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The X-ray structure of the Primary Adducts formed in the Reaction between Cisplatin and Cytochrome c Giarita Ferraro,a Luigi Messorib and Antonello Merlinoa,c*
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x In the present study, the interactions between cisplatin and cytochrome c are investigated. Based on high-resolution Xray diffraction data, two monometalated species, i.e. cyt cPt(NH3)2 and cyt c-Pt(NH3)2Cl, are found to be the main adducts that form in the reaction between the protein and the drug. Both monodentate and bidentate platinum coordination to the protein is observed, with platinum binding either to Met65 or to Met65 and Glu61, simultaneously.
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Cisplatin is one of the most important platinum-based anticancer agents in clinical use [1]. Its mechanism of action is believed to rely mostly on tight platinum(II) binding to DNA and consequent DNA damage [2]; the latter, if not repaired, ultimately leads to the activation of apoptotic death pathways in cancer cells. Although the DNA binding properties are definitely important for the antitumor activity of this Pt compound, the interactions with proteins also play an important role, as they are associated to drug effectiveness, to side effects and also to cisplatin resistance processes [3]. In this framework, assigning the exact sites for cisplatin binding in proteins turns crucial for a deeper understanding of its overall pharmacological profile [4]. Recent investigations pointed out that cisplatin accumulates in the mitochondria [5] causing release of cytochrome c from mitochondria into the cytoplasm; in turn, cytochrome c release triggers apoptotic death of cancer cells [6]. Cytochrome c is a small (104 residues), highly conserved and well-characterized electron transfer protein which has been extensively used to study the interactions occurring between proteins and metallodrugs [7]. Despite several reports on the interactions of cisplatin with cytochrome c are available in the literature [7], the protein binding sites for Pt and the nature of Pt fragments involved in cytochrome c recognition have not been unambiguously characterized. Electrospray ionization mass spectrometry data showed that the cytochrome c–Pt(NH3)2(H2O) monoadduct is the main adduct formed in the cytochrome c/cisplatin reaction and that Met65 is the primary Pt binding site [7b]. According to results from liquid chromatography coupled with LTQ-MS [7c] multiple binding sites (Met80, Glu61/Glu62/Thr63, and Met65) were proposed for cisplatin on cytochrome c. Using Fourier
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transform ion cyclotron resonance mass spectrometry, Pt(NH3)Cl was found to be the main Pt-drug fragment and four binding sites (Met65, Met80, His18, and His33) for cisplatin on cytochrome c were identified [7d]. However, two of these sites appear somewhat questionable since Met80 and His18 are also involved in the anchoring of the heme iron. Based on these arguments, we investigate here the interactions of cisplatin with cytochrome c from the structural point of view. Crystals of horse heart cytochrome c-cisplatin adduct were obtained by soaking experiments: crystals of cytochrome c in a new crystal form (space group P3), grown by hanging drop vapour diffusion method using a reservoir solution of 3.5 M (NH4)2SO4 and 0.6 M NaNO3, and protein concentration 30 mg mL-1, were soaked for 24 h in a solution of 0.005 M cisplatin in 2.0 M (NH4)2SO4, 0.4 M NaNO3 (about 1:3 protein to metal ratio). Although the soaking severely cracks the cytochrome c crystals, X-ray diffraction data could be collected on the adduct formed between the protein and cisplatin at 2.19 Å resolution, using a Saturn944 CCD detector equipped with CuKα X-ray radiation from a Rigaku Micromax 007 HF generator (see supplementary information for further details). The overall structure, which contains six molecules in the asymmetric unit (molecules A-F hereafter), has been determined by the molecular replacement method using the program Phaser [8] and the coordinates of the structure of cytochrome c determined at 1.94 Å resolution by Bushnell et al. (PDB code 1HRC, [9]) as search model. The structure has been refined to an R-factor of 0.231 (Rfree=0.282) using Refmac5 [10] (see SI for further details). The structure of the cytochrome c-cisplatin adduct is shown in Figure 1. Notably, the general conformation of the protein is not significantly affected by cisplatin binding. The Cα r.m.s. deviations of cytochrome c-cisplatin adduct models from those of ligand-free cytochrome c molecule in the starting model are within the range 0.3-0.4 Å. Besides few modifications in the position of the side chains of surface residues, the most significant structural differences in the structure of cytochrome c observed upon cisplatin binding are located close to the Pt binding sites. The electron density maps are well defined in all the six molecules of the asymmetric unit, including the regions of the heme groups (Figure 2). Inspection of the difference Fourier electron-density map (Fo-Fc) clearly revealed five >8σ peaks
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which are located close to Met65, in five out of the six molecules in the a. u. (Figure 3). These peaks are straightforwardly attributed to cisplatin fragments.
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Figure 1. Overall structure of the asymmetric unit (a.u) of the cytochrome c-cisplatin adduct. The six molecules of cytochrome c (molecule A…F) in the a.u. are colored in green, cyan, purple, yellow, pink and grey, respectively. Met80 and His18 of each chain shown are shown together with the heme groups. Cisplatin fragments are shown as spheres, whereas the residues that coordinate the platinum centre are also reported as stick. Coordinates and structure factors of cytochrome c-cisplatin adduct were deposited in the Protein Data Bank under the accession code 4RSZ.
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observed here in the case of molecules A and F was also found [11]. A bidentate Pt binding mode was suggested to occur also in the complex between cisplatin and insulin [13]. On the basis of the reported results it is very likely that Met residues, particularly when assisted by Asp/Glu residues, may represent preferential binding sites for cisplatin on proteins; accordingly, bidentate binding of cisplatin fragments on the protein surface appears to be a structural motif more common and more general than previously believed. In this framework, it should also be recalled that cisplatin binding to thioether sulfur in Met was suggested to play a crucial role in drug metabolism, in its binding to serum proteins [14] and its transfer to DNA [1518]. On the other hand, these results are in contrast with the idea, mainly based on X-ray diffraction data collected on lysozymecisplatin [19] and Cu,Zn superoxide dismutase-cisplatin adducts [20], that histidine plays a major role in cisplatin binding to proteins [21].
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Figure 2. Heme binding motif with Met80 and His18 axially ligated to the heme iron in the molecule A of cytochrome c in the cytochrome c-cisplatin adduct. 2Fo-Fc electron density map is contoured at 1.5σ level and colored in grey. The water molecule and the network of hydrogen bonds involving the side chains of Tyr67, Thr78 and Asn52, believed to play a role in stabilizing the oxidized state of iron [22], are also evidenced.
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The coordination geometry around the Pt center is different in the various molecules in the a. u. In molecules A and F, Pt(II) is coordinated to SD atom of Met65, to OE2 atom of Glu61 and to the N atoms of two ammonia ligands (Figure 3A and B). In molecules B and D, Pt(II) is coordinated to SD atom of Met65, to a chloride ion and to the two N atoms of the ammonia ligands (Figure 3C and D). In molecule E, the Pt ion coordinates the SD atom of Met65 while the other ligands cannot be modelled due to poor electron density (Figure S1). In the molecule C no cisplatin fragment is found (Figure S2). Occupancy factors of Pt ion obtained in conventional structural refinements and validated on the basis of the observation of residual electron density maps (see SI for further details) are 0.50/0.50/0.40/0.40/0.50 for molecules A, B, D, E and F, respectively. Cisplatin binding to SD atom of a Met was already observed in the X-ray structure of the cisplatin adduct of bovine pancreatic ribonuclease [11] and in the low resolution structure of the adduct formed between cisplatin and Na+/K+ ATPase [12]. Interestingly, in the former complex, a bidentate binding mode similar to those 2 | Journal Name, [year], [vol], 00–00
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Figure 3. Details of the binding sites of cisplatin on molecules A (panel A), F (panel B), B (panel C) and D (panel D) of cytochrome c in the cytochrome c-cisplatin adduct. The Pt ion is coordinated to Met65. The binding of cisplatin to cytochrome c is associated with the loss of chloride ions. 2Fo-Fc electron density maps are contoured at 5σ (red) and 0.8σ (grey) level. Structural refinements have suggested an occupancy value for the Pt atoms in the range 0.4-0.5, suggesting a quite relevant degree of protein metalation.
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In conclusion, we have reported here, for the first time, the Xray structure of a cytochrome c-cisplatin adduct. On the basis of crystallographic results the following considerations can be made: i) cisplatin mainly produces monometalated cytochrome c adducts where a [Pt(NH3)2Cl]+ fragment is coordinated to Met65 (in a monodentate fashion) or a [Pt(NH3)2]2+ fragment is simultaneously bound to Met65 and Glu61 (in a bidentate fashion). Thus, the anchoring mode of platinum fragments to Met65 presents a high degree of variability; Pt binding can be assisted by the side chain of an acidic residue. Interestingly, Glu side chains (Glu152, Glu169) were also found close to the Met side chains involved in the cisplatin recognition in the low resolution X-ray structure of the adduct formed with Na+/K+ ATPase [12]. This finding supports previous observations that cisplatin has a slight preference for S donors compared to N donors [11, 15], being in contrast with previous reports suggesting that a high number of histidine residues in a protein importantly contribute to cisplatin binding to proteins [21]. ii) Remarkably, coordination of the cisplatin fragment to Met65 does not affect the overall conformation of the protein and of the heme cavity. Quite unexpectedly, no other cisplatin binding sites could be detected on cytochrome c surface, even at very low occupancy.
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Altogether these results substantiate previous suggestions that different patterns of protein platination are possible within the same protein system [11] and that different binding modes (monodentate or bidentate) are allowed even within the same cisplatin binding site. The authors thank G. Sorrentino and M. Amendola for technical assistance. L.M. acknowledges Beneficentia Stiftung (Vaduz, Liechtenstein) and AIRC (IG-12085) for financial support.
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Notes and references a
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Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, Via Cintia, I-80126, Napoli, Italy. Fax: +39081674090; Tel: +39081674276; E-mail: [email protected] b Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy. c CNR Institute of Biostructures and Bioimages, Vioa Mezzocannone 16, I80126, Napoli, Italy †Electronic Supplementary Information (ESI) available: See DOI: 10.1039/b000000x† [1] (a) Z.H. Siddik. Oncogene 2003, 22, 7265-79; (b) L. Kelland. Nature Reviews Cancer 2007, 7, 573-84; (c) M.H. Green. J. Nat Cancer Inst. 1992, 84, 306–312; (d) B.J.S. Sanderson, L.R. Ferguson, W.A. Denny. Mutat. Res-Fundam. Mol. Mech. Mut. 1996, 355, 59–70; (e)
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O. Rixe, W. Ortuzar, M. Alvarez, R. Parker, E. Reed, K. Paull, T. Fojo. Biochem. Pharmacol. 1996, 52, 1855–1865. [2] (a) R.C. Todd, S.J. Lippard. Metallomics. 2009, 1, 280-91; (b) D. Wang, S. J. Lippard. Nature Reviews Drug Discovery 2005, 4, 30720; (c) X. Zhai, H. Beckmann, H.-M. Jantzen, J.M. Essigmann. Biochemistry 1998, 37, 16307–16315; (d) A. Eastman. Pharmacol. Ther. 1987, 34, 155–166; (e) M. Ohndorf, M.A. Rould, Q. He, C.O. Pabo, S. J. Lippard. Nature 1999, 399, 708–712; (f) A. Casini, C. Gabbiani, G. Mastrobuoni, L. Messori, G. Moneti, G. Pieraccini. Chem. Med. Chem. 2006, 1, 413-417. [3] E. Raymond, S. Faivre, S. Chaney, J. Woynarowski, E. Cvitkovic. Mol Cancer Ther 2002, 1, 227–235. [4] (a) A. Casini, J. Reedijk, J. Chemical Science 2012, 3, 3135-3144; (b) C.A. Rabik, M.E. Dolan, Cancer Treatment Reviews 2007, 33, 923; (c) O. Pinato, C. Musetti, C. Sissi. Metallomics, 2014, 6, 380-95; (d) F. Arnesano, G. Natile. Pure Appl. Chem. 2008, 80, 12, 2715– 2725. [5] G. Kursunluoglu, H. A. Kayali, D. Taskiran. Cell Biochem. Biophys. 2014, 69(3):707-16. [6] (a) H. Kojima, K. Endo, H. Moriyama, Y. Tanaka, E.S. Alnemri, C.A. Slapak, B. Teicher, D. Kufe, R. Datta. Journal of Biological Chemistry 1998, 273, 16647-16650; (b) V. M. Gonzalez, M.A. Fuertes, C. Alonso, J.M. Perez. Molecular Pharmacology 2001, 59, 657-663; (c) M.S. Park, M. De Leon, P. Devarajan. Journal of the American Society of Nephrology 2002, 13, 858-865. [7] (a) A. Casini, C. Gabbiani, G. Mastrobuoni, R. Z. Pellicani, F. P. Intini, F. Arnesano, G. Natile, G. Moneti, S. Francese, L. Messori. Biochemistry 2007, 46 (43), 12220-12230; (b) T. Zhao, F.L. King. J Am Soc Mass Spectrom. 2009, 20, 1141-7; (c) E. Moreno-Gordaliza; B. Cañas; M. A. Palacios; M. Gomez-Gomez, M. Milagros. Talanta 2012, 88, 599-608; (d) N. Zhang, Y. Du, M. Cui, J. Xing, Z. Liu, S. Liu. Anal Chem. 2012, 84, 6206-12. [8] A. J. McCoy R.W. Grosse-Kunstleve, P.D. Adams, M. D. Winn, L. C. Storoni, R.J. Read. J. Appl. Cryst. 2007, 40, 658-674. [9] G.W. Bushnell, G.V. Louie, G.D. Brayer. J. Mol. Biol 1990, 214, 585–595. [10] G. N. Murshudov, A. Vagin, E.J. Dodson, Acta Crystallogr. Sect. D 1997, 53, 240–255. [11] L. Messori, A. Merlino. Inorg Chem 2014, 53, 3929-31. [12] M. Huliciak, L. Reinhard, M. Laursen, N. Fedosova, P. Nissen, M. Kubala. Biochemical Pharmacology 2014, 92, 494-498. [13] E. Moreno-Gordaliza, B. Canas, M. A. Palacios, M. M. GomezGomez. Anal. Chem. 2009, 81, 3507-16. [14] A. Y. Ivanov, J. Christodoulou, J. A. Parkinson, Kevin J. Barnham, A. Tucker, J. Woodrow‖, P. J. Sadler. J Biol Chem 1998, 273, 14721-14730. [15] J. Reedijk, Proc. Natl. Acad. Sci. 2003, 100, 3611-3616. [16] Miller, S.E.; House, D.A. Inorg. Chimica Acta 1989, 161, 1, 131– 137. (b) B. Wu, P. Droge, C. A. Davey. Nat. Chem. Biol. 2008, 4, 110. (c) J. Reedijk. Chem. Rev. 1999, 99, 2499. [17] (a) Lempers, E. L. M.; Inagaki, K.; Reedijk, J. Inorg. Chim. Acta 1988, 152, 201. (b) Lempers, E. L. M.; Reedijk, J. Inorg. Chem., 1990, 29, 217. (c). van Boom, S. S. G. E; Reedijk, J. J. Chem. Soc. Chem. Commun. 1993, 1397. [18] J. Reedijk, Platinum Metals Rev. 2008, 52, 2–11. [19] (a) J. R. Helliwell, S. W. M. Tanley. Acta Cryst. 2013, D69, 121125; (b) A. Casini, G. Mastrobuoni, C. Temperini, C. Gabbiani, S. Francese, G. Moneti, C.T. Supuran, A. T. Scozzafava, L. Messori. Chem. Commun. 2007, 2, 156-158; (c) S. W. M. Tanley, A. M. M. Schreurs, L. M. J. Kroon-Batenburg, J. Meredith, R. Prendergast, D. Walsh, P. Bryant, C. Levy, J. R. Helliwell. Acta Cryst. 2012, D68, 601-612; (d) S. W. M. Tanley, A. M. M. Schreurs, L. M. J. KroonBatenburg, J.R. Helliwell. Acta Cryst. 2012, F68, 1300-1306. [20] V. Calderone, A. Casini, S. Mangani, L. Messori, P. L. Orioli. Angew. Chem. 2006, 45, 1267-9. [21] T. Karasawa, M. Sibrian-Vazquez, R. M. Strongin, P. S. Steyger. Plos One 2013, 8, 6, e66220. [22] M. De March, N. Demitri, R. De Zorzi, A. Casini, C. Gabbiani, A. Guerri, L. Messori, S. Geremia. Journal of Inorganic Biochemistry 2014, 13, 58-67.
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This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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The X-ray structure of the Primary Adducts formed in the Reaction between Cisplatin and Cytochrome c Giarita Ferraro,a Luigi Messorib and Antonello Merlinoa,c*
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x In the present study, the interactions between cisplatin and cytochrome c are investigated. Based on high-resolution Xray diffraction data, two monometalated species, i.e. cyt cPt(NH3)2 and cyt c-Pt(NH3)2Cl, are found to be the main adducts that form in the reaction between the protein and the drug. Both monodentate and bidentate platinum coordination to the protein is observed, with platinum binding either to Met65 or to Met65 and Glu61, simultaneously.
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Cisplatin is one of the most important platinum-based anticancer agents in clinical use [1]. Its mechanism of action is believed to rely mostly on tight platinum(II) binding to DNA and consequent DNA damage [2]; the latter, if not repaired, ultimately leads to the activation of apoptotic death pathways in cancer cells. Although the DNA binding properties are definitely important for the antitumor activity of this Pt compound, the interactions with proteins also play an important role, as they are associated to drug effectiveness, to side effects and also to cisplatin resistance processes [3]. In this framework, assigning the exact sites for cisplatin binding in proteins turns crucial for a deeper understanding of its overall pharmacological profile [4]. Recent investigations pointed out that cisplatin accumulates in the mitochondria [5] causing release of cytochrome c from mitochondria into the cytoplasm; in turn, cytochrome c release triggers apoptotic death of cancer cells [6]. Cytochrome c is a small (104 residues), highly conserved and well-characterized electron transfer protein which has been extensively used to study the interactions occurring between proteins and metallodrugs [7]. Despite several reports on the interactions of cisplatin with cytochrome c are available in the literature [7], the protein binding sites for Pt and the nature of Pt fragments involved in cytochrome c recognition have not been unambiguously characterized. Electrospray ionization mass spectrometry data showed that the cytochrome c–Pt(NH3)2(H2O) monoadduct is the main adduct formed in the cytochrome c/cisplatin reaction and that Met65 is the primary Pt binding site [7b]. According to results from liquid chromatography coupled with LTQ-MS [7c] multiple binding sites (Met80, Glu61/Glu62/Thr63, and Met65) were proposed for cisplatin on cytochrome c. Using Fourier
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transform ion cyclotron resonance mass spectrometry, Pt(NH3)Cl was found to be the main Pt-drug fragment and four binding sites (Met65, Met80, His18, and His33) for cisplatin on cytochrome c were identified [7d]. However, two of these sites appear somewhat questionable since Met80 and His18 are also involved in the anchoring of the heme iron. Based on these arguments, we investigate here the interactions of cisplatin with cytochrome c from the structural point of view. Crystals of horse heart cytochrome c-cisplatin adduct were obtained by soaking experiments: crystals of cytochrome c in a new crystal form (space group P3), grown by hanging drop vapour diffusion method using a reservoir solution of 3.5 M (NH4)2SO4 and 0.6 M NaNO3, and protein concentration 30 mg mL-1, were soaked for 24 h in a solution of 0.005 M cisplatin in 2.0 M (NH4)2SO4, 0.4 M NaNO3 (about 1:3 protein to metal ratio). Although the soaking severely cracks the cytochrome c crystals, X-ray diffraction data could be collected on the adduct formed between the protein and cisplatin at 2.19 Å resolution, using a Saturn944 CCD detector equipped with CuKα X-ray radiation from a Rigaku Micromax 007 HF generator (see supplementary information for further details). The overall structure, which contains six molecules in the asymmetric unit (molecules A-F hereafter), has been determined by the molecular replacement method using the program Phaser [8] and the coordinates of the structure of cytochrome c determined at 1.94 Å resolution by Bushnell et al. (PDB code 1HRC, [9]) as search model. The structure has been refined to an R-factor of 0.231 (Rfree=0.282) using Refmac5 [10] (see SI for further details). The structure of the cytochrome c-cisplatin adduct is shown in Figure 1. Notably, the general conformation of the protein is not significantly affected by cisplatin binding. The Cα r.m.s. deviations of cytochrome c-cisplatin adduct models from those of ligand-free cytochrome c molecule in the starting model are within the range 0.3-0.4 Å. Besides few modifications in the position of the side chains of surface residues, the most significant structural differences in the structure of cytochrome c observed upon cisplatin binding are located close to the Pt binding sites. The electron density maps are well defined in all the six molecules of the asymmetric unit, including the regions of the heme groups (Figure 2). Inspection of the difference Fourier electron-density map (Fo-Fc) clearly revealed five >8σ peaks
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which are located close to Met65, in five out of the six molecules in the a. u. (Figure 3). These peaks are straightforwardly attributed to cisplatin fragments.
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Figure 1. Overall structure of the asymmetric unit (a.u) of the cytochrome c-cisplatin adduct. The six molecules of cytochrome c (molecule A…F) in the a.u. are colored in green, cyan, purple, yellow, pink and grey, respectively. Met80 and His18 of each chain shown are shown together with the heme groups. Cisplatin fragments are shown as spheres, whereas the residues that coordinate the platinum centre are also reported as stick. Coordinates and structure factors of cytochrome c-cisplatin adduct were deposited in the Protein Data Bank under the accession code 4RSZ.
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observed here in the case of molecules A and F was also found [11]. A bidentate Pt binding mode was suggested to occur also in the complex between cisplatin and insulin [13]. On the basis of the reported results it is very likely that Met residues, particularly when assisted by Asp/Glu residues, may represent preferential binding sites for cisplatin on proteins; accordingly, bidentate binding of cisplatin fragments on the protein surface appears to be a structural motif more common and more general than previously believed. In this framework, it should also be recalled that cisplatin binding to thioether sulfur in Met was suggested to play a crucial role in drug metabolism, in its binding to serum proteins [14] and its transfer to DNA [1518]. On the other hand, these results are in contrast with the idea, mainly based on X-ray diffraction data collected on lysozymecisplatin [19] and Cu,Zn superoxide dismutase-cisplatin adducts [20], that histidine plays a major role in cisplatin binding to proteins [21].
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Figure 2. Heme binding motif with Met80 and His18 axially ligated to the heme iron in the molecule A of cytochrome c in the cytochrome c-cisplatin adduct. 2Fo-Fc electron density map is contoured at 1.5σ level and colored in grey. The water molecule and the network of hydrogen bonds involving the side chains of Tyr67, Thr78 and Asn52, believed to play a role in stabilizing the oxidized state of iron [22], are also evidenced.
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The coordination geometry around the Pt center is different in the various molecules in the a. u. In molecules A and F, Pt(II) is coordinated to SD atom of Met65, to OE2 atom of Glu61 and to the N atoms of two ammonia ligands (Figure 3A and B). In molecules B and D, Pt(II) is coordinated to SD atom of Met65, to a chloride ion and to the two N atoms of the ammonia ligands (Figure 3C and D). In molecule E, the Pt ion coordinates the SD atom of Met65 while the other ligands cannot be modelled due to poor electron density (Figure S1). In the molecule C no cisplatin fragment is found (Figure S2). Occupancy factors of Pt ion obtained in conventional structural refinements and validated on the basis of the observation of residual electron density maps (see SI for further details) are 0.50/0.50/0.40/0.40/0.50 for molecules A, B, D, E and F, respectively. Cisplatin binding to SD atom of a Met was already observed in the X-ray structure of the cisplatin adduct of bovine pancreatic ribonuclease [11] and in the low resolution structure of the adduct formed between cisplatin and Na+/K+ ATPase [12]. Interestingly, in the former complex, a bidentate binding mode similar to those 2 | Journal Name, [year], [vol], 00–00
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Figure 3. Details of the binding sites of cisplatin on molecules A (panel A), F (panel B), B (panel C) and D (panel D) of cytochrome c in the cytochrome c-cisplatin adduct. The Pt ion is coordinated to Met65. The binding of cisplatin to cytochrome c is associated with the loss of chloride ions. 2Fo-Fc electron density maps are contoured at 5σ (red) and 0.8σ (grey) level. Structural refinements have suggested an occupancy value for the Pt atoms in the range 0.4-0.5, suggesting a quite relevant degree of protein metalation.
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In conclusion, we have reported here, for the first time, the Xray structure of a cytochrome c-cisplatin adduct. On the basis of crystallographic results the following considerations can be made: i) cisplatin mainly produces monometalated cytochrome c adducts where a [Pt(NH3)2Cl]+ fragment is coordinated to Met65 (in a monodentate fashion) or a [Pt(NH3)2]2+ fragment is simultaneously bound to Met65 and Glu61 (in a bidentate fashion). Thus, the anchoring mode of platinum fragments to Met65 presents a high degree of variability; Pt binding can be assisted by the side chain of an acidic residue. Interestingly, Glu side chains (Glu152, Glu169) were also found close to the Met side chains involved in the cisplatin recognition in the low resolution X-ray structure of the adduct formed with Na+/K+ ATPase [12]. This finding supports previous observations that cisplatin has a slight preference for S donors compared to N donors [11, 15], being in contrast with previous reports suggesting that a high number of histidine residues in a protein importantly contribute to cisplatin binding to proteins [21]. ii) Remarkably, coordination of the cisplatin fragment to Met65 does not affect the overall conformation of the protein and of the heme cavity. Quite unexpectedly, no other cisplatin binding sites could be detected on cytochrome c surface, even at very low occupancy.
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Altogether these results substantiate previous suggestions that different patterns of protein platination are possible within the same protein system [11] and that different binding modes (monodentate or bidentate) are allowed even within the same cisplatin binding site. The authors thank G. Sorrentino and M. Amendola for technical assistance. L.M. acknowledges Beneficentia Stiftung (Vaduz, Liechtenstein) and AIRC (IG-12085) for financial support.
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Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, Via Cintia, I-80126, Napoli, Italy. Fax: +39081674090; Tel: +39081674276; E-mail: [email protected] b Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy. c CNR Institute of Biostructures and Bioimages, Vioa Mezzocannone 16, I80126, Napoli, Italy †Electronic Supplementary Information (ESI) available: See DOI: 10.1039/b000000x† [1] (a) Z.H. Siddik. Oncogene 2003, 22, 7265-79; (b) L. Kelland. Nature Reviews Cancer 2007, 7, 573-84; (c) M.H. Green. J. Nat Cancer Inst. 1992, 84, 306–312; (d) B.J.S. Sanderson, L.R. Ferguson, W.A. Denny. Mutat. Res-Fundam. Mol. Mech. Mut. 1996, 355, 59–70; (e)
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