Translocation of platinum anticancer drugs by human copper ATPases ATP7A and ATP7B

Article abstract of DOI:10.1002/anie.201307718

Cisplatin, carboplatin, and oxaliplatin are widely used anticancer drugs. Their efficacy is strongly reduced by development of cell resistance. Down-regulation of CTR1 and up-regulation of the Cu-ATPases, ATP7A and ATP7B, have been associated to augmented drug resistance. To gain information on translocation of Pt drugs by human Cu- ATPases, we performed electrical measurements on the COS- 1 cell microsomal fraction, enriched with recombinant ATP7A, ATP7B, and selected mutants, and adsorbed on a solid supported membrane. The experimental results indicate that Pt drugs activate Cu-ATPases and undergo ATP-dependent translocation in a fashion similar to that of Cu. We then used NMR spectroscopy and ESI-MS to determine the binding mode of these drugs to the first N-terminal metal-binding domain of ATP7A (Mnk1).

Full text of DOI:10.1002/anie.201307718

Angewandte
Chemie
DOI: 10.1002/anie.201307718
Vectorial Displacement
Translocation of Platinum Anticancer Drugs by Human Copper
ATPases ATP7A and ATP7B**
Francesco Tadini-Buoninsegni, Gianluca Bartolommei, Maria Rosa Moncelli, Giuseppe Inesi,
Angela Galliani, Marilꢀ Sinisi, Maurizio Losacco, Giovanni Natile, and Fabio Arnesano*
Abstract: Cisplatin, carboplatin, and oxaliplatin are widely
used anticancer drugs. Their efficacy is strongly reduced by
development of cell resistance. Down-regulation of CTR1 and
up-regulation of the Cu-ATPases, ATP7A and ATP7B, have
been associated to augmented drug resistance. To gain
information on translocation of Pt drugs by human Cu-
ATPases, we performed electrical measurements on the COS-
1 cell microsomal fraction, enriched with recombinant ATP7A,
ATP7B, and selected mutants, and adsorbed on a solid
supported membrane. The experimental results indicate that
Pt drugs activate Cu-ATPases and undergo ATP-dependent
translocation in a fashion similar to that of Cu. We then used
NMR spectroscopy and ESI-MS to determine the binding
mode of these drugs to the first N-terminal metal-binding
domain of ATP7A (Mnk1).
A
TP7A and ATP7B share 54% sequence identity and are
associated, respectively, with Menkes and Wilsonꢀs diseases.
Both ATPases are 160–170 kDa proteins with eight trans-
membrane (TM) domains and six cytosolic N-terminal metal-
binding domains (NMBD). N- and P-domains are involved in
ATP binding and hydrolysis. The actuator (A)-domain
interacts with the ATP-binding domain and is required for
conformational changes during ATP hydrolysis (Figure 1).[1]
For many P-type ATPases, the ion transport requires passing
through the membrane portion of the protein.[2] It has been
demonstrated that, similarly to Cu, Pt binds to the CXXC
Figure 1. Structures of platinum complexes (top), topology of P-type
Cu-ATPases ATP7A/ATP7B (middle), and diagram of a microsome
containing ATP7A/ATP7B adsorbed on an SSM (bottom). When charge
displacement occurs, a compensating current I(t) flows along the
external circuit (the blue spheres represent electrons) to keep constant
the potential difference (DV) applied across the whole system.
RE=reference electrode.
[*] A. Galliani, Dr. M. Sinisi, Dr. M. Losacco, Prof. G. Natile,
Prof. F. Arnesano
Department of Chemistry, University of Bari “Aldo Moro”
via E. Orabona 4, 70125 Bari (Italy)
E-mail: fabio.arnesano@uniba.it
motifs of the NMBDs of ATP7B and that such interaction
mediates cancer cell resistance to cisplatin.[3,4]
Homepage: http://www.chimica.uniba.it
Recently, the solid supported membrane (SSM) technique
was employed to investigate charge movements (Cu+) in
recombinant human Cu-ATPases.[5–7] The addition of ATP to
microsomes enriched with recombinant ATP7A/B and ad-
sorbed on a SSM is followed by an electrogenic event
recorded as a current transient (see Figure 1; see also the
Supporting Information). The current transient is due to the
flow of electrons along the external circuit toward the
electrode surface, and is required to compensate for the
potential difference across the vesicular membrane produced
by the displacement of positive charges (attributed to
vectorial translocation of bound Cu+) into the microsome
upon ATP utilization. The current recorded by the SSM
method is a measure of the rate of change of the trans-
membrane potential, and is not sensitive to stationary
currents. Therefore, only electrogenic steps within the first
Dr. F. Tadini-Buoninsegni, Dr. G. Bartolommei, Prof. M. R. Moncelli
Department of Chemistry “Ugo Schiff”, University of Florence (Italy)
Prof. G. Inesi
California Pacific Medical Center Research Institute, San Francisco
(USA)
[**] We thank the Universities of Bari and Florence, the Ente Cassa di
Risparmio di Firenze, the Consorzio Interuniversitario di Ricerca in
Chimica dei Metalli nei Sistemi Biologici (CIRCMSB), the Italian
Ministero dell’Universitꢀ e della Ricerca (PON 01078, PRIN
2010M2JARJ, PRIN 2009WCNS5C, PON01_00937, PRIN
20083YM37E), and the European Commission (COST Actions
CM0902 and CM1105) for support. Expression of recombinant
ATP7A, ATP7B, and respective mutants was supported by the USA
National Institutes of Health (RO301-69830) from the NHLBI to
G.I.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201307718.
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catalytic cycle are measured, whereas steady-state events
after the first cycle are not detected.
observed in the cases of CuCl2, cisplatin (Figure 2c), and
oxaliplatin (Figure 2d). Therefore, current measurements
point to a comparable movement of Cu- or Pt-related
charge through ATP7A/B upon ATP utilization. Importantly,
no current transient was detected if the ATP (100 mm) jump
was performed with a buffer solution not containing CuCl2 or
a Pt complex.
In the present work, we first examined the effect of
Zeiseꢀs salt (K[PtCl3(C2H4)]), which is characterized by very
labile chloride ligands owing to the strong labilizing effect of
ethylene, and then extended the investigation to the aquated
species of cisplatin and oxaliplatin (cis-[Pt(NH3)2(H2O)(SO4)]
and cis-[Pt(R,R-dach)(H2O)(SO4)], dach = 1,2-diaminocyclo-
hexane) pre-incubated for 30 min in KCl (300 mm). In the
presence of chloride cis-[Pt(NH3)2(H2O)xCl2Àx] and cis-[Pt-
(R,R-dach)(H2O)xCl2Àx] species are formed, however, for the
time of the experiment (< 90 min), the monoaqua and diaqua
species are still present in relevant amounts (ca. 50% of the
total) and thus they may represent the active form of the
drugs in vivo.
The SSM setup was initially tested for Cu translocation.
Microsomes containing ATP7A/B adsorbed on the SSM were
incubated in a buffer solution containing CuCl2 (5 mm) for ca.
30 min (in the presence of excess dithiothreitol, Cu2+ is
reduced to Cu+). Then the solution was exchanged with an
identical buffer solution containing CuCl2 (5 mm) and, in
addition, ATP (100 mm), and the current transient measured
(Figure 2, black lines and columns). After this preliminary
experiment, the system was flushed with a buffer solution
containing a Pt complex (5 mm) and then incubated for
30 min. The solution was then exchanged with an identical
buffer solution containing a Pt complex (5 mm) and, in
addition, ATP (100 mm), and the current transient detected
(Figure 2, red lines and columns). A similar overall charge
was translocated in all cases (Figure 2, black and red
columns), but the current transient detected in the case of
Zeiseꢀs salt (Figure 2a,b) exhibits a slower decay than those
To check if there was interference between the two
metallic species, we carried out an ATP concentration jump in
the presence of both CuCl2 and Pt complex, with each at 5 mm
concentration: no current transient was detected (Figure 2,
green lines and columns). A cross-check experiment was
performed by the addition of bathocuproine disulfonate
(BCS; 1 mm) to the buffer solution containing CuCl2 and
the Pt complex. Following an ATP jump, a current transient
was recovered (to ca. 60–70% of the charge measured in the
presence of 5 mm Pt complex alone; Figure 2, blue lines and
columns). Thus, chelating Cu+ with BCS allows Pt-related
charge translocation by ATP7A/B to be partially restored.
To further investigate Pt-related charge movements upon
ATP utilization, analogous experiments were performed
using specific mutants of ATP7A and ATP7B. First, the
effect of Asp1044 mutation in ATP7A was analyzed. Asp1044
is the residue receiving the ATP g-phosphate at the catalytic
site in the cytosolic P-domain of ATP7A, to form the aspartyl
phosphorylated intermediate. Its mutation produces catalytic
inactivation of the ATPase. We found that the D1044A
mutant yields no current transient upon ATP jump either in
the presence of CuCl2 (5 mm) or in the presence of a Pt drug
(5 mm; Supporting Information, Figure S1A). This result
indicates that the ATP-induced movement of Cu or Pt-
related charge through the ATPase is directly correlated to
the formation of the acyl-phosphate intermediate by ATP
consumption.
We then considered the effects of site-directed mutations
C575A/C578A of the CXXC motif in the 6th NMBD of
ATP7B, which was shown to be critical for Cu-related charge
displacement.[5] The C575A/C578A double mutant was found
to be catalytically inactive,[5] therefore no current transient
was detected following an ATP jump in the presence of a Pt
drug (5 mm), as well as in the presence of CuCl2 (5 mm;
Figure S1B). Thus, it was concluded that C575A/C578A
mutation in the 6th NMBD interferes with both Cu and Pt
binding, thereby preventing ATP-dependent charge move-
ments.
Having found that the 6th NMBD of ATP7B was essential
for charge movements, we focused our investigation on the
interaction of one of these domains with Pt substrates. All
NMBDs share similar characteristics and we chose the first
domain of ATP7A, Mnk1. Fully 15N-labeled and selectively
13C-cysteine-labeled Mnk1 (15N,13C-Cys Mnk1) was bacteri-
ally expressed with a recognition site for the restriction
protease Factor Xa and a (His)6 tag at the C-terminus, which is
crucial for its purification by metal-affinity chromatography.
After cleavage of the tag, four amino acids (IEGR) of the
restriction site were left at the C-terminus of the protein. The
N-terminal methionine was partially processed in E. coli,
yielding a 76-amino acid protein (numbering the first codon
(Met) as residue 1). Therefore, the ESI-MS spectrum of
Figure 2. Current measurements on ATP7A (a, c, and d) and ATP7B
(b) in the presence of Zeise’s salt (a and b), aquated cisplatin (c) and
oxaliplatin (d). Representative current transients induced by ATP
(100 mm) concentration jumps in the presence of: CuCl2 (5 mm; black
lines), Pt complex (5 mm; red lines), a mixture of CuCl2 and Pt complex
(5 mm each; green lines), and a mixture of CuCl2 and Pt complex (5 mm
each) with BCS (1 mm; blue lines). The insets show the charges
related to ATP-induced current transients obtained under the various
experimental conditions (the same color code is used for lines and
columns). Error bars are a standard deviation of six measurements on
the same SSM.
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apoMnk1 is formed by two series of multi-
ply charged states corresponding to the
protein with and without the first Met
residue (Figure S2).
When cisplatin is added to apoMnk1
(100 mm) in equimolar amounts under anae-
robic conditions, ESI-MS spectra of the
reaction mixture show a new set of signals;
their intensity increases over time, indicat-
ing the formation of adducts between Mnk1
and the Pt complex. The mass difference
between the corresponding charged states
indicates binding of a {Pt(NH3)2Cl}+ and
a {Pt(NH3)2}2+ fragment to the protein. In
the early stage of the reaction, the mono-
dentate adduct {Pt(NH3)2Cl}+-Mnk1 is by
far the major species. After 24 h of incuba-
tion, the signals corresponding to the che-
late adduct {Pt(NH3)2}2+-Mnk1 become the
most intense (Figure 3a,b).
Similar behavior was observed in the
reaction of apoMnk1 with oxaliplatin, how-
ever the reaction appears to be slower: after
3 h of incubation only weak signals relative
to the monodentate adduct {Pt(R,R-dach)-
(oxalateH)}+-Mnk1 are present, together
with those of apoMnk1; after 24 h, the
apoMnk1 signals decrease while intense
Figure 3. ESI-MS spectra of 15N,13C-Cys Mnk1 treated with 1 molequiv of cisplatin (a and
b) or oxaliplatin (c and d) after 3 (a and c) and 24 h (b and d) of incubation. Peaks
corresponding to a +8 charged state are shown. Each species comprises two pairs of
signals corresponding to Mnk1 with and without Met1 as the first residue.
signals relative to the chelate adduct {Pt(R,R-dach)}2+-Mnk1
appear (Figure 3c,d).
1
The starting H,15N-HSQC spectrum of 15N-labeled cis-
platin incubated with apoMnk1 shows signals of the dichlor-
ido species (cis-[PtCl2(15NH3)2], d 15N = À67 ppm) and mono-
aqua species (cis-[PtCl(15NH3)2(H2O)]+, d 15N = À66 and
À83 ppm for the 15NH3 trans to Cl and to H2O, respectively).[8]
After 3 h of incubation, the intensity of these cross-peaks
decreases simultaneously with the appearance of two cross-
peaks relative to the intermediate species characterized by
one 15NH3 group trans to a Cl atom and one 15NH3 group trans
to a S atom (d 15N ca. À66 and À38 ppm, respectively). After
24 h of incubation, only two cross-peaks are present in the
spectrum that are characteristic of 15NH3 trans to S atoms (d
15N ca. À43 and À36 ppm, respectively), thus indicating
a complete loss of chloride ions from the Pt complex
(Figure 4a–c).
To prove that cysteine residues of the Cu+-binding motif,
CXXC, are involved in Pt coordination, 1H,13C-HSQC spectra
were recorded on 15N,13C-Cys Mnk1 incubated with one
equivalent of cisplatin (Figure S3A–C). After 3 h of incuba-
tion, the original cross-peaks of apoMnk1 nearly disappear,
with the concomitant appearance of new cross-peaks for a-
CH and b-CH2 groups of Cys15 and Cys18, that correspond to
a monodentate adduct with the Pt drug (Figure S3B). A
different set of Cys signals, which correspond to formation of
the chelate adduct, is observed after 24 h (Figure S3C). The
1H,13C-HSQC spectra also display natural-abundance e-CH2
cross-peaks of lysines, which all resonate in the same
frequency region. These latter signals do not experience any
relevant change upon reaction with cisplatin (Figure S4).
Figure 4. 2D 1H,15N-HSQC spectra of cis-PtCl2(15NH3)2 incubated at
pH 7.0 with 1 mol equiv of apoMnk1 (a–c) and Cu+-Mnk1 (d–f) at 0 h
(a and d; blue), 3 h (b and e; red), and 24 h (c and f; green) after
mixing. Cross-peaks are assigned to 15NH3 groups trans to O, Cl, or S
donor atoms, respectively. Cross-peaks belonging to the same species
are connected by a straight line. The reaction of cisplatin with
apoMnk1 is shown at the top.
To explore the exchange behavior of the chelate adduct,
after 24 h incubation of 15N,13C-Cys Mnk1 with one equivalent
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of unlabeled cisplatin, a second equivalent of 15N-labeled
cisplatin was added. After an additional 24 h (and up to
several days of incubation) there is no evidence of 15NH3
by cysteines, we propose that the complex that translocates
bears the ammine groups.
The lack of charge translocation following mutation of the
catalytic Asp1044 of ATP7A or the CXXC motif in the 6th
NMBD of ATP7B clearly demonstrates that the Pt-related
charge movement is dependent on formation of a phosphory-
lated intermediate, as well as conformational adjustments
analogous to those required for Cu-related charge transloca-
tion.
The presence of both Cu and Pt in the buffer solution
abolishes charge movements, indicating that simultaneous
occupancy of activating sites by the two metal ions prevents
catalytic activation, as has been previously observed.[11]
Interestingly, the use of a Cu chelator (BCS) partially restores
charge movement, thus demonstrating direct involvement of
the Pt drug.
Copper binding to CXXC motifs of NMBDs triggers
protein conformational changes that weaken the interactions
between the N-terminus and the ATP-binding domain, and
favor catalytic phosphorylation.[12] Based on the NMR experi-
ments performed with 15N-labeled cisplatin, we propose the
mechanism depicted at the top of Figure 4 for the reaction of
cisplatin with one such NMBD. The drug initially forms
a monodentate adduct ({Pt(NH3)2Cl}+-Mnk1) with the pro-
tein. This is an early adduct that may be relevant for Pt-
related charge translocation. On a longer timescale, Pt
releases the second chloride while keeping the two ammines,
thus forming the chelate adduct ({Pt(NH3)2}2+-Mnk1). Once
the chelate adduct is formed, no Pt exchange can occur at the
CXXC motif at a detectable rate.
At variance with what occurs with the MXXM motifs of
CTR1,[13] the S atoms of cysteines do not labilize the ammine
ligands of the Pt drug that are trans to them. This surprising
difference was already noted in a previous paper from our
group reporting the interaction of Pt drugs with Atox1, which
is structurally similar to Mnk1.[14] It is possible that the protein
exerts a protective effect, as witnessed by the strong shielding
in the 1H dimension of the cross peaks of the cisplatin-Mnk1
adducts. Cisplatin is able to readily displace Cu+ bound to the
CXXC motif and coordinates directly in a chelating fashion,
however the displaced Cu ion destabilizes the Pt-Mnk1
structure. An analogous unfolding behavior has previously
been observed for the structurally similar Atox1.[15]
1
signals trans to the S atoms, and the H,13C-HSQC spectrum
of the protein is not further perturbed, which indicates that no
exchange occurs between the second equivalent of cisplatin
and the Pt already chelated by the two Cys residues of Mnk1
in the chelate adduct ({Pt(NH3)2}2+-Mnk1). Since in the
cytosol there is a relevant concentration of glutathione (GSH,
ca. 5 mm), we wanted to explore if the reaction between
cisplatin and apoMnk1 would take place also in the presence
of (10 fold) excess GSH. Results showed that Mnk1 competes
successfully with GSH for complexation to platinum (Fig-
ure S5).
One equivalent of 15N-labeled cisplatin was added to
preformed Cu+-Mnk1 adduct. Both ESI-MS (Figure S6) and
NMR measurements (Figure 4d–f) indicate that the Pt drug
can bind to Cu+-Mnk1, as found for the apoprotein, but in the
case of the holoprotein there is no evidence of monodentate
adduct formation. Instead, the chelate adduct (both 15NH3
trans to S atoms) is already formed after 3 h of incubation,
thus indicating that Cu+ can catalyze Pt drug chelation by two
Cys residues with a complete loss of chlorido ligands. Notably,
the chemical shifts of the ammine ligands of cisplatin are the
same as those observed in the former experiment with
apoMnk1 after 24 h incubation. Also the 1H,13C-HSQC
spectrum of 15N,13C-Cys Cu+-Mnk1, after 3 h of incubation
with cisplatin, shows a great decrease in intensity of the
signals of the holoprotein, while the signals of {Pt(NH3)2}2+-
Mnk1 appear (compare Figure S3E with Figure S3D,C),
clearly indicating that cisplatin can displace Cu+ bound to
CXXC motifs, thus potentially interfering with cellular Cu
homeostasis. Furthermore, after 24 h of incubation, a remark-
able reduction in the intensity of 15NH3 and 15N,13C-Cys Mnk1
signals (Figure 4 f; see also Figure S3F) is observed, accom-
panied by the coalescence of amide signals in the middle of
1
the H,15N-HSQC spectrum, a typical sign of protein unfold-
ing (Figure S7). Notably, the {Pt(NH3)2}2+-Mnk1 chelate
adduct alone unfolds at a much slower rate (Figures S3C
and S7C), thus indicating that the Cu+ ion displaced by
cisplatin promotes protein unfolding.
Taken together, our measurements clearly support ATP-
induced Pt-related charge translocation in ATP7A/B. The
charge translocation occurs with rates analogous to that of Cu
in the case of aquated cisplatin or oxaliplatin. Numerical
integration of the ATP-induced current transients in the
presence of CuCl2 or Pt complex yields similar values for the
translocated charge (Figure 2, black and red columns), thus
indicating that an equal amount of positive charge is displaced
by ATP7A/B upon ATP utilization within a single catalytic
cycle. Analysis of bacterial Cu-ATPase, that is CopA from A.
fulgidus[9] and L. pneumophila,[10] suggested that the transport
stoichiometry could probably be two Cu+ ions per hydrolyzed
ATP. Thus, assuming the same stoichiometry (2Cu+ per ATP)
in the case of human ATP7A/B, our measurements indicate
that one Pt species bearing two positive charges, or two Pt
species bearing a single positive charge, is/are translocated
following utilization of one ATP molecule. Based on the
stability of the ammine groups, which are not trans-labilized
In conclusion, our studies show that cisplatin and oxali-
platin can activate the catalytic cycle of ATP7A/B, leading to
charge movement. Cisplatin can react with the ATPase N-
terminal extension, initially forming quite long-lasting mono-
dentate adducts, which then evolve into stable and unreactive
chelate adducts. It is likely that translocation by ATPases (at
short incubation times) and sequestration in the N-terminal
extension (at long incubation times) may contribute to
cisplatin resistance in vivo, with the predominance of either
phenomenon depending on dosage and time of administra-
tion. Resistance would be certainly higher under conditions of
ATPase protein overexpression.
Received: September 2, 2013
Revised: October 10, 2013
Published online: December 27, 2013
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[7] F. Tadini-Buoninsegni, G. Bartolommei, M. R. Moncelli, K.
Keywords: ATPases · charge measurements · copper ·
NMR spectroscopy · platinum drugs
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