Peculiar features in the crystal structure of the adduct formed between cis-PtI2(NH3)2 and hen egg white lysozyme

Article abstract of DOI:10.1021/ic402611m

The reactivity of cis-diamminediiodidoplatinum(II), cis-PtI ₂(NH₃)₂, the iodo analogue of cisplatin, with hen egg white lysozyme (HEWL) was investigated by electrospray ionization mass spectrometry and X-ray crystallography. Interestingly, the study compound forms a stable 1:1 protein adduct for which the crystal structure was solved at 1.99 A resolution. In this adduct, the Pt^II center, upon release of one ammonia ligand, selectively coordinates to the imidazole of His15. Both iodide ligands remain bound to platinum, with this being a highly peculiar and unexpected feature. Notably, two equivalent modes of Pt^II binding are possible that differ only in the location of I atoms with respect to ND1 of His15. The structure of the adduct was compared with that of HEWL-cisplatin, previously described; differences are stressed and their important mechanistic implications discussed.

Full text of DOI:10.1021/ic402611m

Communication
pubs.acs.org/IC
Peculiar Features in the Crystal Structure of the Adduct Formed
between cis-PtI2(NH3)2 and Hen Egg White Lysozyme
Luigi Messori,*,† Tiziano Marzo,† Chiara Gabbiani,‡ Amparo A. Valdes,§ Adoracion G. Quiroga,§
and Antonello Merlino*,⊥
†Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
‡Department of Chemistry and Industrial Chemistry, University of Pisa, Via Risorgimento 35, 56126 Pisa, Italy
§Department of Inorganic Chemistry, Universidad Autonom
́ ́
a de Madrid C/Francisco Tomas y Valiente 7, 28049 Madrid, Spain
⊥Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, Via Cintia,
I-80126 Napoli, Italy
S
* Supporting Information
In 2007, Casini et al. determined the X-ray structure of the
A B S T R A C T : T h e r e a c t i v i t y o f c i s -
HEWL−cisplatin derivative obtained by soaking experiments on
diamminediiodidoplatinum(II), cis-PtI2(NH3)2, the iodo
analogue of cisplatin, with hen egg white lysozyme
(HEWL) was investigated by electrospray ionization
mass spectrometry and X-ray crystallography. Interestingly,
the study compound forms a stable 1:1 protein adduct for
which the crystal structure was solved at 1.99 Å resolution.
In this adduct, the PtII center, upon release of one
ammonia ligand, selectively coordinates to the imidazole of
His15. Both iodide ligands remain bound to platinum, with
this being a highly peculiar and unexpected feature.
Notably, two equivalent modes of PtII binding are possible
that differ only in the location of I atoms with respect to
ND1 of His15. The structure of the adduct was compared
with that of HEWL−cisplatin, previously described;
differences are stressed and their important mechanistic
implications discussed.
pregrown HEWL crystals, at 1.9 Å resolution. The structure
revealed selective platination of the only HEWL histidine residue,
i.e., His15, at the imidazole ND1 atom (at the so-called right-
handed site).5a PtII has an occupancy equal to 0.3 and is also
bound to the N atoms of two ammonia molecules. The fourth
ligand was not detected (Figure S1a in the Supporting
Information, SI). This result was in substantial agreement with
the ESI MS spectra of HEWL−cisplatin derivatives, which
showed two peaks of similar intensity at 14569 and 14605 Da,
formally corresponding to either a [Pt(NH3)2Cl]+ fragment or
intact cisplatin bound toHEWL. Asimilar situation was described
by Dyson and co-workers in the case of the cisplatin−transferrin
system and interpreted in terms of a two-step cisplatin-to-protein
binding process.6
More recently, cocrystallization experiments conducted by
Helliwell and co-workers revealed that cisplatin binds HEWL by
coordinating either the ND1 or the NE2 atom (i.e., it can bind at
the so-called “right-handed” and “left-handed” sites) of the
imidazole ring of His15,5b,c after release of one chlorine ligand
(Figure S1b in the SI).
isplatin [cis-diamminedichloroplatinum(II)] is a leading
Canticancer drug in widespread clinical use for the treatment
of several types of malignancies.1 Since the discovery of cisplatin,
research has been largely focused on the characterization of
platinum−DNA adducts according to the concept that DNA is its
primary biological target: these studies indicated the N7 atom of
guanine as the preferential platination site.2 Interestingly,
bifunctional adducts involving two adjacent nucleobases are
predominantly formed by cisplatin3 that are believed to represent
the major DNA lesions, ultimately leading to apoptotic cancer
cell death. Yet, nucleobases are not the only biological targets for
platinum drugs. Cys, Met, and His residues in proteins are
alternative reactive sites to which platinum can efficiently
coordinate.4 The interaction between cisplatin and proteins is
underscored by a number of crystallographic and electrospray
ionization mass spectrometry (ESI MS) studies that include
determination of the complexes between this molecule and hen
egg white lysozyme (HEWL),5 transferrin,6 the copper
chaperone Atox-1,7 hemoglobin,8 cytochrome c,9 ubiquitin,10
DNA polymerase,11 metallothionein,12 and superoxide dismu-
tase.13
Although cisplatin remains one of the most effective
chemotherapeutic agents for cancer treatment (in particular for
testicular and ovarian cancers), it shows some major drawbacks,
such as cumulative toxicities of nephrotoxicity and ototoxicity,
inherent or treatment-induced resistance, and relevant patient
toxicity. This has provided the motivation for developing novel
metal complexes (either platinum or nonplatinum) as prospec-
tive anticancer agents with a different mechanism of action.
Recently, a number of cis- and trans-diamminediiodidoplatinum
15
compounds [such as cis-PtI2(ammine)2,14 trans-PtI2(ammine)2
and trans-PtI2(ammine)(ammine′)216] showing unconventional
reactivity have been synthesized and their cytotoxicities toward a
variety of human tumor cell lines evaluated.
As a model for the interaction of iodido analogues of
platinum(II) drugs with proteins16 and for a direct comparison
of the results with cisplatin, the cisplatin analogue cis-PtI2(NH3)2,
previously synthesized (see the SI for details)17 and charac-
Received: October 16, 2013
Published: November 20, 2013
© 2013 American Chemical Society
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Inorganic Chemistry
Communication
terized, was chosen to perform a detailed investigation of its
reaction with HEWL by X-ray crystallography and ESI MS.
Crystals of the adduct formed between HEWL and cis-
PtI2(NH3)2 were obtained by soaking experiments, in which
pregrown HEWL crystals were incubated for 2 months with an
excess of the platinum drug. Representative yellow crystals of the
resulting adduct are reported in Figure 1.
Figure 3. Details of the binding site of PtII in HEWL−cis-PtI2(NH3)
showing the Pt ion bound to His15. 2Fo−Fc electron density maps are
contoured at the 1σ level. For the sake of clarity, just one of the two
different modes of binding of cis-PtI2(NH3)2 to HEWL is shown.
Figure 1. Crystals of the adduct between HEWL and cis-PtI2(NH3)2.
These crystals were subjected to X-ray diffraction data
collection, and the structure of the adduct was solved at 1.99 Å
resolution. Details of crystallization, data collection, and structure
refinement are given in the SI. The overall structure of the adduct
between HEWL and cis-PtI2(NH3)2 (Figure 2), which refines to
Rfactor = 18.4 (Rfree = 23.4), is very similar to that of the native
protein (PDB code 193L): the root-mean-square deviation in
positions of CA atoms is 0.3 Å.
Figure 4. Two modes of binding of cis-PtI2(NH3)2 to HEWL. I atoms are
colored in purple, and N atoms are colored in blue.
In the former mode of binding, the NH3 group is close to the N
and OD1 atoms of Asn93 (distances 3.8 and 4.5 Å, respectively);
in the latter, the NH3 group is solvent-exposed and interacts with
two water molecules, which, in turn, are in contact with Arg14,
whereas the I atom occupies the position close to Asn93 (Figure
4a,b). Within these assumptions, the His(N)−Pt distance is 2.1
Å, whereas the Pt−I and Pt−N(NH3) distances are, on average,
both close to 2.4 Å.
Remarkably, the present structure shows that the PtII ion binds
the protein with an occupancy value close to 1.0. To the best of
our knowledge, this is the highest occupancy ever observed for
platinum in adducts between proteins and cisplatin-like drugs.
No other significant modifications of the electron density map
were observed, ruling out the presence of additional (secondary)
binding sites. This finding supports the view that interaction of
this platinum drug with HEWL is highly selective for His15.
Independent valuable information on the same system was
subsequently gained through application of ESI MS (see the SI
for details). ESI MS is indeed a powerful and informative tool to
characterize the interactions of metal-based drugs with small
model proteins. Precise information may be derived on the
stoichiometry and kinetics of binding as well as on the exact
nature of the protein-bound metal fragments.4b,15,18 HEWL
dissolved in a suitable buffer (20 mM ammonium acetate buffer,
pH 6.8) was treated with a 3:1 excess of a fresh solution of the
study compounds, and ESI MS spectra were measured after
increasing time intervals. Deconvoluted ESI MS spectra recorded
at the indicated time intervals (6, 24, 48, and 72 h) are shown in
Figure 5. The progressive formation of adducts is well witnessed
by the appearance of a number of peaks of higher molecular mass
than the native protein. Interestingly, the dominant peak within
those attributed to platinum adducts shows a value of 14769.63
Da, which perfectly matches the mass of an adduct in which a
single [PtI2NH3] fragment is bound to the protein. This
observation offers further and independent support to the
above-reported crystallographic results.
Figure 2. Ribbon representation of the structure of the adduct formed
between HEWL and cis-PtI2(NH3)2. The side chain of His15, which is
solvent-accessible, being situated on the protein surface, is shown along
with platinum, ammonium, and I ligands. For sake of clarity, just one of
the two different modes of binding of cis-PtI2(NH3)2 to HEWL is shown.
The structure has been deposited in the Protein Data Bank under the
accession code 4MR1.
In agreement with that found in the case of cisplatin by Casini
et al.,5a platination occurs at the ND1 atom of the imidazole ring
of His15, i.e., at the right-handed site. The local environment of
the protein-bound PtII center was identified by observation of a
very clear electron density map (Figure 3). Notably, the PtII ion is
coordinated to the ND1 atom of His15, two I atoms, and a N
atom in a classical square-planar geometry. Refinement of the
temperature factors of platinum ligands and inspection of the
residual Fo−Fc electron density maps indicate that two equivalent
modes of platinum binding are present in the crystal structure,
which differ only in the location of the I atoms with respect to the
ND1 of His15 (Figure 4). The existence of two alternative modes
of binding was further supported by inspection of the anomalous
scattering map, which confirmed the presence of anomalous
signals of I and Pt atoms in the binding moieties (Figure S2).
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ACKNOWLEDGMENTS
■
We thank Beneficentia Stiftung and COST Action CM1105 for
financial support, G. di Martino for her help in the crystallization,
G. Sorrentino and M. Amendola for technical assistance. A.G.Q.
and A.A.V thank the Minstero de Economia y Competitividad for
Grant SAF2012-34424.
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ASSOCIATED CONTENT
* Supporting Information
■
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(15) Messori, L.; Cubo, L.; Gabbiani, C.; Alvarez-Valdes
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Materials and analytical techniques, crystallization, X-ray data
collection, structure solution, and refinement, scheme of cisplatin
binding to HEWL at the right-handed site, and anomalous
electron density map contoured at 4.0σ. This material is available
free of charge via the Internet at http://pubs.acs.org.
Michelucci, E.; Pieraccini, G.; Ríos-Luci, C.; Leon, L. G.; Padron
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: luigi.messori@unifi.it.
*E-mail: antonello.merlino@unina.it.
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Author Contributions
The manuscript was written through contributions of all authors.
Notes
The authors declare no competing financial interest.
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