International Journal of Biological Macromolecules 307 (2025) 141705

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International Journal of Biological Macromolecules

journal homepage: [Link]/locate/ijbiomac

Structure difference of Jack bean urease and Helicobacter pylori urease on

binding interactions with quercetin
Yanni Li a , Shuai Guo b, Hui Zou c,*, Yilun Chen c,*
a
School of Pharmacy, Binzhou Medical University, Yantai 264003, Shandong Province, China
b
Department of Emergency, First Peoples Hospital of NingYang, Taian, Shandong, China
c
College of Food Science and Engineering, Key Laboratory of Food Nutrition and Healthy in Universities of Shandong, Shandong Agricultural University, Taian, China

A R T I C L E I N F O A B S T R A C T

Keywords: Urease catalyzes the hydrolysis of urea to carbamate and ammonia, leading to nitrogen loss, environmental
Jack bean urease pollution, and health issues, so numerous compounds have been screened for urease inhibition using Jack bean
Helicobacter pylori urease urease (JBU) and H. pylori urease (HPU) without consideration their structure difference. Previous studies have
Dimer structure
shown that the same inhibitor can exhibit distinct inhibitory effects on JBU and HPU, but limited papers focus on
Quercetin
Binding interactions
the effects mechanism. In this study, we systematically investigated the thermodynamic and kinetic properties of
JBU and HPU binding with quercetin, focusing on the structural effects on both commonly studied ureases. The
results revealed that quercetin inhibited both JBU and HPU activities, with IC50 values of 16.76 ± 0.77 μM and
36.17 ± 0.73 μM, respectively. Inhibition was identified as noncompetitive for JBU and mixed-competitive for
HPU. Quercetin interacted with both JBU and HPU with quenching rate constants (Kq) of 3.72 ± 0.18 × 1013
M− 1 s− 1 for JBU and 0.28 ± 0.04 × 1013 M− 1 s− 1 for HPU. Molecular docking revealed that quercetin mainly
bound to the flap region of JBU, inhibiting its function, and the JBU-quercetin complex had high binding stability
and low binding free energy.

1. Introduction natural substrate of ureases or interacting with allosteric sites, particu­

larly the mobile flap, which contains a helix-turn-helix motif and plays a
Ureases (EC [Link]) are nickel-dependent enzymes that widely exist crucial role in regulating substrate influx and product efflux [8,9].
in bacteria, yeasts, fungi, plants, and mollusks [1,2]. These enzymes Despite their overall structure similarity, ureases are composed of
catalyze the conversion of inorganic nitrogen into protein amino acids different polypeptide chains and self-assemble into distinct quaternary
and other nitrogen-containing compounds, often leading to adverse structures. The most extensively studied urease, Jack bean urease (JBU),
environmental and health consequences. Specifically, urease activity in forms oligomers of a single-chain polypeptide (α), typically assembling
soil or the rumen can rapidly hydrolyze urea to ammonia, resulting in into a dimer of trimers ((α₃)₂). In contrast, the urease from the human
inefficient urea utilization, excessive ammonia release, and significant pathogen H. pylori (HPU), essential for colonizing the gastric mucosa,
environmental and economic challenges [3]. Ureolytic bacteria such as consists of two types of polypeptide chains (α, β) and assembles into a
Helicobacter pylori (H. pylori), Proteus mirabilis, Klebsiella pneumoniae, and dodecamer with tetrahedral symmetry ([[αβ]3]4). Several studies have
Staphylococcus aureus utilize urease to maintain pH homeostasis, reported that identical inhibitors exhibit different binding affinities and
creating favorable conditions for the development of respiratory, inhibitory effects when interacting with JBU and HPU. For example,
gastrointestinal, and urinary tract infections [4,5]. sanguinarine demonstrated mixed-type inhibition toward HPU and non-
Given these adverse effects, urease inhibitors are extensively competitive inhibition toward JBU [10], and epiberberine displayed
employed in agriculture to enhance nitrogen utilization and in the uncompetitive inhibition against HPU and competitive inhibition
pharmaceutical industries to treat gastric and urinary tract infections against JBU [11]. Additionally, baicalin and scutellarin, the major fla­
[6,7]. A wide range of natural and synthetic compounds have demon­ vonoids in Scutellaria baicalensis, have been demonstrated to act as non-
strated urease inhibitory activity [1]. These inhibitors typically targeted competitive inhibitors for HPU and competitive inhibitors for JBU [12].
the two Ni2+ ions located in the active site by either mimicking the These findings indicate that differences in urease structure significantly

* Corresponding authors.
E-mail addresses: zouhui@[Link] (H. Zou), cylun@[Link] (Y. Chen).

[Link]
Received 15 November 2024; Received in revised form 21 February 2025; Accepted 1 March 2025
Available online 7 March 2025
0141-8130/© 2025 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
Y. Li et al. International Journal of Biological Macromolecules 307 (2025) 141705

Fig. 1. Lineweaver-Burk plots of ureases activity in the presence of quercetin. (A) JBU. (B) HPU.

through a 0.45 μm membrane and loading HPU onto a Hitrap-DEAE

Table 1
column. The final purification recovered approximately 35.69 % of
Kinetic parameters of quercetin with JBU and HPU.
the original HPU activity, resulting in a specific activity of nearly 185 U/
Type of Quercetin concentration Km (mM) Vmax (mM/min) mg protein. SDS-PAGE analysis was performed to examine the subunit
urease (μM)
composition of JBU and HPU [17]. As shown in Fig. S1, JBU (A)
0 1.47 ± 0.16a 1.01 ± 0.07a exhibited a single distinct band at 90.8 kDa corresponding to the
6.25 1.46 ± 0.01a 0.73 ± 0.03b
JBU α-subunit, while HPU (B) displayed two bands, one at 26.5 kDa for the
12.5 1.47 ± 0.10a 0.52 ± 0.02c
25 1.45 ± 0.05a 0.31 ± 0.01d
α-subunit and another at 61.7 kDa for the β-subunit, as also reported in
0 0.32 ± 0.01c 0.64 ± 0.01a previous studies [2,36,37].
6.25 0.35 ± 0.01bc 0.58 ± 0.02a
HPU
12.5 0.38 ± 0.01ab 0.51 ± 0.01b 2.3. Enzyme assay
25 0.42 ± 0.03a 0.42 ± 0.02c

Km Michaelis-Menten constant, Vmax maximum velocity of enzymatic activity; The urease inhibitory activity of quercetin was assessed using the
Values with different letters in the same column represent statistically signifi­ Berthelot reaction with minor modifications [18]. Briefly, the reaction
cant differences (p < 0.05). mixtures consisted of 200 μL of 0.5 mg/mL JBU or HPU, 200 μL of
quercetin at serial concentrations (6.25, 12.5, 18.75, 25, 37.5, 50, 75,
affect binding interactions, although the underlying inhibitory mecha­ 100 μM for JBU and 12.5, 25, 37.5, 50, 75, 100, 200, 400 μM for HPU),
nisms remain incompletely understood. and 500 μL of urea (30 mM). The mixtures were incubated at 37 ◦ C to
Natural urease inhibitors have recently garnered attention due to allow the enzymatic reactions to proceed. After 30 min, the reaction was
their lower toxicity and higher efficacy. As a physiologically active terminated by adding 500 μL of phenol reagent (containing 0.05 g/L
compound, quercetin has been extensively studied and confirmed to be sodium nitroprusside and 1 % phenol) and 600 μL of alkali reagent
one of the most effective inhibitors [13,14]. In addition to its antimi­ (containing 0.1 % NaOCl and 0.5 % NaOH). The absorbance was sub­
crobial properties, quercetin has potential as a valuable anti-Helicobacter sequently measured at 625 nm, and urease activity was quantified as
pylori agent [15]. Consequently, the present study aims to investigate IC50 values, as previously described [19]. A phosphate buffer (0.01 M
the binding characteristics of two ureases, JBU and HPU, with quercetin, K₂HPO₄, 1 mM EDTA, and 0.01 M LiCl) at pH 8.2 was used for all assays.
a natural inhibitor. To achieve this, both theoretical and experimental
approaches were employed, and the thermodynamic and kinetic pa­ 2.4. Determination of inhibition type
rameters were computed and analyzed. The binding interactions be­
tween quercetin and both ureases were further examined and discussed. Lineweaver–Burk plots were constructed to determine the inhibition
type of quercetin on JBU and HPU [10]. The procedure followed was
2. Materials and methods similar to the determination of IC50. The reaction mixtures consisted of
200 μL of JBU or HPU (0.5 mg/mL), 500 μL of urea at varying concen­
2.1. Materials trations (0.313, 0.625, 1.25, 2.5, 5, and 10 mM), and 200 μL of either
quercetin or phosphate buffer. For each substrate concentration, quer­
JBU was sourced from Sigma-Aldrich Co. (St. Louis, MO, USA), while cetin was tested at three different concentrations (6.25, 12.5, and 25
HPU was purified from Helicobacter pylori 26,695 in our laboratory. Urea μM). After 30 min of incubation, the enzymatic reaction was terminated
was obtained from Solarbio Biotech Co., Ltd. (Beijing, China), and by adding 500 μL of phenol reagent and 600 μL of alkali reagent. The
quercetin standard was purchased from Shanghai Yingxin Laboratory absorbance at 625 nm was measured and recorded as the reaction ve­
Equipment Co., Ltd. (Shanghai, China). All other reagents were of locity (V). The reciprocal of velocity (1/V) was plotted against the
analytical grade. reciprocal of substrate concentration (1/[S]) for each quercetin con­
centration. Based on the regression equations derived from the Line­
weaver–Burk plots, the Michaelis constant (KM) and maximum velocity
2.2. Purification of HPU and SDS-PAGE analysis of JBU and HPU (Vmax) were calculated, allowing for the identification of the inhibition
type.
HPU was extracted and purified from H. pylori 26,695 using the
methodology outlined by Liu et al. [16]. In this study, classic ion ex­
change chromatography was employed for HPU purification. The pro­
cess involved filtering the supernatant of lysed bacterial cultures

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Y. Li et al. International Journal of Biological Macromolecules 307 (2025) 141705

Fig. 2. Fluorescence spectrum and quenching analysis of JBU and HPU in the presence of quercetin. (A, B) Fluorescence spectra diagram. (C, D) Stern Volmer plots
diagram. (E, F) The double-logarithm plots diagram. KSV is the Stern-Volmer quenching constant, Kq is the fluorescence quenching rate constant, K is the binding
constant, and n is the number of binding sites.

microplate reader, with an emission range of 300 to 360 nm in 5 nm

Table 2 increments and an excitation wavelength set at 280 nm. The Stern-
Fluorescence quenching parameters of quercetin with JBU and HPU. Volmer relationship was derived based on the provided equations,
Type of urease Kq (1013 L/mol × s) Ksv (105 L/mol) K (107 L/mol) n with peak emission intensity observed at 315 nm [38].
JBU 3.72 ± 0.18 3.72 ± 0.18 1.62 ± 0.28 1.34 /
F0 F = 1 + Ksv [Q] = 1 + Kq × τ0 [Q]
HPU 0.28 ± 0.04 0.28 ± 0.04 1.82 ± 0.30 1.37

lg[(F0 ▬F)/F ] = lg K + nlg[Q]

2.5. Fluorescence quenching analysis
F0 and F represent the fluorescence intensities without and with
quercetin, respectively. [Q] denotes the inhibitor concentration, Kₛᵥ is
Fluorescence quenching assays were employed to quantify the
the Stern-Volmer quenching constant, Kq is the fluorescence quenching
inhibitor-urease binding affinities using fluorescence spectroscopy with
rate constant, K is the binding constant, and n represents the number of
black 96-well plates [20]. JBU or HPU was added either alone or in the
binding sites.
presence of quercetin solutions to achieve final inhibitor concentrations
ranging from 0 to 12.5 μM after 10 min of stabilization. The fluorescent
signal from each well was measured using a BioTek Synergy H1

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Y. Li et al. International Journal of Biological Macromolecules 307 (2025) 141705

Fig. 3. Isothermal titration calorimetry for the association of quercetin with the JBU and HPU. (A, B) Typical raw titration curve diagram. (C, D) are the integration
of the thermogram yielded binding isotherm). Ka is the association constant, ΔH is enthalpy, ΔS is entropy, ΔG is Gibbs free energy change.

2.6. Isothermal titration calorimetry (ITC) measurements fitting followed a one-site binding model to determine the association
(Kₒₙ) and dissociation (Koff) rate constants. The association constant (KA)
The interaction energy between quercetin and urease was measured was calculated as Kₒₙ/Koff, and the dissociation constant (KD) as 1/KA.
using isothermal titration calorimetry with a Nano ITC [21]. Urease (40
μM) and the inhibitor (0.5 mM) were dissolved in 20 mM phosphate 2.8. Molecular docking
buffer, filtered, degassed, and equilibrated prior to testing. Both urease
and the inhibitor were placed in the calorimeter cell and dosing syringe, Molecular docking was performed using AutoDock 4.2 coupled with
respectively. At 25 ◦ C, 20 injections of the inhibitor (2.5 μL each) were AutoDock Tools version 1.5.6 [23]. The 3D structure of quercetin was
administered every 150 s. The reaction heat (ΔJ/s) was recorded using drawn, and the structures of JBU (3LA4) and HPU (1E9Y) were obtained
Nano ITC software, with curve fitting based on a one-site binding model. from the Protein Data Bank ([Link]). Two ureases were pro­
This enabled the calculation of thermodynamic parameters, including cessed using AutoDock Tools to add hydrogens, merge no-polar hydro­
the association constant (Kₐ), enthalpy (ΔH), and entropy (ΔS). The gens, and calculate the total charge. The Grid box was set to cover the
Gibbs free energy (ΔG) was then computed as: ΔG = ΔH − TΔS. protein catalysis site, the force field of AD4 with genetic algorithm pa­
rameters was employed for the docking calculations, 20 poses were then
2.7. Surface plasmon resonance (SPR) measurement generated and the protein-ligand complex with the lowest scores was
selected separately for interaction analysis using PLIP online server (http
The binding kinetics were evaluated using single-cycle kinetics on a s://[Link]/plip-web/plip) [24].
Biacore T200 system at 25 ◦ C. The analysis employed a Series Sensor
Chip CM5 (Cytiva, Marlborough, MA) and a running buffer consisting of 2.9. Molecular dynamic simulation
PBS 10× with 0.05 % Tween 20 [22]. JBU or HPU was immobilized on
the chip using an amine coupling kit, achieving a coupling level of The detailed interaction mechanism between quercetin and urease
14,000 RU. The interaction was then assessed with serial dilutions of was investigated using molecular dynamics simulations [25]. The
inhibitor solutions at concentrations of 1 mM, 333 μM, 111 μM, 37 μM, quercetin poses with the lowest scoring values were selected and pro­
12.3 μM, and 4.1 μM (for JBU), or 200, 100, 50, 25, 12.5, and 6.25 μM cessed through the Antechamber Python Parser Interface (ACPYPE) to
(for HPU), all at 25 ◦ C. Measurements were taken with a flow rate of 10 generate the corresponding topology. Molecular dynamics simulations
μL/min for 120 s in the running buffer, consisting of an association phase were carried out using Gromacs 2020.5, with parameters optimized
of 120 s and a dissociation phase of 600 s, except during the regeneration using the Amber 99-ILND force field and the TIP3P water model. After
step, which used a flow rate of 30 μL/min. Data collection and curve neutralizing the system with Cl− ions and minimizing the energy using

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Y. Li et al. International Journal of Biological Macromolecules 307 (2025) 141705

Fig. 4. Surface plasmon resonance assay of quercetin with JBU and HPU. (A, B) The typical raw subtracted binding curve. (C, D) Equilibrium binding analysis. Kon is
the association rate constant, and Koff is the dissociation rate constant. KA is the association constant, and KD is the dissociation constant, where KD = 1/KA.

the steepest descent method, the system was subjected to a 1 ns NVT

± standard error, and the significant differences between groups were
(300K) simulation with position restraints, followed by 50 ns NPT mo­
calculated using the Tukey test in one-way analysis of variance
lecular dynamics simulations. During the simulations, the temperature
(ANOVA).
was maintained at 300 K using a V-rescale thermostat for temperature
coupling, and long-range electrostatic interactions were treated using
3. Results and discussion
the Ewald PME method. Bond length constraints were applied using the
LINCS algorithm with a 2 fs time step, and all other parameters were set
3.1. Urease inhibition type determination
to default. The stability of the urease-quercetin complex was evaluated
by analyzing the root-mean-square deviation (RMSD), hydrogen bond
To evaluate the urease inhibition activity of quercetin, we co-
formation, and binding energy.
incubated it with JBU and HPU. Quercetin significantly inhibited the
activities of both JBU and HPU in a dose-dependent manner. The IC50
2.10. MMPBSA calculation and alanine scanning mutagenesis values, which represent the concentrations of quercetin required to
inhibit 50 % of urease activity, were 16.76 ± 0.77 μM for JBU and 36.17
To identify the effects of flap and key residues on the binding ± 0.73 μM for HPU (Fig. S2). Notably, quercetin exhibited a significantly
interaction between quercetin and urease, the binding energies were stronger inhibitory effect on JBU compared to HPU (p < 0.05, data not
calculated using molecular mechanics Poisson-Boltzmann surface area shown).
(MMPBSA) method inherent in Ambertools [26], for providing insights The Lineweaver-Burk analysis was performed to determine the in­
into the thermodynamic contributions of the interaction, including van hibition type. The following inhibition types were considered: non-
der Waals energy (Vdw), and Coulombic interaction energy (Cou) et al. competitive inhibition (with an unchanged Km and reduced Vmax),
The amino acids with higher binding values were mutated with ALA competitive inhibition (with an increased Km and unchanged Vmax),
with PyMOL, and molecular dynamics simulations were then conveyed anti-competitive inhibition (with a reduced Km and reduced Vmax), and
to analyze the binding interaction characteristics change using the same mixed-type competitive inhibition (with a reduced Km and increased
method of Section 2.9. Vmax) [27]. As shown in Fig. 1 and Table 1, the Km and Vmax values in the
absence of quercetin were 1.47 ± 0.16 mM and 1.01 ± 0.07 mM/min for
2.11. Statistical analysis JBU, and 0.32 ± 0.01 mM and 0.64 ± 0.01 mM/min for HPU. For JBU,
an increase in quercetin concentration resulted in a constant Km and a
Each experiment’s analysis was conducted using Origin 8.0 software decrease in Vmax, with the regression lines intersecting the X-axis,
(OriginLab, Northampton, USA). The data were presented as the mean

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Y. Li et al. International Journal of Biological Macromolecules 307 (2025) 141705

Fig. 5. Binding interactions of quercetin with JBU (PDB 3LA4) and HPU (PDB 1E9Y). (A, C) The complex structure of JBU-quercetin and HPU-quercetin. (B, D) The
binding site and interaction details of quercetin with JBU and HPU.

indicating non-competitive inhibition. This suggests that quercetin tar­ available for quercetin binding in both JBU and HPU [29].
gets the allosteric site of urease. For HPU, an increase in quercetin
concentration led to an increase in Km and a decrease in Vmax, with the 3.3. Thermodynamic parameters analysis
regression lines intersecting at the second quadrant, suggesting mixed
competitive inhibition. This implies that quercetin binds directly to both ITC experiments provide a comprehensive thermodynamic profile,
the active and allosteric sites of urease [28]. including the association constant (Ka), enthalpy change (ΔH), and en­
tropy change (ΔS) under specific conditions. As shown in Fig. 3, the Ka
3.2. Fluorescence quenching analysis values for JBU and HPU were 4.52 × 104 M− 1 and 1.48 × 104 M− 1,
respectively. These results indicate that quercetin binds to both JBU and
The intrinsic fluorescence of the protein is attributed to amino acid HPU with moderate affinity. The thermodynamic parameters for both
residues such as tryptophan, tyrosine, and phenylalanine. Variations in ureases revealed negative ΔG and ΔH values, suggesting that the in­
fluorescence intensity or wavelength reflect interactions between the teractions were exothermic and spontaneous. For JBU, the interaction
protein and its ligand. Fig. 2 and Table 2 displayed the fluorescence was primarily driven by entropy (|TΔS| > |ΔH|), indicating that the
spectra of JBU and HPU at various quercetin concentrations, along with process was entropy-dominated. The minimal ΔH value for the JBU-
the computed Stern-Volmer and double-logarithmic plots for quenching quercetin interaction suggests that hydrogen bonding played a key
interactions. As quercetin concentration increased, the fluorescence in­ role in the binding mechanism [30]. In contrast, for HPU, the negative
tensity decreased, while the maximum emission wavelength remained ΔS value indicates that the binding process was enthalpy-driven, with
relatively unchanged. This trend suggests that quercetin interacts with Vdw interactions and hydrogen bonds contributing to the overall
both JBU and HPU without significantly altering the urease structure. interaction [31,32].
Furthermore, the quenching constants (Kq) for the interaction of
quercetin with JBU and HPU were determined to be 3.72 × 1010 M− 1 s− 1 3.4. Kinetic parameters analysis
and 0.28 × 1010 M− 1 s− 1, respectively. These values exceed 2 × 1010
M− 1 s− 1, indicating a static quenching mechanism. Therefore, quercetin To further investigate the kinetic differences between the two ure­
and JBU/HPU interaction is a non-covalent binding interaction. A ases and quercetin, the association rate (Kon) and dissociation rate (Koff)
double logarithmic regression analysis was employed to estimate the of quercetin were measured using SPR. The kinetic data were well-fit to
number of binding sites (n) and the binding constant (K). As shown in the steady-state affinity model, as shown in Fig. 4. The Kon and Koff rates
Table 2, the binding constant (K) for the JBU-quercetin interaction was were determined to be 42.91 M− 1 s− 1 and 9.20 × 10− 3 s− 1 for JBU, and
1.62 × 107 M− 1, with a corresponding binding site number (n) of 1.34. 102.21 M− 1 s− 1 and 7.66 × 10− 3 s− 1 for HPU, respectively. These results
Similarly, for HPU-quercetin, K was 1.82 × 107 M− 1, with n equal to suggested that the interactions between quercetin and JBU/HPU were
1.37. The calculated K values (both in the range of 107) are significantly characterized by slow association and dissociation kinetics. The calcu­
higher than 105, suggesting a strong interaction between quercetin and lated association constant (KA) values were 4.66 × 103 M− 1 and 1.33 ×
both ureases. The values of n indicate that a single binding site is 104 M− 1 for JBU and HPU, respectively, indicating that both JBU and

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Y. Li et al. International Journal of Biological Macromolecules 307 (2025) 141705

Fig. 6. Molecular dynamics simulation assay of the interaction of quercetin with JBU and HPU. (A, B) RMSD profile of free JBU/HPU (Dark red) and quercetin-JBU
complex (Light red) for 50 ns. (C) The number of hydrogen bonds of quercetin-JBU complex (Light red) and quercetin-HPU complex (Light blue). (D) The binding
energy of quercetin-JBU complex (Light red) and quercetin-HPU complex (Light blue). (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)

HPU bind to quercetin with moderately high affinities. This finding noting that, it formed a hydrogen bond with the residue His248, which is
aligned with the results from ITC. Specifically, quercetin demonstrated coordinated with Ni and plays a crucial role in the active site; and a
slow dissociation kinetics from urease, resulting in prolonged target hydrogen bond with His322, which is one of the key residues in the flap
occupancy-a desirable trait for urease inhibitors [33]. [34,35]. We conducted molecular docking studies with luteolin, another
urease inhibitor (Fig. S3, Table S4 and S5), and observed results similar
to those obtained with quercetin. Both compounds interacted with the
3.5. Molecular docking analysis
flap region of JBU and the flap and Ni active centers of HPU. Based on
these findings, we hypothesize that quercetin’s interaction with the flap
The binding sites of quercetin and urease were investigated through
region of JBU and with the residues in the active site of HPU may be
molecular docking. Fig. 5 and Table S1 summarize the docking results,
attributed to its higher binding energy, which hinders access to the
showing that quercetin exhibited binding energies of − 6.68 kcal/mol
active site.
and − 6.16 kcal/mol for JBU and HPU, respectively. Specifically, Fig. 5A
and B, and Table S2 showed quercetin formed hydrogen bonds with the
residues located in the Flap region (involving the residues Val591 (1H- 3.6. Molecular dynamics simulation analysis
bond, 3.0 Å), His594 (1H-bond, 4.1 Å), Leu595 (1H-bond, 2.6 Å),
Asp596 (2H-bonds,3.4 Å, 4.1 Å), Arg597 (1H-bond, 3.0 Å), and Glu598 Molecular dynamics simulations were conducted for 50 ns to
(1H-bond, 3.9 Å)), which indicated that quercetin restricted the flexi­ examine the stability, conformational changes, and internal motions of
bility of the flap but did not interact with the active site, consistent with the JBU-quercetin and HPU-quercetin complexes. These were compared
previous experimental findings (subsection 3.1) [34]. Fig. 5C and D, and with free urease to investigate the mechanism of quercetin binding.
Table S3 showed that quercetin entered the active site of HPU, formed RMSD is a crucial dynamical parameter for assessing the flexibility and
hydrogen bonds with residues of Asp165 (2H-bond, 3.1, 3.4 Å), Asn168 stability of the complex. Typically, a system reaches equilibrium and
(1H-bond, 3.1 Å), His248 (1H-bond, 3.9 Å), His322 (1H-bond, 4.1 Å), stability when the RMSD exhibits low and consistent fluctuations
Arg338 (2H-bond, 2.9, 3.6 Å), and Ala365 (1H-bond, 3.0 Å), hydro­ throughout the simulation, whereas higher fluctuations indicate
phobic interactions with Asn168 (3.4 Å), Ala169 (3.5 Å), and Ala365 reduced stability. In this study, the RMSD values of free JBU, the JBU-
(3.4 Å), and π-stacking interactions with His221 (5.4 Å). It is worth quercetin complex, free HPU, and the HPU-quercetin complex were

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Y. Li et al. International Journal of Biological Macromolecules 307 (2025) 141705

Fig. 7. Energy analysis of JBU-quercetin using MMPBSA method. (A) Energy vs. time profile of MMPBSA calculation. (B) Residue-wise total energy. (C) Van der
Waals energy per residue. (D) Coulombic energy per residue.

calculated and presented in Fig. 6. For JBU, the average RMSD was (Fig. 7) indicated that the two key residues CME592 (− 15.85 kJ/mol)
approximately 0.330 nm, with fluctuations ranging from 0 to 30 nm and HIS593 (− 12.78 kJ/mol) within the flap region showing higher
(Fig. 6A). In contrast, the JBU-quercetin complex stabilized after 10 ns, binding energy. Therefore, we selected the two aforementioned sites for
with an average RMSD of approximately 0.194 nm. For HPU, the mutation, and molecular dynamics simulations were performed after
average RMSD was approximately 0.345 nm, exhibiting negligible var­ separately mutating the two residues (CME592 and HIS593) to alanine
iations beyond 25 nm (Fig. 6B). The RMSD value of the HPU-quercetin (ALA). The results of Fig. 8 showed that the mutations of CME592 and
complex was consistently higher and fluctuated up to 10 ns; however, HIS593 led to an increase in RMSD values, with the CME592 mutant
after that, the RMSD values stabilized with a lower fluctuation rate, showing an RMSD of 0.465 and the HIS593 mutant showing 0.262. Both
showing an average RMSD of 0.237 nm. These results suggest that the of these values were higher than the original RMSD of 0.194, proving
JBU-quercetin complex was more stable than the HPU-quercetin higher ligand instability occurred. In addition, the number of hydrogen
complex. bonds decreased in both mutants, with values of 2.377 and 1.677,
Hydrogen bonds play a critical role in protein-ligand binding and compared to the original value of 2.853. The binding energy of the
stability, contributing to protein-ligand selectivity. As shown in Fig. 6C, mutants also decreased, with values of − 224.919 kJ/mol and −
the number of hydrogen bonds formed between HPU and quercetin 195.267 kJ/mol compared to the original value of − 243.31 kJ/mol. All
ranged from 0 to 4, while the JBU-quercetin complex formed between these results indicated both two key residues in the flap of JBU play a
0 and 7 hydrogen bonds, indicating greater stability throughout the crucial role by binding strongly to quercetin and inhibiting its access to
simulation. Fig. 6D showed that the energy of the JBU-quercetin com­ the active site, thus exerting an inhibitory effect.
plex fluctuated slightly but maintained a stable conformation after 10
ns, with an energy range of − 250 to − 300 kJ/mol. In contrast, the HPU- 4. Conclusion
quercetin complex exhibited more substantial energy fluctuations,
ranging from − 100 to − 250 kJ/mol. These results confirmed that the In summary, both JBU and HPU can be inhibited by quercetin but to
interaction between quercetin and JBU was stronger than that between different degrees. Further research demonstrated that quercetin formed
quercetin and HPU. The primary difference likely arises from the complexes with two ureases, with spontaneous binding. The two urease-
structural nuances between JBU and HPU, particularly the open quercetin complexes displayed slow association and dissociation ki­
conformation of the flap in JBU, compared to the closed conformation netics characterized by enthalpically-entropically driven binding of JBU
observed in HPU [34]. and enthalpically driven binding of HPU. Notably, quercetin was bound
to the flap of JBU in a non-competitive fashion while bound to both the
3.7. Residues effects on the binding interaction of quercetin and urease active site and flap of HPU in a mix-type competitive fashion. The
experimental results suggested that the distinct structures of ureases
MMPBSA calculations, which assess binding affinity and stability, resulted in different thermodynamic and kinetic properties when bind­
provide valuable mechanistic insights into protein–ligand interactions. ing with inhibitors. These differences should be carefully considered
These calculations are essential for the selection of high-affinity candi­ during in silico inhibitor screening.
dates targeting the protein of interest. MMPBSA calculations results

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Fig. 8. Molecular dynamics simulation assay of the interaction of quercetin with mutant JBU. (A) The RMSD profile of quercetin-mutant JBU for 50 ns. (B) The
numbers of hydrogen bonds of quercetin-mutant JBU for 50 ns. (C) The binding energy of quercetin-mutant JBU for 50 ns.

CRediT authorship contribution statement Data availability

Yanni Li: Writing – original draft, Methodology, Data curation. Data will be made available on request.
Shuai Guo: Visualization, Formal analysis. Hui Zou: Writing – review &
editing, Data curation. Yilun Chen: Writing – review & editing, Funding References
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