the space occupied by warfarin at Sudlow site I largely overlaps with that of BLE and BLI, which was found to coincide essentially with the displacement of warfarin by flavones revealed by aforementioned results. On the other hand, we also noted that the Trp214 and Tyr263 residues of HSA are in close proximity to the benzopyrone moieties of both flavones, suggesting the existence of hydrophobic interaction between them. These models thus provide a good structural basis to explain the efficient quenching of HSA fluorescence in the presence of either BLE or BLI, as mentioned above. MD Simulations. To examine the mode of action of HSA-flavone complexation, we subjected the final docking results to explicit-solvent MD simulations. In the AutoDock calculations, we used a semiflexible algorithm, namely, only the ligands were treated as flexible bodies, whereas the protein was treated rigidly; thus, the effect of protein flexibility was neglected. To take this effect into account and to refine the docking results, the behavior of the predicted complexes was studied in a dynamic context. The backbone rmsd time series was calculated during the production phase using the respective initial structure as a reference. As depicted in Figure 8, the HSA-BLE and HSA-BLI structures showed stable rmsds after 7 ns of

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Figure 8. Backbone rmsd of HSA as a function of time during MD simulations.

simulation time. Thus, the last 1 ns trajectories were used for post-MD analysis. Further, we calculated the rmsds for each subdomain (Figure 9). As is shown, the binding location, subdomain IIA, was quite stable during the MD simulations, and the rmsds of subdomains IB and IIIB were larger. In addition, the rmsd of HSA-BLE was larger than that of HSA-BLI, in line with aforementioned observations of CD spectra, which suggested more conformational changes of HSA induced by BLE binding. The refined binding models were obtained by averaging the last 1 ns of the MD trajectories. The resulting structures are shown in Figure 10. According to the analysis of the crystal structure, the interior of Sudlow site I is predominantly nonpolar, and the central binding cavity consists of three hydrophobic compartments. Nevertheless, an outer cluster of basic residues (Lys195, Lys199, Arg218, and Arg222) are present at the entrance of the cavity.5 As shown, both ligands are situated in Sudlow site I and the binding pocket is formed by the packing of six helices. BLE and BLI were both located in the central cavity, at which warfarin is located. The phenyl group (B-ring) is located within the binding pocket, extending into the inner hydrophobic compartment which is formed by several aliphatic residues, including Leu219, Leu238, Leu260, Ile290, and Ala291. The benzopyrone moiety (A- and C-rings) protrudes

Figure 10. Binding sites of (A) BLE and (B) BLI on HSA. Selected residues (green) and both ligands (cyan) are represented by stick models. Hydrogen bonds are shown in orange dot line.

from the hydrophobic cavity and points toward the entrance of Sudlow site I, since the hydrophilic phenolic hydroxyl groups can interact with nearby polar residues. These binding models seem reasonable, since the benzopyrone group is more hydrophilic compared to the phenyl group, and a converse framework (A-ring in the inner and B-ring in the outer) could lead to a decrease in both hydrophobic and polar interactions. It is worth

Figure 9. Backbone rmsd of each subdomain of HSA as a function of time during MD simulations.

Binding Modes of Flavones to HSA

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TABLE 3: Computationally and Experimentally Determined Binding Free Energy Components for HSA-BLE and HSA-BLI Complexesa component

HSA-BLE

HSA-BLI

∆Emm,ele ∆Emm,vdW ∆Gsol,pol ∆Gsol,npol -T∆Sconf ∆Gbind,cal1 ∆Gbind,cal2 ∆Gbind,expt1 ∆Gbind,expt2

-5.88 -32.29 21.28 -4.17 15.39 -5.67 -6.50 -7.62 -8.07

-46.26 -42.15 66.74 -6.23 24.55 -3.35 -7.73 -6.39 -6.52

a ∆Emm,ele, ∆Emm,vdW, ∆Gsol,pol, ∆Gsol,npol, -T∆Sconf, and ∆Gbind,cal1 were calculated using the MM-GBSA approach, based on eqs 1-5; ∆Gbind,cal2 was predicted by AutoDock; ∆Gbind,expt1 was obtained from fluorescence titration assays, calculated from Kd with ∆G ) RT ln Kd; ∆Gbind,expt2 was obtained from displacement of warfarin, calculated from Ki with ∆G ) RT ln Ki. All energy components are in units of kcal/mol.

noting that both the ligands have the pyrone moiety (C-ring) tightly pinned between the nonpolar side chains of Leu238 and Ala291. This was commonly found for Sudlow site I binders.5 The A-, C-, and B-rings are slightly noncoplanar, since hydrophobic interactions twisted the planar molecules against the internal steric interaction. In addition to hydrophobic contacts, the flavones make specific interactions with polar residues nearby, thus stabilizing their polar moieties through hydrogen bonds and electrostatic interactions. To test this, we detected the formation of hydrogen bonds by examining the snapshots during the last 1 ns of equilibrium phase of the simulations in terms of distance and orientation. The results show that two nitrogen atoms of Arg222 solidly formed two hydrogen bonds with the ligand BLE, occupying 74.9 and 39.0% of the last 1 ns. In the case of BLI, it was positioned to make two hydrogen bond interactions with the hydroxyl group of Glu292, and the occupancy percentages were 64.6 and 43.1%. Analysis of Binding Free Energy. Further insight into the forces involved in ligand binding can be obtained by analyzing the free energy contributions. Although AutoDock could be used for constructing binding models, the semiempirical energy function employed is rather crude for estimating binding free energy.46 In this case, the predicted ∆Gbind with AutoDock yielded an incorrect energetic trend for two complexes as compared to experimental binding affinities (Table 3). Therefore, a more physically rigorous method was required. The MMGBSA method is such a promising solution,46 which has been suggested as capable of ranking the relative binding free energy of different ligands47 and widely used in research of protein-ligand interaction.23 Thus, we undertook MM-GBSA calculations on both HSA-BLE and HSA-BLI complexes. The calculated free energy contributions for each system with the MM-GBSA method are presented in Table 3. In both cases, the interaction energy due to electrostatic interaction between HSA and ligands led to favorable binding (∆Emm,ele < 0), whereas the electrostatic component of the solvation free energy is consistently unfavorable (∆Gsol,pol > 0). A more favorable electrostatic contribution (∆Emm,ele) is observed for BLI than that for BLE. This is expected, since BLI is more negatively charged than BLE. However, desolvating a negatively charged moiety also led to a larger penalty, thus resulting in an intriguing observation that the electrostatic component (∆Emm,ele + ∆Gsol,pol) is even more favorable in the HSA-BLE complex (15.4 kcal/mol) than that in the HSA-BLI complex (20.48 kcal/

mol). In each case, the favorable nature of the nonpolar interaction mostly stems from the van der Waals interaction energy (∆Emm,vdW), compared to the nonpolar component of solvation (∆Gsol,npol). Moreover, the data indicate that the favorable contribution increases with the addition of the glycoside moiety, since there is more reduction of solventaccessible surface area during desolvation. An inspection of the free energy components for the HSA-BLE complex reveals that the electrostatic component of the free energy of binding contributes unfavorably to binding (∆Emm,ele + ∆Gsol,pol > 0), whereas the data show that the nonpolar component contributes favorably (∆Emm,vdW + ∆Gsol,npol < 0). This trend holds for the HSA-BLI complex as well. However, the unfavorable electrostatics component (∆Emm,ele + ∆Gsol,pol) in each case is compensated by the highly favorable nonpolar component of the free energy (∆Emm,vdW + ∆Gsol,npol). In total, when quantitatively comparing the nonpolar (∆Emm,vdW + ∆Gsol,npol) with the electrostatic (∆Emm,ele + ∆Gsol,pol) contributions for both complexes, we come to the conclusion that the association between each ligand and HSA is dominated by more favorable nonpolar interactions, which is common for noncovalent association. Normal-mode analysis results obtained through the NMODE module of AMBER 10 indicate that in both complexes, the values of ∆Sconf were negative. Accordingly, entropy was found to contribute unfavorably to binding. The entropy change upon binding (-T∆Sconf) remained more favorable for the HSA-BLE complex, thus totally leading to a higher bind affinity than that of BLI. This may be due to the increase in solute entropy penalty of the flavone moiety upon binding by connecting to the flexible glycoside ring. The results of the MM-GBSA calculation successfully predicted the correct binding affinity trend for these two systems. As listed in Table 3, the ∆Gbind for BLI, derived from MMGBSA, is 2.32 kcal/mol more positive than that for BLE, which is in agreement with the experimentally determined trends (fluorescence titration, 1.23 kcal/mol; displacement of warfarin, 1.55 kcal/mol) and confirms the fact that glycosylation decreases the binding affinity of flavones. On the other hand, comparing the predicted values with the experimental values reveals that the absolute values of predicted ∆Gbind are both underestimated. However, this is expected since several factors (e.g., the energy term of conformational changes and the entropic contribution of the solvents) are not taken into account for the theory basis of MM-GBSA. Thus, in this work, the results of MM-GBSA with differences of ∼2-3 kcal/mol from the results of experiments in absolute value are reasonable. Furthermore, to identify the energetically important residues for flavones binding, we decomposed the binding free energy into each residue for both complexes. The free energy contributions of important residues are presented in Table 4. As is shown, the residues with favorable contributions to the binding free energy in both systems were almost the same, due to their similar binding sites. Moreover, it was observed that nonpolar residues contribute more to the binding free energy than polar residues. On the basis of these results, it can be concluded that hydrophobic interactions play an important role in the HSAflavone binding. Conclusions By using a combination of experimental and computational methods, we presented here reasonable association models for elucidating the modes of action of flavones binding to HSA. Binding properties of two flavones, BLE and BLI, were

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Liu et al.

TABLE 4: Free Energy Contributions of Important Residues of HSA to the Bindings with BLE and BLIa ligand

residue

∆Emm,ele

∆Emm,vdW

∆Gsol,pol

∆Gsol,npol

∆Gbind

BLE

Arg218 Leu219 Leu238 Leu260 Ile290 Ala291 Arg218 Leu219 Leu238 Leu260 Ile290 Ala291 Glu292

-0.48 -0.15 -0.04 0.16 0.09 0.00 -1.67 -0.11 0.14 -0.21 -0.02 0.16 -10.94

-0.90 -1.22 -1.63 -1.05 -2.25 -1.94 -0.66 -0.77 -1.96 -0.80 -1.53 -2.75 -0.55

0.94 0.14 0.18 0.07 0.22 0.41 1.94 0.16 -0.02 0.31 0.53 0.91 10.45

-0.12 -0.15 -0.33 -0.08 -0.21 -0.33 -0.29 -0.13 -0.30 -0.08 -0.16 -0.46 -0.25

-0.56 -1.39 -1.82 -0.90 -2.15 -1.85 -0.68 -0.84 -2.16 -0.79 -1.18 -2.14 -1.30

BLI

a All energy components are in units of kcal/mol and were obtained from free energy decomposition using the MM-GBSA approach.

determined using fluorescence spectroscopy, via measuring changes of the Trp214 fluorescence. Competitive binding and chemical unfolding assays were undertaken, and the results revealed that the binding sites of both flavones are located at Sudlow site I, adjacent to Trp214 and Tyr263. CD spectroscopy was used to investigate the secondary structures and interactions between HSA and flavones. On the basis of these findings, the binding models were mapped by molecular docking and MD simulations, thus providing residue-specific information about the complexes formation. BLE and BLI were found to be located at Sudlow site I near Trp214 and Tyr263, overlapping rooms with ANS and warfarin. Additionally, the model showed the microenvironment of the binding sites of both flavones to be rich in nonpolar residues. To the end, we used post-MD free energy calculations following the MM-GBSA approach to determine the roles of nonpolar, electrostatics, and entropic contributions in binding. The results suggested hydrophobic interactions play an important role in the HSA-flavone binding. Glycosylation was found to decrease the ability of the flavone binding to HSA, due to more unfavorable electrostatic component of the solvation free energy and more unfavorable entropy penalty. Both experimental and computational data fit each other well. Provided that binding affinities for drugs to HSA range from 1 to 100 µM, BLE could be classified as a relatively high-affinity binder for HSA and BLI a moderate-affinity one. However, via the cleavage of the glycoside moiety, BLI could also be converted into a relatively high-affinity binder in vivo. Therefore, it is worth noting, as evidenced by our work, that when BLE or BLI was administrated with other Sudlow site I drugs (e.g., warfarin), special attention should be paid. Because the displacement of the drug from the binding site may occur, thus increasing its effective concentration along with an enhancement of potency, it can be toxic sometimes. For decades, pharmaceutical interactions with HSA have been of great importance in terms of drug discovery, because HSA is a key determinant for pharmacokinetic properties of many drugs. As a result, much effort has been exerted, and many approaches have been utilized in this field. Our work presented here, as well as recent reports from other groups,24,25 suggests that MD-based computational methods, especially when combined with other experimental means, could be valuable tools for understanding interactions between HSA and druglike ligands.

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