Exploring the interaction of myricetin with human alpha-2-macroglobulin: biophysical and in-silico analysis

Abstract

Myricetin (MYR) is a bioactive secondary metabolite found in plants that is recognized for its nutraceutical value and is an essential constituent of various foods and beverages. It is reported to exhibit a plethora of activities, including antioxidant, antimicrobial, antidiabetic, anticancer, and anti-inflammatory. Alpha-2-macroglobulin (α2M) is a major plasma anti-proteinase that can inhibit proteinases of both human and non-human origin, regardless of their specificity and catalytic mechanism. Here, we explored the interaction of MYR-α2M using various biochemical and biophysical techniques. It was found that the interaction of MYR brings subtle change in its anti-proteolytic potential and thereby alters its structure and function, as can be seen from absorbance and fluorescence spectroscopy. UV spectroscopy of α2M in presence of MYR indicated the occurrence of hyperchromism, suggesting complex formation. Fluorescence spectroscopy reveals that MYR reduces the fluorescence intensity of native α2M with a shift in the wavelength maxima. At 318.15 K, MYR binds to α2M with a binding constant of 2.4 × 10³ M⁻¹, which indicates significant binding. The ΔG value was found to be − 7.56 kcal mol⁻¹ at 298.15 K, suggesting the interaction to be spontaneous and thermodynamically favorable. The secondary structure of α2M does not involve any major change as was confirmed by CD analysis. The molecular docking indicates that Asp-146, Ser-172, Glu-174, and Tyr-180 were the key residues involved in α2M-MYR complex formation. This study contributes to our understanding of the function and mechanism of protein and flavonoid binding by providing a molecular basis of the interaction between MYR and α2M.

Introduction

Flavonoids constitute a class of phytochemicals that are ubiquitously found in the plant kingdom. Flavonoids are more prevalent in the bark, root, epidermis of leaves, and skin of fruits. Secondary plant metabolites have been widely used as a source of natural molecules and play a vital role against environmental stresses and pathogens. Flavonoids are important in various clinical applications such as the pharmaceutical and perfume industries [1]. Moreover, these compounds have potential health benefits such as anti-inflammatory, anti-tumor, antibacterial, antiviral, anti-obesity and protection against cardiovascular, neurological, and hepatocellular damage [2].

Myricetin (MYR) is a bioactive plant secondary metabolite, originally isolated from the bark of the tree Myricarubra (Chinese bayberry), belonging to the family Myricaceae, with a molecular formula C₁₅H₁₀O₈ [3, 4]. MYR (3, 5, 7, 3′, 4′, 5′-hexahydroxyflavonol) is a versatile dietary phytochemical flavonol having a multitude of pharmacological functions in humans. The antioxidant activity of MYR is attributed to its hydroxylation pattern positioned at 3′,4′,5′ of ring-B of the pyrogallol group [5, 6]. The daily dietary intake of MYR in the western diet ranges between 0 and 30 mg with a median of 10 mg [7]. MYR consumption has been suggested in treating cancer by initiating cell cycle arrest and reactive oxygen species (ROS) dependent mitochondrial-dependent apoptosis in A549 lung cancer cells [8]. Moreover, MYR has been used in the treatment and prevention of neurodegeneration due to ischemia [9]. Evidence suggests natural products (NPs), mostly of plant origin, have been shown to possesses beneficial effects against a variety of diseases including cancer, cardiovascular diseases, Alzheimer’s, and human immunodeficiency virus (HIV) [10]. Recently, a variety of NPs were screened using an in-silico drug discovery method and an enzymatic assay, and only a limited number of those NPs were shown to be SARS-CoV-2 inhibitors. Among them, MYR was considered as a robust SARS-CoV-2 inhibitor, targeting the viral main protease (Mpro) to inhibit viral replication, suggesting MYR might be a promising COVID-19 therapeutic option [11].

Alpha-2-macroglobulin (α2M) belongs to a multifunctional protein family consisting of alpha-1-macroglobulin (α1M), alpha-2-macroglobulin (α2M), complement components (C₃, C₄, and C₅), and pregnancy zone proteins (PZP), sharing conserved structural and functional features throughout evolution [12]. It is constitutively abundant in the biological fluids of both vertebrates and invertebrates. α2M is a ubiquitous, tetrameric pan-proteinase inhibitor of the innate immune system, and has a remarkable ability to inhibit an incredibly diverse range of proteinases (serine, threonine, and metallo-proteinases) regardless of its catalytic site and specificity. α2M also forms stable complexes with hormones, inflammatory cytokines, interleukins, growth factors and drugs thereby affecting the distribution, elimination, and pharmacokinetic properties of these biomolecules [13]. The α2M traps a variety of proteinases via its unique bait and trap mechanism in its cage-like structure formed by dimer of dimmers [14]. The protein possesses an inherent ability to trap the proteinases released by cells during inflammation and hence, modulates the extracellular proteolytic activity brought through fibrinolysis and coagulation [13]. α2M has also been documented to trap proteinases originating from non-human sources [15].

The interaction of MYR with plasma proteins has been widely studied; however, the interaction of MYR with α2M has not been reported. The mechanism of MYR interaction with α2M was therefore believed to be worth investigating using a variety of spectroscopic techniques, including UV–visible, steady-state fluorescence, synchronous fluorescence, far UV CD measurements, and FRET. A molecular docking study was also used to identify the binding site of MYR on α2M. Additionally, the investigation of MYR and α2M interaction is a critical topic as the complex formation has therapeutic advantages.

Materials and methods

Materials

Trypsin, soybean trypsin inhibitor (STI), and N-benzoyl-DL-arginine-p-nitroanilide (BAPNA), Sephacryl S300HR were purchased from Sigma Chemical Co. (St. Louis, MO). MYR (CAS Num: 529–44-2) was purchased from SRL, India. All other chemicals were analytical grade of highest purity and were purchased from Merck.

Methods

Purification of α2M

Isolation of human α2M was performed using previous published sources and standardized in our laboratory [16, 17]. Freshly discarded human blood was collected from the Blood Bank, Department of Pathology, JN Medical College and Hospital, Aligarh. After centrifugation and separation of blood plasma, ammonium sulfate fractionation was performed and it was further dialyzed. Protein purification was carried out by gel-filtration chromatography on sephacryl S300HR column pre-equilibrated using the same sodium phosphate buffer (50 mM), pH 7.4. The dialyzed sample was loaded onto the column and fractions of 3.0 ml were collected. A 5% nondenaturing PAGE was run using tris–glycine buffer (pH 8.3) [18] and a single α2M protein band was detected after staining the gel with 0.15% CBBR-250 for 30 min, followed by washing and destaining.

Sample preparation

MYR stock solution (1 mM) was prepared in DMSO (dimethyl sulfoxide) and later diluted using 50 mM sodium phosphate buffer (pH 7.4) to achieve the desired concentration (2–20 μM). MYR was initially dissolved in DMSO due to its low solubility, and the appropriate quantity of the stock solution was then diluted with sodium phosphate buffer 50 mM (pH 7.4). The total amount of DMSO in the solution was 0.06% of the total volume. The purified human α2M (20 μM) was treated with increasing concentrations of MYR (2–20 μM).

Activity assay

For the activity assay, native α2M was taken as a reference, which exhibits 100% activity. Increasing concentrations of MYR (5–20 μM) were incubated with α2M (20 μM). All the samples were treated with trypsin (0.1%) for 15 min. After that, STI (0.1%) was added, and incubation was performed for 15 min. Finally, BAPNA (5 μg/11 ml) was added. Upon incubation for 30 min at 37 °C, the activity was measured at 410 nm [19].

Instrumentation

The (UV-Vis) spectral measurements were recorded on a Shimadzu double-beam UV spectrophotometer UV-1800 (Japan) using a quartz (1 × 1 cm) cuvette. Normalization of the obtained fluorescence data was performed using a/e-UV-Vis-IR spectral analysis software. CD spectra were analyzed in the far UV region on a JASCO J-1500 spectropolarimeter equipped with a Peltier temperature controller, maintaining the sample temperature at 25 °C.

UV-Vis spectroscopy

The α2M (20 μM) was titrated against an increasing concentration of MYR (2–20 μM) [20]. An alteration in absorbance spectra of α2M ranging between 240 and 350 nm was recorded with a screening speed of 1000 nm/min. A corresponding MYR solution without α2M was used as a blank and subtracted.

Steady-state fluorescence quenching

The steady-state fluorescence measurements were carried out after exciting protein at 280 nm and emission spectra were recorded between 285 and 385 nm. The spectra of α2M alone (20 μM) and a mixture of α2M and MYR at varying concentrations (2–20 μM) were recorded. Slit widths for both excitation and emission were set at 5 nm.

Synchronous fluorescence

Synchronous fluorescence was conducted to study alteration in the microenvironment of the fluorophore [Δλ = 60 nm (Trp), Δλ = 15 nm (Tyr)] on interaction with MYR. The fluorescence spectra of α2M (20 μM) were recorded with increasing concentrations of MYR (2–20 μM).

Fluorescence resonance energy transfer (FRET)

The UV–Vis absorption and fluorescence emission spectra of MYR and α2M were recorded in the range of 250–400 nm, respectively. The calculation of energy efficiency €, foster distance (R₀), distance between donor and acceptor was carried out using Photochem CAD software.

Circular dichroism spectroscopy

CD spectra of α2M (20 μM) were observed in with 10 μM and 20 μM of MYR. The baseline spectrum of 50 mM sodium phosphate buffer (pH 7.4) was subtracted, and each spectrum was obtained from the mean of 3 scans with a scanning speed of 500 nm/min.

Molecular docking

The 3D crystal structures of α2M (PDB ID: 4acq) and MYR were retrieved from the protein data bank and PubChem, respectively. The preparation of MYR and α2M was performed using MGL Tools. In the docking process, water molecules and ions that were present were deleted. Using the MGL Autodock Tool, a grid map with three-dimension grid points (40 × 40 × 40) and a grid spacing of 0.375 Å was created. The grid’s center was determined to be X = 9.757, Y = 18.303, and Z =  − 23.688. Once the preparations were complete, the docking studies were performed using AutodockVina [21] and binding energy was obtained. Visualization and analysis of the results were carried out using Pymol and Ligplus, respectively.

Statistical analysis

The data is presented as the mean ± SD and each experiment was repeated three times.

Results

Assessment of anti-proteinase activity of α2M in presence MYR

To examine the effect of MYR on α2M, the trypsin inhibitory assay was carried out using varying concentrations of MYR (Fig. 1A). Native α2M exhibited maximum activity and was taken as a reference. With the addition of increasing concentration of MYR (5–20 μM), the activity of α2M declined in a gradual manner. As depicted in Fig. 1B, at 10 μM of MYR concentration, an inhibitory activity of α2M decreases approximately by 11% of its original potential. Upon doubling the MYR concentration (20 μM), the protein activity declines by approximately 28% of its original activity (i.e., around 72% of residual activity of α2M was present). This suggests that MYR causes a subtle alteration in the α2M activity upon interaction. Thus, it can be concluded that exposure to micromolar concentrations of MYR induced alteration in the functional status of α2M.

Fig. 1.

A Molecular structure of MYR. B Effect of increasing concentration of MYR (5–20 μM) on inhibitory activity of human α2M (20 μM)

UV-vis spectroscopy

UV-Vis absorption spectroscopy provides information about the nature of the interaction between flavonoids and proteins. Proteins exhibit two characteristic absorption bands, one at a lower wavelength (180–230 nm), due to n → π ∗ transitions in the peptide bonds and the other at a higher wavelength (230–300 nm) almost entirely due to π → π ∗ transitions. The absorption bands in the wavelength range 230–300 nm correspond to the cumulative contribution of aromatic amino acid residues. There is a weak absorption band near 260 nm due to disulfide. Figure 2 depicts the UV–Vis absorption spectra of human α2M in the presence and absence of MYR. Human α2M showed a characteristic peak at 280 nm due to the presence of fluorophores (Tyr, Trp, and Phe). Fluorophores are of critical importance during interaction studies, as it ensures the complex formation between biomolecule and ligands. The absorbance of human α2M (20 μM) increased with increasing concentrations of MYR (2–20 μM), suggesting complex formation between α2M and MYR.

Fig. 2.

Absorption spectra of MYR alone and native human α2M (20 μM) gradually titrated with MYR (2–20 μM) at room temperature

Steady-state fluorescence quenching measurements

Florescence spectroscopy was employed to probe the ligand–protein binding interaction due to its sensitivity, convenience, and reliability. The protein intrinsic fluorescence is primarily due to aromatic fluorophores, Tyr, Trp, and Phe. The α2M was excited at 280 nm and the fluorescence intensity was measured as a function of the increasing concentration of MYR (2–20 µM). As depicted in Fig. 3, α2M exhibits a strong fluorescence emission peak at around 330 nm which shows a progressive decrease with increasing concentration of MYR (2–20 μM). MYR fluorescence shows weak fluorescence in aqueous solution at around 300 nm. Quenching of fluorescence is followed by a simultaneous peak shift (hypsochromic shift) of about 2 nm in the emission maxima of the lower wavelength region. A hypsochromic shift suggests an alteration in the polarity of aromatic moieties. The microenvironment of Tyr and Trp residues becomes more hydrophobic (buried inside a hydrophobic core) upon interaction with MYR.

Fig. 3.

Representative emission spectra of human α2M in the absence and in the presence of an increasing concentration MYR. Intrinsic fluorescence spectra of human α2M in the absence and presence of increasing concentrations of MYR (2–20 μM)

Mode of quenching

The fluorescence data were studied by the linear Stern–Volmer equation (Eq. 1). The slope of the Stern–Volmer plot was used to obtain the Stern–Volmer quenching constant, defined by

$$\frac{F_0}{F}=K_{\mathit{SV}}\left[Q\right]+1=K_q{\tau }_0\left[Q\right]+1$$ 1

where F_o and F represent fluorescence intensities in the absence and presence of MYR, K_sv is the Stern–Volmer quenching constant, [Q] is the concentration of MYR, K_q is the bimolecular rate constant, and τ₀ is the average life time of tryptophan fluorescence (10⁻⁸ s). Furthermore, K_q can be calculated using the equation (Eq. 2)

$$K_q=\frac{K_{\mathit{SV}}}{{\tau }_0}$$ 2

The Stern–Volmer plot between F_o/F and the molar concentration of the MYR (1:1) was linear (Fig. 4A), suggesting that only single type of quenching occurred. The linear regression results using Eq. 1 shown in Table 1, suggest that the K_sv of α2M and MYR were positively correlated with the increasing temperature. From Fig. 4A and Table 1, it is evident that the fluorescence quenching mechanism between α2M and MYR was due to dynamic quenching. In dynamic quenching, the bimolecular quenching constants are likely to increase with raising the temperature and vice-versa in static quenching. Since higher temperature lowers the stability of complex formation and bimolecular quenching constants.

Fig. 4.

A The linear Stern–Volmer plot of F_o/F vs MYR at different temperatures 298.15 K, 308.15 K, and 318.15 K. B Modified Stern–Volmer plot of log [(F0/F)-1] and log [MYR] at different temperatures 298.15 K, 308.15 K, and 318.15 K. C Van’t Hoff plot for temperature dependence of K_b

Table 1.

Binding and thermodynamic parameters of α2M-MYR system

Temp (K)	Ksv (M−1)	Kq (M−1 s−1)	Kb (M−1)	N	ΔS (Kcalmol−1 K−1)	ΔH (Kcal mol−1)	ΔG (Kcal mol−1)
298.15	5.61 × 104	0.561011	0.3 × 103	0.99	0.0844	17.4	− 7.56
308.15	6.91 × 104	0.69 × 1011	1.5 × 103	0.99	-	-	− 8.40
318.15	8.84 × 104	0.84 × 1011	2.4 × 103	0.99	-	-	− 9.24

The bimolecular quenching rate constant (K_q) is calculated using the obtained K_sv values. The fluorescence lifetime of the biopolymer in the absence of quenchers was found 10⁻⁸ s. It is apparent from Table 1, the values were found to increase with an increased in temperature, confirming the dynamic mode of quenching between α2M-MYR. The maximum scatter collision-quenching constant (K_q) for various quenchers with biopolymers is approximately 2 × 10¹⁰ L/ mol s⁻¹. Thus, the α2M-MYR quenching rate constant is found to be greater than the quenching constant of biomolecules.

Binding constant and number of binding sites

The binding constant (K_b) for the α2M-MYR interaction can be determined by using the modified Stern–Volmer equation (Eq. 3) as shown below.

$$log=\frac{F_0-F}{F}=\log K_b+nlog[Q]$$ 3

where n and K_b are the number of binding sites and the binding constant, respectively. The values of n and K_b can be obtained from the linear plot of log [(F₀-F)/F] vs log [Q] (Fig. 4B).

The results in Table 1, show that the values of the binding constant increase with an increase in temperature, suggesting that the quenching between α2M and MYR was dynamic. The binding stoichiometry is almost equivalent to unity, inferring a single class of binding site available in α2M for MYR.

Thermodynamic parameters

Thermodynamic parameters for α2M-MYR system were obtained from the Vant Hoff plot (Fig. 4C) using the following equations (Eqs. 4 and 5)

$$ln{K}_b=-\frac{{\mathrm{\Delta }H}^0}{2.303RT}+\frac{\mathrm{\Delta }S}{2.303R}$$ 4

$$\mathrm{\Delta }G=RTln{K}_b$$ 5

where K_b represents the binding constant at different temperatures (298.15 K, 308.15 K, and 318.15 K), R represents the gas constant (1.987 calmol⁻¹ K⁻¹), and T is the corresponding temperature in Kelvin. The values of enthalpy change ∆H, ∆S, and ∆G were calculated using the above equations and listed in Table 1. The relation between the change in enthalpy (∆H) and the change in entropy (∆S), which could ascertain the mode of interaction, is listed in Table 2. As seen in Tables 1 and 2, the values ∆H and ∆S were positive, indicating that hydrogen bonding and hydrophobic forces perform a principle role in the binding of α2M and MYR. The negative value of ∆G denotes an exothermic and a thermodynamically favorable interaction between α2M-MYR complexes.

Table 2.

The relationship between interaction mode and thermodynamic parameters

∆H > 0, ∆S > 0	Hydrophobic force
∆H < 0 or ∆H = 0, ∆S > 0	Electrostatic interaction
∆H < 0, ∆S < 0	Van der Waals interaction or hydrogen bond

Synchronous fluorescence

Synchronous fluorescence spectroscopy provides information related to the alteration in milieu of the fluorophore (Trp and Tyr) found in the protein during interaction with the ligand. Any alteration in protein structure near these residues is observed by taking its corresponding emission maxima (λ_max) and a peak shift is observed due to the alteration in the polarity of the microenvironment around Tyr and Trp residues. Figure 5A and B show the synchronous fluorescence spectra of α2M at different concentrations of MYR (2–20 µM). During the measurement of synchronous fluorescence spectra, the simultaneous scanning of both the excitation and emission monochromators is preferred rather than scanning one of them. Synchronous fluorescence spectroscopy characterized the alteration in microenvironment around Trp residues when the Δλ = 60 nm while synchronous fluorescence spectroscopy characterized the alteration in the microenvironment around Tyr residues when the Δλ = 15 nm. Figure 5A shows the synchronous fluorescence spectra of α2M when the Δλ was 60 nm in the absence and presence of increasing MYR concentrations (2–20 μM). A decline in fluorescence intensity with no shift in λ_max was observed, suggesting a stable microenvironment around Trp for α2M. Similarly, Fig. 5B illustrates the synchronous fluorescence spectra of α2M when the Δλ was 15 nm in the absence and presence of increasing MYR concentrations (2–20 μM). A decline in fluorescence intensity but no shift in λ_max was observed, indicating that the microenvironment around Tyr for α2M remains unaltered. The finding supports the binding of MYR to α2M; however, no significant change in polarity is observed as no peak shift was reported.

Fig. 5.

Representative Synchronous fluorescence spectrum of α2M-MYR A at Δλ = 60 nm B at Δλ = 15 nm

Fluorescence resonance energy transfer (FRET)

FRET is a sensitive spectroscopic measurement in which a donor fluorophore in an excited electronic state can non-radiatively transfer its excitation energy to an adjacent acceptor chromophore through long-range dipole–dipole interactions. During complex formation, FRET evaluates the effectiveness of energy transfer and the distance between a donor and an acceptor molecule. According to Forster’s non-radiative energy transfer theory, energy can only be transferred when (i) the donor molecule has intrinsic fluorescence, (ii) the donor’s emission spectrum and the acceptor’s absorption spectrum have spectral overlap, (iii) the distance between the two is between 2 and 8 nm, and (iv) the donor’s transition dipole is properly oriented. FRET is associated with dynamic quenching mechanism as decrement in the fluorescence intensity of excited-state donor fluorophores is observed. The overlapping spectra of α2M (donor) and MYR (acceptor) are depicted in Fig. 6. According to Forster’s theory, the efficiency of the energy transfer (E_FRET) can be computed as the inverse sixth power of the distance between the donor and acceptor molecules (r) in Eq. 6.

$$E_{\mathit{FRET}}=1+\frac{r}{R_0}$$ 6

where R₀ represents the Forster distance at which the transfer efficiency between donor and acceptor is 50%. R₀ is calculated using Eq. 7 as follows:

$$R_0^6=8.8\times {10}^{-25}{K}^2{N}^{-4}\phi J$$ 7

where k² is the spatial dipole orientation factor, n represents the refractive index of the medium, φ represents donor fluorescence quantum yield in the absence of the acceptor, and J is the spectral overlap integral between the absorption spectrum of MYR and the emission spectrum of α2M given by Eq. 8

$$J=\frac{\sum F(\lambda )\epsilon (\lambda )\lambda 4\mathrm{\Delta }\lambda }{\sum F(\lambda )\mathrm{\Delta }\lambda }$$ 8

where F (λ) is the corrected fluorescence intensity of the α2M at wavelength λ, and Ɛ (λ) is the molar absorption coefficient of the MYR at wavelength λ. Efficiency of energy transfer (E) represents the extent of spectral overlap between α2M and MYR, which was determined by using Eq. 9. The distance (r) between α2M and MYR can also be estimated using Eq. 9:

$$E_{\mathit{FRET}}=1+\frac{F}{F_0}=\frac{R_0^6}{R_0^6+r^6}$$ 9

where F and F_o represent fluorescence emission intensities of α2M in the presence and absence of MYR respectively. Using the above Eqs. (6–9), the values of R₀, r, E, and J were found to be 3.84 nm, 2.63 nm, 0.9050, and 1.73 × 10⁻¹⁴ cm⁶mmol⁻¹, respectively (Table 3). The “r” value is within the range of 2–8 nm, suggesting a high probability of energy transfer between α2M-MYR systems.

Fig. 6.

Spectral overlap of the fluorescence emission of α2M (curve a) with the absorption spectra of MYR (curve b). c(α2M) = c(MYR) = 20 μM

Table 3.

Energy transfer parameters for α2M (20 μM) binding with MYR (20 μM)

Sample	J (cm6/mmol)	E	Ro (nm)	r (nm)
α2M-MYR	1.73 × 10−14	0.9050	3.84	2.63

Circular dichroism measurement

CD spectroscopy is a sensitive tool employed to estimate structural variation in the secondary and tertiary structure of proteins induced by the ligand. Far-UV (190–250 nm) CD spectra allow the characterization of protein secondary structure, while near-UV (250–300 nm) CD spectra help to elucidate the modifications in the tertiary structure of proteins. Any alteration within the secondary structure of α2M after its interaction with MYR can be analyzed in the far-UV region of CD spectra. The secondary structure of α2M is mainly β-helical (70%). Figure 7 depicts far-UV CD spectra of α2M over an interval of 200–250 nm in the absence and presence of varying concentrations of MYR. In absence of MYR, α2M had one negative peak (215 nm) in the CD profile, which resembled the characteristic peaks of β-helix in proteins. As shown in Fig. 6, the addition of 10 μM of MYR to α2M (20 μM) caused a slight decrease in the negative ellipticity of α2M and hence a decrease in the β-helical content of protein without discernible alteration in the peak’s position or form. On further addition of MYR (20 μM) to α2M (20 μM), a decrease in negative ellipticity is observed. Thus, it is evident that the interaction of MYR with α2M causes a slight change in the native structure of proteins.

Fig. 7.

Far-UV CD spectra of human α2M in the presence of 1:0, 2:1, and 1:1 molar ratio of α2M to MYR

Molecular docking

Molecular docking explores the nature of small molecules in the binding pocket of a target protein, polypeptide, or DNA. The molecular docking studies were performed to predict the binding affinity, bonds involved during the binding process, and the formation of the best energetically ranked conformation between a ligand and macromolecule. The greater the negative value of binding energy, the greater the binding affinity of MYR with the protein. Results of molecular docking revealed that MYR interacts with the monomer of α2M (Fig. 8). The key amino acid components involved in the binding interaction of MYR with protein includes Asp-146, Ser-172, Glu-174, and Trp-180. As depicted in Fig. 9, MYR forms two hydrogen bonding interactions with the amino acid residue Glu 174 at a distance of about 2.75 and 2.90 Å through its oxygen atom, and one hydrogen bond is observed with Asp-146, Tyr-180, and Ser-172 residues of α2M at a distance of about 3.10 Å, 2.82 Å, and 2.86 Å, respectively, as shown by green dotted lines. Consequently, hydrogen bonding and hydrophobic interactions are the principle interaction forces associated with MYR-α2M interaction. The binding energy obtained from AutodocVina software for MYR-α2M interaction was −7.3 kcal/mol, supporting the spontaneity of the reaction.

Fig. 8.

Representative Molecular docking of human α2M with MYR. A Tetrameric structure of human α2M (PDB Id: 4acq). B Surface view of human α2M monomer binds with MYR in its pocket. C Cartoon model representing amino acid residues involved in human α2M-MYR interaction

Fig. 9.

Ligplot depicting amino acid residues involved in human α2M-MYR interaction. (The black, blue, and red colors in the figure depicts carbon, nitrogen, and oxygen, respectively)

Discussion

Flavonoids are plant compounds that are prevalent in the human diet and considered beneficial due to their potential health benefits, including their anti-inflammatory, anticancer, anti-fungal, antibacterial, and antioxidative properties [22–24]. In particular, MYR exhibits effective therapeutic potential, particularly for the treatment of cancer, type 2 diabetes mellitus, liver damage, cardiovascular diseases, obesity, and osteoporosis [25, 26]. MYR has the ability to reduce anxiety, enhance cognition, and may be able to prevent neurodegenerative conditions like Alzheimer’s disease. Dihydromyricetin (DMY) and MYR are used as nutritional supplements due to their capacity as antioxidants and other health benefits. MYR consequently has the potential to be employed as a pharmaceutical and nutraceutical ingredient [27, 28].

Moreover, the interactions between plasma proteins and flavanols, including MY, DMY, quercetin, and kaempferol have been extensively studied [29–31]. α2M is a plasma protease inhibitor that is mostly recognized as an emergency-type protease or backup inhibitor that acts when other specific anti-proteinases are used up or overwhelmed [13]. This abundant plasma protein in humans is frequently regarded as a physiological guardian to prevent broad-spectrum proteolysis induced by endogenous or exogenous proteinases. It effectively entraps active proteases and acts as a vehicle for quick receptor-mediated clearance from the extracellular environment. Additionally, α2M is also performs other functions like binding and transportation of hormones, metal ions, and cytokines [32].

The present study is the first attempt to explore the α2M-MYR molecular interaction and characterization. The α2M-MYR interaction results in the reduction in the activity of α2M with an increase in the concentration of MYR. The structural and conformational changes were observed using a number of biophysical techniques. The UV–Vis absorption spectra showed enhancement in the maximum absorption peak (hyperchromic shift) which can be attributed to the binding of α2M to MYR and the formation of a α2M- MYR complex [29]. The fluorescence spectra of α2M exhibit concentration-dependent decrease in fluorescence intensity (quenching) along with hypsochromic shift [33]. The quenching mechanism was found to be dynamic in nature as confirmed by the value of K_sv, K_q, and K_b calculated at three different temperatures 298.15 K, 308.15 K, and 318.15 K (Table 1). The interaction of rutin with beta-lactoglobulin has been shown to follow a similar dynamic process of fluorescence quenching [34, 35]. The synchronous fluorescence spectra illustrate a diminution in fluorescence intensity without a shift in the position of peak. A similar phenomenon exists in the interaction between 7-geranyloxycoumarin (marmenol) and bovine serum albumin (BSA) [36]. The FRET results further validate the dynamic mode (R₀ > r) of quenching [37]. The CD spectra reveal a minor change (decrease) in the beta-helical content of native α2M, confirming an alteration in the secondary structure of α2M [38]. The quercetin-α2M interaction was shown to cause an identical change in the secondary structure of α2M [20].

Thermodynamic parameters ΔG, ΔH, and TΔS (Table 1) associated with α2M-MYR complex were recorded using fluorescence quenching results. The value of enthalpy and entropy were found to be positive, indicating the reaction is endothermic in nature. Also, positive value of ΔH and ΔS suggests the involvement of hydrogen bonding and hydrophobic interactions as major binding forces during α2M and MYR interactions. However, a negative value of ΔG confirms that the interaction process is exothermic and thus thermodynamically favorable (spontaneous) [39, 40]. The result of thermodynamic parameters at 37 °C is consistent with that of molecular docking. Molecular docking, a computer-assisted tool, provide a useful information related to the binding mode of a ligand within the known 3D structure of proteins [41]. Molecular docking unveils key residues Asp-146, Ser-172, Glu-174, and Trp-180 that are associated throughout α2M-MYR interaction. Hydrophobic interaction and hydrogen bonding are the two principal forces that play a significant role in stabilizing the α2M-MYR complex. Binding energy (− 7.3 kcal/mol) revealed via in-silico study also supports the reaction’s spontaneity.

Conclusion

Myricetin is an essential dietary nutrient found in fruits, vegetables, tea, and wine are among the rich sources of MYR, that supports immunity and exhibits health-promoting benefits. α2M being the major abundant plasma anti-proteinase that is found in the blood of healthy people at levels higher than 1.5 mg/ml and is a key transporter of proteins, hormones, metal ions, and pharmaceutical drugs. Once MYR binds to α2M its structure as well as anti-proteinase activity is altered. Several biophysical approaches, including UV/visible absorption spectroscopy, fluorescence quenching investigations, synchronous measurements, FTIR, CD, and molecular docking, were used to demonstrate the conformational and structural changes induced in α2M by binding to MYR. Therefore, considering the functional importance of α2M in the removal of different exogenous and endogenous proteinases as well as its role as a carrier of metabolites and molecules, any alteration in its structure or/and function is likely to have a multidimensional effects.

Author contribution

SA: data collection; analysis and interpretation of results; manuscript draft; revised manuscript. HA: analysis and interpretation of results; revised manuscript; approved the final version of the manuscript. MKZ: analysis and interpretation of results; revised manuscript; funding acquisition. MKG: funding acquisition; manuscript draft. FHK: study conception and design; analysis and interpretation of results; approved the final version of the manuscript.

Funding

MKG acknowledges the financial support (RSP-2023-R-393), King Saud University, Riyadh, Saudi Arabia. MKZ acknowledges ICMR for providing Research Associate fellowship.

Data availability

The data generated in the study will be made available on reasonable request from the corresponding author.

Declarations

Ethical approval

NA.

Informed consent

NA.

Conflict of interest

The authors declare no competing interests.