1) The study investigated the interactions of the anticancer drug cisplatin with a mutated form of the copper transport protein Atox1 where all three cysteine residues were replaced with alanine (3Cys 3Ala-Atox1) using CD spectroscopy. 2) The results showed that cisplatin did not bind to the mutated Atox1 as evidenced by no changes in CD signals upon addition of cisplatin. 3) However, both the mutated Atox1 and mutated Atox1 mixed with cisplatin showed unfolding over time in far-UV CD, suggesting cisplatin may bind to other amino acids besides cysteine in Atox1.

1. UMEÅ UNIVERSITY, Department of Chemistry, SE-901 87 Umeå Sweden
Mutated Atox 1 and its interactions with the anticancer drug Cisplatin
Protein Structure and Function, Autumn 2011
Group B
Devulapally Praneeth Reddy
Gaurav Dutta Dwivedi
Mana Zamenzarrabi
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2. Abbreviations:
Cys............................................................................................................................cysteine
Ala.............................................................................................................................Alanin
Cu..............................................................................................................................Copper
CisPt...................................................................Cisplatin (Cis-diamminedichloroplatinum)
TClPt.....................................................................................................Tetrachloroplatinate
WT......................................................................................................................Wild type
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3. Abstract:
Cisplatin acts as an effective chemotherapeutic agent in treating diverse class of solid tumors. The
primary response to this drug is strong; however, at later stage the tumor cell usually initiates the
resistance towards cisplatin.
In this study, we use spectroscopic methods to see the interaction of Cisplatin and discuss the role of
cysteines in cisplatin binding to a mutated Atox 1 where all three cysteines have been replaced with
alanine (3 Cys 3 Ala-Atox1) . We observed that by adding Cu to (3Cys 3Ala) Atox 1 no Cu binding was
detected in near-UV CD region. In the thermal unfolding experiment, temperature facilitates the
denaturation of both WT Apo and the mutated one. However in WT at 20°C Apo alone and with CisPt
almost possess the same signals as associated to mutated Atox 1. Lacking of Cysteine residues
interrupts the binding between the Atox 1 and metals (Cu, CisPt, and TClPt).
In future research Knowledge of these binding interactions should support in the development of
future anticancer strategies. In addition, they may use the knowledge gained from this research to
continue to look for Cisplatin analogs less eager to bind to proteins, avoiding side-effects and
resistance.
Introduction:
It was found in 1965 that cisplatin inhibits the cell division (1); so, that is why nowadays platinum-based
drugs become prominent in cancer treatment. Among cancer therapeutics, cisplatin carries out its
antitumor activity by binding to DNA and interfering with replication and transcription process as a
result of forming a stable chelate. (2)
It has been suggested that in cell, cisplatin (Cis-diamminedichloroplatinum) loses its chloride groups
due to hydrolysation. Since water is a better leaving group than chloride, cisplatin can binds to DNA. (3)
Platinum-based drugs have limited effect and after sometime they lose their anticancer effect,
namely, cells display resistance to them. (4)
Studies have shown that some copper transporting proteins play a role in cisplatin resistance. There
are several pathways for uptake and transport of copper in the cell. Here, the pathway with Atox1 and
ATP7A/B, linked to Golgi network is considered. ATP7A/B proteins contain domains which are
structurally similar to Atox 1. (2) (4) (5)
It is believed that cisplatin interacts with the copper binding site of these proteins (Cys 12 and Cys 15
of Atox 1) and being transported out of the cell or accumulated by them. One evidence which supports
Cu-transferring proteins involvement in cell resistance is the escalating number of these proteins in
resistant cell. (6) Cisplatin binding to the Cu-binding site of Atox 1 has been shown in publications by
focusing on crystal structure of these proteins and in the very recent article by obtaining in-cell NMR (7)
(8)
.
There is a possibility that cisplatin has other binding sites as well, since platinum can form a bond with
methionine, histidine and even the third cysteine residue of Atox 1. (9) (10)
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4. The main goal of this project is to identify the role of cysteines in cisplatin binding to Atox 1. A
mutated form of Atox 1 where all three cysteines have been exchange to alanine will be studied (3 Cys
3 Ala-Atox1). The results will be compared to wild type Atox 1.
Figure1: (A) Structure of Atox 1 (3IWL). The three cysteines are marked.
(B) Structure of Tetrachloroplatinum 12 and Cisplatin 13.
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5. Materials and methods:
Protein preparation: The mutated protein 3cys 3Ala- Atox1 was kindly provided by Maria Espling
(Umea University).
CD Spectroscopy:
All the experiments were performed by JASCO J-810 CD-spectrophotometer at room temperature
(20 ). Cu (20mM in H2O), CisPt (6.7mM in H2O), Buffer (50mM Nacl, 20mM MES, 0.25mM DTT, pH 6),
3Cys 3Ala-Atox 1 (2.2mM) and TClPt (24.1mM) were used as a stock solution. CisPt solution was
freshly prepared. In titration experiments 0-5 fold of CisPt or TClPt was added to premixed Cu-3Cys
3Ala-Atox 1. In another set of titration experiments 0-5 folds of Cu was added to 1:1 CisPt-3Cys 3Ala-
Atox 1 solution. CD signals were optimised by subtracting the buffer from each spectrum. Far- UV CD
time dependences were performed for mut-Atox1 and Cu+mut- Atox 1 +5 eq CisPt for both in three
days. Thermal unfolding experiment was done on mut-Atox 1 and for mut-Atox 1 +5 eq. CisPt (20-70
0
C).
Results:
As it is demonstrated in fig. 2A no change in CD signal is observed by adding 1 eq.Cu to 3Cys 3Ala-Atox
1. While, changes in CD intensity of the wild type, apo and holo Atox1 are quite recognisable.
Therefore by removing Cys residue, no Cu binding was detected in near-UV CD.
It is clear from fig. 2B that CisPt, even in higher concentrations does not bind to the mutated Atox 1
pre mixed with 1eq Cu. In contrast, CD signals become more negative by increasing the CisPt
concentration in wild type.
Like the previous study on WT Atox 1, order of adding metals (Cu/Pt) to Atox 1 does not make any
difference in CD signals intensity and overall result in near-UV titration (fig 2C) . Here there is no
change in signal upon addition of Cu/Pt to the mutated protein.
Fig. 2D, indicates titrations of TClPt to 3Cys 3Ala-Atox1 (premixed with 1eq.Cu) in near-UV CD follows
the same pattern as titration with CisPt and CuCl2, namely no metal binding was detected. Not even
this highly reactive platinum substance induced new near-UV CD signals.
In fig. 3A and 3B which CD signals were detected over time (0-72h), are clearly shown that
apo+5eq.CisPt and Cu+5eq.CisPt will be unfolded during time but unfolding in Cu samples are more
significant compare to Apo based on far-UV detection. In both cases CisPt might bind to mutated
protein (Atox1) which does not have cysteine residue; therefore, cysteine amino acids are not the only
binding site for CisPt. With reservation for that 3Cys 3Ala Atox1+1eq.Cu or pure 3Cys 3Ala Atox1
without added CisPt were not tested. Cu that not has a site to bind is reactive and might be
responsible for protein unfolding.
In the matter of thermal unfolding, CisPt mediates higher denaturation of WT. But for mutated protein
alone and with CisPt presents nearly the same signal. (fig4)
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6. Figure 2: Near-UV CD-monitored titrations of Cu and Pt to 3Cys 3Ala-atox1(pH6,20°c).(A) 50 µM 3Cys
3Ala-Atox 1 (1:1), Cu; (B) CisPt (up to five fold excess as indicated) added to 50 µM 3Cys 3Ala-Atox 1
pre mixed with 50 µM Cu; (C) Cu (up to fivefold excess as indicated ) added to 50 µM 3Cys 3Ala-Atox 1
premixed with CisPt (1:1). Tetrachloroplatinum (TClPt) was used in one titration to further test the
system because of its higher reactivity than cisplatin. 0-5 eq TClPt added to 1:1 premixed 3Cys 3Ala-
atox1:Cu.
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7. -
Figure 3: Far-UV CD spectra as a function of time (0 h to 72h). Time points are (before) 0, 1, 2, 3, 5, 24,
48 and 72h. (a) 3cys 3 ala (50 μM) +5eq.cisplatin. (b) 3 cys 3 ala Atox1 (50 μM) pre mixed with 1 eq.Cu
and added 5 eq cisPt and incubated at 20 °C.
Figure 4: (A) CD changes at 220nm (at 20°c) plotted as a function of time. (B) Thermal unfolding
3Cys3Ala-Atox1 (50 μM) alone and mixed with 5eq Cisplatin.
Discussion:
While there are diverse mechanisms which are involved in CisPt movement within the cell, we have a
good reason to believe that there is a linkage between CisPt resistance and the human Cu transport
system11. In the previous studies it was found that CisPt binds to apo-Atox1 in solution, as was
expected from the crystal structure work.
We investigated interactions of CisPt with a mutated form of (3Cys 3Ala) Atox1 (both pure and
premixed with Cu) looking at Cu and Pt binding in solution using the CD spectroscopy. Our solvent
environment and parameters were selected to mimic the cell environment.
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8. It is also evident from the previous work that CisPt binds to the holo form (Cu WT-Atox1) without loss
of Cu—in its place, both metals seems to bind to WT Atox 1 metal binding site, but it was not the same
for mutated Atox1. Even by increasing the concentration of Cu in (3Cys 3Ala) Atox 1 no Cu binding was
observed in near-UV CD. (fig 2) CisPt titration did not give new signals as for WT we reach to a
conclusion that in near –UV CD changes for WT Atox 1depends on Cysteines.
In far-UV CD, both Apo/Holo Atox1+5eq.CisPt (previous study), Mut+5eq.CisPt , and
Mut+1eq.Cu+5eq.CisPt were unfolded over time, mutated +5eq.CisPt in solution shows slow protein
unfolding at 20 °C (Fig. 3) but stability for pure mut-Atox 1 is unknown while WT Apo was stable, So we
cannot conclude stability of mutated protein from this result. Also we suspected about the
involvement of other binding sites which may be Methionine or Histidine. Unfolding gets more
extensive when adding Cu in both WT and mutant (Fig. 4A). Probably because Cu cannot bind and its
high reactivity induces unfolding. Since pure Cu-3Cys3Ala Atox1 was not tested we cannot conclude
CisPt binding, but suspected about some interactions with other amino acids. This has to be studied in
the future.
We also performed thermal stability of (3Cys 3Ala) Atox 1 alone and with cisPt which was monitored
by CD at 220 nm (Fig. 4). In the matter of thermal unfolding, CisPt mediates additional denaturation of
WT apo and holo. In case of mutated Atox 1 there were rather the same but small changes in the CD
signals can be detected which may be due to unfolding and exposure to the solvent cisPt has more
chance to binds to other amino acids like Methionine/Histidine. Since there was some problem with
CD temperature controller we could not continue this experiment up to 90 . But from up and down
scan we can say that mutated protein is not stable alone or with CisPt.
Acknowledgements:
We are extremely grateful to Maria Espling for supervising the project in Protein Structure and
Function as well as reading the manuscript; she has given her valuable feedback throughout the
project and necessary correction as and when needed. We are also deeply indebted to Magnus Wolf-
Watz for his guidance and help during the project.
References:
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9. copper chaperone Atox1 and promotes unfolding in vitro, Proc Natl Acad Sci U S A. 2011 April 26;
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