Cisplatin Binding Sites in Human H-Chain Ferritin - Inorganic

Jul 24, 2017 - Human Ft is composed of 24 subunits of two types, denoted as H ..... through contributions of all authors who approve the final version...
2 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/IC

Cisplatin Binding Sites in Human H‑Chain Ferritin Giarita Ferraro,† Silvia Ciambellotti,‡ Luigi Messori,‡ and Antonello Merlino*,†,§ †

Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, Via Cintia, I-80126 Naples, Italy ‡ Dipartimento di Chimica, Università di Firenze, Via Della Lastruccia 3, Sesto Fiorentino, 50019 Firenze, Italy § Institute of Biostructures and Bioimages, Naples, Italy S Supporting Information *

ABSTRACT: The aim of this work is to identify the cisplatin binding sites on human Hchain ferritin. High-resolution X-ray crystallography reveals that cisplatin binds four distinct protein sites, that is, the side chains of His136 and Lys68, the side chain of His105, the side chain of Cys90 and the side chain of Cys102. These Pt binding sites are compared with those observed for the adduct that cisplatin forms upon encapsulation within horse spleen Lchain ferritin (87% identity with human L-chain ferritin).

■

INTRODUCTION Since its fir st in t ro d uct i o n in t h e cl in ics , cis diamminedichloroplatinum(II) (cisplatin) had a major impact in the treatment and prognosis of various solid tumors, such as testicular, ovarian, oral, head, neck, stomach, cervical, small-cell lung, and bladder cancers.1 However, the use of cisplatin is significantly limited by a few severe side effects, for example, systemic toxicity and nephrotoxicity, and by intrinsic or acquired resistance.2 The development of innovative delivery systems with tumor-targeting potential is thus highly desirable to expand the activity and the applications of this drug. In this respect, different strategies have been devised to increase tumor selectivity of cisplatin, like the use of controlled-release polymers, of polymeric carriers, of inorganic nanoparticles and liposomes. In this scenario, attempts are made to incorporate anticancer drugs within ferritin (Ft) nanocages with the aim to develop new endogenous delivery nanosystems that selectively target cancer cells. Cisplatin-loaded Ft nanocages are interesting from the pharmaceutical point of view, since they possess a uniform and stable structure, are basically nontoxic for normal cells, highly soluble in the bloodstream, and thermostable. Nanometer cisplatin core Ft particles were constructed using horse spleen Ft,3−5 pig pancreas Ft,6 and Aplysia juliana hepatopancreas Ft,7 but the most interesting encapsulated nanocages are those obtained using the human protein,8 being the most compatible with the host immune system. Human Ft is composed of 24 subunits of two types, denoted as H and L chains,9 which assemble to form a spherical cage with inner and outer diameters of ∼8 and 12 nm.10,11 In the H chain, iron(II) oxidation by dioxygen occurs at a dinuclear © 2017 American Chemical Society

ferroxidase center including residues Glu27, Glu62, His65, and Glu107.12 The L chain does not manifest ferroxidase activity but retains nucleation sites for mineral core formation. H-chain (HuHFt) and L-chain (HuLFt) Ft are specifically recognized by TfR1 and Scara5, respectively; these two surface receptors are overexpressed in several tumor cells.13,14 Thus, cisplatin-loaded H-chain Ft (HuHFt-cisplatin) or L-chain Ft (HuLFt-cisplatin) can be internalized selectively in tumor cells, through a receptor-mediated endocytosis mechanism. HuHFt was selectively targeted on different tumor tissues: in a screening of 474 clinical samples it was found that it specifically binds to the nine most common solid tumors.15 It was demonstrated that cisplatin-loaded Fts are cytotoxic toward pheochromocytoma, gastric cancer, and melanoma cells4−8 and that Pt uptake is higher for cisplatin-loaded Ft than for free cisplatin.6 The molecular basis for preferential cisplatin delivery to cancer cells by HuHFt and HuLFt is still unknown. However, it is now evident that the molecular mechanisms of cell apoptosis induced by HuHFt-cisplatin are different from those induced by free cisplatin4 and that HuHFt is able to enter nuclei as the whole intact cage, without the use of a nuclear localization signal.16 Pt atoms are probably released inside cells following protein degradation. While there are studies on the use of Ft in the transport of cisplatin, there have been just a few reports exploring the nature of the interactions involved when this drug binds the protein carrier.17 Received: April 27, 2017 Published: July 24, 2017 9064

DOI: 10.1021/acs.inorgchem.7b01072 Inorg. Chem. 2017, 56, 9064−9070

Article

Inorganic Chemistry We have previously reported the X-ray structure of the horse spleen L-chain apoFt-cisplatin adduct, obtained by the cage disassembly reassembly procedure:3 in this case cisplatin binds close to the side chain of His132, on the inner surface of the cage. Detailed information on the cisplatin binding sites of HuHFt are still missing. Here we report the crystal structure of the complex formed in the reaction between HuHFt and cisplatin. The effect of the presence of cisplatin on the ability of the protein to bind iron(II) and on the ferroxidase activity of the protein is also evaluated.

■

RESULTS AND DISCUSSION HuHFt Binds Cisplatin, without the Need of Cage Disassembly. First, we studied the binding of cisplatin to HuHFt in solution at physiological pH. Intrinsic fluorescence data were collected on the protein upon addition of increasing amounts of cisplatin and excitation at 280 nm (Figure 1). The

Figure 2. CD spectra of HuHFt and of a HuHFt-cisplatin sample dialyzed upon 24 h of incubation of the protein in the presence of cisplatin in 1:10 protein-to-metal ratio. Spectra were acquired at 20 °C using a protein concentration of 0.05 mg mL−1 in a 10 mM sodium phosphate buffer, pH 7.4.

does not reach completion even at 90 °C. The presence of cisplatin apparently enhances the thermal stability of HuHFt, since at 90 °C the adduct retains a slightly higher secondary structure content than the ligand-free protein (Figure S3). Altogether these findings point out that HuHFt binds the drug, even in the absence of the disassembly/reassembly process that was earlier applied to encapsulate cisplatin inside Ft cages.3 In any case cisplatin binding to the protein does not significantly alter the secondary structure content and the overall structure of HuHFt, while slightly affecting its thermal stability. Structure of HuHFt-cisplatin. To determine the location of the Pt binding sites on HuHFt structure, we tried to grow crystals of the adduct using both soaking and cocrystallization procedures. Crystals of the adduct were first grown by cocrystallization experiments, but they diffract X-ray just at very low resolution (>10 Å). Crystals of the HuHFt-cisplatin adduct suitable for X-ray diffraction experiments were then obtained by the soaking procedure: HuHFt crystals were soaked in a solution of reservoir containing 2.5 mM of cisplatin for three weeks. Soaking of metallodrugs into preformed protein crystals is commonly used to study the formation of many protein−metallodrug adducts,19−21 although this procedure might exclude the potential binding sites that are involved in the crystal packing contacts. The structure of the HuHFt-cisplatin adduct was solved at 2.05 Å resolution, using the PDB code 4Y08, without ligands, as starting model22 and compared with that of cisplatin-free protein, solved by us at 1.74 Å resolution and already described by other authors.22,23 The structure of HuHFt is almost complete; exceptions are two residues at the N-terminal region and six residues at the Cterminal tail, for which no electron density is observed. The final model contains 1917 non-H atoms, including six Cl− and eight Mg2+, and refines to R and Rfree values of 0.146 and 0.191. The model of the adduct contains 1724 non-H atoms, including six Cl−, five Mg2+, and four cisplatin fragments (with three Pt atoms adopting two alternative positions), and refines to R and Rfree values of 0.162 and 0.206. Both structures present more than 98% of the residues in the most favored region of the Ramachandran plot (0 outliers for HuHFt and 1 for HuHFtcisplatin adduct). Details of crystallization, data collection, and refinement statistics are reported in Table 1. The structures

Figure 1. Fluorescence spectra of HuHFt upon cisplatin titration. Excitation at 280 nm, protein concentration = 0.05 mg mL−1 in 10 mM sodium phosphate pH 7.4.

occurrence of large and progressive fluorescence quenching strongly suggests cisplatin binding to the protein. Afterward, fluorescence spectra of HuHFt in the presence of increasing amounts of cisplatin were recorded upon excitation at 295 nm, which selectively excites Trp (Figure S1). As expected on the basis of previous literature data,18 the fluorescence emission maximum of HuHFt peaks at 325−329 nm, upon excitation at 280 or 295 nm, respectively. The strong quenching observed upon cisplatin addition suggests formation of stable adducts with this drug. Then, a sample of HuHFt was incubated for 24 h in the presence of a cisplatin-to-protein molar ratio of 10/1 and dialyzed to remove the excess of the drug. Inductively coupled plasma atomic emission spectroscopy (ICP-OES) determinations performed on the dialyzed sample pointed out an average Pt content of 1.35−1.95 atoms per subunit. Circular dichroism (CD) spectra on the dialyzed HuHFtcisplatin sample were registered to assess possible changes in the secondary structure of the protein induced by cisplatin binding. Spectra of HuHFt and of the HuHFt-cisplatin adduct are indeed very similar, although the free protein appears to possess a slightly larger content of α-helical structure, as judged by the presence of deeper minima at 208 and 222 nm in the CD spectrum (Figure 2). The CD signal at 222 nm was followed as a function of temperature to compare the stability of the adduct with that of HuHFt (Figure S2). As expected, the protein is highly thermostable, and the denaturation process 9065

DOI: 10.1021/acs.inorgchem.7b01072 Inorg. Chem. 2017, 56, 9064−9070

Article

Inorganic Chemistry were deposited in the Protein Data Bank under the access codes 5N26 and 5N27 for HuHFt-cisplatin and HuHFt, respectively. Table 1. Data Collection and Refinement Statistics HuHFta PDB code space group unit-cell parameters a = b = c (Å) Ft monomers per a.u. resolution (Å) observed reflections unique reflections completeness (%) Rmerge Rpim CC1/2 CC* I/σ(I) multiplicity refinement resolution (Å) reflections in working set reflections in test set R/Rfree (%) non-H atoms occupancy of Pt ions B-factor of Pt ions (Å2) overall B-factor deviations f rom ideality rmsd bonds (Å) rmsd angles (Å) a

HuHFt-cisplatina

5N27 F432

5N26 F432

182.96 1

183.07 1

105.6−1.74 (1.77−1.74) 333 941

105.7−2.05 (2.09−2.05) 132 369

27 459

16 819

99.9 (98.4)

98.5 (91.8)

0.114 (0.733) 0.032 (0.404) (0.627) (0.837) 18.0 (1.8) 12.2 (3.8)

0.143 (0.588) 0.053 (0.236) (0.878) (0.955) 17.8 (3.7) 7.9 (6.9)

105.6−1.74 26 057

105.7−2.05 15 963

1378

837

14.6/19.1 1917

19.9

16.2/20.6 1724 0.50, 0.50, 0.40, 0.20, 0.40, 0.25, 0.40 45.9, 45.0, 55.9, 50.9, 47.7, 56.1, 51.5 19.4

0.020 1.75

0.019 1.92

Figure 3. Cartoon representation of HuHFt-cisplatin monomer and 24-mer. Cisplatin binding sites are evidenced.

provided by Mangani and co-workers22 and by Mikami and coworkers.23 The structure of the HuHFt-cisplatin adduct presents four cisplatin binding sites. Pt atoms are bound close to (i) both the ND1 atoms of His136 and the NZ atom of Lys68 (Figure 4A) at the inner surface of the cage, (ii) the ND1 atom of His105, with the His side chain and Pt adopting two distinct conformations, respectively (Figure 4B), (iii) the SG atom of Cys90 and (iv) the SG atom of Cys102 side chains at the outer cage surface (Figure 4B). Close to both these cysteines two alternative positions of the Pt atoms were observed. The presence of bound Pt atoms was verified by inspection of anomalous (Figure 4C,D), 2Fo-Fc (Figure 4A,B), and Fo-Fc electron density maps. Geometric parameters of the Pt ligands and Pt-ligand distances are listed in Table 2. Data indicate that Pt and its ligands adopt the expected square-planar geometry, although distorted. Occupancy and B-factors of Pt atoms are within the ranges of 0.20−0.50 and 45.0−56.1 Å2, respectively. Occupancy of Pt was adjusted to obtain the lowest negative or positive electron density peaks in residual Fo-Fc electron density maps. At the main binding sites (occupancy = 0.50), close to side chains of His136 and Lys68 and to ND1 atom of His105, a Pt(NH3)22+ fragment and a Pt(NH3)2(OH2)2+ fragment were modeled, respectively. Thus, as in the case of the adduct that cisplatin forms with cytochrome c,24 both monodentate and bidentate modes of binding are observed. In the other Pt binding sites the electron density is not sufficiently well-defined to allow a complete description of the metal coordination sphere. However, anomalous map (Figure 4D) suggests that two alternative cisplatin fragments bind close to Cys90 and Cys102, probably occupying alternative positions. With exception of His136 (His132 in the L chain), the residues involved in cisplatin binding to HuFt are not conserved in the L chain and in other ferritins. This indicates that these binding sites cannot be used to recruit cisplatin in other ferritins. These results are also supported by the comparison between the structure of HuHFt-cisplatin and that of the adduct formed when the drug is encapsulated within horse spleen L-chain Ft,3 which indicates significant differences in the Pt binding sites. Binding to His side chains has been frequently observed in the reaction between cisplatin and proteins.17,25−29 Pt binding to Lys side chains has been recently described for the thaumatin−cisplatin adduct,30 whereas the interaction with Cys residues has been already found in the complex of cisplatin with human superoxide dismutase.31

Values in parentheses refer to highest resolution shell.

The overall structures of HuHFt and of the HuHFt-cisplatin adduct are very similar. The root-mean-square deviation (rmsd) in positions of carbon α atoms between the two models is 0.10 Å. Both structures conserve the typical features of ferritin: each Ft chain has a largely helical structure, composed of a four-helix bundle with a short fifth helix at the C-terminus (Figure 3). A comparison of the local residue environment was performed by calculating rmsd per residue. The major differences (>0.2 Å) reside in the regions encompassing N-terminal tail (residues 3− 5) and residues 89−92. Experimental data indicate that HuHFt-cisplatin structure contains a Mg2+ ion in the ferroxidase site (Figure S4A), that is, close to Glu27 and His65, whereas HuHFt contains also an additional Mg2+ site close to Gln58 and Glu61 (Figure S4B). Notably the position occupied by Mg2+ ions in HuHFt ferroxidase site is not exactly the same observed in the Febound structure of HuHFt22 (Figure S4C). Additional Mg2+ and Cl− ions are found on the protein surface. The positions of these ions are in agreement with the indications previously 9066

DOI: 10.1021/acs.inorgchem.7b01072 Inorg. Chem. 2017, 56, 9064−9070

Article

Inorganic Chemistry

Table 2. Selected Geometric Parameters at Cisplatin Binding Sites distance (Å) Pt1-ND1 His136 Pt1-NZ Lys136 NZ Lys68-Pt1-ND1 His136 ND1 His136−Pt1-N1 NZ Lys68−Pt1-N2 Pt2 (A)-ND1 His105 (A)a ND1 His105 (A)-Pt2(A)-N1 ND1 His105 (A) -Pt2(A)-O Pt2 (B)-ND1 His105 (B)a Pt3(C)-SG Cys102 Pt3(A)-SG Cys102 Pt4 (A)-SG Cys90 (A)a Pt4 (B)-SG Cys90 (B)a a

angle (deg)

2.06 2.06 82.0 97.0 97.2 1.93 84.9 94.2 2.05 2.38 2.30 2.05 2.40

His105 and Cys90 adopt two alternative conformations.

Figure 5. Fluorescence quenching upon Fe2+ binding to HuHFt and HuHFt-cisplatin. Normalized fluorescence was calculated as I/Io from spectra registered upon excitation at 280 nm, protein concentration = 0.3 mg mL−1 in 10 mM sodium phosphate buffer, pH 7.4. Similar results were obtained using protein concentration = 0.5 mg mL−1. Figure 4. Cisplatin binding sites in HuHFt-cisplatin adduct showing the Pt centers bound to ND1 atom of His136 and NZ atom of Lys36 (A), to ND1 and NE2 atom of His105 and to side chains of Cys90 and Cys102 (B). 2Fo-Fc electron density maps are contoured at 3σ (violet) and 1σ level (marine blue). Anomalous electron density map allowing the identification of Pt binding sites is shown at 2σ level in panels C and D and is reported in red. 2Fo-Fc electron density map of these sites is compared to those observed for HuHFt in Figure S1.

His136 and Lys68, to the ferroxidase site that indeed affect the catalytic reaction.

■

CONCLUSIONS The goal of this work was to identify the cisplatin binding sites on the human H-chain ferritin and to characterize them from the structural point of view. This is the first report of a structure for human H-chain ferritin in complex with an antitumor metallodrug. Data indicate that the protein is able to bind cisplatin, even without performing the cage disassembly− reassembly procedure that was used in the past to encapsulate the drug inside Ft cages. It is inferred that cisplatin could soak within the Ft cage through the C3 or the C4 channels.32 Cisplatin binds the protein close to side chains of His105, His136, and Lys68 and close to the side chains of Cys90 and of Cys102, and it does not affect greatly the overall iron binding capability of the protein. However, we found that cisplatin binding completely abolishes the ferroxidase activity of HuHFt, probably due to competition at the level of the ferroxidase site. Comparison of the present data with those obtained for the reaction of cisplatin with horse spleen L-chain Ft (87% identity with human L-chain Ft) suggests that the two Ft chains have a different reactivity with this drug; thus, different outcomes as delivery systems may be predicted. It can be speculated that the

Cisplatin Binding to HuHFt Does Not Affect the Iron Binding Capability of the Protein but Significantly Affects Its Catalytic Activity. To investigate the effect of cisplatin on the iron binding ability of HuHFt, fluorescence spectra of HuHFt and HuHFt-cisplatin containing different amounts of Fe2+ and upon excitation at 280 nm were compared (Figure 5). Results suggest that the ability of HuHFt to bind iron is not greatly affected by the presence of Pt atoms bound to the protein. Additionally, the ferroxidase activity of HuHFt and HuHFtcisplatin was comparatively analyzed. The catalytic activity of ferritin treated with cisplatin (protein subunit/cisplatin molar ratio 1:10) was completely lost as shown in Figure 6 from the comparison of kinetics (diferric peroxo and diferric oxo species formation) with respect to the cisplatin-free protein. Probably, this is due to the proximity of bound-Pt ions, in positions 9067

DOI: 10.1021/acs.inorgchem.7b01072 Inorg. Chem. 2017, 56, 9064−9070

Article

Inorganic Chemistry

curves were recorded using 0.05 mg mL−1 protein solutions in 10 mM sodium phosphate buffer at pH 7.4. Before the measurements the instrument was calibrated with an aqueous solution of d-10(+)-camphorsulfonic acid at 290 nm. ICP-OES data were collected on the HuHFt-cisplatin adduct, prepared incubating for 24 h the protein in the presence of cisplatin in 1:10 protein-to-metal ratio (and successively dialyzed), using an ICP-OES Optima 2000 (PerkinElmer, Europe) at Department of Chemistry of University of Florence. To assess the catalytic activity after the reaction with 10 equiv of cisplatin per protein subunit, a size-exclusion chromatography was applied to exclude the presence of unreacted cisplatin and to exchange the buffer of protein solution into 200 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 200 mM NaCl pH 7.0. The kinetics of formation of diferric peroxo and diferric oxo species were followed as the change in absorbance at 650 and 350 nm, respectively, after the rapid addition of two Fe2+ ions per monomer (freshly prepared solution in 1 mM HCl) to the protein samples (2.12 mg mL−1).32,33 These measurements were conducted through a stopped-flow spectrophotometer (SX.18MV stopped-flow reaction analyzer; Applied Photophysics, Leatherhead, England) at room temperature. Crystals of HuHFt suitable for X-ray diffraction studies were grown at 293 K by hanging drop vapor diffusion method, using 2.0 M MgCl2, 0.1 M bicine pH 9.0 as reservoir. Protein crystallizes at a concentration of 18 mg mL−1. The adduct with cisplatin was obtained by soaking procedure: crystals of HuHFt were soaked in a solution consisting of 2.0 M MgCl2, 0.1 M bicine pH 9.0 with 2.5 mM cisplatin. Crystals were kept in this solution for three weeks. Both HuHFt and HuHFtcisplatin crystals were fished with a nylon loos and flash-frozen at 100 K in a nitrogen gas produced by an Oxford Cryosystem (and maintained at 100 K during the data collection) using glycerol (25%) as a cryoprotectant. X-ray diffraction data were collected on a Saturn944 CCD detector at the CNR Institute of Biostructures and Bioimages, Naples, Italy, using Cu Kα X-ray radiation from a Rigaku Micromax 007 HF generator. Data were processed and scaled using HKL2000.34 Data collection statistics are reported in Table 1. Structure Resolution and Refinement. The structure of HuHFt and of the adduct with cisplatin were solved by molecular replacement using the PDB file 4Y08,22 without water molecules and other ligands, as starting model. Inspection of the electron density maps calculated using this model clearly reveals the presence of many sites attributable to ions and water molecules. Pt binding sites were identified by analyzing the Fourier difference (Figures 3A,B) and anomalous electron density maps (Figures 3C,D) and by comparing the electron density maps of the adduct with those obtained for cisplatin-free HuHFt (Figure S3). Occupancy of Pt atoms was defined by minimizing the presence of residual positive and negative peaks of electron density around the Pt center in the Fo-Fc electron density map. Cisplatin ligands were defined as described in the Supporting Information. Cisplatin fragments are identified in the coordinates deposited in the PDB as “CPT”. However, we have not used the CPT definition given by PDB to refine the structure, since it is not correct. Mg2+ ions were identified comparing the features of the electron density maps of our structures with those already described in literature.22,23 Considering that the protein is in the apo form, a Mg2+ ion was located in the ferroxidase site. Conventional structural refinements were performed with REFMAC5.7;35 model building and map inspections were performed using COOT.36 Refinement statistics are reported in Table S1. Structure validation was performed using Whatcheck.37 Coordinates, structure factors, anomalous maps, and PDB validation reports have been provided to the Editor and referees, for their use. Coordinates and structure factors were deposited in the Protein Data Bank under the accession codes 5N26 and 5N27, for HuHFt-cisplatin and HuHFt, respectively.

Figure 6. Loss of HuHF ferroxidase activity after the treatment with cisplatin (10 equiv per protein subunit). The plots show the reaction kinetics of formation of the intermediate diferric peroxo and of the products diferric oxo species, as the change in absorbance at 650 and 350 nm during 5 s (upper and lower panels, respectively) after the addition of two Fe2+ ions per protein subunit. The curves represent the mean with its errors of three replicates experiments.

presence of four distinct Pt binding sites on human H-chain Ft structure can allow this molecule to vehicle a large amount of Pt to cancer cells, thus representing a smart delivery system for Pt-based drugs.

■

EXPERIMENTAL SECTION

Sample Preparation and Solution Characterization, Crystallization, and X-ray Diffraction Data Collection. HuHFt was expressed and purified as reported in previous papers [see, e.g., ref 22]. Intrinsic fluorescence of HuHFt and of HuHFt in the presence of increasing amount of cisplatin was measured at 25 °C with a Varian Cary Eclipse spectrophotometer operated with the Cary Eclipse Bio software package. Experimental conditions: excitation wavelength: 280 or 295 nm; scan rate: 30 nm/min; data interval: 0.5 nm; protein concentration: 0.05 mg mL−1 in 10 mM sodium phosphate, pH 7.4. Fluorescence measurements were reported in Figure 1 of the main text and in Figure S1. Fluorescence spectroscopy was also used to monitor modification of emission of HuHFt and HuHFt-cisplatin during iron loading upon addition of increasing amounts of Fe2+. In all these experiments HuHFt-cisplatin adduct was formed upon incubation of the protein in the presence of cisplatin in 1:5 protein subunit to molar ratio, followed by extensive dialysis to remove the excess of the drug. Fluorescence spectra in this case were collected at 0.3 or at 0.5 mg mL−1. CD spectra of HuHFt and of HuHFt-cisplatin adduct, prepared incubating for 24 h the protein in the presence of cisplatin in 1:10 protein-to-metal ratio (and successively dialyzed), were recorded on a JASCO J-710 spectropolarimeter equipped with a Peltier thermostatic cell holder (model PTC-348WI). Both spectra and thermal unfolding 9068

DOI: 10.1021/acs.inorgchem.7b01072 Inorg. Chem. 2017, 56, 9064−9070

Article

Inorganic Chemistry

■

(11) Harrison, P. M.; Hempstead, P. D.; Artymiuk, P. J.; Andrews, S. C. In Metal Ions in Biological Systems; Sigel, A., Sigel, H., Eds.; Marcel Dekker, Inc.: New York, 1998; Vol. 35, pp 435−477. (12) Theil, E. C. Ferritin: structure, gene regulation, and cellular function in animals, plants, and microorganisms. Annu. Rev. Biochem. 1987, 56, 289−315. (13) Li, L.; Fang, C. J.; Ryan, J. C.; Niemi, E. C.; Lebrón, J. A.; Björkman, P. J.; Arase, H.; Torti, F. M.; Torti, S. V.; Nakamura, M. C.; Seaman, W. E. Binding and uptake of H-ferritin are mediated by human transferrin receptor-1. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 3505−10. (14) Li, J. Y.; Paragas, N.; Ned, R. M.; Qiu, A.; Viltard, M.; et al. Scara5 is a ferritin receptor mediating non-transferrin iron delivery. Dev. Cell 2009, 16, 35−46. (15) Fan, K.; cao, C.; Pan, Y.; Lu, D.; Yang, D.; Feng, J.; Song, L.; Liang, M.; Yan, X. Magnetoferritin nanoparticles for targeting and visualizing tumour tissues. Nat. Nanotechnol. 2012, 7, 459−464. (16) Zhang, L.; Li, L.; Di Penta, A.; Carmona, U.; Yang, F.; Schöps, R.; Brandsch, M.; Zugaza, J. L.; Knez, M. H-Chain Ferritin: A Natural Nuclei Targeting and Bioactive Delivery Nanovector. Adv. Healthcare Mater. 2015, 4, 1305−1310. (17) Messori, L.; Merlino, A. Cisplatin binding to protein: a structural prospective. Coord. Chem. Rev. 2016, 315, 67−89. (18) Levi, S.; Santambrogio, P.; Albertini, A.; Arosio, P. Human ferritin H-chains can be obtained in non-assembled stable forms which have ferroxidase activity. FEBS Lett. 1993, 336, 309. (19) Merlino, A. Interactions between proteins and Ru compounds of medicinal interest: a structural prospective. Coord. Chem. Rev. 2016, 326, 111−134. (20) Merlino, A.; Marzo, T.; Messori, L. Protein Metalation by Anticancer Metallodrugs: A Joint ESI MS and XRD Investigative Strategy. Chem. - Eur. J. 2017, 23, 6942−6947. (21) Messori, L.; Merlino, A. Cisplatin binding to proteins:molecular structure of the RNase A adduct. Inorg. Chem. 2014, 53, 3929−3931. (22) Pozzi, C.; Di Pisa, F.; Bernacchioni, C.; Ciambellotti, S.; Turano, P.; Mangani, S. Iron binding to human heavy-chain ferritin. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2015, 71, 1909−1920. (23) Masuda, T.; Goto, F.; Yoshihara, T.; Mikami, B. The universal mechanism for iron translocation to the ferroxidase site in ferritin, which is mediated by the well conserved transit site. Biochem. Biophys. Res. Commun. 2010, 400, 94−9. (24) Ferraro, G.; Messori, L.; Merlino, A. The X-ray structure of the primary adducts formed in the reaction between cisplatin and cytochrome c. Chem. Commun. (Cambridge, U. K.) 2015, 51, 2559− 2561. (25) Tanley, S. W. M.; Schreurs, A. M. M.; Kroon-Batenburg, L. M. J.; Helliwell, J. R. Room-temperature X-ray diffraction studies of cisplatin and carboplatin binding to His15 of HEWL after prolonged chemical exposure. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2012, 68, 1300−1306. (26) Tanley, S. W. M.; Schreurs, A. M. M.; Helliwell, J. R.; KroonBatenburg, L. M. J. Experience with exchange and archiving of raw data: comparison of data from two diffractometers and four software packages on a series of lysozyme crystals. J. Appl. Crystallogr. 2013, 46, 108−119. (27) Calderone, V.; Casini, A.; Mangani, S.; Messori, L.; Orioli, P. L. Structural investigation of cisplatin-protein interactions: selective platination of His19 in a cuprozinc superoxide dismutase. Angew. Chem., Int. Ed. 2006, 45, 1267−1269. (28) Ferraro, G.; Massai, L.; Messori, L.; Merlino, A. Cisplatin binding to human serum albumin: a structural study. Chem. Commun. (Cambridge, U. K.) 2015, 51, 9436−9439. (29) Casini, A.; Mastrobuoni, G.; Temperini, C.; Gabbiani, C.; Francese, S.; Moneti, G.; Supuran, C. T.; Scozzafava, A.; Messori, L. ESI mass spectrometry and X-ray diffraction studies of adducts between anticancer platinum drugs and hen egg white lysozyme. Chem. Commun. (Cambridge, U. K.) 2007, 2, 156−158.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b01072. Notes on the interpretation of the electron density maps (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Antonello Merlino: 0000-0002-1045-7720 Author Contributions

The manuscript was written through contributions of all authors who approve the final version of the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank G. Sorrentino and M. Amendola for technical assistance, M. Severi and A. Pratesi for ICP-OES measurements at Dept. of Chemistry of Univ. of Florence. L.M. thanks Beneficentia Stiftung, ITT (Istituto Toscano Tumori), Ente Cassa Risparmio Firenze (ECR), AIRC (IG-16049) for financial support. CIRCMSB consortium is also acknowledged. SC acknowledges a post-doctoral grant by Fondazione Cassa di Risparmio di Firenze (n. 2013.0494) provided by FiorGen.

■

REFERENCES

(1) Wang, D.; Lippard, S. J. Cellular processing of platinum anticancer drugs. Nat. Rev. Drug Discovery 2005, 4, 307−320. (2) Rosenberg, B. Platinum complexes for the treatment of cancer. Interdiscip. Sci. Rev. 1978, 3, 134−147. (3) Pontillo, N.; Pane, F.; Messori, L.; Amoresano, A.; Merlino, A. Cisplatin encapsulation within a ferritin nanocage: a high-resolution crystallographic study. Chem. Commun. (Cambridge, U. K.) 2016, 52, 4136−4139. (4) Xing, R.; Wang, X.; Zhang, C.; Zhang, Y.; Wang, Q.; Yang, Z.; Guo, Z. Characterization and cellular uptake of platinum anticancer drugs encapsulated in apoferritin. J. Inorg. Biochem. 2009, 103, 1039− 1044. (5) Yang, Z.; Wang, X.; Diao, H.; Zhang, J.; Li, H.; Sun, H.; Guo, Z. Encapsulation of platinum anticancer drugs by apoferritin. Chem. Commun. (Cambridge, U. K.) 2007, 7, 3453−3455. (6) Ji, X. T.; Huang, L.; Huang, H. Q. Construction of nanometer cisplatin core-ferritin (NCC-F) and proteomic analysis of gastric cancer cell apoptosis induced with cisplatin released from the NCC-F. J. Proteomics 2012, 75, 3145−57. (7) Zhu, B.; Huang, L.; Huang, H. Q. Cloning analysis of ferritin and the cisplatin-subunit for cancer cell apoptosis in Aplysia juliana hepatopancreas. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 2012, 156 (2), 95−103. (8) Falvo, E.; Tremante, E.; Fraioli, R.; Leonetti, C.; Zamparelli, C.; Boffi, A.; Morea, V.; Ceci, P.; Giacomini, P. Antibody-drug conjugates: targeting melanoma with cisplatin encapsulated in protein-cage nanoparticles based on human ferritin. Nanoscale 2013, 5, 12278− 12285. (9) Arosio, P.; Adelman, T. G.; Drysdale, J. W. On ferritin heterogeneity. Further evidence for heteropolymers. J. Biol. Chem. 1978, 253, 4451−4458. (10) Harrison, P. M.; Arosio, P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta, Bioenerg. 1996, 1275, 161−203. 9069

DOI: 10.1021/acs.inorgchem.7b01072 Inorg. Chem. 2017, 56, 9064−9070

Article

Inorganic Chemistry (30) Russo Krauss, I.; Ferraro, G.; Merlino, A. Cisplatin-Protein Interactions: Unexpected drug binding to N-Terminal amine and lysine side chains. Inorg. Chem. 2016, 55, 7814−7816. (31) Banci, L.; Bertini, I.; Blazevits, O.; Calderone, V.; Cantini, F.; Mao, J.; et al. Interaction of cisplatin with human superoxide dismutase. J. Am. Chem. Soc. 2009, 134, 7009−7014. (32) Theil, E. C.; Turano, P.; Ghini, V.; Allegrozzi, M.; Bernacchioni, C. Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C4 cage axes. JBIC, J. Biol. Inorg. Chem. 2014, 19, 615−622. (33) Bernacchioni, C.; Ghini, V.; Pozzi, C.; Di Pisa, F.; Theil, E. C.; Turano, P. Loop electrostatics modulates the intersubunit interactions in ferritin. ACS Chem. Biol. 2014, 9, 2517−25. (34) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276, 307. (35) Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. REFMAC 5 for the refinement of macromolecular crystal structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 355−367. (36) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−2132. (37) Hooft, R. W. W.; Vriend, G.; Sander, C.; Abola, E. E. Errors in protein structures. Nature 1996, 381, 272.

9070

DOI: 10.1021/acs.inorgchem.7b01072 Inorg. Chem. 2017, 56, 9064−9070