Coordination Chemistry Reviews: Luigi Messori, Antonello Merlino

Coordination Chemistry Reviews 315 (2016) 6789
Contents lists available at ScienceDirect
Coordination Chemistry Reviews

journal homepage: www.elsevier.com/locate/ccr
Review
Cisplatin binding to proteins: A structural perspective

Luigi Messori a , Antonello Merlino b,c,
a
Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, FI, Italy
Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte SantAngelo, Via Cintia, I-80126 Napoli, Italy
c
CNR Institute of Biostructures and Bioimages, Via Mezzocannone 16, I-80126 Napoli, Italy
b
Contents
1.
2.
3.
4.
5.
6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
A gallery of molecular structures for cisplatin-protein derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
2.1.
Superoxide dismutases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
2.2.
Lysozyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
2.3.
RNase A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
2.4.
Atox-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
2.5.
Cytochrome c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2.6.
Glutaredoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2.7.
Na+ /K+ -ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
2.8.
HSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Recurrent structural features in cisplatin-protein adducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.1.
Nature of the interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.2.
Binding selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.3.
Pt/Protein stoichiometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
3.4.
Effects of Pt binding on the overall protein conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
3.5.
Physicochemical characteristics of cisplatin binding regions and extension of cisplatin-protein interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Methodological limits of crystallography and future challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Toward a unied picture of protein platination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
a r t i c l e
i n f o
Article history:
Received 12 December 2015
Accepted 27 January 2016
Available online 4 February 2016
Keywords:
Cisplatin
Metallodrugs
Bioinorganic chemistry
a b s t r a c t
The interactions of clinically established anticancer Pt-based drugs with proteins play crucial
roles in Pt cellular uptake and biodistribution, as well as in determining side effects and
resistance, thus affecting the overall pharmacological prole of this class of drugs. Here, we summarize a number of recent crystallographic studies of cisplatin/protein adducts that contribute
unveiling the molecular basis for cisplatin-protein recognition. Details of each molecular structure are carefully and comparatively described; common trends and regularities occurring in the
analyzed adducts are highlighted. Analysis of the structural features of its protein derivatives, integrated with selected results arising from the application of other biophysical methods on strictly
Corresponding author at: University of Naples Federico II, Department of Chemical Sciences, Complesso Universitario, di Monte SantAngelo, I-80126 Via Cintia, Napoli,
Italy. Tel.: +39 081674276; fax: +39 081674090.
E-mail address: antonello.merlino@unina.it (A. Merlino).
http://dx.doi.org/10.1016/j.ccr.2016.01.010
0010-8545/ 2016 Elsevier B.V. All rights reserved.
68
L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789
Medicinal chemistry
Antitumor agents
Platinum
Platinated proteins
related systems, allows an overall elucidation of the protein platination process and offers a more
comprehensive understanding of the mode of action of cisplatin and its parent Pt-based drugs.
Abbreviations:
a.u., asymmetric unit
Atox-1, a copper chaperone protein
ATP7A, Menkes disease protein
ATP7B, Wilsons disease protein
beSOD, superoxide dismutase from
bovine erythrocyte
CA, carbon alpha atom
CD, circular dichroism
Cox17, Cytochrome c oxidase Copper
Chaperone
Ctr1, Copper transport protein 1
Cyt c, horse heart cytochrome c
cyt c, cytochrome c
DMSO, dimethyl sulfoxide
DNA, deoxyribonucleic acid
ESI-MS, electrospray mass
spectrometry
Grx, glutaredoxin
GSH, glutathione
HEWL, hen egg white lysozyme
HSA, human serum albumin
hSOD, human superoxide dismutase
MD, molecular dynamics
Na+ /K+ -ATPase, sodium/potassium
pump dependent adenosine
triphosphatase
ND1, atom of the side chain of His
NMR, nuclear magnetic resonance
spectroscopy
oxPfGrx-1, oxidized Plasmodium
falciparum Glutaredoxin-1
Occupancy, proportion of sites lled by
atoms
PAGE, polyacrylamide gel
electrophoresis
PDB, protein data bank
PEG, polyethylen glycole
PfGrx-1, Plasmodium falciparum
glutaredoxin-1
Rmsd, root mean square deviation
RNase A, bovine pancreatic
ribonuclease
SOD, superoxide dismutase
Space group, description of the
symmetry of the crystal
ssRNA, single-stranded RNA
QM/MM, quantum
mechanism/molecular mechanics
1010 m
1 A,
1. Introduction
Since the end of 1970s, cisplatin [cis-Pt(Cl2 (NH3 )2 ] (Fig. 1) has
been widely used in the clinics for cancer therapeutics, in particular to treat and even cure a few solid tumors that manifest a high
chemo-sensitivity toward platinum drugs, such as testicular and
ovarian cancers [14].
The in-vivo molecular mechanism of cisplatin, which behaves
as a classical prodrug, involves most probably its aquation and
subsequent DNA binding [57]; in turn, Pt binding induces large
Fig. 1. Structure of cisplatin.
2016 Elsevier B.V. All rights reserved.
structural modications of the DNA double helix, ultimately

leading to cancer cell apoptosis [4]. Although DNA is the commonly accepted primary target for cisplatin [8,9], interactions
between cisplatin and a variety of intracellular biomolecules
(in particular thiol-rich or His-rich) are also very important
owing to the high reactivity and afnity of Pt compounds
toward S- and N-donors [10]. Indeed, the process of proteincisplatin recognition is reputed crucial in determining cisplatin
transport, its cellular uptake, biodistribution and toxicity prole
[11,12].
After injection into the bloodstream, most of the platinum (65
to 98%) deriving from cisplatin is associated with proteins [13], in
particular to hemoglobin [14], serum albumin [1517] and transferrin [18,19]. In addition, a signicant portion of Pt is bound to
-glutamyl-cysteine-glycine (glutathione, GSH) [20] and/or other
cysteine-rich biomolecules [21] like a few small proteins of the
metallothionein family [22]. Cisplatin may enter cells with the help
of proteins belonging to the so called copper trafcking system
[23] including members of the Copper TRansport (Ctr) protein family, like Ctr1 [24,25] and possibly its homologue Ctr2 [26], which
are both responsible for copper cellular uptake. The ATPase copper pumps ATP7A (Menkes disease protein) and ATP7B (Wilsons
disease protein) are most likely responsible for sequestration and
efux of cisplatin [25,27,28]; the copper chaperone Atox1 possibly
plays a key role in delivering cisplatin to ATPases [29]. Since Atox1
translocates to the nucleus in response to copper exposure, this latter protein might be also involved in the delivery of cisplatin to DNA
[30]. Cox17, a copper chaperone associated with copper transfer to
mitochondrial proteins, seems to be involved in cisplatin transfer
to mitochondria [31].
Therefore, exploring how cisplatin interacts with proteins is
now indispensable for understanding in more depth the mechanisms of protein-drug recognition, for studying the inherent or
acquired cisplatin resistance processes, for the design of new therapeutic agents possibly manifesting a reduced toxicity toward
slowly proliferating or terminally differentiated cells. Several techniques have been used so far to characterize cisplatin binding sites
on proteins. Nuclear magnetic resonance spectroscopy (NMR) has
been often applied to locate the cisplatin binding sites on human
serum albumin (HSA) [32], cytochrome c (cyt c) [33], transferrin
[34], Ctr1[35], Atox-1 [3639], ATPase [40] and on the metalbinding domain of Menkes disease protein [35,40] and Wilsons
disease protein [41]. Conversely, mass spectrometry strategies,
like top-down mass spectrometry [42] or electrospray ionization mass spectrometry (ESI-MS) [42], combined with proteomics
approaches, have represented powerful tools to identify platinated
proteins and to determine the cisplatin binding sites on proteins
[4347,31].
Using the above mentioned approaches the interactions of cisplatin with HSA [48], insulin [49], cyt c [50], calmodulin [51,52],
myoglobin [53] and hemoglobin [54], ubiquitin [44,55] [56], copper chaperone Atox1 [57], Cox17 [31], ribonuclease A (RNase A)
[58], insulin growth factor [59], transferrin [19], 2-macroglobulin,
1-anti-trypsin, apolipoprotein A1 and A2 [60], bovine erythrocyte superoxide dismutase [61], Sp1 Zinc nger protein [62,63],
2-microglobulin [64] were investigated and described extensively. Recently, mass spectrometry analysis carried out after gel
electrophoresis and Coomassie blue staining allowed the identication of several cisplatin-binding proteins, including myosin
IIA, glucose-regulated protein 94, heat shock protein 90, calreticulin, valosin containing protein, and ribosomal protein L5
[65]. Computational methods (Quantum Mechanics/Molecular
Mechanics (QM/MM) calculations and molecular dynamics (MD)
simulations) were also exploited to unveil the structural determinants of cisplatin recognition by Ctr1 [6668] and Atox-1
[35,69].
The interaction of cisplatin and related Pt-based drugs with proteins has been already reviewed in the past [1,12,7073]. However,
during the last few years, the results obtained on these systems in
solution through spectroscopic and spectrometric methods have
been effectively complemented by a substantial amount of new
crystallographic data, which provide not only the exact location and
nature of metallic fragments bound to the protein but also details on
the interactions that cisplatin fragments establish with the nearby
protein residues. Accordingly, in this review article, we describe the
structural outcome of Pt metalation associated with cisplatin binding to proteins where such information is directly available from
X-ray crystallography data. Table 1 shows an overview of the structures of the protein-cisplatin adducts discussed here. Notably, early
structural studies were mostly performed by analyzing the reactivity of cisplatin with relatively small model proteins; more recently,
these investigations have been extended to systems of higher complexity and greater physiological relevance such as HSA, the major
plasma protein.
69
Fig. 2. Ribbon representation of the molecular model of the beSOD-cisplatin adduct

(PDB code 2AE0) [74]. The fourth ligand of Pt in the cisplatin fragments is indicated
as x.
2. A gallery of molecular structures for cisplatin-protein

derivatives
Here, the available high resolution molecular structures for
protein derivatives of cisplatin are systematically analyzed and discussed. In total, more than 40 distinct structures will be considered,
as reported in Table 1. All the described structures have appeared
between 2006 and 2015 thus lling, in a relatively short time lapse,
a surprising gap in the general knowledge. Cisplatin derivatives
suitable for X-ray structure analysis have been obtained with a variety of small model proteins such as cyt c, RNase A, lysozyme but
also with proteins of greater mass and importance such as superoxide dismutases (SODs) and HSA. In the case of lysozyme several
different models are available, obtained under a variety of experimental conditions. In general, the high resolution of the available
structures allows elucidation of the binding interactions of the platinum center(s) with the protein side chains and also, in most cases,
determination of the exact nature of the Pt fragment bound to the
protein and identication of the coordination geometry around the
Pt center. Below the individual structures are analyzed in detail.
2.1. Superoxide dismutases
SODs are dimeric metalloenzymes that catalyse the dismutation
of superoxide anion radicals into molecular oxygen and hydrogen
peroxide. Bovine erythrocyte superoxide dismutase (beSOD) was
the rst protein to be studied from the structural point of view in
complex with cisplatin [74].
Crystals of beSOD-cisplatin, obtained after incubation of the protein at a 1:10 protein:metal ratio for two weeks at 4 C, diffracted
X-ray at 1.8 A resolution [74]. The structure of beSOD-cisplatin (PDB
code 2AE0) revealed that cisplatin binding does not induce any
global protein conformational changes when compared with the
structure of beSOD (PDB code 1SXS); Pt selectively binds His19,
which is the primary anchoring site for the drug (Fig. 2).
The occupancy of the Pt centre close to His19 is slightly different for the two chains of the beSOD dimer with values of 0.7 and
0.6, in chain A and B, respectively. B-factors for the Pt atoms are
in the range 1316 A 2 , only slightly larger than the value observed
in average for the protein atoms, which is as low as 7.3 A 2 . Unexpectedly, in this structure, clear evidence is offered for ammonia
release from cisplatin. The Pt atom is bound to ND1 of His19 with a
Two chloride ions with a PtCl distance of
bond length of 2.2 0.1 A.
2.32.5 0.1 A coordinate to Pt. The fourth coordination position is
probably occupied by a very weakly bound water molecule, which
has not been included in the nal model (Fig. 3). The angle between
the two chlorides and the Pt centre lies between 93.5 and 97.1 . A
70
Table 1
Structures of cisplatin-protein adduct reported in the Protein Data Bank.
Protein
Resolution ()
PDB code
Data collection
temperature (K)
Metal
binding site
Reference
Bovine Cu/Zn SOD

Plasmodium falciparum Glutaredoxin 1
Human Cu/Zn SOD
Hen egg white lysozyme (HEWL)
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
HEWL
RNase A
1.80
1.87
1.97
1.70
2.28
3.00
1.80
3.00
2.10
2.10
1.80
2.00
2.80
2.90
0.98
1.12
1.42
1.69
1.70
1.70
1.70
1.70
2.10
1.70
3.00
1.69
1.59
1.90
2.00
0.98
1.85
1.95
1.85
2AE0
4N10
4N11
4N0Z
3RE0
4DDB
4DDC
4G49
4G4A
4G4B
4GCB
4GCC
4GCD
4GCE
4MWK
4MWM
4MWN
4OWB
4DD0
4DD1
4DD4
4DD6
4OW9
3TXG
3TXK
3TXF
3TXB
2I6Z
4YEN
4YEO
4Z46
4ZEE
4OT4
100
100
100
100
100
100
100
295
295
295
100
300
300
300
294
200
294
295
100
100
295
295
295
295
277
295
295
100
295
294
100
100
100
[74]
To be published
To be published
To be published
[78]
[85]
[85]
[84]
[84]
[84]
[175]
[175]
[175]
[175]
[89]
[89]
[89]
[87]
[85]
[85]
[85]
[85]
[85]
[178]
[178]
[178]
[178]
[83]
[90]
[90]
[86]
[86]
[97]
RNase A
1.95
4RTE
100
Human copper chaperone Atox-1 (dimer)
2.14
3IWX
113
His19
His49
Cys29
His49
Cys111
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
His15
Met29
Met29/Gln28
Met29
His105
His119
Cys12
Cys15
Human copper chaperone Atox-1 (dimer)

Human copper chaperone Atox-1 (monomer)
2.14
1.60
4YEA
3IWL
113
113
Human copper chaperone Atox-1 (monomer)
1.60
4YDX
113
Cytochrome c
2.19
4RSZ
100
HSA
3.16
4S1Y
100
careful inspection of the structure revealed a tight contact between

a Cl atom and the main-chain carbonyl oxygen atom of Thr17 (the
distance is 3.2 0.1 A).

The unexpected reactivity of cisplatin with beSOD, involving the
release of the ammonia ligands, was tentatively explained in terms
of a specic protein polarizable microenvironment that is likely
formed when the protein reacts with the drug. Theoretical calculations reveal that this peculiar situation greatly decreases the
thermodynamic trans inuence, enhances the kinetic trans effect
at the platinum center and thus favours the release of (at least)
one ammonia ligand [7577]. Losing of ammonia ligands instead
of chlorides has been also observed when studying the reaction
between cisplatin and the eight-residue long peptide MTGMKGMS,
which mimics one of the extracellular methionine-rich motifs of
yeast Ctr1 [67].
Interestingly, cisplatin binding to beSOD signicantly differs
from what observed when the drug reacts with the human
Cys12
Cys15
Cys12
Cys15
Met65
Glu61
Met298,
Met329,
Met548,
His288,
His305
[111]
[121]
[90]
[121]
[90]
[108]
[159]
enzyme [78]. In this latter case the primary cisplatin binding

site is a cysteine residue (Cys111), which is replaced by a Ser
residue in beSOD (beSOD His19 is an Asn residue in hSOD)
(Fig. 4). In particular, the interaction of cisplatin with hSOD
takes place through Pt-coordination to the sulfur atoms of the
Cys111 residues of the two subunits of the dimer. Furthermore,
in hSOD-cisplatin, at variance with what is observed in the case of
beSOD-cisplatin adduct, the coordination sphere of Pt(II) presents
a more usual chemical environment: beyond the sulfur atom of
Cys111, the Pt(II) coordination sphere is completed by two NH3
molecules and one Cl ion (Fig. 5). Structural analyses reveal
that there is no space to host two cisplatin moieties at the same
time.
The structure of hSOD-cisplatin adduct presents two dimers
in the asymmetric unit (a.u), i.e. two hSOD dimers constitute the
part of the crystallographic unit cell which can be used to generate the whole crystallographic cell by using the symmetry of
Fig. 3. Cisplatin binding site in beSOD [74]. Pt is coordinated by the NE2 atom
of His19 and by two chloride ions and probably by a weakly coordinated water
molecule, which is not shown in the Fig. since it is not included in the deposited
molecular structure. In the Fig. the residue Thr17 and Asp50 are also shown. The
fourth ligand of Pt in the cisplatin fragment is indicated as x.
71
Fig. 6. Cisplatin binding site in HEWL. Pt is bound to ND1 atom of His15 with a
Two N atoms from ammonia molecules (PtN distances
bond length of 2.1 0.2 A.
are coordinated to the Pt centre. No electron density has been detected
of 2.0 0.2 A)
for the fourth Pt ligand (PDB code 2I6Z) [83]. The apparent dissociation constant for
HEWL-cisplatin adduct has been evaluated to be KD = 1.691.81 103 M [86].
observed, ruling out the presence of additional cisplatin binding

sites on protein structures.
2.2. Lysozyme
Fig. 4. Ribbon representation of the molecular structure of the hSOD-cisplatin

adduct (PDB code 3RE0) [78].
Fig. 5. Cisplatin binding site in hSOD [78]. Pt is coordinated by the SG atom of Cys111
of the two chains. Cys111 face each other at the dimeric interface, so that there is no
space to accommodate two cisplatin fragments at the same time. Pt coordination is
completed by two ammonia molecules and a chloride ion.
the space group. In each of the four chains of this structure, Pt(II)
atom has an occupancy factor = 0.4. PtSG distances are in the range
2.22.4 0.3 A.
Quite unexpectedly, in both beSOD-cisplatin and hSOD-cisplatin
structures, no other signicant peaks of the electron density were
Hen egg white lysozyme (HEWL) has been widely studied

since its rst characterization by Alexander Fleming [79]. Under
physiological conditions, the protein exists as a monomer containing 129 residues, corresponding with a molecular mass of
approximately 14 kDa. HEWL catalyzes the hydrolysis of the 1,4-linkages of peptidoglycans comprising the cell walls of bacteria. The
native structure of this relatively small globular enzyme is folded
into an -domain (residues 138, and 86129) and a domain
(residues 3985), containing primarily -helical and -sheet secondary structures, respectively. The scaffold of HEWL has been
considered particularly suitable to probe the fundamental interactions of proteins with metal complexes [8082].
HEWL-cisplatin is undoubtedly the most studied protein adduct
of the drug. The rst X-ray structure of HEWL-cisplatin complex was solved at 1.9 A resolution in 2007 [83]. HEWL-cisplatin
crystals were obtained at 277 K in a typical soaking experiment:
tetragonal HEWL crystals (space group P43 21 2) grown in 50 mM
NaCl and 5 mM sodium acetate buffer pH 6.5 have been incubated three days in a drop containing the reservoir solution and
cisplatin (1:10 protein:metal ratio). This structural determination
highlighted that the Pt centre (occupancy = 0.3) is bound to the ND1
atom of His15 atom and to the nitrogen atoms from two ammonia ligands (Fig. 6). The fourth Pt ligand is not detectable: it might
correspond with a loosely bound/disordered platinum-coordinated
chloride or to a water molecule. This result is in good agreement
with ESI MS spectra of HEWL-cisplatin derivatives that gave two
well resolved peaks at 14,569 and 14,605 Da, corresponding with
either a [Pt(NH3 )2 Cl]+ fragment or intact cisplatin bound to HEWL,
respectively [83].
More recently, co-crystallization experiments conducted by
Helliwell and co-workers revealed that cisplatin binds HEWL using
dimethyl sulfoxide (DMSO), but not using aqueous media, by coordinating both the ND1 and the NE2 atom (i.e. it can bind at the both
so-called right-handed and left-handed sites) of the imidazole
ring of His15 [84,85] (Fig. 7AC). In this bis-cisplatin adduct, the
binding of the Pt center to the His is associated with the release of
72
Fig. 7. Cisplatin binding site in HEWL in the X-ray structures of the adduct solved by Tanley et al. [84], PDB code 4DD4 (panel A) [85], 4DD6 (panel B) [85], 4DDB (panel
C)[85]. The rst structure has been obtained in the presence of DMSO and using glycerol as cryoprotectant, the other two structures in the presence of DMSO at pH 4.7 and
6.5, respectively, and cryocooled using paratone as cryoprotectant. In all cases, Pt is coordinated by both ND1 and NE2 atoms of His15. Similar results have been obtained in
the presence of N-acetylglucosamine (NAG), which is a natural substrate of HEWL (PDB code 4DDC), and after prolonged exposure to cisplatin in both aqueous (PDB code
4G49) and DMSO media (PDB code 4G4A) [84]. The geometry of cisplatin ligands in these structures is under revision by Helliwells group.
one Cl ligand (Fig. 7A and B) or of one ammonia ligand (Fig. 7C).

Bis-cisplatin adducts were also observed upon reacting cisplatin
with ubiquitin [44,55] and horse heart myoglobin [53]. Similar
results emerged from further structural characterizations of HEWLcisplatin adducts that were obtained after prolonged exposure of
crystals to cisplatin (714 months) in both aqueous and DMSO
media [84].
For the binding of two Pt ions to occur to the imidazole ring
of a His side chain, both the ND1 and NE2 atoms have to be
sp2 -hybridized with nitrogen lone pairs in the plane of the imidazole ring. This implies that the hydrogen of one of the imidazole
N atoms has to be removed. This situation occurs in crystals of
HEWL-cisplatin being greatly facilitated by the crystallization conditions, which involve a high concentration of chloride and acetate
ions.
Subsequently, the Helliwell group also studied the relative binding afnities of cisplatin and carboplatin to HEWL His15 side chain.
Using the same molar concentration of cisplatin and carboplatin (a
three-fold excess with respect to HEWL) these authors determined
which of these anticancer compounds manifests a larger afnity
for binding to HEWL His15 [70]. Five independent structures were
rened with resolutions falling in the 1.83.5 A range:one at 100 K

and four at 300 K. Both the 100 and 300 K X-ray diffraction data sets
showed evidence for greater binding of cisplatin over carboplatin
at both the ND1 and NE2 atoms of His15 side chain, particularly
close to ND1 atom of His15, where a clear anomalous difference
electron density is observed for the Cl atoms in the 100 K structure.
In the same work the effect of a high X-ray radiation dose (up
to 1.8 MGy) on the stability of the platinated protein adducts [70]
was also assessed. Data revealed that cisplatin does not dissociate
from the His15 side chain with the adduct remaining stable over
prolonged exposure to X-ray irradiation.
Interestingly, crystallographic studies of carboplatin binding to
His15 in HEWL revealed a partial chemical conversion of this drug
to cisplatin due to the high sodium chloride concentration used in
the crystallization conditions [87]. In these structures, unrestrained
values for Pt to His15 nitrogen distances are within the range
The occupancy factor for the Pt centre is within the
2.12.6 0.1 A.
range 0.530.71 and 0.400.59 for the two sites.
Conversion of carboplatin to transbromoplatin was observed in
the structure of HEWL cocrystallized in the presence of the Pt drug
and NaBr: in this structure, at the binding site close to ND1 atom
of His15, there are two large anomalous difference density peaks
at distances of 2.5 0.1 A from Pt centre, which were assigned to
Br atoms [88]. Similar results, i.e. conversion to the respective trans
halide derivative, transiodoplatin, were obtained for both cisplatin
and carboplatin in crystals grown in NaI [87]. These structures,
solved using monoclinic crystals with two molecules in the a.u.
(space group P21 ), also show the presence of an additional Pt compound binding site in a packing crevice.
Recently, by using X-ray diffraction data collected for the adduct
at different temperatures (150 K, 200 K and 295 K) on triclinic crys
tals, which diffract at ultra-high and high resolutions (from 0.98 A),
Tanley and Helliwell were also able to observe several platinum
binding modes at His15 and multiple conformations of His15 side
chain [89]. Overall, these results reveal a versatile binding of cisplatin to the His15 side chain.
A re-interpretation of crystallographic data collected at 150 K
for triclinic crystals of HEWL coand at atomic resolution (0.98 A)
crystallized with cisplatin and of data collected on tetragonal HEWL
crystals stored in DMSO media and in the presence of cisplatin for
14 months was proposed very recently [90].
This analysis reveals that in the former structure, the Pt binding
site close to His15 exhibits a large exibility, since two conformers coexist with low Pt occupancy. In this structure, a [Pt(NH3 )Cl2 ]
fragment binds NE2 atom of His15. Thus, as in the case of the
beSOD-cisplatin structure, cisplatin should release an ammonia
ligand upon binding. Additional Pt2+ ions are also present on the
surface of HEWL, close to N-terminal Lys1 and at about 2.9 0.1 A
from ND1 of His15. In the latter structure (HEWL crystals stored in
DMSO media and in the presence of cisplatin), a [PtCl3 ] fragment
is bound at the ND1 atom of His15, whereas a [PtCl2 ] fragment is
bound to NE2 atom of His15 and to a NH1 of Arg14 side chain at
the same time. Interestingly, these data indicate that Pt centre can
bind the N atoms of the side chain of an Arg residue, in agreement
with what recently suggested by Sadler and co-workers [47].
Altogether, these data suggest an intrinsic difculty in the interpretation of the electron density maps associated with cisplatin
binding to protein side chains; indeed, they show that cisplatin
binding sites manifest an unusual exibility and reveal that different fragments can bind proteins, also at the same binding site.
These observations point out that much caution should be taken
in the interpretation of the electron density maps of complexes
between proteins and metallodrugs (see also [91]).
While carboplatin and cisplatin concur for the same HEWL binding site (His15), a different behavior is observed when competition
experiments were carried out using cisplatin and oxaliplatin [86].
In fact, oxaliplatin binds the side chain of Asp119 [92] so that a
bis-Pt adduct with both cisplatin and oxaliplatin fragments bound
to HEWL can be formed. In this case both the Asp119 and His15
binding sites are occupied [86].
HEWL was also used as a model to study the interaction of iodide
analogues of cisplatin with proteins [93]. In this framework, it is
interesting to mention the case of cis-Pt(NH3 )2 I2 , which surprisingly was recently found to be more cytotoxic than cisplatin in
cisplatin-sensitive cancer cells and to overcome cisplatin resistance
in cisplatin-resistant cancer cells [94].
Studies in solution demonstrated that cis-Pt(NH3 )2 I2 interacts
with proteins releasing different Pt ligands at diverse pHs [94]. In
particular, when the reaction was carried out at neutral to basic pH,
the potential drug reacts with proteins as expected on the basis of
the cisplatin behavior; i.e. it forms adducts releasing one iodido
ion [94]. On the contrary, at acidic pH, the Pt compound releases
an ammonia ligand, whereas it retains the iodido ions [95]. This
observation was conrmed by crystallographic data showing that
a [Pt(NH3 )I2 ] fragment binds ND1 atom of His15 [93]. In this structure, peculiar structural features were observed: two alternative
73
Fig. 8. Iodido analogue of cisplatin (cis-Pt(NH3 )2 I2 ) binding site in HEWL crystals at

acidic pH (PDB code 4MR1) [93] The two alternative conformations of the [Pt(NH3 )I2 ]
fragment are reported in panel A and B.
I atoms are coloured in violet. (For interpretation of the references to color in this
gure legend, the reader is referred to the web version of this article.)
modes of binding for the [Pt(NH3 )I2 ] fragment were found at ND1
atom of His15 (Fig. 8). The comparison between the structure of
HEWL-cis-Pt(NH3 )2 I2 and that of the adduct that the same protein
forms with cisplatin suggests that ND1 is the kinetically preferred
binding site for Pt-based drugs; the drugs could bind NE2 atom
of the same His in a second step. This suggestion is conrmed by
further crystallographic studies of adducts between the protein
and other potential Pt-based anticancer agents that preferentially
bind the ND1 atom of His15 [96]. Data are in good agreement with
the experimental nding that structures of HEWL-carboplatin from
crystals obtained under the same experimental conditions with different Pt compound soaking times reveal that carboplatin binds
rst at ND1 atom and then at both the ND1 and NE2 atom of His15
[88].
The structure of the adduct that cis-Pt(NH3 )2 I2 forms with HEWL
at neutral to basic pH remains to be determined.
2.3. RNase A
On the basis of the X-ray crystallographic studies above discussed, it may be inferred that cisplatin binding takes place
predominantly to a single protein site, close to imidazole rings
of solvent exposed His residues so that a high number of His in
a protein could importantly contribute to cisplatin binding [65].
However, the structural results obtained for adducts of cisplatin
with bovine pancreatic ribonuclease (RNase A) are exemplary for
a different situation. Indeed, the structure of the primary adduct
formed in the reaction between cisplatin and RNase A (RNase
A-cisplatin adduct) represents the rst high resolution X-ray structure for a protein adduct where cisplatin is bound close to a
74
methionine side chain [97]. The Met-Pt binding is of general interest, since it features a kind of reactivity that may occur between
cisplatin and a variety of biological targets. Reedijk suggested that
the Met-cisplatin adduct could play a key role in the transfer of
cisplatin fragments from proteins to DNA [98,99]. Met-Pt intermediates can form in the cell playing a major role in the mechanism
of action of anticancer Pt compounds. In this respect, it is worthy
to recall that binding of cisplatin to Met activates the ligand in the
trans position [100] and that platination rates of both GMP and DNA
are substantially enhanced when assaying Met adducts of trans-Pt
compounds (the same adduct with cisplatin reacts slightly faster
with GMP but slower with DNA)[101].
RNase A is one of the rst proteins whose structure was determined by X-ray crystallography; accordingly it has been used as a
model system in pioneering studies in many elds of protein chemistry, including enzymology, ultra-high resolution crystallography
and chemical synthesis [102].
RNase A cleaves and hydrolyzes the phosphodiesteric bond at
the 5 -ribose of single-stranded RNA (ssRNA) in two distinct steps
[102]. In the former, the His12 side chain extracts a proton from
the 2 -OH of ssRNA, thus facilitating its attack on phosphorus atom,
whereas the His119 side chain protonates the 5 -O, facilitating its
release. In the latter step, a water molecule attacks the 2 ,3 -cyclic
phosphodiester, producing a phosphate monoester on C3 of the
ribose sugar unit on RNA.
The RNase A-cisplatin structure, solved at 1.85 A resolution,
reveals several details of the drug-protein interaction. It is appropriate to mention the way in which crystals of the RNase A-cisplatin
adduct were formed and treated, since this procedure can be in
principle used on crystals of other enzymes. Crystals of the RNase
A-cisplatin adduct were obtained by soaking procedure. Monoclinic
RNase A crystals have been grown by hanging drop vapour diffusion
method using 20% (w/v) polyethylene glycol (PEG) 4 K and 20 mM
sodium citrate buffer at pH 5.0 as precipitant. Soaking was carried out for 3 h at 298 K with RNase A crystals in drops to which
a solution of cisplatin (1:10 protein to metal ratio) dissolved in
the reservoir solution had been added. Soaked crystals, which presented signicant cracks on their surfaces, were removed from the
drop in a nylon loop, dehydrated at air and then frozen at 100 K.
This dehydration procedure [103] removes the need for cryoprotectants to be used, ruling out any effect that the cryoprotectants
might produce on cisplatin binding to the protein [104].
The comparison of the protein structure in the RNase A-cisplatin
adduct with previously determined ligand-free RNase A structures
from isomorphous crystals (PDB code 1JVT) [105] shows that the
binding of the drug does not induce any signicant structural variation (Carbon alpha (CA) root mean square deviation (rmsd) in the
The Pt drug binds close to Met29 in both the
range 0.370.67 A).
molecules present in the a. u. (chains A, B) (Fig. 9).
Met29 was already identied as a Pt binding site in the complex formed between RNase A and PtCl4 2 [106]. Different binding
modes are observed in the two chains: the Pt center is anchored to
the protein either in a monodentate (molecule A) or in a bidentate
fashion (molecule B) (Fig. 10A and B). In particular, in molecule A,
three ligands originating from cisplatin remain bound to the Pt centre: a chloride ion and two ammonia molecules. The Pt coordination
geometry is nearly square planar as it is usually found for Pt(II) complexes. In molecule B, the Pt coordination sphere is completed by
the presence of the side chain of residue Gln28. This nding has
provided, for the rst time, structural evidence for the existence
of a bidentate mode of cisplatin binding to protein residue side
chains. Bidentate binding was previously suggested on the basis
of mass spectrometry data collected on insulin-cisplatin [107] and
ubiquitin-cisplatin [44,45,55] adducts and subsequently observed
also in the structures of cyt c-cisplatin [108] and glutaredoxincisplatin adducts (see below).
Fig. 9. The asymmetric unit content of the RNase A-cisplatin adduct. The two
molecules in the a. u. are colored in gray and cyan, respectively [97].
Data are also in very good agreement with ESI-MS spectrum of

the RNase A-cisplatin derivative that exhibits a peak at 13,909 Da,
which well corresponds with the binding of a [Pt(NH3 )2 ]2+ moiety
to RNase A (with concomitant release of the two chloride ligands) and a peak at 13968 Da, which should correspond with a
[Pt(NH3 )2 Cl]+ fragment + Na+ bound to the protein [58].
In molecule A, on the basis of the inspection of residual peaks
in the Fo-Fc electron density map, the existence of two alternative
modes of binding for cisplatin was proposed. The occupancy of the
Pt centre close to Met29 is slightly different for the two molecules
in the a.u. with values of 0.75/0.25 for the alternative conformation observed in molecule A and 0.55 for molecule B, respectively.
B-factors for the Pt atoms are in the range 28.734.8 A 2 , in line
with the average B-factor of the overall structure (27.8 A 2 ). Unrestrained PtS distances are 2.32.5 0.2 A for the A and B molecule
respectively.
The overlay of the two molecules in the asymmetric unit of
the RNase A-cisplatin structure reveals that the cisplatin fragments have different orientations (Fig. 12A and B). The variability
observed in the orientations of the cisplatin fragment within Met29
site suggests that the coordinated fragment possesses a high exibility.
Notable features of the RNase A-cisplatin structure are the
details of the interactions between the Pt ligands and the protein.
In particular, in the main cisplatin binding site of molecule A one of
the two ammonia ligands is hydrogen bonded to carbonyl oxygen
of Ser32, whereas the Pt-coordinated water molecule is hydrogen
bonded to the side chain of Arg33 (Fig. 10A). In the alternative cisplatin binding the NH3 ligand is hydrogen bonded to the side chain
OD1 atom of Asp14. In molecule B one of the two ammonia ligands
is hydrogen bonded to two water molecules, whereas the other is
in direct contact with the side chain OD2 atom of Asp14 (Fig. 10B).
These interactions may be an important feature, potentially stabilizing the protein adduct. In this respect it is important to highlight
the role of the leaving and non-leaving/carrier groups in platinum
drugs-protein interaction in general, as depending on their nature
(bulkiness, hydrophobicity etc.), conguration and structure. The
ligands may drive the complex toward a different kind of reactivity,
as suggested in other papers [93,95,109].
The structure of RNase A-cisplatin adduct above described was
recently compared with those of the adducts that the same protein forms with carboplatin and oxaliplatin, under comparable
experimental conditions [58]. This comparative structural analysis
suggests that cisplatin and carboplatin behave similarly when they
react with proteins, since both drugs mainly binds Met29 side chain,
while oxaliplatin shows a signicantly different behavior, as this
75
Fig. 10. Cisplatin binding site in the two molecules of the a. u. of the RNase A-cisplatin adduct [97]. In molecule A, Pt is coordinated by the SD atom of Met29, to two ammonia
ligand and a water molecule that replaces one chloride ligand (monodentate fashion) (panel A). In molecule B, Pt centre is bound to SD atom of Met29, NE2 atom of Gln28
ion and two ammonia molecules (bidentate fashion) (panel B). The binding to this site induces disordering of residues 1622, in particular in chain B. The overall binding
constant for RNase A-cisplatin complex is estimated to be K = 5.6 103 M1 [110].
drug also binds Asp14 and side chains of His105 and His119. The
different adduct formation observed when cisplatin, carboplatin
and oxaliplatin react with RNase A [58,97] is in line with the results
obtained with HEWL [70,86,92]. These structural differences that
are probably related to a different kinetics of metallodrugprotein
binding may have a more general signicance and may be at the
basis of the different pharmacological and toxicological prole of
the three drugs.
RNase A has been also used to study the way by which cisplatin induces the formation of protein dimers [111], since this
molecule is often employed as a prototype in experiments of protein aggregation [112]. The reactions between RNase A and cisplatin
at four distinct cisplatin/protein molar ratios comprised between
2.5 and 15 (0.20 M sodium phosphate pH 6.7) were monitored by
gel electrophoresis and chromatographic studies with the aim to
gain structural information on the aggregation state of the various RNase A-cisplatin adducts (Fig. 11). 24 h incubation of the
bovine enzyme in the presence of an excess of cisplatin leads
to formation of a platinated monomer (PtM, whose structure
has been solved by X-ray crystallography at 1.85 A resolution),
a platinated dimer (PtD), a platinated trimer (PtT) and a few
higher oligomers, whose structural and functional features are distinct when compared with those of the previously characterized
swapped dimers, trimers and higher oligomers of the same protein
[112116]. Oligomerization yield depends on the cisplatin amount
used. Puried monomeric, dimeric and trimeric platinated RNase
A species were characterized from the structural and biochemical point of view. The proteins are correctly folded, as judged
by circular dichroism (CD) spectra, and retain a limited level of
ribonuclease activity. Platinated dimer and platinated trimer are
slightly more active than RNase A with respect to double stranded
DNA.
The structure of PtM, which has been solved under different
experimental conditions (and in a different space group) [111]
when compared with that of the RNase A-cisplatin adduct above
described [97], reveals that the protein can bind cisplatin at three
different binding sites: close to Met29 (occupancy factor = 0.75),
as previously observed [97], but also close to His105 and His119
side chains (occupancy factor = 0.40.5) (Fig. 12). The tris-cisplatin
adduct has been also observed in solution upon reaction between
cisplatin and ubiquitin [44]. As also found in the RNase A-cisplatin
Fig. 11. Electrophoretic behavior of the isolated PtT, PtD and PtM compared with
native RNase A. PtM, PtD and PtT were analysed side-by-side with untreated
RNase A, to PolyAcrylamide Gel Electrophoresis (PAGE) under both denaturing (A)
and native (B) conditions at 4 C [111]. As expected, RNase A migrates as a single
species, with electrophoretic mobility clearly higher than those displayed by dimeric
and trimeric platinated forms, both under native and denaturing conditions. The
different reaction ratios (1:5, 1:10 and 1:15) did not affect charge exposition of the
products and, consequently, the electrophoretic mobility of platinated oligomers.
Courtesy of Prof. G. Gotte, University of Verona, Italy.
structure [97], in the main binding site Pt centre is bound to SD atom

of Met side chain retaining two ammonia molecules and a chloride
ion as ligands (Fig. 15A); it is likely that two distinct conformations
of the cisplatin fragment coexist in this region.
76
Fig. 12. Cartoon representation of PtM [111].
In the other two (minor) binding sites, the Pt center is bound

to a N atom of His105 or His119 side chains after releasing of a
chloride ion (Fig. 13B and C) [111]. Since His119 is in the active
site of the protein, these data could partly explain the reduction of
the enzymatic activity of RNase A in the presence of cisplatin. The
comparison between the structure of PtM [111] and that of the
main adduct that forms in the reaction between cisplatin and RNase
A above described [97] suggests that the protein metalation process
may manifest a large degree of kinetic variability depending on the
applied experimental conditions.
The structures of PtD and PtT have not yet been solved, but
CD data indicate that they should adopt the same folding of the
platinated monomer. The two chains in the dimer could be held
together by the covalent binding of protein side chains belonging to different RNase A molecules to the same Pt atom. In this
respect, it should be reminded that the high trans effect of Met
sulfur atom can lead to possible displacement of Pt ligands, thus
favoring the formation of protein cross-linking. The ability of a platinum complex to cross-link residues from different chains has been
already observed for the human Ctr1 copper transporter [117]. Furthermore, it has been proved that Pt from cisplatin can cross-link
His6 of angiotensin II and His12 of bombesin [51] and residues
Met51Met71/Met72, Met109Met124, Met109Met144, and
Glu127 or Asp129Met144Met145 of apo-calmodulin [51,118].
The ability of cisplatin to induce the formation of protein dimers
was also observed in the case of Atox-1 (see below) and HSA
[73,119] and suggested for insulin [47].
Altogether these results indicate that:
(1) different patterns of protein platination are possible within the
same protein system (mono-adduct, bis-cisplatin adduct, triscisplatin adduct),
(2) different binding modes (monodentate or bidentate) are
allowed even within the same cisplatin binding site,
(3) ligands coordinated to the Pt atom can ne tune the interactions
between cisplatin and proteins,
(4) the number of binding sites and the kind of adducts formed
in the reaction between cisplatin and proteins may depend
critically on the experimental conditions used to study the
formation of the adduct and may manifest large kinetics variability,
(5) cisplatin is able to induce the formation of higher oligomers of
proteins via crosslinking.
Fig. 13. Cisplatin binding sites in PtM. (A) Met29, (B) His105, (C) His119. It is
notable that in PtM, cisplatin fragment retains one chlorine. This is not surprising since crystals of this adduct were grown in 3.0 M sodium chloride and 30%
ammonium sulfate [111].
Fig. 14. Cartoon representation of the monomeric structure of the copper chaperone
Atox1 in complex with cisplatin [124].
2.4. Atox-1
Cisplatin may take advantage of cellular copper-transport proteins to enter and to be exported from the cell [120122]. Copper
chaperone Atox1 is a 68 residues protein consisting of a compact ferredoxin-like 1 2 3 4 5 6 structure, which binds Cu(I)
through a conserved CXXC motif located at the solvent-exposed
1-1 loop and then delivers it to the N-terminal binding domains
(MBDs) of active membrane transporters ATP7A (Menkes disease
protein) and ATP7B (Wilsons disease protein). Atox-1 binds cisplatin and regulates its accumulation in the cells [41,123]. It is
possible that Atox-1 delivers the drug to both ATP7B and ATP7A,
since these two proteins are linked to tumor resistance to cisplatin.
77
Two structures of Atox-1 complexed with cisplatin were

reported. In both structures Pt is bound to the cysteines in the Cu
binding site. In the former structure, determined at 1.6 A resolution
(PDB code 3IWL) [124,125], there is one Atox-1 chain in the a. u.,
in contrast to previous metal-bridged dimeric structures solved in
the presence of Cu(I), Cd(II), and Hg(II) (Fig. 14).
A mono-adduct is formed, where the Pt centre loses both chlorides and ammonia ligands. In particular, the resulting naked Pt(II)
ion binds to N and S atoms of Cys12, to S atom of Cys15 from the
CXXC motif. A tris(2-carboxyethyl)phosphine (TCEP) molecule with
a P-Pt distance of 2.5 0.1 A completes the coordination sphere of
the metal. The two Cys sulfur atoms occupy trans positions with
respectively. TCEP is
SGPt distances of 2.3 0.1 and 2.4 0.1 A,
hydrogen bonded to the side chain of Lys60, an evolutionary conserved residue which is believed to play a key role in metal transfer
from Atox1 to target proteins (Fig. 15A). The TCEP molecule is used
during the protein purication process that precedes the crystallization trials.
These results are consistent with ESI-MS spectra showing a
species corresponding with Atox1 plus a single Pt(II) ion and a TCEP
molecule [124].
A re-interpretation of the crystallographic data (PDB code
4YDX), recently performed, conrms the correct assignments of the
electron density maps around the Pt centre [90] (Fig. 15B).
However, since TCEP molecules are absent in physiological environments, it is plausible that under these experimental conditions
the [Pt(NH3 )2 ]2+ moiety binds to Cys12 and Cy15 residues of Atox1
keeping the two ammonia ligands, as suggested by in solution
NMR data collected on Atox-1-cisplatin adduct by Calandrini and
coworkers [35]. These authors also provided a structural model
for this adduct by hybrid Car-Parrinello density functional theorybased QM/MM simulations [35]
In the other structure, a dimeric Atox1-cisplatin adduct,
[(Atox1)2 -cisplatin] (PDB code 3IWX, 2.14 A resolution), was found
(Fig. 16). This structural determination is interesting, since experiments revealed that the metal (Cu(I)- Cd(II))-bridged Atox1 dimer
plays a key role in metal transfer between the Atox1 protein
and its target metal binding domains of ATP7B [125127]. Atox1cisplatin crystals contain a single dimer in the asymmetric unit.
The overall arrangement of the two chains in the dimer is similar to that observed for (Cu(I), Cd(II) or Hg(II))-bridged Atox1
dimers (PDB codes 1FEE, 1FE0 and1FE4, [128] with a rmsd of 0.34 A
for CA atoms when compared with Cu(I)-bridged Atox1 dimer.
The overall fold of the two chains of the dimer is similar to that
Fig. 15. Details of cisplatin binding site in Atox-1-cisplatin adduct rened by Boal et al. [124] (A) and. by Shabalin et al. [90] (B). The two structures have exactly the same
orientation. Only minor differences can be noticed. The occupancy factor for Pt ion is 0.75 (B-factor 17.7 A 2 ) in the formed structure, whereas it is 0.90 (B-factor anisotropically
rened) in the latter one.
78
Fig. 16. Cartoon representation of the dimeric structure of the copper chaperone
Atox1 in complex with cisplatin, [(Atox1)2 -cisplatin] [124]. In this structure the
existence of the Pt binding site close to Cys residues has been recently questioned
[90].
Fig. 18. The asymmetric unit content of the Cyt c-cisplatin adduct [108]. The six
molecules in the asymmetric unit (molecules A. . .F) are colored in green, cyan, purple, yellow, pink and gray, respectively. Cisplatin binding sites and heme groups are
shown. (For interpretation of the references to color in this gure legend, the reader
is referred to the web version of this article.)
In the
of monomeric Atox1-cisplatin complex (CA rmsd = 0.30 A).
(Atox1)2 -cisplatin structure, the Pt(II) ion bridges the two Atox1
molecules by binding to Cys15 from each metal binding site with
respectively (Fig. 16).
Cys(S)-Pt distances of 2.3 0.1 and 2.1 0.1 A,
In this model, the two Cys12 residues are too far away to directly
coordinate the Pt centre (Fig. 17A). The two additional Pt coordination sites are occupied by ammonia ligands. The geometry around
the metal centre is nearly square planar with the two Cys15 oriented trans to one another. The adduct is further stabilized by
hydrogen bonds between the ammonia molecules and atoms of the
side chains of residues Thr11 and Cys12. Retention of the ammonia
ligands is in agreement with ESI-MS data.
To further characterize the structure of Atox-1-cisplatin complexes, molecular dynamics simulations were also performed and
compared with MD data obtained for modeled complexes of the
same protein with carboplatin and oxaliplatin [129]. Data reveal
that the three drugs can react with both monomeric and dimeric
Atox1 forming binary and ternary adducts stabilized by a covalent
interaction of the Pt center with the protein [129].
Recently, a re-interpretation of the dimeric (Atox-1)2 -cisplatin
structure has been proposed. In this structure no cisplatin fragment is bound to the protein, whereas a Cu+ ion is bound at the
metal binding site close to Cys12 and Cys15 (Fig. 17B) [90]. This
result is indirectly supported by the observation that Cu-bound
form of Atox-1 is still able to bind cisplatin, thus suggesting that
the Pt binding site is distinct from that of Cu+ [127,130] and by
results by Palm et al., suggesting that cisplatin forms a Cu-Pt Atox-1

complex, where both copper and cisplatin are bound simultaneously to the protein, with the two metals close enough for
possible d-d electronic interactions [131]. In any case, the structural characterization of the dimetal CuPt Atox1 complex deserves
further study.
2.5. Cytochrome c
Cytochrome c (cyt c) is a small electron-transfer protein that
is bound to the outer surface of the inner mitochondrial membrane. This protein has been often used to study protein metalation
processes, mainly because it is small (104 residue, 12 kDa), very stable in solution and commercially available at low cost. However,
studies on the interaction between cyt c and metallodrugs are also
important since cyt c is a likely target for anticancer agents being
a crucial factor in apoptosis. During the execution of apoptosis,
cyt c interacts with the phospholipid cardiolipin and forms a complex with peroxidase activity. Subsequent oxidation of cardiolipin
induces the opening of permeability pores at the outer mitochondrial membrane, which allows the release of cytochrome c from
mitochondria into cytoplasm. In turn, this process triggers a cascade of events ultimately leading to cell apoptosis. It was recently
reported that cisplatin accumulates in mitochondria causing the
release of cyt c into the cytosol [132]. Thus, cyt c turns out to be
essential for activation of apoptosis of cancer cells [133135].
Fig. 17. Metal binding site close to Cys12 and Cys15 in the structure of [(Atox1)2 -cisplatin]. The third cysteine (Cys41) and the methionine (Met10) are conserved residues
constituting the MxCxxC motif (panel A) [124]. A re-interpretation of the electron density map of this binding site suggests that this site is occupied by Cu+ instead of Pt2+
(panel B) [90].
79
Fig. 19. Cisplatin binding site in four out of the six molecules of the asymmetric unit of the Cyt c-cisplatin adduct. In molecules A and F, Pt is coordinated by both the SD atoms
of Met65 and OE2 atom of Glu61, to an ammonia molecule and a chloride ligand (bidentate fashion). In molecules B and D, Pt(II) is coordinated by the SD atom of Met65, to
a chloride ion and to the two N atoms of the ammonia ligands (monodentate fashion). There is no evidence of electron density corresponding with Pt ligands located in the
molecule C. In E, the Pt ion binds the SD atom of Met65 while the other ligands cannot be modeled due to poor electron density [108].
In spite of the several studies reported so far on the interactions of cisplatin with cyt c, controversial opinions still existed
on the Pt protein binding sites and on the nature of the cisplatin moiety bound to the protein. According to results from
ESI-MS experiments carried out using an aqueous solution containing cyt c and cisplatin in a 1:4 to 1:16 molar ratios, the
cyt c[Pt(NH3 )2 (OH2 )]2+ monoadduct is the main product of the
reaction; Met65 being identied as the primary Pt binding site
[136]. Liquid chromatography coupled with LTQ-MS [137] experiments suggested the existence of multiple cisplatin binding sites
on cyt c (Met80, Glu61/Glu62/Thr63, and Met65). Fourier transform ion cyclotron resonance mass spectrometry data pointed out
that [Pt(NH3 )Cl]+ is the main Pt-drug fragment bound to cyt c
and that this fragment binds close to Met65, Met80, His18, and
His33 side chains [50]. However, the possibility that cisplatin could
bind Met80 and His18 seems rather remote, since the side chains
of these residues are also involved in the anchoring of the heme
iron. In order to gain additional information on the interaction
between cisplatin and cyt c, crystals of horse heart cytochrome ccisplatin adducts (Cyt c-cisplatin hereafter) were grown and X-ray
diffraction data collected on these crystals at 2.19 A resolution. Cyt
c-cisplatin crystals belong to the space group P3 and contain six
molecules in the asymmetric unit (molecules A-F) (Fig. 18). The
cisplatin diffusion into the crystals, while it gradually loses chloride ligands, is slow and the pathways to the six molecules in the
asymmetric unit are (geometrically) different. As a result, in the
structure of the Cyt c-cisplatin adduct, the six independent Cyt
c molecules manifest a different degree of platination and form
80
Superimposition of the various structures reveals that, although

there are substantial differences in the relative orientation of the
side chain residues involved in cisplatin binding, Pt basically occupies the same position in the majority of Cyt c structures.
Upon comparing structures of the ligand-free Cyt c (molecule
C) with those of the cisplatin-bound molecules, no signicant
structural variations are noticed. This result might be explained
by considering the rather rigid structure of Cyt c and by taking
into account that the six molecules have been rened using noncrystallographic symmetry restraints.
In conclusion, the results of this analysis suggest that Cyt c
retains a rather rigid structure in the presence of cisplatin in the
crystal state; furthermore, coordination of the cisplatin fragment to
Met65 does not affect either the overall protein fold or the structure
of the heme cavity.
Fig. 20. Superimposition of the ve molecules of Cyt with cisplatin bound to Met65
from the asymmetric unit of the structure of Cyt-cisplatin. Pt centres are indicated
as gray spheres.
different adducts with cisplatin. In particular, inspection of the

electron maps shows that a total of ve Pt binding sites can be
identied.
A [Pt(NH3 )2 Cl]+ fragment is bound to SD atom of Met65 in chains
B and D, whereas the Met is assisted by the side chain of Glu61
in molecules A and F, where a [Pt(NH3 )2 ]2+ fragment is bound to
the protein. Molecule C does not present any cisplatin bound to its
chain. In molecule E, a Pt centre is bound to Met65 side chain, but no
other information on its ligands can be deduced from the electron
density map (Fig. 19). Notably, Met65 was earlier proposed to be
the primary binding site in the reaction between cisplatin or PtCl4 2
and tuna cytochrome c [138].
Interestingly, the presence of a Glu side chain close to the coordinating Met residues was also observed in the low resolution X-ray
structure of the adduct formed in the reaction between cisplatin
and Na+ /K+ ATPase (see below).
The unusual features of the Cyt c-cisplatin adduct, i.e. the presence of multiple copies of Cyt c complexed with the drug and of
one cisplatin-free Cyt c molecule under the same experimental
conditions, offer an ideal opportunity to unveil the structural modications induced by cisplatin binding and to analyze the exibility
of protein molecules in the presence of cisplatin. In particular the
comparison between the ve different structures of the adduct can
provide information about the mobility of the Cyt c chain in the
presence of the drug, whereas the comparison between the unligated Cyt c and the ve structures of Cyt c-cisplatin complexes
may highlight the structural changes specically induced by the
presence of cisplatin, that are not affected by differences in the
experimental conditions potentially leading to artifacts and controversial results.
The positions of the Pt center in the ve Cyt c-cisplatin molecules
in the asymmetric unit are comparatively shown in Fig. 20.
2.6. Glutaredoxin
Glutaredoxins (Grx), also called thioltransferases, are small
thermostable and evolutionarily conserved thiol-disulde oxidoreductases that are ubiquitously distributed in bacteria, eukarya,
archaea and plants [139,140]. These enzymes play a central role
in redox homeostasis as dithiol reductants, being involved in trypanothione biosynthesis. They are also involved in the formation
of deoxyribonucleotides, regulation of transcription factor binding activity, S-glutathionylation of proteins, iron sulfur-cluster
biogenesis and signal transduction [141]. Glutaredoxins are members of the thioredoxin-fold superfamily; other members of this
superfamily are thioredoxins, tryparedoxin, and protein-disulde
isomerases [140,141]. These proteins share a conserved structure
which consists of a central four-stranded -sheet surrounded by
three or ve -helices (the thioredoxin-fold). Classical dithiol
glutaredoxins have an active site characterized by the presence of a
CPYC (Cys-Pro-Tyr-Cys) motif; the rst Cys in this sequence is critical for protein-disulde and mixed protein-glutathione disulde
reduction. The structure of malaria parasite Plasmodium falciparum
Glutaredoxin 1 (PfGrx-1) in complex with cisplatin has been solved
both in the oxidized and reduced state (Fig. 21). Notably, the structures of the two complexes are signicantly different. The structure
of the oxidized form of PfGrx-1 (ox-PfGrx-1), solved at 1.97 A resolution and deposited with pdb code 4N10, reveals that cisplatin
binds close to His49, adopting two distinct orientations. In one of
the two cisplatin fragment conformers the NH3 , Cl and OH2 Pt ligands are involved in an intricate network of hydrogen bonds with
water molecules, the side chain of residues Asn19 and His47 and
the main chain atoms of residues Asn45 and Ser46 (Fig. 22).
Analysis of cisplatin binding site in the structure of the reduced
form, solved at 1.97 A resolution (PDB code 4N11), shows that cisplatin is bound to the reduced Cys29, i.e. at the N-terminal cysteine
of the Cys-Pro-Tyr-Cys motif, adopting three different conformations (Fig. 23). In this structure, the Cys29Cys32 disulphide bridge
Fig. 21. Cartoon diagrams of reduced (panel A) and oxidized (panel B) forms of PfGrx-1 in complex with cisplatin.
81
2.7. Na+ /K+ -ATPase
Fig. 22. Cisplatin binding site in the oxidized PfGrx-1-cisplatin adduct. Pt centre is
coordinated by the ND1 atom of His49, to a Cl ion, by an ammonia molecule and a
water molecule. This type of binding is unusual since it suggests that cisplatin binds
to the protein releasing one ammonia molecule.
Fig. 23. The active site Cys-Pro-Tyr-Cys motif in the reduced form of PfGrx-1 in
complex with cisplatin.
is broken before cisplatin binding. The rupture of the Cys29Cys32

disulphide bridge has been also observed in the ultra-high resolution structure of PfGrx-1 solved at 0.95 A resolution.
In one of the three conformers the Pt centre completes its coordination sphere with the N atom of Lys26. Lys26 has been modeled in
multiple conformations also in the structure of the wild-type protein. The ribbon representation of one of three cisplatin orientations
in the cisplatin binding site of PfGrx-1 is reported in Fig. 23.
The same authors also solved an additional structure of the
PfGrx-1-cisplatin adduct. In this structure the cisplatin fragment
is partly hydrolyzed. A chloride ion, an ammonia molecule and a
loosely bound water molecule (distance between the Pt atom and
coordinate the Pt centre.
the oxygen is = 2.9 0.1 A)
The binding of this fragment is stabilized by a wide network of
hydrogen bonds directly formed by Pt ligands with protein residues
or mediated by water molecules.
The sodium/potassium pump Na+ /K+ -dependent adenosine

triphosphatase (Na+ /K+ -ATPase) is a transmembrane protein
present in all animal cells and responsible for maintaining the
potassium and sodium gradients and for controlling the volume
of the cell. Na+ /K+ -ATPase malfunction is associated with numerous pathological states [142]. The structure of Na+ /K+ -ATPase is
composed of two subunits:- and -subunit [143,144]. The subunit (110 kDa) is responsible for ATP hydrolysis and cation
transport; it is formed by 10 transmembrane helices (TM1-TM10)
and three cytoplasmic domains (actuator (A), phosphorylation (P)
and nucleotide binding (N) domain). The -subunit (55 kDa) is
important for enzyme folding and maturation into the plasma
membrane; it has a cytoplasmic N-terminus, a single transmembrane segment and a glycosylated ectodomain [145,146].
Cisplatin can covalently bind to Na+ /K+ -ATPase inhibiting its
function, with serious consequences in the kidney [147]. Early
studies suggested that at low drug concentration cisplatin binds
the protein through carbonyl groups, whereas at high concentration drug-protein binding extends to polypeptide C O and C N
groups, without signicantly altering the overall enzyme structure
[148].
Crystals of Na+ /K+ -ATPase-cisplatin were obtained through the
soaking procedure by adding small grains of cisplatin powder
(1 mg) directly to a drop containing crystals of Na+ /K+ -ATPase
complexed with ouabain, grown using 1517% (w/v) PEG 2 K
monomethyl ether, 10% (v/v) glycerol, 200 mM MgCl2 , 100 mM
MES (titrated with N-methyl-d-glucamine, pH 6.2), 100 mM urea
and 5% (v/v) t-butanol [149]. Two Na+ /K+ -ATPase-cisplatin crystals
(Na+ /K+ -ATPase-cisplatin) originating from two different crystallization drops and soaking procedures were analysed. Crystals of
Na+ /K+ -ATPase-cisplatin adduct present two Na+ /K+ -ATPase
heterotrimers in the asymmetric unit, like those of the complex Na+/K + -ATPaseouabain (PDB code 3N23 [150], Fig. 24) and
diffract X-rays at 7.4 A and 7.9 A resolution, respectively. Although
the data are collected at low resolution, the authors, thanks to
anomalous scattering of Pt centre captured using X-ray diffraction
data collected at the absorption edge of platinum, are able to identify the position of the metal atom in the anomalous difference
Fourier map, which only shows the location of atoms specically
absorbing the radiation used to collect the data. In particular, the
analysis of the two datasets revealed six and four prominent peaks
(>6) in the anomalous difference Fourier map, three of which
are in common, thus identifying seven cisplatin binding sites in
Na+ /K+ -ATPase, which are predominantly cytoplasmic. These sites
are denoted as Sites I-VII.
Notably, all cisplatin binding sites on Na+ /K+ -ATPase are located
in the proximity of Met or Cys residues: Met151, Met171, Met666,
Met157, Cys457 and Met463 in the -chain, Met268 and Met121
in the -chain. Site I-V are located in the -chain. Site I is localized
close to Met151 side chain, at the cytoplasmic interface of TM2 and
the P-domain. The binding of cisplatin at this site could be stabilized
by the side chains of Glu152, Lys155, Lys352 and Lys736. In fact, Glu
was already shown to be able to strengthen cisplatin binding to a
Met side chain in the Cyt c-cisplatin adduct and coordination of
Pt to Lys appears to be possible upon migration of Pt atom from
the side chain of a Met [47]. Site II is located close to Met171 side
chain. This is on the surface of the A-domain in close proximity
to Glu169 side chain. Site III is at Met666. This residue is located
on the surface of the P-domain and has Asp662, Asp665, Gln670,
and Asp573 side chain as neighbors. Site IV is close to the Met157
side chain, at the unstructured region that connects TM2 to the Adomain. The binding to this site could be assisted by the side chain
of Arg191. Site V is between the Cys457 and Met463 side chains on
the surface of the N-domain.
82
Fig. 25. The overall structure of HSA-cisplatin adduct [159].
Fig. 24. The structure of Na+ /K+ -ATPase (PDB code 2ZXE or 3N23). -subunit is
shown in green, -subunit in cyan and -subunit in gray. Residues involved in the
cisplatin binding sites are represented as spheres. The overall binding constant for
Na+ /K+ -ATPase-cisplatin complex is estimated to be K = 1.93 104 M1 [148].
Sites VI and VII are observed at the glycosylated, extracellular

ectodomain of the -subunit. Site VI is close to Met268 side chain.
This residue, which is exposed to solvent, is in close proximity to
the C-terminal carboxylate and to side chain of Asp269. Site VII is
at the Met121. The binding to this site could be assisted by the side
chains of Asp119 or Asp120.
Interestingly, Glu (Glu152, Glu169), Gln and Asp side chains
were found close to some Met side chains involved in the cisplatin
recognition [145].
The identied cisplatin binding sites suggest that the antitumor
drug can inhibit the Na+ /K+ -ATPase function either by obstructing
the N-terminal ion exchange pathway or by hampering the conformational variations that are associated with enzymatic catalysis
[149].
2.8. HSA
Human serum albumin (HSA) is the most abundant protein
(about 50 mg L1 in healthy patient) in blood plasma. It is a 66.5 kDa
protein (585 residues) that plays many indispensable physiological roles [151]. The most outstanding function of HSA is that it
serves as a depot protein and a transport protein for many exogenous and endogenous compounds. Moreover, it can hold some
ligands in a strained orientation and renders potential toxins harmless by transporting them to disposal sites. Furthermore, it has
an important role in maintaining the osmotic pressure needed for
proper distribution of body uids between intravascular compartments and body tissues [152154]. HSA structure consists of three
similar -helical domains (IIII). Each domain comprises two subdomains, called A and B, which are formed by six and four -helices,
respectively. Domains I, II and II assemble asymmetrically in a
heart-like shape, with dimensions of about 80 80 30 A [155].
HSA has been exploited as the carrier conjugate of various anticancer drugs, including cisplatin. Reaction between cisplatin and
HSA is also believed to be the main route for Pt binding in human
blood plasma.
The structure of HSA in complex with cisplatin has been elusive
for many years, although interactions between the protein and the
Pt drug have been extensively studied [48,156]. Recently, we were
able to obtain crystals of the HSA-cisplatin adduct upon 24 h of
soaking of HSA crystals in a 0.005 M solution of cisplatin and to solve
the structure at 3.16 A resolution (Fig. 25). Contrary to expectations
[157,158], Cys34 does not seem to be involved in the drug recognition by the protein: cisplatin binding to HSA leads to simultaneous
platination of His105, His288, Met298, Met329 and Met548. In particular, the molecular model of the HSA-cisplatin adduct (PDB code
4S1Y) revealed that, under the investigated experimental conditions, cisplatin mainly binds HSA close to His105 (subdomain IA)
and Met329 (subdomain IIB) (Fig. 26). Additional minor binding
sites are found close to His288, Met298 (domain II, in the loop
traversing the two subdomains to link them together) and Met548
(subdomain IIIB) side chains.
Further structural data have been then obtained at lower resolu collecting data on a crystal of HSA that was incubated
tion (3.89 A)
in a 0.005 M solution of cisplatin for three months. The low resolution of the data does not allow to obtain detailed information
on the cisplatin fragment bound to the protein, although new Pt
binding sites can be identied in the proximity of both His67 and
His247 side chains and close to the side chain of His535 [159], close
to binding sites already identied in previous works [48].
Altogether these data indicate that HSA possesses at least seven
different binding sites for cisplatin.
The results of these structural analyses can be discussed in the
wide controversial literature available on the HSA/cisplatin system.
The number of cisplatin binding sites identied by X-ray crystallography ts within the range obtained by experimental methods
which is within 0.710.2 ([1] and references therein). Met298,
Met329 and Met548 have been identied as cisplatin binding
residues also in previous studies, particularly by multidimensional
liquid chromatography coupled to tandem mass spectrometry
(LCMS/MS) studies and by ESI-Q-TOF mass spectrometry [48].
Met298 has been also considered signicant in the recognition of
l-thyrozine, 3,3 ,5,5 -tetraiodothyronine [160], and in the binding
of Ru-based anticancer agents [161].
83
Fig. 26. Details of the binding sites of cisplatin on HSA structure in the HSA-cisplatin adduct [159]. The metal centre binds the side chains of His105 (A) and Met329 (B). Pt
completes its coordination sphere with a chloride ion and two ammonia ligands. Two NH3 ligands bound to the same Pt centre have been also observed by NMR experiments
on HSA-cisplatin adduct [32]. The overall binding constant for HSA-cisplatin complex is estimated to be K = 8.5 102 M1 [164].
However, in the same experiments additional cisplatin binding

sites can be identied: Cys34, His67, Cys124, His128, Tyr148 or
Tyr150, His247, Asp375 or Glu376 [48,60,162].
His105 has been identied to play a role in the neutral-to-basic
(NB) HSA transition [163], thus suggesting that the cisplatin binding could have a role in the allosteric regulation of the protein.
Other controversial results come from Neault et al. which suggest the existence of a direct coordination of the Pt cation to the
polypeptide C N group and close to Tyr residues on the basis of IR
data [164].
The differences between the binding sites observed in our
structure and previous data can be easily explained considering
the different conditions used to perform the experiments. In this
respect, it should be underlined that it has been demonstrated that
cisplatin binding to HSA is a slow process that depends on both
time of incubation and cisplatin/protein ratio and concentration
[164]. It is useful to recall that kinetics of adduct formation can vary
sensibly for different proteins. For example myoglobincisplatin
interaction is faster than blood proteins-cisplatin binding, since,
the percentage of the 1:1 myoglobin:cisplatin adduct reaches
40% in 6 h [53], whereas the protein binding in the plasma is
completed in 35 h [165]. Furthermore, longer incubation time
helps the formation of bis-cisplatin adducts [53]. Kinetic constants obtained for the reaction between HSA and cisplatin
range from 0.850 to 90 104 M1 . The dissociation rate constant
for HSA-cisplatin adduct has been evaluated to be KD = 0.65 s1
[1].
3. Recurrent structural features in cisplatin-protein

adducts
Careful comparative inspection of the above reported molecular structures allows the identication of a few general trends
concerning the nature, the selectivity, the stoichiometry and the
conformational effects of Pt protein interactions, that are summarized below. In addition, it is possible to dene the physicochemical
characteristics of the cisplatin binding regions and the extent of
cisplatin-protein interface.
3.1. Nature of the interaction

In all X-ray structures of cisplatin protein adducts solved up to
now, Pt is invariantly anchored to donor atoms from protein side
chains through relatively strong coordinative bonds. At variance,
non-covalent binding of cisplatin fragments/molecules to proteins
has never been described so far in crystallographic studies. Formation of coordinative bonds from the Pt center to protein donors
requires previous release of at least one Pt ligand, in most cases the
weak chloride ligand, although exceptions to this general rule are
possible. Indeed, peculiar environmental conditions may indeed
favour even the release of NH3 ligands. In turn, coordinative Pt binding to proteins is often stabilized by networks of hydrogen bonds
that Pt ligands form with protein atoms or with solvent molecules
bound to protein atoms. This observation suggests that Pt ligands
may have a major role in directing the Pt center to its ultimate binding site. Remarkably, in a few cases, release of the original Pt ligands
is complete so that only the naked Pt center is found associated
with the protein.
3.2. Binding selectivity
From inspection of the above structures it is evident that Pt
coordination manifests a quite large selectivity for a few protein side chains. Thus, the various Pt fragments, i.e. [Pt(NH3 )2 ]2+ ,
[Pt(NH3 )2 Cl]+ , [Pt(NH3 )Cl]+ , [Pt(NH3 )Cl2 ] and their aquated derivatives, preferentially coordinate to Met, His, or Cys side chains.
However, as free cysteines are not frequently present on protein
surfaces cisplatin binding is mostly limited to His and Met residues
usually solvent exposed. No structural evidence has been obtained
so far for Pt coordination to Ser or Thr residues as it was earlier
claimed by Dyson and cowokers for the case of the interaction of
cisplatin with transferrin [18].
The cisplatin fragments can coordinate protein side chains
either in a monodentate fashion (as in the case of the binding
sites observed in HSA-cisplatin, HEWL-cisplatin, Atox-1-cisplatin,
ox-PfGrx-1 and in molecules B and D of Cyt c-cisplatin) or in a bidentate fashion, i.e. binding simultaneously to two side chains (as in
molecule B of RNase A-cisplatin adduct, in molecule A and E of
84
Cyt c-cisplatin adduct and in the structure of the complex between

reduced PfGrx-1 and cisplatin).
Cisplatin binding to Met and His residues can be assisted by
the side chain of a neighbor residue. Generally, cisplatin fragments
bound to proteins are in contact with 12 water molecules and
with no more than 2 residue side chains, beyond those involved in
the covalent binding (Table 2). Cisplatin fragments form hydrogen
bonds with Asp, Glu and Gln side chains preferentially, although
hydrogen bonds with the main chain atoms of Ser, Lys and Leu
have been also observed. Other residues involved in the formation
of the cavity needed to accommodate the cisplatin fragments are
Ala, Tyr, Gly, Thr, Asn, Ile, Val and Phe. The lowest number of neighbor residues of cisplatin fragments are seen for the binding to the
side chain of His105 of RNase A, where the cisplatin molecule is
completely solvent exposed.
Notably, the anchoring mode of cisplatin fragments to protein
side chains presents a high degree of conformational exibility. In
the RNase A-cisplatin and in the ox-PfGrx-1-cisplatin structures,
alternative modes of binding of cisplatin fragment have been found.
In the ultrahigh resolution structure of HEWL-cisplatin from triclinic crystals different conformations of His side chain responsible
for cisplatin recognition were identied.
3.3. Pt/Protein stoichiometry
Cisplatin can produce monometalated (as in the case of the cyt
c structure), bis-metalated (as in the case of HEWL structure) and
even polymetalated adducts (as in the case of the RNase A-cisplatin
adduct crystallized using protein puried upon incubation in excess
of cisplatin and in the case of the HSA-cisplatin adduct). Remarkably, in specic cases, cisplatin can coordinate side chains from
two different protein molecules, inducing the formation of protein aggregates, dimers or higher rank oligomers. In this case a
Pt/protein stoichiometry of 1:2 can be found.
3.4. Effects of Pt binding on the overall protein conformation
Typically, coordination of cisplatin-derived fragments does not
signicantly affect the overall conformation of the proteins, producing in most cases only localized structural changes. However,
cisplatin binding can alter the active site geometry, inhibiting the
activity of enzymes by competitive inhibition (as in the case of the
RNase A-cisplatin adduct). Obviously, strong and often irreversible
enzyme inhibition is observed when Pt is bound to a catalytically
important protein residue.
3.5. Physicochemical characteristics of cisplatin binding regions
and extension of cisplatin-protein interface
A comprehensive analysis of the structural features of the cisplatin binding sites and the comparison between the different
protein regions involved in the binding of cisplatin allow a deep
understanding of the overall features needed to make a surface
patch capable of recognizing the drug. The binding of cisplatin in
the deposited X-ray structures solved up to now1 is often observed
in large cavities, which are formed at the interface between two
or more protein molecules. For example, the cisplatin binding site
close to Cys111 in hSOD is located in a huge cavity (Area = 1143 A 2 ,
Volume = 2712 A 3 , Fig. 27) formed at the interface between two
1
Although the protein data bank contains several proteins entries that include
a cisplatin molecule, many of these are multiple structures of the same protein.
To obtain statistically relevant information on the physical-chemical features of
cisplatin binding sites, a non-redundant dataset has been analyzed. This dataset
includes the entries reported in Table 2.
Fig. 27. Ribbon representation of the asymmetric unit content of the X-ray structure
of the hSOD-cisplatin adduct [78].
SOD dimers; in the structure of RNase A-cisplatin (pdb code 4OT4),

one out of the two cisplatin binding sites, i.e. that in molecule A,
is in a cavity characterized by a surface of 507 A 2 and a volume of
712 A 3 , at the interface between the two molecules present in the
asymmetric unit.
Cisplatin binding sites are typically located on protein surface
segments characterized by the presence of side chains (of a Met, Cys
or His) with a solvent accessibility ranging from 13.6 to 91.6 A 2 ,
with a mean value of 51.8 25.5 A 2 . Just three out of the 22 side
chains involved in cisplatin binding considered in the present study
have a solvent accessibility <25 A 2 .
Cisplatin binding is preferentially observed at loop regions or
at the end of secondary structure elements. Cisplatin binding is
sometimes associated with an enhancement of protein exibility
which can also result in the disordering of a large portion of the
protein structure.
4. Methodological limits of crystallography and future
challenges
The limits of our description are those typical of crystallographic studies of proteins (for detailed reports see [166,167]).
Protein crystal structure analysis is limited in general by a relatively poor resolution compared with crystal structure analysis of
small molecules. For this reason, ligand-Pt distances and protein
side chain atom-Pt distances should be quoted with caution. In the
present review, the errors associated with Pt-ligand distances were
evaluated considering the Diffraction Precision Index (DPI), calculated using Online DPI server (http://cluster.physics.iisc.ernet.in/
is required for high precidpi/) [168]. Atomic resolution (>1.0 A)
sion, but the majority of structures of protein-cisplatin adducts
were determined at medium-to-high resolution (see Table 1). To
date, just a single structure of an adduct formed in the reaction
between cisplatin and a protein was solved at atomic resolution, i.e. HEWL-cisplatin. The collection of data on protein-cisplatin
adducts at atomic resolution is highly desirable [169]. Using data
85
Table 2
Structural features of selected cisplatin-protein adducts.
PDB Code
Cisplatin
number in the
PDB le
Pt binding site
Residues or water
molecules forming
H-bonds with
cisplatin
Neighbour Residues
Cisplatin
fragment
identied by
mass
spectrometry
studies
40T4 (RNasi A)
[97]
200
Met29
Pt(NH3 )2 (OH2 )
Asp14
Asp53
2 wat
Asp14
Ser59Asp14
Gln28Ser32Asp53Ala56
Pt(NH3 )2 2+
Pt(NH3 )2 Cl+
[58]
201A
(alternative to
200)
201B
Met29
Pt(NH3 )X2
Met29/Gln28
Pt(NH3 )2
Asp14
2 wat
Asp14Tyr25
501A
His119
Pt(NH3 )2 Cl
6 wat
Gln11His12Val118Phe120
502A
503A
His105
Met29
Pt(NH3 )2 Cl
Pt(NH3 )2 Cl
504A
(alternative to
503A)
Met29
PtX3
1 wat
Asp14
Gln28
1 wat
not evaluated due
to the absence of Pt
ligands
284A
His19
PtCl2 X
287B
His19
3REO (hSOD) [78]
201B
202B
201C
(alternative to
201B)
Cys111
Asp109
Cys111
4N11 (PfGrx-1) To be
published
204A
Cys29Lys26
4N1O (PfGrx-1) To
be published
203A
Met59Gln63?
204A
205A
(alternative to
204A)
2I6Z [83]
4GCB [175]
(HEWL)
4S1Y (HSA) [159]
4RTE (RNasiA)
[111]
2AE0 (Cu/ZnSOD)
[74]
Cisplatin fragment
identied by X-ray
crystallographic
studies
Ala56Ser59Ser16Tyr25
Pt(NH3 )2 2+
Pt(NH3 )2 Cl+
[58]
Ser16Asp14Tyr25Gln28
Ser32
Ser32Gly31Thr17
PtCl2 X
Thr17Gly31Ser32
Pt(NH3 )2 Cl,
{PtCl(NH3 )2 }2
{Pt(NH3 )2 Cl}3 ,
{Pt (NH3 )2 Cl}4
[61]
Pt(NH3 )2 Cl
Pt(NH3 )2 Cl
Pt(NH3 )2 Cl
Leu106
Leu106Ile151Ile113
Glu24
Leu106Ile113
Pt(NH3 )2 Cl
In three different
orientations
Lys26
Val75Tyr31Pro30Glu28
3 wat
His49
His49
PtCl(X) or
PtCl(X)2
Pt(NH3 )Cl2
Pt(NH3 )Cl2
1 wat
2 wat
Asn19Met48
Val50Met48Asn19
300A
213A
His15
His15
Pt(NH3 )2 X
Pt(NH3 )2 Cl
Wat
Thr89
601A
His105
Pt(NH3 )2 Cl
605A
His288
PtX3
604A
Met298
PtX3
602A
Met329
Pt(NH3 )2 Cl
not evaluated due

to the low
resolution of the
crystallographic
data
not evaluated due
ligands and to the
low resolution of
the
crystallographic
data
not evaluated due
ligands and to the
low resolution of
the
crystallographic
data
not evaluated due
to the low
resolution of the
crystallographic
data
[Pt(NH3 )2 Cl2 ]
[Pt(NH3 )2 Cl]+
2[Pt(NH3 )2 Cl]+
[83]
[48]
86
Table 2 (Continued)
PDB Code
4RSZ (Cyt c) [108]
3IWL (Atox-1) [124]
Cisplatin
number in the
PDB le
Pt binding site
603A
Met548
PtX3
not evaluated due

ligands and to the
low resolution of
the
crystallographic
data
202D
Met65
Pt(NH3 )2 Cl
Glu61, Glu62,
Glu92
203B
202A
203F
202E
Met65
Met65/Glu61
Met65/Glu61
Met65
Pt(NH3 )2 Cl
Pt(NH3 )2
Pt(NH3 )2
PtX3
Glu61, Glu92
69A
Cys12
Cys15
TCEP
Pt
Cisplatin fragment
identied by X-ray
crystallographic
studies
Residues or water
molecules forming
H-bonds with
cisplatin
Neighbour Residues
Cisplatin
fragment
identied by
mass
spectrometry
studies
Pt(NH3 )2 (OH2 )2+

Pt(NH3 )Cl+
[136]
Glu92
Glu92
not evaluated due
ligands
Thr11
[124]
X = undened.
at atomic resolution it would be, in principle, possible to model the

dynamic behavior of the adducts. As structural data are obtained
from samples that are in their crystal state, a relevant loss of
information on the dynamic aspects of the investigated interactions invariantly and unavoidably occurs [170]. Obtaining dynamic
information on the adducts is important to acquire a better description of the protein-ligand interaction and to establish accuracy of
structural data with more condence. Dynamic information from
crystallographic data could be gained when several different structural models of the same protein will become available [171,172],
although results obtained using this comparison should be considered with caution, since the disposition of protein chains in the
crystal and crystal lattice forces could deeply affect protein dynamic
features.
Other points that merit attention are the conditions under
which crystals were obtained and the protocol used to collect Xray diffraction data and to rene the structure. In fact, the way
in which diffraction images are collected and models are rened
have a strong inuence on the quality of the nal structures and on
the type and magnitude of their associated errors. Structural data
from crystallographic studies are often obtained under experimental conditions that contain high concentrations of salts, polymers
or organic molecules, thus being very far from the physiological ones [173]. Furthermore, in order to reduce radiation damage
[174], diffraction data are often collected at 100 K, though data
at room temperature could be more indicative of the real structural features of the adducts. Data collection temperatures for
the structures of the cisplatin-protein adduct solved to date, are
reported in Table 1. Inspection of Table 1 indicates that the majority of protein-cisplatin structures were solved under cryogenic
conditions, although there are a small number of structures of
HEWL-cisplatin that were solved at room temperature. Upcoming
developments of room temperature data collection systems at synchrotron will allow the acquisition of room temperature diffraction
data in a near future.
Regarding the renement procedure, the inclusion of a ligand
in a molecular structure requires the denition of topology and
parameter les that describe the chemistry of the ligand. It should
be noted that for a long time the standard ligand for cisplatin (CPT)
did not include appropriate restraints (in the ligand dictionary, CPT
had tetrahedral geometry restraints!). The RCSB, PDBe and PDBj

versions for CPT description also varied and even included NH2 as
ligand descriptor rather than NH3 !
Another critical point is the Pt ligand assignments. The identication of Pt ligand is rather challenging. Anomalous diffraction
data are routinely used to discriminate Cl from N, since Cl
has 17 electrons and N has 7 electrons. However, the anomalous scattering signal for Cl is very weak, especially when data
are collected using Cu K radiation. Useful data are obtained
when X-ray diffraction images are collected at high resolution
and with high redundancy. In these cases, the denition of the
details of Pt ligands can be assessed with good condence. In
this respect, particularly informative seems the case of pdb code
4GCB [175], which was rened using data with exceptional redundancy. As a result, the chloride anomalous diffraction difference
map peaks are highly signicant (see Fig. 1a in Ref. [175]) and
so chemical assignments of cisplatin ligands are very clear. If
Cl and N atoms can be discriminated by deep inspection of
electron density maps and of B-factor values, much more difcult is the discrimination between N atom of ammonia ligand
and O atom of water molecules, in the aquated cisplatin fragments [Pt(NH3 )2 OH2 ]. In this respect, neutron crystallography data
might be very useful [176] to resolve doubtful structural assignments.
5. Toward a unied picture of protein platination
Overall, even considering the limits reported in the previous
paragraph, comparative analysis of the above crystallographic data
offers a rather exhaustive description of the Pt-protein adducts
formed, at least from the structural point of view. Indeed, several
relevant features could be elucidated including the coordinative
nature of platinum-protein interaction, the number, identity and
location of Pt binding sites on the protein surface, the nature of
protein bound Pt fragments and also the consequences of Pt binding on the overall protein fold. However, to improve the general
picture of the platination process and gain some dynamic information, crystallographic data may be advantageously integrated
and complemented with results arising from the application of
other biophysical techniques in solution such as NMR, UVvisible
87
surprising because of the intrinsic differences of the two experimental approaches.

3. Changes in the bound metallic fragments: Remarkably, some
interesting time-dependent effects were identied whereby the
protein bound metallic fragments may undergo further transformations. Typically, these transformations may be related to
further release (aquation) of the chloride ligands and associated
mass shift. Even more importantly it was observed that metallic
fragments may be removed from protein binding upon competition with stronger ligands, e.g. glutathione, implying that the
Pt-protein interactions are potentially reversible under physiologically relevant conditions [45,55].
Fig. 28. Representative examples of ESI-MS spectra collected for a cisplatin-protein

adduct: spectra of RNase A treated with 3 104 mol L1 cisplatin after 24 h (A), 72 h
(B), 168 h (C) of incubation at 37 C under the following experimental conditions:
metal:protein ratio = 3:1, buffer: 20 mmol L1 ammonium acetate at pH 6.8.
absorption spectroscopy, CD, intrinsic uorescence and mass spectrometry. We will not enter here into details of such analysis and we
will not cover the extensive literature already existing on these topics; we will just make some examples that seem to us particularly
meaningful.
Highly instructive is in our opinion the information descending from the application of mass spectrometry methods. To this
respect, it is worthy reminding that electrospray ionization mass
spectrometry (ESI-MS) experiments have been often compared
and integrated with crystallographic data on strictly related systems. Indeed, for the small model proteins cyt c, HEWL and RNase
A, which behave excellently in mass spectrometry experiments,
extensive ESI-MS data were collected in our laboratories that nicely
complement structural information from crystallographic studies.
A few exemplary ESI-MS spectra for these systems are represented
in Fig. 28.
The most relevant results that we could derive from ESI-MS
investigations of cisplatin-protein adducts are detailed below.
1. Kinetics of adduct formation: Formation of cisplatin/protein
adducts is relatively slow and may take hours or even several
days or weeks to reach completion, upon protein incubation in
the presence of cisplatin at room temperature in the standard
buffer. This observation derives from time course ESI-MS experiments where adduct formation is repeatedly monitored through
inspection of the relative intensities of the peaks of adducts
with respect to the peak of the native protein, on samples
taken at regular time intervals. Slowness in adduct formation is
most likely related to the time necessary for the cisplatin aquation/activation process.
2. Nature of the metallic fragments: The mass shifts of the formed
adducts compared with the native protein can be measured very
accurately; in some cases protein binding of metallic fragments
well match those observed in the crystallographic studies, in
other cases the fragment identied is different, but this is not
Now, putting together the structural information coming out

from crystallographic studies with independent information deriving from spectrometric investigations a rather exhaustive picture of
the protein platination process induced by cisplatin in small model
proteins may be drawn.
We assume that cisplatin under physiological conditions, undergoes a progressive activation process consisting of the slow
replacement of one/two chloride ligands with one/two water
molecules; the process is relatively slow, but may be facilitated
by non-covalent interactions with the protein surface and by
the microenvironment. Upon activation, cisplatin is converted
to a more reactive cationic species with a strong tendency to
bind and coordinate proteins. Typically, Pt binding occurs almost
exclusively at few solvent exposed protein side chains, mostly
the imidazole group of His, the thioether group of Met and
the thiol group of free cysteines. Afterward, the protein bound
Pt fragment may undergo a further evolution, losing other ligands e.g. the second chloride in the case of the monoaquated
fragment, but even ammonia ligands, possibly creating additional coordinative bonds to the protein. The interaction may
be favored by a rearrangement of the network of hydrogen
bonds.
Remarkably, Pt coordination to selected protein side chains
only causes some local structural changes; it hardly affects
the overall protein conformation. Coordinative binding of the
metallic fragment gives rise to a relatively strong and stable protein modication; however, the bound Pt fragment may
be removed by treatment of the cisplatin protein adduct with
strong platinum ligands. In this respect, it is useful to note
that the transfer of Pt from adducts formed with ubiquitin and
horse heart myoglobin to biological nucleophiles was veried
[45,55].
This implies that the interaction is potentially reversible. Finally,
it is remarkable that upon challenging serum albumin, a far bigger and more complex protein, with cisplatin the same schemes of
interaction observed with small model proteins are basically conserved and platination occurs at the same side chains with similar
modalities.
A nal note. Remarkably, in all mentioned crystallographic
and solution studies, cisplatin derivatives were typically prepared by challenging the drug with individual puried proteins.
However, in real cellular systems, cisplatin is simultaneously
challenged with a large variety of different proteins. It follows
that in real systems cisplatin is in the condition to bind a huge
number of different proteins in such a way that its biological actions will be most probably the result of hundreds of
distinct Pt protein interactions and of their functional consequences. Such biological effects will arise either by activating
or repressing specic signaling pathways but also by blocking selected enzymes, when platination of that specic enzyme
is relevant. In any case the binding motifs for cisplatin are
mostly conserved and largely correspond with those identied
and characterized in single puried proteins as documented by
88
recent multidimensional protein identication technology (MUDPIT) studies of Pt protein interactions in protein cellular extracts
[177].
6. Concluding remarks
Since adducts with proteins are important in dening the therapeutic proles of cisplatin, it is imperative to understand the basic
principles that govern the formation of these protein-metallodrug
complexes. Pt metalation of protein by cisplatin has been studied
for a long time, but only very recently details on cisplatin binding to
protein side chains could be elucidated. Here, we have summarized
the most signicant progresses recorded in the structural characterization of protein-cisplatin adducts. We have examined the
known structures of protein-cisplatin adducts to establish the key
characteristics of the cisplatin modes of binding to proteins. This
analysis provides clues as to what features on a protein target might
make it suitable or ideal for cisplatin binding. A rather exhaustive
description, at the atomic level, of the protein platination process
induced by cisplatin has thus been gained. The knowledge of the
general structural features of cisplatin binding sites may inspire
new efforts to improve the pharmacokinetic/pharmacodynamic
prole of this important drug. In addition, these observations could
help the prediction of cisplatin binding sites on target proteins or
could be used to design cisplatin variants that target more selectively specic protein sites. Data can be also used for interpreting
the results of experimental studies carried out so far on protein
systems for which a 3D model is known.
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Coordination Chemistry Reviews: Luigi Messori, Antonello Merlino

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Coordination Chemistry Reviews 315 (2016) 6789

Contents lists available at ScienceDirect

Coordination Chemistry Reviews

Cisplatin binding to proteins: A structural perspective

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

Fig. 1. Structure of cisplatin.

2016 Elsevier B.V. All rights reserved.

structural modications of the DNA double helix, ultimately

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

Fig. 2. Ribbon representation of the molecular model of the beSOD-cisplatin adduct

2. A gallery of molecular structures for cisplatin-protein

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

Bovine Cu/Zn SOD

Human copper chaperone Atox-1 (dimer)

Human copper chaperone Atox-1 (dimer)

Human copper chaperone Atox-1 (monomer)

careful inspection of the structure revealed a tight contact between

distance is 3.2 0.1 A).

enzyme [78]. In this latter case the primary cisplatin binding

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

observed, ruling out the presence of additional cisplatin binding

Fig. 4. Ribbon representation of the molecular structure of the hSOD-cisplatin

Hen egg white lysozyme (HEWL) has been widely studied

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

one Cl ligand (Fig. 7A and B) or of one ammonia ligand (Fig. 7C).

rened with resolutions falling in the 1.83.5 A range:one at 100 K

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

Fig. 8. Iodido analogue of cisplatin (cis-Pt(NH3 )2 I2 ) binding site in HEWL crystals at

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

Data are also in very good agreement with ESI-MS spectrum of

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

structure [97], in the main binding site Pt centre is bound to SD atom

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

Fig. 12. Cartoon representation of PtM [111].

In the other two (minor) binding sites, the Pt center is bound

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

Two structures of Atox-1 complexed with cisplatin were

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

results by Palm et al., suggesting that cisplatin forms a Cu-Pt Atox-1

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

Superimposition of the various structures reveals that, although

different adducts with cisplatin. In particular, inspection of the

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

2.7. Na+ /K+ -ATPase

is broken before cisplatin binding. The rupture of the Cys29Cys32

The sodium/potassium pump Na+ /K+ -dependent adenosine

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

Fig. 25. The overall structure of HSA-cisplatin adduct [159].

Sites VI and VII are observed at the glycosylated, extracellular

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

However, in the same experiments additional cisplatin binding

3. Recurrent structural features in cisplatin-protein

3.1. Nature of the interaction

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

Cyt c-cisplatin adduct and in the structure of the complex between

SOD dimers; in the structure of RNase A-cisplatin (pdb code 4OT4),

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

3REO (hSOD) [78]

4S1Y (HSA) [159]

not evaluated due

L. Messori, A. Merlino / Coordination Chemistry Reviews 315 (2016) 6789

4RSZ (Cyt c) [108]