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Interaction of Au(III) and Pt(II) complexes with

Na/K-ATPase: experimental and theoretical study
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Cite this: Metallomics, 2018,

10, 1003 of reaction stoichiometry and binding sites†
Ana V. Vujačić Nikezić, ‡a Goran V. Janjić, ‡b Aleksandra M. Bondžić, a

Božidarka L. Zarić, a Dragana D. Vasić-Anićijević, a Tatjana G. Momić a

Vesna M. Vasić *a

The present paper deals with investigation of the interaction between selected simple structure Au(III)
([AuCl4], [AuCl2(dmso)2]+, [AuCl2(bipy)]+) and Pt(II) ([PtCl2(dmso)2]) complexes with Na/K-ATPase as the target
enzyme, using an experimental and theoretical approach. Reaction stoichiometries and binding constants for
these enzyme/complex systems were determined, while kinetic measurements were used in order to reveal
the type of inhibition. Based on the results obtained by quantum mechanical calculations (electrostatic
surface potential (ESP), volume and surface of the complexes) the nature of the investigated complexes was
characterized. By using the solvent accessible surface area (SASA) applied on specific inhibitory sites (ion
channel and intracellular domains) the nature of these sites was described. Docking studies were used to
determine the theoretical probability of the non-covalent metal binding site positions. Inhibition studies
implied that all the investigated complexes decreased the activity of the enzyme while the kinetic analysis
indicated an uncompetitive mode of inhibition for the selected complexes. Docking results suggested that
the main inhibitory site of all these complexes is located in the ion translocation pathway on the extracellular
Received 18th May 2018, side in the E2P enzyme conformation, similar to the case of cardiac glycosides, specific Na/K-ATPase
Accepted 27th June 2018 inhibitors. Also, based on our knowledge, the hydrolyzed forms of [AuCl4] and [PtCl2(dmso)2] complexes
DOI: 10.1039/c8mt00111a were investigated for the first time by theoretical calculations in this paper. Thereby, a new inhibitory site
situated between the M2 and M4 helices was revealed. Binding in this site induces conformational changes in
rsc.li/metallomics the enzyme domains and perturbs the E1–E2P conformational equilibrium, causing enzyme inhibition.

Significance to metallomics
It is well known that cisplatin and the metal based compounds possess anticancer properties. Nevertheless, the mechanism of their reaction is not well
understood, with the exception of cisplatin. Since recently Na/K-ATPase was shown to be a new target for the testing of potential anticancer compounds, the
experimental and theoretical elucidation of the interaction of various Au(III) and Pt(II) complexes with Na/K-ATPase were performed in this work. Understanding
of these mechanisms enables the structure-based design of novel metal complexes with anticancer properties capable of interfering with cellular pumps and
enhances the role of metal complexes as anticancer active compounds.

1. Introduction (Fig. 1), which catalyzes adenosine triphosphate (ATP) hydrolysis.

This process is the driving force for maintaining the ion gradient
The sodium potassium pump (Na/K-ATPase) is a hetero- across the cell membrane by transporting 3 Na+ out and 2 K+ ions
transmembrane protein, a complex of a, b and FXYD subunits into the cell.1–3 It is critical for the resting membrane potential,
the electrical activity of muscles and nerves, Na+-coupled
Vinča Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia. transport, and osmotic balance as well as cell volume regula-
E-mail: evasic@vin.bg.ac.rs, anavu@vin.bg.ac.rs, aleksandrab@vin.bg.ac.rs, tion by exchanging potassium and sodium ions.4,5 In addition,
bzaric@hotmail.com, dragana.vasic87@gmail.com, momict@vin.bg.ac.rs Na/K-ATPase has a critical role in cell survival, proliferation,
Institute of Chemistry, Metallurgy and Technology, University of Belgrade,
adhesion and migration.6
Belgrade, Serbia. E-mail: janjic_goran@chem.bg.ac.rs
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
Since proteins have been proven to be possible targets for
c8mt00111a antitumor metal complexes, the evaluation of their involvement
‡ These authors have equally contributed to the paper. in the overall mechanism of action of anticancer metallo-drugs

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Scheme 1 Structural formula of selected complexes: [AuCl4], [AuCl2-

bipy]+, [AuCl2(dmso)2]+, and [PtCl2(dmso)2].

which depend on the properties of the central metal ion. In our

Fig. 1 Structure of Na/K-ATPase. previous work the stoichiometry of some Au(III) complex–
enzyme interactions was determined and the locations of the
binding sites of the investigated complexes in the enzyme
is of great importance.7–9 Recently Na/K-ATPase has become a structure were predicted.17,18 Moreover, docking studies of
new target for testing potential anticancer compounds’ activity Na/K-ATPase inhibition induced by binuclear Au(III) complexes
since it is highly expressed in some human cancer cells.10–16 revealed binding sites of the complexes in the E1 and E2P
Hence, selective binding of the anticancer compounds to its enzyme conformation located in the ion translocation pathway,
isoforms could be a promising strategy in the therapy of similar to the case of cardiac glycosides, specific Na/K-ATPase
different cancers. Since the metal complexes from the Pt(II) inhibitors.14,17,41
and Au(III) group possess anticancer activity a lot of attention This paper deals with the effects of charge, ligand and the
has been devoted to the examination of their interactions with central metal ion of a series of simple isoelectronic square planar
this enzyme.17–26 In addition, it is known that cisplatin nephro- Au(III) and Pt(II) chloro complexes: ([AuCl4], [AuCl2(dmso)2]+,
toxicity is related to the inhibition of kidney Na/K-ATPase [AuCl2(bipy)]+ and [PtCl2(dmso)2]) (Scheme 1) and their hydro-
activity. However, this side effect of cisplatin can be reduced using lytic products on Na/K-ATPase inhibition in order to get a better
certain sulfhydryl containing compounds without suppression insight into the mechanism of their molecular interactions with
of its antitumor activity, indicating the reversible inhibition of this enzyme. As these complexes strongly differ according to
Na/K-ATPase by cisplatin.27–30 their inhibitory potency and chemical characteristics (charge,
Many Au(III) based complexes synthesized in order to over- charge distribution, hydrolytic properties, reduction potential,
come the toxic effects of Pt(II) drugs act as reversible non- and volume),20,42 the aim of this work was to characterize
specific inhibitors of Na/K-ATPase.8,17–20,31 Moreover, various complex–protein interactions by evaluating reaction stoichio-
organometallic Au(III) complexes also induce cytotoxic effects,32–34 metries and to predict possible binding sites for complexes.
and extensive studies have been performed on their therapeutic The obtained results could be further exploited for the structure-
applications as anticancer agents.35–37 based design of the metal complexes with anticancer properties
In general, despite numerous inhibition studies, the mechanism capable of interfering with cellular pumps.
of interaction of Na/K-ATPase with Au(III) and Pt(II) drugs has not
been completely clarified. The molecular basis for the biological
action of Pt(II) and Au(III) complexes with this physiologically 2. Materials and methods
important enzyme is most probably their strong and selective
targeting of thiol and imidazole groups of proteins, which is 2.1. Chemicals
associated with the loss of protein function.8,38,39 The analysis The chemicals for determination of Na/K-ATPase activity (stannous
of the cisplatin-Na/K-ATPase adduct crystal structure suggested chloride and ammonium molybdate) were purchased from Merck
that cisplatin binds to Met151 which can block the N-terminal KGaA, Darmstadt, Germany. H[AuCl4], Na/K-ATPase from the
pathway for transported cations, and also to Met171 which can porcine brain cortex (specific activity of 0.1 IU mg1 solid), and all
hinder the interaction of cytoplasmic domains during the commercially available chemicals, dimethyl sulfoxide (DMSO), ATP,
catalytic cycle.40 However, compared to cisplatin, Au(III) com- NaCl, KCl, MgCl2, HCl and Tris–HCl were obtained from Sigma
plexes inhibit the sodium pump with higher power and in Aldrich Co., St. Louis, MO. [AuCl2(dmso)2]Cl and [AuCl2(bipy)]Cl
general react faster with biomolecules.21 Consequently, the (where bipy = 2,20 -bipyridine)31,32 and [PtCl2(dmso)2]42,43 were
difference between the Au(III) and Pt(II) inhibitory potency and synthesized according to the published procedures. Structures of
the reaction rate could be ascribed to various binding sites the complexes are presented in Scheme 1.

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2.2. Sample preparation 2.5. Apparatus

1  10 M stock solutions of the Au(III) complexes in dimethyl The spectrophotometric measurements were performed using a
sulfoxide were prepared and stored in a freezer at 20 1C. Lambda 35 UV-Vis Spectrometer, Perkin Elmer, Inc., Waltham,
A 2.4  103 M stock solution of [PtCl2(dmso)2] complex in MA, USA.
0.1 M HClO4 was prepared shortly before use by dissolving in
0.1 M HClO4 (Merck, p.a.) as a supporting electrolyte, in order 2.6. Molecular docking
to ensure the stability of the complex and eliminate hydrolysis High-resolution crystal structures of Na/K-ATPase in E1 (PDB
to the greatest possible extent. Working solutions were pre- code: 4HQJ)48 and E2P conformation (PDB code: 4HYT)49 were
pared by diluting the stock solutions with deionized water to extracted from the Protein Data Bank and used for the docking
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the desired concentrations. In all the experiments, a fresh study. The water molecules and other ligands were removed
enzyme stock solution in 50 mM Tris–HCl buffer at pH 7.4 from the structure. The metal complexes were optimized with
was used. The Na/K-ATPase concentration for a stock solution the B3LYP method, using the 6-311++g** basis set for non-
was determined using the Markweel method44 which is a metal atoms and the lanl2dz basis set for metals, in the
modified Lowry method.45 The enzyme concentration was kept Gaussian09 program.50 The structures of the selected protein
constant while the concentrations of the metal complexes were were prepared for docking studies in the AutoDockTools
increased. Before the acquisition of the spectra, the solution program, while the docking studies were done with the Auto-
was incubated for 5 minutes at 37 1C after the addition of the Dock program.51 The AutoDock program provides a protocol
metal complex. that includes molecular docking of the drug molecule on the
anticancer target (basic, with selective receptor flexibility, or
2.3. Na/K-ATPase assay with explicit hydration), prediction of binding and active sites,
Na/K-ATPase activity was determined in a standard incubation and virtual screening of target biomolecules.52
medium (200 ml), containing 50 mM Tris–HCl (pH 7.4), 100 mM Some single bonds of metal complexes were set to be
NaCl, 20 mM KCl, 5 mM MgCl2, 2 mM ATP and protein (2 mg rotatable, while all protein residues were kept rigid. The tested
solid per ml) in the presence or absence (control) of the desired complexes move freely during the docking run (AutoDock
concentration of the complex. Incubation mixtures were pre- virtual screening used 100 runs per compound) inside a grid
incubated for 10 min at 37 1C in the presence or absence of the box, containing the whole protein. Visual analysis of structures
inhibitor or water (control). The preincubation time in the and calculation of solvent accessible surface area (SASA, 300 grid
presence of inhibitor was quite enough (15 min for Au(III) and points per atom and radius probe of 1.4 Å) were carried out with
30 min for Pt(II) complexes) to induce the maximal inhibition the program BIOVIA Discovery Studio.53
for the given complex, as determined in the independent
experiment. The reaction was started by the addition of ATP,
allowed to proceed for 15 min, and interrupted by the addition 3. Results and discussion
of the ice cold HClO4 and immediate cooling on ice. The inorganic 3.1. Absorption spectra of the complexes in the reaction
orthophosphate (Pi) liberated from the hydrolysis of ATP was mixture
measured using a modified spectrophotometric procedure based The absorption spectra of 1  105 M Au(III) and 2  104 M
on the stannous chloride method, by reading the absorbance at Pt(II) complexes were recorded in the absence and the presence
690 nm.46 The results are expressed as the mean percentage of 1  106 M enzyme, under the conditions for proper enzyme
of enzyme activity relative to the corresponding control value. functioning, as described in the Material and Methods section.
All experiments were performed in triplicate. The complexes were added into the reaction mixture in their
unhydrolyzed forms (Scheme 1). Absorption spectra were
2.4. Kinetic measurements recorded during the time needed to complete the enzyme
Kinetic experiments were carried out according to the slightly reaction. Within this time interval and in the absence of
modified method of Phillips47 using the commercial Na/K-ATPase protein the absorption spectra did not change. Before enzyme
from the porcine cerebral cortex. The initial velocities were addition, the Au(III) complexes exhibited an absorption band in
measured in the same incubation medium as a function of rising the range from 300–370 nm (Fig. 2, black lines), characteristic
concentrations of MgATP2 (0.1–5.0 mmol l1). The measure- for the Au(III) chromophore8 while the Pt(II) complex showed the
ments were performed in the absence and presence of metal maxima at about 210 nm and 270 nm. Under the applied experi-
complexes while maintaining the concentrations of the other ions mental conditions, complexes [PtCl2(dmso)2]+ and [AuCl2bipy]+
(Na+, K+ and Mg2+) constant. The experimental data were fitted to were stable over 24 h,54 and [AuCl2(dmso)2]+ for about 10 min,
the Michaelis–Menten equation by nonlinear regression analysis while the [AuCl4] complex immediately underwent spontaneous
using an OriginPro9 computer program. Maximum enzymatic hydrolysis.55 The presence of at least 0.1 M Cl ions in the
velocity (Vmax) and Michaelis–Menten constant (Km) values were Na/K-ATPase medium assay was expected to partially prevent
determined by a computer-generated best fit of the data to a Line- the spontaneous hydrolysis at pH 7.4.56 The successive proto-
weaver–Burk plot and expressed in mmol Pi per h per mg protein and lytic constants for a stepwise hydrolysis of [AuCl4] are known57
mmol per l of ATP, respectively. Results are given as mean  SEM. but there are no available literature data for [Au(DMSO)2Cl2]+,

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Fig. 2 Absorption spectra of 1  105 M Au(III) and 2  104 M Pt(II) complexes in the reaction mixture in the absence (black line) and presence (blue line)
of 1  106 M Na/K-ATPase. (A) [AuCl2bipy]+, (B) [AuCl2(dmso)2]+, (C) [AuCl4] and (D) [PtCl2(dmso)2]. Referent solution: 50 mM Tris–HCl (pH 7.4), 100 mM
NaCl, 20 mM KCl, 5 mM MgCl2 and 1  106 M Na/K-ATPase.

[Au(bipy)Cl2]+ and [Pt(DMSO)2Cl2]. In general, the first step of confirming the affinity of Au(III) for fast interaction with the
chloro-complex hydrolysis is usually the reversible substitution enzyme. However, [PtCl2(dmso)2] absorption spectra were taken
of one chloride ligand from the metal ion coordination sphere using a high complex concentration because of its low extinction
by the entering nucleophile. In the second reversible step, the coefficient, and the changes of absorption spectra after adding
second chloride ligand is more easily displaced by another the protein at a given concentration are negligible.
nucleophile.56 In the solution containing 1  105 M [AuCl4]
under the experimental conditions the species [AuCl(OH)3],
[AuCl2(OH)2], [AuCl3(OH)], and [Au(OH)4] are in equilibrium.55 3.2. Inhibition and kinetic analyses of the interaction of the
Their concentrations are 5.15  106 M, 2.60  106 M, 1.84  metal complexes with Na/K-ATPase
107 M, and 2.10  106 M, respectively. The other problem In order to determine the stoichiometry of the interaction
concerning the complex stability is the spontaneous Au(III) between the selected complexes with different inhibitory
reduction, and the multidentate ligand, as bipyridine in the case power20,58 and Na/K-ATPase, the inhibition of the enzyme
of [AuCl2(bipy)]+ can hinder this reaction.8 induced by each complex was followed in the presence of
After the addition of 1  106 M Na/K-ATPase, changes of 1  106 M enzyme. The complex ions were added to the
the absorption spectra were observed, indicating an interaction reaction mixture until the complete inhibition of enzyme activity
between the complexes and the enzyme (Fig. 2A–D). The spectral was achieved in their excess. The saturation curves, which
changes appeared immediately after the enzyme addition, thus represent the percent of enzyme inhibition as dependent on

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Fig. 3 (A) Inhibition of Na/K-ATPase and (B) Scatchard analysis of saturation curves in the presence of [AuCl4] (blue), [AuCl2(dmso)2]+ (red),
[AuCl2(bipy)]+ (wine), and [PtCl2(dmso)2] (green). Enzyme concentration: 1  106 M.

the total complex concentration, typical for complex–protein (N1 and N2) and the dissociation constants, Kd (Table 1). The
interactions, were constructed and are shown in Fig. 3A. results obtained using these two models are in accordance.
Previous results indicated that some sulfhydryl compounds To characterize the type of Na/K-ATPase inhibition induced
are capable of recovering the inhibited enzyme activity27–30 by the Au(III) and Pt(II) complexes, Lineweaver graphs (1/v0 vs.
indicating the reversible process of inhibition. In that case, it 1/[MgATP]) obtained by Michaelis–Menten curves (data not
is possible to analyze the binding process using two methods, shown) for selected complex concentrations (near IC50 values)
the two sites binding pharmaceutical model59 and Scatchard were constructed (Fig. 4). All complexes exerted the uncompe-
analysis60 which have been developed for the description of the titive type of inhibition pointing out that their binding,
interaction between a biological macromolecule and ligand. although away from the active site, causes structural distortion
These methods were successfully applied to the data obtained of the active and allosteric sites of the complexed enzyme and
from the saturation curves and the results are presented in inactivates the catalysis. This leads to a decrease in both Km
Table 1. and Vmax values (Table 2). The uncompetitive inhibitor may
The concentration of the bound and free complex was bind the free enzyme or enzyme–substrate complex that
obtained from the saturation graph presented in Fig. 3A. exposes the inhibitor binding site.
Fig. 3B shows the Scatchard graph which represents the depen- Since the inhibition of the enzyme was uncompetitive, the
dence of the ratio between bound and free ligand concentra- value of Ki was calculated from the data presented in Fig. 4.
tion (Cbound/(CfreeCenzyme)) vs. bound complex concentration Values of 1/Km and a/Km obtained from the intersection of
(Cbound/Cenzyme) per mole of the enzyme. Concave-up shaped the replot with the abscissa were used for calculating the
plots were obtained in all cases, suggesting the presence of concentration-dependent value, a, which was further used for
multiple classes of binding sites. Points of intersection of the calculating Ki from eqn (1):
tangents obtained from Scatchard graphs with the x-axis give
the total number of complex ions bound per mol of the enzyme a = 1 + [I]/Ki (1)
(Ntot), presented in Table 1. The conclusion can be made that
where [I] represents the inhibitor concentration.
Au(III) and Pt(II) complex ions exert an affinity toward at least
two sets of binding sites on the Na/K-ATPase.60 In order to
confirm the obtained results the two sites binding pharmaceutical 3.3. Molecular modelling
model was also applied.59 It gave us the maximal number of In order to rationalize the reaction stoichiometry, type of
bound complexes per mol of the enzyme for each binding site inhibition, inhibitory potency and location of potential binding

Table 1 Binding parameters for the interaction of Na/K-ATPase with selected complexes obtained applying two sites binding and the Scatchard model

Method AuCl4 AuCl2(dmso)2 AuCl2bipy PtCl2(dmso)2
Two sites binding N1 0.83  0.17 1.11  0.09 0.99  0.07 1.96  0.90
N2 1.36  0.24 3.05  0.05 3.47  0.47 5.16  0.17
Kd1 (M1) (1.38  0.48)  108 (4.03  0.52)  109 (4.70  0.48)  107 (3.44  0.48)  107
Kd2 (M1) (2.55  0.15)  107 (1.45  0.19)  106 (6.33  0.33)  105 (7.73  0.25)  105
R2 0.9940 0.9950 0.9970 0.9915

Scatchard Ntot 2.4 4.5 4.15 6.2

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by calculating the values of the electrostatic surface potential

on the van der Waals radius (ESP), volumes and surface areas
(Table 3).50 Taking into account the hydrolysis of Pt(II) and
Au(III) chloro complexes in the working mixture,42,56 the
[MeCln(OH)pL] species (Me is the metal ion, L is the mono-
dentate or bidentate ligand (dmso or bipy), n and p are the
numbers of Cl or OH ligands) were considered in the
following studies and the results are presented in Table 3.
The replacement of chloro with hydroxo ligands in the
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coordination sphere of metal ions induced the decrease of

electronic density on both metal centres and consequently
the increase of ESP values. Hence, the Pt(II) ion in the
[PtCl2(dmso)2] complex has a more negative ESP value than
the Au(III) ion in the [AuCl2(dmso)2]+ complex. The neutral
Fig. 4 Lineweaver–Burk linearization of Michaelis–Menten curves, in the
charge of the [PtCl2(dmso)2] complex in synergism with the
absence and presence of the investigated complexes. suitable ESP value of the Pt(II) ion could facilitate the inter-
action of this complex with positively charged amino acid
residues in its environment. On the other hand, these inter-
Table 2 Parameters of Na/KATPase inhibition induced by the selected actions are weaker in the case of the positively charged Au(III)
Au(III) and Pt(II) complexes complex due to the lower ESP value of the Au(III) ion and the
Complex Km (mM) Vmax (mM h1 mg1) Ki (M) increased repulsion force between them. Based on these results
the binding of the Au(III) complex in the vicinity of the
Control 0.81  0.15 0.0959  0.0069 —
[AuCl4] 0.34  0.01 0.0361  0.0004 3.66  107 negatively charged amino acid residues is expected.
[AuCl2(dmso)2]+ 0.33  0.04 0.0343  0.0019 3.42  106 Au(III) complexes with bipyridine or dimethyl sulfoxide
[AuCl2bipy]+ 0.44  0.07 0.0509  0.0026 5.97  105 ligands have higher surface areas and volumes than the hydro-
[PtCl2(dmso)2] 0.67  0.09 0.0786  0.0040 2.36  105
lyzed species of [AuCl4] (Table 3). The bigger size and square
Ki – inhibitory constant. planar geometry of these complexes facilitate the possibility of
achieving contact with a higher number of amino acid residues
sites, computational studies were performed. Since Au(III) and in the binding site of Na/K-ATPase. The values of the surface
Pt(II) chloro complexes undergo stepwise hydrolysis, various area and volumes for Pt(II) complexes with the same ligands as
hydrolyzed species in equilibrium in the reaction mixture are the Au(III) complex are almost equal, indicating that only the
responsible for enzyme inhibition.42,56,61–63 Today it is still con- ligand size affects these parameters (Table 3). Nevertheless, it
troversial whether hydrolyzed forms of anticancer metallo drugs should be noted here that by geometry optimizations it is
are also responsible for the anticancer activity. However, some possible to obtain several optimal structures that correspond
theoretical studies on cisplatin hydrolysis supported the possibility to local or global minima. The geometry of the optimized
of the reactivity of hydrolysis products with the nucleobases.64 structure largely depends on the level of theory and the initial
3.3.1. Theoretical characterization of the investigated com- geometry of the structure used for optimization. Hence,
plexes. The studied complexes were characterized theoretically depending on the method of theory used for optimization of
the complex structure, different values of surface areas and
volumes could be obtained.18
Table 3 Calculated ESP values, volume and surface area for the selected
Au(III) and Pt(II) complexes
Table 4 The distribution of side chain solvent accessibility (in Å2) as a
Metal complex ESP for metal ions Volume (Å3) Surface (Å2)
function of amino acid nature and enzyme conformation
[AuCl2bipy]+ 0.027 208.2 234.2
[AuCl(OH)bipy]+ 0.174 373.6 280.2 Amino acid residues
[Au(OH)2bipy]+ 0.402 369.4 278.9
Conformation Negative Positive Polar Nonpolar Sa (Å2)
[AuCl2(dmso)2]+ 0.009 210.1 257.3 Ion exchange channel
[AuCl(OH)(dmso)2]+ 0.129 346.6 260.9 E2P 504.8 0 647.5 2134.1 3 286.4
[Au(OH)2(dmso)2]+ 0.334 343.3 259.7 E1 478.1 0 611.9 2170.8 3 260.8
Db 26.7 0 35.6 36.7 25.6
[AuCl4] 0.562 106.2 139.5
[AuCl2(OH)2] 0.503 98.6 137.9 Intracellular domains
[AuCl(OH)3] 0.599 89.5 125.1 E2P 4854.6 2444.0 7256.5 5784.1 20 339.2
[Au(OH)4] 0.735 80.6 113.1 E1 4613.0 4552.1 5296.5 5868.0 20 329.6
Db 241.6 2108.1 1960.0 83.9 9.6
[PtCl2(dmso)2] 0.252 210.4 252.1
a b
[PtCl(OH)(dmso)2] 0.155 343.6 257.7 Total solvent accessibility. Difference in SASA values between two
[Pt(OH)2(dmso)2] 0.039 340.4 256.7 considered conformations.

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Table 5 Estimated binding energies (in kcal mol1) for the energetically most favourable binding sites of selected metal complexes in the E1 and E2P Na/
K-ATPase conformations

Estimated binding energy (kcal mol1)

E1 conformation E2P conformation
Metal complex
N Location Energy Location Energy
[AuCl2bipy]+ 1 Channelb 4.81 Channelb 5.06
2 P–A domains 4.57 N–P domains 4.22
3 N domain 4.21 P domain 4.21

[AuCl(OH)bipy]+ Channelb 4.78 Channelb 5.43

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2 Channelb 4.78 b subunitd 4.64
3 A domain 4.15 b subunitd 4.42

[Au(OH)2bipy]+ 1 Channelb 4.8018 Channelb 5.06

2 Channelb 4.2018 Channelb 4.22
3 A domain 4.1518 a–b subunits 4.21

[AuCl2(dmso)2]+ 1 b subunitd 3.47 Channelb 3.67

2 b subunitd 3.14 Channelb 3.36
3 Channelb 2.95 P domain 2.93
4 Channelb 2.89 N domain 2.93

[AuCl(OH)(dmso)2]+ 1 N domain 3.28 N–P domains 3.26

2 Channelb 3.26 Channelb 3.18
3 Channelb 2.80 Channelb 3.17
4 N domain 2.68 N–P domains 2.86

[Au(OH)2(dmso)2]+ 1 N domain 3.10 N–P domains 3.11

2 Channelb 2.98 Channelb 2.90
3 Channelb 2.74 A domain 2.65
4 b subunitd 2.71 N–P domains 2.54

[AuCl4] 1 b subunitd 2.30 M2–M4c 2.06

2 M2–M4c 1.84 b subunitd 2.00

[AuCl3(OH)] 1 b subunitd 2.37 M2–M4 2.48

2 b subunitd 2.25 M2–M4 2.31

[AuCl2(OH)2] 1 b subunitd 2.63 Channelb 5.78

2 M2–M4c 1.34 Channelb 4.87

[AuCl(OH)3] 1 b subunitd 2.60 M2–M4c 2.76

2 M2–M4c 1.77 M2–M4c 2.31

[Au(OH)4] 1 b subunitd 2.17 M2–M4c 2.50

2 M2–M4c 1.30 M2–M4c 2.34

[PtCl2(dmso)2] 1 b subunitd 2.41 Channelb 2.54

2 b subunitd 2.37 Channelb 2.53
3 P domain 2.37 P domain 2.53
4 A–P domains 2.33 P–A domains 2.27
5 A–N domains 2.30 N–A domains 2.26
6 A–N–P domains 2.26 N domain 1.96
7 N–P domains 2.20 N–P–A domains 1.90

[PtCl(OH)(dmso)2] 1 b intracellular side 3.43 M2–M4c 4.29

2 b intracellular side 2.71 N domain 2.66
3 b subunitd 2.70 P domain 2.39
4 M2–M4c 2.67 A domain 2.36
5 P domain 2.63 b intracellular side 1.67
6 M8–M9e 2.21
7 b subunitd 2.14

[Pt(OH)2(dmso)2] 1 M2–M4c 2.69 M2–M4c 2.32

2 b intracellular side 1.94 M2–M4c 2.03
3 b subunitd 1.94 g intracellular side 1.94
4 b intracellular side 1.81 A domain 1.92
5 b subunitd 1.64 b subunitd 1.83
6 M2–M4c 1.56 A domain 1.71
7 b subunitd 1.48 Channelb 1.62
Binding sites number. b The extracellular side of the Na/K exchange channel. c M2 and M4 a-helices. d Complexes bonded in the extracellular
part of the b subunit. e Complexes bound in the transmembrane part of the b subunit, the loop between the M8 and M9 a-helices.

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Fig. 6 Binding sites in E2P conformation of Na/K-ATPase for

[PtClOH(dmso)2] (A) and [AuCl(OH)3] (B) complexes.

The results indicated that the surface of the ion channel,

which is the most attractive for complex binding both for Au(III)
and Pt(II) compounds, is covered with the negatively charged
and predominantly nonpolar amino acid residues. Neverthe-
less, there are no significant differences in the charge and
polarity distribution on the surface of the ion channel between
two major conformations of the enzyme, indicating an almost
equal possibility for binding there.
On the other hand, results obtained for domains by SASA
calculations indicated that negatively charged amino acid
residues are situated on their surface, particularly in the E2P
enzyme conformation (Table 4). Therefore, positively charged
complexes are expected to bind there, but the comparison of
the results obtained in the case of the ion channel and domains
suggests that negatively charged complexes would preferably
bind in the domains because of a certain amount of positively
charged amino acid residues located in that region. Never-
theless, the binding in the channel is possible due to the large
solvent accessibility of the polar and nonpolar side chains. The
binding of negative species in the channel is slightly more
pronounced for the E2P conformation, due to the larger solvent
accessibility of polar residues. Moreover, neutral Pt(II) com-
Fig. 5 Binding sites in E1 and E2P conformation of Na/K-ATPase for
plexes are suitable for the interactions, both with neutral and
dimethyl sulfoxide and bipyridine Au(III) and Pt(II) complexes.
charged amino acid residues, and could be bound both in the
ion channel and on the domains.
3.3.2. Analysis of the accessible surface of residues in 3.3.3. Docking studies. Docking studies were performed to
selected inhibitory sites of Na/K-ATPase. The SASA (Solvent predict the possible binding sites of the selected complexes and
Accessible Surface Area Approximation) method was used for their hydrolyzed species on Na/K-ATPase in its E1 and E2P
the quantification of changes in the solvent accessibility of conformations. For analysis of the estimated binding energies,
certain amino acids during the enzyme conversion between two the number of predicted binding sites according to the experi-
major conformations, as a function of their nature (polar, mentally obtained N values (Table 2) was taken into account. Their
nonpolar, positively and negatively charged). Thereby, regions locations were denoted by numbers from 1 to 7, according to the
of the enzyme-containing inhibitor binding sites, which are in binding energy decrease (Table 5). For better visualization, the
accordance with the experimentally determined type of inhibi- positions of notable binding sites for [AuCl2bipy]+, [AuCl2(dmso)2]+
tion (Table 2), such as the extracellular part of the ion exchange and [PtCl2(dmso)2] in the Na/K-ATPase structure in the E1 and E2P
channel and the intracellular domains, were used to perform conformations are presented in Fig. 5, as examples. The docking
the calculations.17,18,40 The location and sequence of analyzed results for all other complexes are presented in Fig. S2–S4 (ESI†).
amino acids is shown in Fig. S1 (ESI†) while the distribution of In general, the most preferable binding sites for non-hydrolyzed
side chain solvent accessibility is shown in Table 4. Au(III) and Pt(II) complexes are located at an extracellular part of the

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Fig. 7 Binding mode of the [AuCl(OH)3] and [PtCl(OH)(dmso)2] complex between M2 and M4.

b subunit, and the transmembrane part (ion exchange channel and (cytoplasmic) P, A and N domains. It must be noticed that these
several transmembrane helices) as well as in the intracellular results are in accordance with the kinetically obtained type of

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inhibition (Table 1) and also with our previous study of the 3.3.5. Binding sites of hydrolyzed Au(III) and Pt(II) species.
interaction between Na/K-ATPase and certain mononuclear Au(III) Binding sites of negatively charged [AuCl4] and its hydroxo
complexes with pyridine ligands and their binuclear analogues.17,18 derivatives, as well as of the hydrolyzed Pt(II) complex are
Moreover, the crystallographic study of cisplatin binding to an expected to differ compared to those of positive Au(III) and
enzyme in E1 and E2P conformations revealed the binding sites in neutral Pt(II) complexes. The docking results confirmed our
the cytosolic domains as well as in the extracellular part of the assumption since they indicated the main inhibitory site
enzyme.40 located on the intracellular side between the M2 and M4
3.3.4. Correlation between binding energy and ligand– a-helix (Fig. 6).
macromolecule complex stability. From the results presented Based on the analysis of amino acid residues at this binding
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in Table 5 it is obvious that the binding energy depends on a location, the conclusion can be made that Lys146 (sequence
central metal ion as well as on its ligand surrounding. Phe139 to Phe 154 of M2 a-helix) and Lys342 (sequence Leu330
[AuCl2bipy]+ has the highest binding energy among Au(III) to Arg346 of M4 a-helix) are responsible for binding of negative
compounds for both enzyme conformations, due to the and neutral complexes due to the strong hydrogen bond
additional p–p interactions in which the bipyridine ligand is formation between their NH3 groups and OH groups of com-
involved.18 In general, the binding affinities of the investigated plexes (Fig. 7). These bindings induce conformational changes
complexes toward the enzyme are similar for both E1 and E2P in enzyme domains and the block E1–E2P conformational
conformations (Table 5), although their positions differ due to equilibrium, thus influencing the protein functionality and
the structural differences, charge, volume and accessibility of causing enzyme inhibition.40
certain amino acids.
Based on SASA calculations, the conclusion can be made
that the penetration of positively charged Au(III) complexes to 4. Conclusion
the extracellular side of the Na/K ion channel is directed by the
electrostatic interactions between the negatively charged amino The reaction mechanism between selected Au(III) ([AuCl4],
acid residues situated at the channel entry and these complexes [AuCl2(dmso)2]+, [AuCl2(bipy)]+) and Pt(II) ([PtCl2(dmso)2])
(Fig. S5, ESI†). Binding of the complex in the extracellular side chloro complexes with Na/K-ATPase was clarified using experi-
of the ion channel and in the intracellular part (cytoplasmic mental and theoretical studies. The kinetic analysis indicated
domains) in both enzyme conformations blocks the transport that all investigated Au(III) complexes, as well as Pt(II) complex,
of Na+ and K+ ions and confirms the uncompetitive enzyme inhibited the enzyme uncompetitively. SASA approximation
inhibition obtained experimentally for Au(III) complexes and applied on the specific inhibitory sites (ion channel and
the neutral Pt(II) complex. However, low values of complex intracellular domains) of Na/K-ATPase revealed that the sur-
volume and surface area could facilitate its fitting in the Na/K face of the ion channel is predominantly covered with
ion channel.18 negatively charged amino acid residues, while the positive,
By comparing the locations of the [AuCl2(dmso)2]+ complex negative and neutral charge of amino acid residues on the
in the E1 and E2P conformations of the enzyme, the conclusion surface of the domains are equally distributed. Docking
can be made that the inhibitory action of this complex at its studies, which considered the enzyme–parent complex ion
lower concentrations is related to the E2P conformation of the system revealed the main inhibitory site located in the extra-
enzyme, whereas, in the E1 conformation inhibition is possible cellular side of the ion channel. On the other hand, a new
at higher complex concentrations (binding at sites (3) and (4), inhibitory site was revealed for hydrolyzed forms of [AuCl4]
Table 5). In the case of the [AuCl2bipy]+ complex lower values of and [PtCl2(dmso)2] complexes. The location of this site is
volume and surface area (Table 3) compared to [AuCl2(dmso)2]+ situated on the intracellular side of the transmembrane part
and the planar geometry of the bipyridyl ligand facilitate its of the enzyme, between M2 and M4 helices. Based on the
better fitting in the Na/K ion channel. However, the binding analysis of amino acid residues at this binding location, it was
energy of the non-charged Pt(II) complex is generally lower found that Lys146 i Lys342 are responsible for binding these
compared to the positively charged gold complexes, even for species due to the strong hydrogen bond formation between
the same ligands (Table 5). This finding is in accordance with their NH3 groups and the OH groups of the species. Formation
the experimentally determined Ki values given in Table 2. The of these hydrogen bonds blocks E1–E2P conformational
predicted locations of binding sites (Fig. 5) indicated that the equilibrium, thus inducing enzyme inhibition. Thereby, for
inhibition of Na/K-ATPase by this complex is related to its E2P both adapted enzyme conformations similar results were
conformation. SASA calculations for this neutral complex also obtained. The theoretically and experimentally obtained
coincide with the results obtained by docking studies, suggest- results in this paper enable a deeper insight into the ligand–
ing its interactions both with neutral and charged amino acid macromolecule reaction mechanism. The characterization of
residues. As previously mentioned, the [PtCl2(dmso)2] complex the non-covalent complex binding behaviour obtained in this
inhibited Na/K-ATPase uncompetitively (Table 2) which is in study can be used in the rational design of compounds with
accordance with the positions of its binding sites, which do not potential anticancer activity and also as a tool for the elucida-
lead to the conformational changes that may influence the tion of the mechanisms for processes affecting Na/K-ATPase
binding of ATP to the active site of the enzyme.40 biological function.

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Conflicts of interest Na,K-ATPase Isozymes in Colorectal Cancer and Liver

Metastases, Front. Physiol., 2016, 7, 9.
The authors declare that they have no competing interests 13 H. Y. Wang and G. A. O’Doherty, Modulators of Na/K-
regarding the publication of this paper. ATPase: a patent review, Expert Opin. Ther. Pat., 2012, 22,
14 F. Lefranc, T. Mijatović, Y. Kondo, S. Sauvage, I. Roland,
Acknowledgements O. Debeir, D. KrstiĆ, V. Vasić, P. Gailly, S. Kondo, G. Blanco
This work was financially supported by the Ministry of Education, and R. Kiss, Targeting the a-1 subunit of the sodium pump
Science and Technological Development of the Republic of Serbia, (the Na+/K+-ATPase) to combat glioblastoma cells, Neurosur-
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Project no. 172023. Numerical simulations were run on the gery, 2008, 62, 211–222.
PARADOX supercomputing facility at the Scientific Computing 15 T. Mijatović, L. Ingrassia, V. Facchini and R. Kiss, Na+/K+-
Laboratory of the Institute of Physics Belgrade, supported in part ATPase a subunits as new targets in anticancer therapy,
by the Ministry of Education, Science and Technological Devel- Expert Opin. Ther. Targets, 2008, 12, 1403–1417.
opment of the Republic of Serbia. 16 T. Mijatović, I. Roland, E. Quaquebeke, B. Nilsson, A. Mathieu,
F. Vynckt, F. Darro, G. Blanco and V. Facchini, The a1 subunit of
the sodium pump could represent a novel target to combat non-
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