Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

2.4-Å Crystal Structure of the Asymmetric Platinum Complex {Pt(ammine)(cyclohexylamine)}2+ Bound to a Dodecamer DNA Duplex

2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês

10.1074/jbc.m206979200

ISSN

1083-351X

Autores

Adam P. Silverman, Weiming Bu, Seth M. Cohen, Stephen J. Lippard,

Tópico(s)

Ferrocene Chemistry and Applications

Resumo

cis-trans-cis-Ammine(cyclohexylamine)diacetatodichloroplatinum(IV) is an oral analog of the platinum anti-cancer drug cisplatin that is currently in phase III clinical trials. Its active form, {Pt(ammine)(cyclohexylamine)}2+, binds to DNA similarly to cisplatin, forming intra- and interstrand cross-links between adjacent purine bases. Since {Pt(ammine)(cyclohexylamine)}2+ contains two different ligands, it can form two isomeric 1,2-d(GpG) intrastrand cross-links. Here we report the 2.4-Å resolution x-ray crystal structure of the major adduct between {Pt(ammine)(cyclohexylamine)}2+ and a DNA dodecamer, using the same sequence as previously reported for crystal structures of cisplatin-DNA (Takahara, P. M., Rosenzweig, A. C., Frederick, C. A., and Lippard, S. J. (1995)Nature 377, 649–652) and oxaliplatin-DNA (Spingler, B., Whittington, D. A., and Lippard, S. J. (2001) Inorg. Chem. 40, 5596–5602). Both duplexes in the asymmetric unit contain 1,2-intrastrand cross-links in which the cyclohexylamine ligand is directed toward the 3′-end of the platinated strand. The chair conformation of the cyclohexyl group is clearly resolved. Platination distorts the duplex, resulting in a global bend angle of about 38o and a dihedral angle between platinated guanine bases of ∼31o. Both end-to-end and end-to-groove packing interactions occur in the crystal lattice, the latter positioned in the minor groove across from the site of the platinum cross-link. A high degree of homology observed between this structure and the previously reported platinum-DNA structures suggests that these platinum complexes distort the DNA duplex in a very similar manner. These results suggest that differences in activity between these drugs are unlikely to result from gross conformational distortions in DNA structure following platinum intrastrand cross-link formation. cis-trans-cis-Ammine(cyclohexylamine)diacetatodichloroplatinum(IV) is an oral analog of the platinum anti-cancer drug cisplatin that is currently in phase III clinical trials. Its active form, {Pt(ammine)(cyclohexylamine)}2+, binds to DNA similarly to cisplatin, forming intra- and interstrand cross-links between adjacent purine bases. Since {Pt(ammine)(cyclohexylamine)}2+ contains two different ligands, it can form two isomeric 1,2-d(GpG) intrastrand cross-links. Here we report the 2.4-Å resolution x-ray crystal structure of the major adduct between {Pt(ammine)(cyclohexylamine)}2+ and a DNA dodecamer, using the same sequence as previously reported for crystal structures of cisplatin-DNA (Takahara, P. M., Rosenzweig, A. C., Frederick, C. A., and Lippard, S. J. (1995)Nature 377, 649–652) and oxaliplatin-DNA (Spingler, B., Whittington, D. A., and Lippard, S. J. (2001) Inorg. Chem. 40, 5596–5602). Both duplexes in the asymmetric unit contain 1,2-intrastrand cross-links in which the cyclohexylamine ligand is directed toward the 3′-end of the platinated strand. The chair conformation of the cyclohexyl group is clearly resolved. Platination distorts the duplex, resulting in a global bend angle of about 38o and a dihedral angle between platinated guanine bases of ∼31o. Both end-to-end and end-to-groove packing interactions occur in the crystal lattice, the latter positioned in the minor groove across from the site of the platinum cross-link. A high degree of homology observed between this structure and the previously reported platinum-DNA structures suggests that these platinum complexes distort the DNA duplex in a very similar manner. These results suggest that differences in activity between these drugs are unlikely to result from gross conformational distortions in DNA structure following platinum intrastrand cross-link formation. cis-Diamminedichloroplatinum(II), cisplatin, 1The abbreviations used are: cisplatin, cis-diamminedichloroplatinum(II); carboplatin, cis-diammine-1,1-cyclobutanedicarboxylatoplatinum(II); CyNH2, cyclohexylamine; dach, 1,2-diaminocyclohexane; HMG, high-mobility group; HMGB1, high-mobility group box protein 1; HMGB1a, box A of HMGB1; TG*G*A, AG*G*C, DNA duplexes containing a cisplatin 1,2-intrastrand cross-link at the bases marked with an asterisk; HPLC, high performance liquid chromatography; RMSD, root mean squared deviation is a paradigm for the treatment of testicular and other germ-cell cancers (1Cohen S.M. Lippard S.J. Prog. Nuc. Acid Res. Mol. Biol. 2001; 67: 93-130Crossref PubMed Scopus (546) Google Scholar). The diminished activity of cisplatin against several other cancers, the acquired immunity developed by many tumors, and unpleasant side effects caused by the drug have led to a search for improved platinum chemotherapeutics (2Chu G. J. Biol. Chem. 1994; 269: 787-790Abstract Full Text PDF PubMed Google Scholar). Since the 1970's, thousands of new platinum anti-cancer drug candidates have been developed and screened (3Hambley T.W. Coord. Chem. Rev. 1997; 166: 181-223Crossref Google Scholar, 4Ziegler C.J. Silverman A.P. Lippard S.J. J. Biol. Inorg. Chem. 2000; 5: 774-783Crossref PubMed Scopus (44) Google Scholar). Fewer than 30 compounds have undergone clinical trials, however, and none has surpassed the efficacy of cisplatin for treating testicular cancer. In fact, only one other drug,cis-diammine-1,1-cyclobutanedicarboxylatoplatinum(II) (carboplatin), has been approved for use in the United States. Cisplatin and several of its cytotoxic analogs are depicted in Fig.1. Platinum anti-cancer drugs bind to DNA, forming a variety of intrastrand and interstrand adducts, the most abundant of which are 1,2-intrastrand cross-links between the N7 atoms of two adjacent guanine bases (5Jamieson E.R. Lippard S.J. Chem. Rev. 1999; 99: 2467-2498Crossref PubMed Scopus (2620) Google Scholar). X-ray crystallographic studies of cisplatin bound to a dodecamer DNA duplex indicate that the {Pt(NH3)2d(GpG)}2+ adduct induces the duplex to bend toward the major groove, resulting in significant widening of the minor groove (6Takahara P.M. Rosenzweig A.C. Frederick C.A. Lippard S.J. Nature. 1995; 377: 649-652Crossref PubMed Scopus (737) Google Scholar, 7Takahara P.M. Frederick C.A. Lippard S.J. J. Am. Chem. Soc. 1996; 118: 12309-12321Crossref Scopus (407) Google Scholar). An NMR solution structure shows appreciably more bending and a larger dihedral angle between the cross-linked guanine bases than observed in the crystal structure (8Gelasco A. Lippard S.J. Biochemistry. 1998; 37: 9230-9239Crossref PubMed Scopus (299) Google Scholar). In the complex formed between domain A of high-mobility group box protein 1 (HMGB1a) and a cisplatin-DNA d(GpG) cross-link, a phenylalanine residue of the protein intercalates into the widened minor groove at the site of platination (9Ohndorf U.-M. Rould M.A. He Q. Pabo C.O. Lippard S.J. Nature. 1999; 399: 708-712Crossref PubMed Scopus (528) Google Scholar). The binding of HMG-domain proteins to cisplatin adducts potentiates the activity of the drug by shielding the DNA from nucleotide excision repair and possibly by diverting the protein from its native function (10Zamble D.B. Mu D. Reardon J.T. Sancar A. Lippard S.J. Biochemistry. 1996; 35: 10004-10013Crossref PubMed Scopus (311) Google Scholar, 11Vichi P. Coin F. Renaud J.-P. Vermeulen W. Hoeijmakers J.H.H. Moras D. Egly J.-M. EMBO J. 1997; 16: 7444-7456Crossref PubMed Scopus (154) Google Scholar, 12Zhai X. Beckman H. Jantzen H.-M. Essigmann J.M. Biochemistry. 1998; 37: 16307-16315Crossref PubMed Scopus (87) Google Scholar). cis-trans-cis-Ammine(cyclohexylamine)diacetatodichloroplatinum(IV) is a platinum(IV) compound related to cisplatin. It can be administered orally and has been in phase III clinical trials (13Ferrante K. Winograd B. Canetta R. Cancer Chemother. Pharmacol. 1999; 43: S61-S68Crossref PubMed Scopus (91) Google Scholar, 14Wong E. Giandomenico C.M. Chem. Rev. 1999; 99: 2451-2466Crossref PubMed Scopus (1761) Google Scholar). The mechanism of action of this compound is similar to that of other mononuclear platinum anti-cancer drugs following conversion tocis-ammine(cyclohexylamine)dichloroplatinum(II) by intracellular reducing agents, such as glutathione. The activated species, {Pt(NH3)(NH2Cy)}2+(NH2Cy is cyclohexylamine), forms two 1,2-d(GpG) adducts with DNA, termed 3′ and 5′ orientational isomers (15Hartwig J.F. Lippard S.J. J. Am. Chem. Soc. 1992; 114: 5646-5654Crossref Scopus (107) Google Scholar), according to the positioning of the substituted amine with respect to the direction of the platinated strand. These isomers occur in an ∼2:1 ratio, the more abundant isomer having the cyclohexylamine ligand directed toward the 3′-end of the platinated strand (15Hartwig J.F. Lippard S.J. J. Am. Chem. Soc. 1992; 114: 5646-5654Crossref Scopus (107) Google Scholar). The differences in activity among platinum drugs may involve variations in their DNA binding properties. Very few studies have been performed to explore this possibility. Recent work has demonstrated that both the nature of the ligands on the platinum atom and the bases immediately flanking the d(GpG) adduct can affect the binding of HMGB1 to platinated DNA (16Wei M. Cohen S.M. Silverman A.P. Lippard S.J. J. Biol. Chem. 2001; 276: 38774-38780Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These studies indicated that HMGB1a differentially recognizes the adducts of platinum complexes containing different carrier ligands and that HMGB1a binds to {Pt(NH3)(NH2Cy)}2+ adducts in TG*G*A and AG*G*C sequences about half as well as cisplatin adducts. It has also been demonstrated that {Pt(NH3)(NH2Cy)}2+ adducts are more effective than those of oxaliplatin and cisplatin in blocking translesion synthesis past the site of damage (17Vaisman A. Lim S.E. Patrick S.M. Copeland W.C. Hinkle D.C. Turchi J.J. Chaney S.G. Biochemistry. 1999; 38: 11026-11039Crossref PubMed Scopus (122) Google Scholar). This property may be one reason for its success in early clinical trials. Structural studies have provided important information about the interactions between platinum compounds and DNA. The crystal structures of cisplatin (6Takahara P.M. Rosenzweig A.C. Frederick C.A. Lippard S.J. Nature. 1995; 377: 649-652Crossref PubMed Scopus (737) Google Scholar, 7Takahara P.M. Frederick C.A. Lippard S.J. J. Am. Chem. Soc. 1996; 118: 12309-12321Crossref Scopus (407) Google Scholar) and oxaliplatin (18Spingler B. Whittington D.A. Lippard S.J. Inorg. Chem. 2001; 40: 5596-5602Crossref PubMed Scopus (203) Google Scholar) bound to homologous DNA sequences reveal several interesting differences and provide some insight into the effects of carrier ligands on the structure of platinum-DNA adducts. The most significant feature of the oxaliplatin-DNA structure that differentiates it from that of the cisplatin-DNA adduct is the presence of a hydrogen bond between the O6 atom of G7 and an amino group of the coordinated 1,2-diaminocyclohexane (dach) ligand. In the present article we report the first x-ray crystal structure of an asymmetric platinum complex, {Pt(NH3)(NH2Cy)}2+, bound to a DNA duplex. For comparison purposes we have employed the same DNA sequence as used for the previous two structures (Fig. 2). The major orientational isomer of this platinum complex was investigated. This work significantly extends the structural information available on platinum-DNA adducts and offers insights into the differences and common features between a variety of platinated DNA structures. The compoundcis-[Pt(NH3)(NH2Cy)Cl2] was prepared by standard methods (19Abrams M.J. Giandomenico C.M. Vollano J.F. Schwartz D.A. Inorg. Chim. Acta. 1987; 131: 3-4Crossref Scopus (47) Google Scholar, 20Barton S.J. Barnham K.J. Habtemariam A. Sue R.E. Sadler P.J. Inorg. Chim. Acta. 1998; 273: 8-13Crossref Google Scholar). Crystallization reagents were obtained from Aldrich, Fluka, and Sigma. Phosphoramidites and reagents for DNA synthesis were purchased from Glen Research. High-performance liquid chromatography (HPLC) was carried out on a Waters 600E system controller using a Waters 486 detector (λ, 260 nm) for preparative runs, and on a Waters 600S controller with a Waters 2487 detector (λ, 260 nm) for analytical runs. Top and bottom strands of the 12-bp deoxyoligonucleotide (top strand sequence 5′-d(CCTCTGGTCTCC)) were prepared (10-μmol scale, 2 syntheses for top strand) by using standard phosphoramidite methods on an Applied Biosystems 392 RNA/DNA synthesizer. After automated synthesis, the oligonucleotides were deprotected with ammonium hydroxide by incubating the crude reaction mixtures at 65 °C for 1 h. The bottom strand was purified by ion-exchange HPLC (Dionex NucleoPac PA-100, 9 mm × 250 mm, 10% acetonitrile, 25 mmammonium acetate, linear gradient from 0.2 to 0.4 m NaCl over 30 min). The product was desalted by dialysis against water using Slide-A-Lyzer cassettes (Pierce). The resulting product was analytically pure as judged by both ion exchange and C18 reverse-phase HPLC. The top strand was platinated with {Pt(NH3)(NH2Cy)}2+. Activated {Pt(NH3)(NH2Cy)}2+ was prepared by mixing 2 equivalents of silver nitrate with 1 equivalent ofcis-[Pt(NH3)(NH2Cy)Cl2] in 1.0 ml of water for 6 h. The mixture was protected from light and centrifuged to remove precipitated silver chloride. The top strand was platinated with 2.2 equivalents of the activated platinum complex in a solution containing 10 mm sodium phosphate (pH 6.8) at 37 °C for 22 h. The product was purified by ion-exchange HPLC (Dionex NucleoPac PA-100, 9 mm × 250 mm, 10% acetonitrile, 25 mm ammonium acetate, linear gradient from 0.2 to 0.4m NaCl over 30 min). Two product peaks were observed and collected separately. The products were then dialyzed against water using Slide-A-Lyzer cassettes. The major product was purified by reverse phase HPLC (C18Vydac Column, linear gradient from 7.5% acetonitrile, 0.0925m ammonium acetate to 17.5% acetonitrile, 0.0772m ammonium acetate over 30 min). The purified platinated deoxyoligonucleotide top strand was then annealed to the purified bottom strand by using 1 equivalent of each strand and 1× annealing buffer (100 mm MgCl2, 100 mm HEPES pH 7, and 500 mm LiCl) in 1 ml of solution. The mixture was heated to 85 °C for 1.0 min, and the solution was allowed to cool to room temperature over 2 h. The annealed product was purified by ion-exchange HPLC (Dionex NucleoPac PA-100, 9 mm × 250 mm, 10% acetonitrile, 25 mm ammonium acetate, linear gradient from 0.2 to 0.4m LiCl over 30 min). The purified double-stranded product was dialyzed against water as before. Finally, the duplex was re-annealed by heating to 80 °C for 1 min, followed by cooling to room temperature over 2 h. The hanging drop vapor diffusion method was used for crystallization (21Drenth J. Principles of Protein X-ray Crystallography. Springer, New York1994Crossref Google Scholar). Initial conditions were obtained from a nucleic acid sparse matrix screen, Natrix (Hampton Research) (22Scott W.G. Finch J.T. Grenfell R. Fogg J. Smith T. Gait M.J. Klug A. J. Mol. Biol. 1995; 250: 327-332Crossref PubMed Scopus (87) Google Scholar). Crystals suitable for diffraction were grown at 4 °C from 3-μl droplets containing 0.2 mm DNA and 0.5× well solution (1× well solution contained 50 mm sodium cacodylate (pH 6.5), 12 mm spermine, 120 mm Mg(OAc)2, and 15% w/v polyethylene glycol (PEG) 4000). The crystallization drops were equilibrated against a 1× well solution at 4 °C. Crystals of approximate dimensions of 0.5 × 0.2 × 0.05 mm appeared after 5–10 days. Crystals were transferred to a cryogenic freezing solution containing 50 mm Na cacodylate (pH 6.5), 12 mm spermine, 120 mmMg(OAc)2, 25% PEG 4000, and 15% glycerol at 4 °C, then mounted in a loop and frozen at 100 K under liquid nitrogen. Crystals were screened for diffraction on a Mar Research detector (mar345 345 mm 150 marpck plate) with CuKα radiation, λ = 1.54179 Å. Several crystals that diffracted to 2.8-Å resolution or better were stored on loops under liquid nitrogen. Data frames from these crystals were auto-indexed to determine unit cell parameters using the HKL program suite (23Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). From the cell constants and lack of systematic absences, the space group was determined to be P1. A high-resolution data set was collected at the Advanced Photon Source at Argonne National Laboratory. A data set with diffraction to 2.4-Å resolution was collected at 100 K on beam line SCD 19-ID (wavelength 0.970 Å). A total of 360 frames of data were taken, with φ varying by 1° per frame, using a 10-s exposure time per frame. The intensities were integrated by using DENZO and scaled with SCALEPACK (23Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). A summary of data collection statistics appears in TableI.Table IData collection and refinement statisticsData CollectionWavelength (Å)0.970 ÅUnit cell parametersa = 31.644 Åα = 79.533°b = 35.638 Åβ = 83.016°c = 46.556 Åγ = 81.355°Space groupP1Z2Resolution range (Å)50–2.4Total reflections42245Unique reflections7337Completeness1-aValues in parentheses for the highest resolution shell (2.49–2.40 Å).(%)96.3 (96.3)R sym1-aValues in parentheses for the highest resolution shell (2.49–2.40 Å).,1-bR sym = Σ‖I - 〈I〉‖/ΣI.(%)9.0% (27.7%)9.2Refinement StatisticsR 1-cR = Σ∥Fo‖ - ‖Fc∥/Σ‖Fo‖. [5267 reflns with Fo > 4ς(Fo)]0.2008R frec1-dR free = R obtained for a test set of reflections (10% of diffraction data).[565 reflns with Fo > 4ς (Fo)]0.2495RMSD bond lengths (Å)0.005RMSD 1,3 distances (Å)0.022RMSD planes (for 504 atoms)0.0003RMSB bonded atoms0.002No. of refined parameters4163No. of restraints48781-a Values in parentheses for the highest resolution shell (2.49–2.40 Å).1-b R sym = Σ‖I - 〈I〉‖/ΣI.1-c R = Σ∥Fo‖ - ‖Fc∥/Σ‖Fo‖.1-d R free = R obtained for a test set of reflections (10% of diffraction data). Open table in a new tab Refinements were carried out with SHELXL97 (24Sheldrick G.M. SHELX97–2:Program for the Refinement of Crystal Structures;. University of Gottingen:, Gottingen1997Google Scholar), using the resolution range 50–2.4 Å. Manual refinement and rebuilding of the structure were performed with XTALVIEW (25McRee D.E. J. Mol. Graphics. 1992; 10: 44-46Crossref Google Scholar). The unit cell dimensions revealed the crystal to be isomorphous with the previously reported cisplatin-DNA structure (6Takahara P.M. Rosenzweig A.C. Frederick C.A. Lippard S.J. Nature. 1995; 377: 649-652Crossref PubMed Scopus (737) Google Scholar,7Takahara P.M. Frederick C.A. Lippard S.J. J. Am. Chem. Soc. 1996; 118: 12309-12321Crossref Scopus (407) Google Scholar), entry 1AIO in the Protein Data Bank (26Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27555) Google Scholar). Two platinated DNA duplexes comprise the asymmetric unit. The platinum atoms, ammine ligands, and water molecules were removed from this structure, and a rigid-body refinement was performed to generate an initial model. The DNA Dictionary for SHELX was used with appropriate restraints for the phosphodiester backbone, sugars, and nucleobases (27Clowney L. Jain S.C. Srinivasan A.R. Westbrook J. Olsen W.K. Berman H.M. J. Am. Chem. Soc. 1996; 118: 509-518Crossref Scopus (174) Google Scholar, 28Gelbin A. Schneider B. Clowney L. Hseih S.-H. Olson W.K. Berman H.M. J. Am. Chem. Soc. 1996; 118: 519-529Crossref Scopus (189) Google Scholar). Large areas of electron density around G6-G7 (Fig. 2) on a sigma-d ∥Fo - Fc∥ map were identified as the {Pt(NH3)(NH2Cy)}2+ moiety, starting coordinates for which were obtained from the Cambridge Structural Database (29Allen F.H. Kennard O. Chem. Des. Autom. News. 1993; 8: 31-37Google Scholar). The complex was manually fit into each of the duplexes using XTALVIEW. For subsequent refinement steps, 1,2- and 1,3-distances for the cyclohexylamine ligand were restrained. Forty-eight waters were added at locations having proper distances for hydrogen bonding interactions and electron densities greater than 2.0ς on a sigma-d ∥Fo - Fc∥ map. Refinement statistics are included in Table I. Geometry parameters were calculated by using the programs 3DNA (30Lu X.-J. Olson W.K. J. Mol. Biol. 1999; 285: 1563-1575Crossref PubMed Scopus (87) Google Scholar) and Curves (31Lavery R. Sklenar H. J. Biomol. Struct. Dyn. 1988; 6: 63-91Crossref PubMed Scopus (920) Google Scholar). The structure has been deposited in the Protein Data Bank, ID number1LU5. The unit cell is isomorphous with that of the previously reported cisplatin-DNA structure (6Takahara P.M. Rosenzweig A.C. Frederick C.A. Lippard S.J. Nature. 1995; 377: 649-652Crossref PubMed Scopus (737) Google Scholar, 7Takahara P.M. Frederick C.A. Lippard S.J. J. Am. Chem. Soc. 1996; 118: 12309-12321Crossref Scopus (407) Google Scholar) and similarly contains two DNA duplexes in the asymmetric unit. These duplexes are labeled molecule A (Fig.3) and molecule B. Two types of hydrophobic packing interactions occur between duplexes in the crystal lattice, end-to-end and end-to-minor groove. These packing interactions are mediated by hydrogen bonds between individual duplexes. The cisplatin-DNA and oxaliplatin-DNA structures exhibit similar packing interactions. In the hydrophobic end-to-minor groove packing, depicted in Fig.4, the end of one molecule packs against the minor groove opposite the platinum-binding site of another molecule. The C1-G24 base pair of molecule A interacts with the G6-C19 base pair of a molecule B helix. A similar interaction occurs between the C1-G24 base pair of molecule B and the G6-C19 base pair of molecule A. End-to-groove packing of this kind is often observed in A-DNA structures (32Frederick C.A. Quigley G.J. Teng M.-K. Coll M. van der Marel G.A. van Boom J.H. Rich A. Wang A.H.-J. Eur. J. Biochem. 1989; 181: 295-307Crossref PubMed Scopus (61) Google Scholar). In the end-to-end packing arrangement, the 3′-end of one molecule stacks against the 3′-end of another such that a pseudo-continuous helix is formed. This type of packing is characteristic of B-DNA structures (33Wang A.H.-J. Teng M.-K. J. Cryst. Growth. 1988; 90: 295-310Crossref Scopus (18) Google Scholar). The crystal structure reveals the asymmetric attachment of thecis-{Pt(NH3)(NH2Cy)}2+fragment to the d(GpG) unit in both molecules A and B. Both maintain full Watson-Crick base pairing despite the distortion of the base steps. Their global bend angles and base step parameters differ slightly as a result of minor differences in local base pair and base step parameters (Tables II andIII). Both helices start with A-DNA conformations at the 5′ end of the platinated strand and gradually become more B-DNA-like, as determined by the slide and sugar puckering parameters (Table IV and Fig.5).Table IIBase pair parameters for molecules A and Bκ2-aBase pair parameters are defined as follows: κ, buckle; ς, opening; ω, propeller twist; Sx, shear;Sy, stretch; Sz, stagger.ςωSxSySzABABABABABABC1 - G2416.611.90.01−1.26−6.88−3.580.650.70−0.24−0.18−0.200.02C2 - G234.389.171.034.78−2.90−2.730.570.50−0.12−0.12−0.21−0.25T3 - A223.634.8614.42.89−12.5−7.52−0.04−0.320.06−0.22−0.17−0.20C4 - G21−1.37−3.102.862.58−1.04−5.050.390.070.060.35−0.240.14T5 - A202.834.28−1.50−0.96−5.20−5.20−0.09−0.14−0.29−0.190.260.17G6 - C1919.521.7−0.994.03−15.0−16.4−0.34−0.260.020.120.180.51G7 - C18−6.91−4.493.510.47−13.2−16.8−0.18−0.140.06−0.03−0.030.27T8 - A17−0.66−11.84.44−2.02−21.4−21.8−0.21−0.61−0.31−0.15−0.66−0.25C9 - G16−2.69−10.91.01−1.47−13.4−6.410.25−0.14−0.35−0.310.110.00T10 - A152.77−7.26−5.150.13−8.94−2.73−0.69−0.66−0.28−0.070.130.47C11 - G140.420.36−6.23−4.54−11.4−11.6−0.05−0.03−0.17−0.010.030.27C12 - G13−6.66−10.12.777.51−3.74−8.030.641.08−0.10−0.300.130.34A-DNA0.000.00−0.85−0.8511.411.40.000.00−0.11−0.110.150.15B-DNA0.000.00−0.38−0.38−1.29−1.290.000.000.010.01−0.02−0.022-a Base pair parameters are defined as follows: κ, buckle; ς, opening; ω, propeller twist; Sx, shear;Sy, stretch; Sz, stagger. Open table in a new tab Table IIIBase step parameters for molecules A and Bρ3-aBase step parameters are defined as follows: ρ, roll; τ, tilt; Ω, twist; Dx, Shift; Dy,slide; Dz, rise.τΩDxDyDzABABABABABABC1-G24/C2-G234.172.68−3.280.5325.826.5−0.99−0.64−2.28−2.323.543.24C2-G23/T3-A222.412.672.40−0.0128.428.40.17−0.44−1.62−1.813.313.48T3-A22/C4-G219.2410.4−1.08−0.2834.032.4−0.330.39−1.44−1.593.413.40C4-G21/T5-A205.045.55−1.67−0.7929.030.9−0.48−0.26−1.68−1.633.233.15T5-A20/G6-C196.233.83−0.33−2.4225.725.6−0.70−0.63−1.36−1.532.942.93G6-C19/G7-C1828.526.21.662.5732.532.71.281.17−1.62−1.733.713.67G7-C18/T8-A173.504.105.804.1634.533.5−0.74−0.75−0.88−1.233.043.25T8-A17/C9-G164.305.18−2.120.8534.837.40.620.56−0.62−0.823.403.26C9-G16/T10-A157.303.540.47−1.6229.028.9−0.49−0.20−0.49−0.573.233.38T10-A15/C11-G14−1.530.984.692.8840.839.30.120.06−0.22−0.383.333.12C11-G14/C12-G136.191.73−1.322.0932.736.20.080.67−0.41−0.303.363.47A-DNA10.810.80.000.0030.930.90.000.00−2.08−2.083.183.18B-DNA−2.80−2.800.000.0035.935.90.000.00−0.62−0.623.343.343-a Base step parameters are defined as follows: ρ, roll; τ, tilt; Ω, twist; Dx, Shift; Dy,slide; Dz, rise. Open table in a new tab Table IVSugar puckering for molecules A and BStepSugar pucker (Mol. A)DNA typeSugar pucker (Mol. B)DNA typeC1C2′-exoAC3′-exoAC2C3′-endoAC3′-endoAT3C3′-endoAC3′-endoAC4C3′-endoAC3′-endoAT5C3′-endoAC3′-endoAG6C3′-endoAC3′-endoAG7C3′-endoAC3′-endoAT8C3′-endoAC2′-exoAC9C4′-exoA/BC4′-exoA/BT10C1′-exoA/BC1′-exoA/BC11C1′-exoBC1′-exoA/BC12O1′-endoBC4′-exoA/B Open table in a new tab Despite the large positive roll caused by the platinum binding to the N7 atoms of G6 and G7, even the hydrogen bonds between the platinated G-C base pairs remain intact (Fig. 6). The minor groove is widened, and the major groove is narrowed significantly. The platinum-nitrogen distances are 2.0 ± 0.1 Å. The dihedral angle between the G6 and G7 nucleobases is 32oin molecule A and 29o in molecule B. These values are slightly greater than those from the previously reported platinated-DNA structures. In molecule A, the platinum atom is displaced 1.2 Å from the plane of the 5′ guanine and 0.61 Å from the plane of the 3′ guanine. The corresponding displacements in molecule B are 1.1 and 0.93 Å, respectively. The platinum atoms are 0.05 and 0.11 Å from the least-squares plane containing the four coordinated nitrogen atoms in molecules A and B, respectively. The cyclohexylamine ligand is well resolved at the 1ς level in an omit map, shown in Fig. 7, and is directed toward the 3′-end of the platinated strand. It adopts a chair conformation and points away from the major groove. The amino group of the NH2Cy ligand forms a hydrogen bond with the O6 atom on G6. The N···O distances is 2.6 Å for molecule A and 2.9 Å for molecule B. The ammine ligand forms a weak hydrogen bonding interaction with the O2P atom on G7, at a distance of 3.1 Å for molecule A and 3.4 Å for molecule B. The ammine ligand is about 4.7 Å from the O-6 atom of G7 in both duplexes, too far to form a hydrogen bond. The five platinated duplexes contained in the three platinum-DNA structures determined in our laboratory possess considerable homology. The root-mean-square deviations (RMSD) between comparable atoms in the various duplexes and platinum binding sites are listed in TableV. All RMSD values are less than 1.0 Å, indicating a great deal of similarity in both the bent duplexes and in the sites of platination. A comparison of selected distances and angles (Table VI) similarly reveals only small variations.Table VRMSD values comparing different platinum-DNA crystal structuresDuplexesRMSD DNA duplex5-aDNA duplexes without platinum bound.RMSD platinum binding site5-bBase-pairs G6-C19 and G7-C18 and the platinum coordination sphere, {Pt(N7)2(N)2}Mol. A5-cMol. A and Mol. B are structures from this work. - Mol. B0.429850.13665Mol. A - cisplatinA0.595720.30431Mol. A - cisplatinB0.568490.32048Mol. A - oxaliplatin0.798580.43382Mol. B - cisplatinA0.451710.24020Mol. B - cisplatinB0.459350.27976Mol. B - oxaliplatin0.644710.47012CisplatinA - cisplatinB0.219980.12702CisplatinA - oxaliplatin0.686080.42241CisplatinB - oxaliplatin0.673900.375925-a DNA duplexes without platinum bound.5-b Base-pairs G6-C19 and G7-C18 and the platinum coordination sphere, {Pt(N7)2(N)2}5-c Mol. A and Mol. B are structures from this work. Open table in a new tab Table VIAngles and distances in the platinum binding sitesParameterMol. A6-aMol. A and Mol. B are the structures from this work.Mol. B6-aMol. A and Mol. B are the structures from this work.Cisplatin ACisplatinBOxaliplatinN7-N7 (Å)2.822.982.892.922.97NL-NL(Å)6-aMol. A and Mol. B are the structures from this work.2.803.092.872.722.72N7-Pt-N7 (°)92.398.599.498.698.6NL-Pt-NL(°)6-bNL refers to the nitrogen in the ammine, cyclohexylamine, or dach ligand.87.699.198.584.184.1G6-G7 dihedral (°)3229262625Global bend angle (°)39373955306-a Mol. A and Mol. B are the structures from this work.6-b NL refers to the nitrogen in the ammine, cyclohexylamine, or dach ligand. Open table in a new tab The crystal packing in the platinum-DNA duplex structures has an effect on the observed symmetry as originally indicated by the homology between the cisplatin and oxaliplatin structures (7Takahara P.M. Frederick C.A. Lippard S.J. J. Am. Chem. Soc. 1996; 118: 12309-12321Crossref Scopus (407) Google Scholar, 18Spingler B. Whittington D.A. Lippard S.J. Inorg. Chem. 2001; 40: 5596-5602Crossref PubMed Scopus (203) Google Scholar). The present results support this idea. The common end-to-minor groove packing significantly affects the geometry at the platination site. The solution structures of cisplatin-DNA and related adducts, solved by NMR spectroscopy, display higher global bend angles than any of the crystal structures (8Gelasco A. Lippard S.J. Biochemistry. 1998; 37: 9230-9239Crossref PubMed Scopus (299) Google Scholar). This difference suggests that the solution form of platinated-DNA differs from that in the crystal lattice. The present structure supports our previous assignment of the major product of DNA platination by {Pt(NH3)(NH2Cy)}2+ as the isomer with the cyclohexylamine oriented toward the 3′-end of the platinated strand (15Hartwig J.F. Lippard S.J. J. Am. Chem. Soc. 1992; 114: 5646-5654Crossref Scopus (107) Google Scholar). The hydrogen bonds observed between the platinum ligands and the DNA strand may contribute to this preference. One hydrogen bond is formed between the NH3 and the 5′ O2P atom, and another occurs between the NH2 group of the cyclohexylamine ligand and O6 of the 3′ guanine base. In the cisplatin-DNA structure, the distance between the 3′-directed ammine ligand and O6 of the 3′ guanine is 3.5 Å. This distance is too long for significant hydrogen bonding, suggesting that such a bond may not form in the minor orientational isomer of the {Pt(NH3)-(NH2Cy)d(GpG)}2+cross-link. Moreover, it is unlikely that the cyclohexylamine amino group would form a hydrogen bond with the 5′ guanine O6 atom in the minor isomer, since the NH3···O6 distance in the major isomer is 4.7 Å. Recent work by the Marzilli group (34Sullivan S.T. Ciccarese A. Fanizzi F.P. Marzilli L.G. J. Am. Chem. Soc. 2001; 123: 9345-9355Crossref PubMed Scopus (57) Google Scholar, 35Carlone M. Fanizzil F.P. Intini F.P. Margiotta N. Marzilli L.G. Natile G. Inorg. Chem. 2000; 39: 634-641Crossref PubMed Scopus (31) Google Scholar) suggested that hydrogen bonding between platinated guanine bases and their associated phosphate groups with the ammine or amine ligands in a {PtL2}2+ complex (L2 is two amines or a diamine) is extremely weak. In the present 3′ orientational isomer, the hydrogen bond with O-6 of the 3′ guanine is quite substantial and, in our previous {Pt(dach)}2+ complex, there was a similarly strong hydrogen bond on the 3′-end involving the amino group of dach and the 3′-phosphate. Crystal packing (see above) may contribute to these effects, or perhaps the conclusion about weak hydrogen bonds in these platinated duplexes is premature. The very small differences observed in the G6-G7 base pair step geometry for the cisplatin-, oxaliplatin-, and {Pt(NH3)(NH2Cy)}2+-DNA structures may signal a common geometric feature that modulates their recognition by minor groove binding proteins. These results also support the hypothesis that the flanking sequence dependence of proteins binding to platinum-DNA adducts is most likely an inherent property of sequence-dependent protein-DNA contacts, not differential distortion around the platination site (16Wei M. Cohen S.M. Silverman A.P. Lippard S.J. J. Biol. Chem. 2001; 276: 38774-38780Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 36Sullivan S.T. Saad J.S. Fanizzi F.P. Marzilli L.G. J. Am. Chem. Soc. 2002; 124: 1558-1559Crossref PubMed Scopus (33) Google Scholar). Most proteins that bind platinum-DNA adducts, such as HMGB1 and TBP, interact with the minor groove. The crystallographic studies from our laboratory and computational studies by Marzilli and co-workers (34Sullivan S.T. Ciccarese A. Fanizzi F.P. Marzilli L.G. J. Am. Chem. Soc. 2001; 123: 9345-9355Crossref PubMed Scopus (57) Google Scholar,35Carlone M. Fanizzil F.P. Intini F.P. Margiotta N. Marzilli L.G. Natile G. Inorg. Chem. 2000; 39: 634-641Crossref PubMed Scopus (31) Google Scholar) have demonstrated that platinum-DNA adducts are nearly structurally homologous, even in the minor groove where most protein-DNA interactions occur. Yet these proteins are able to recognize differentially adducts that contain differing amine ligands (16Wei M. Cohen S.M. Silverman A.P. Lippard S.J. J. Biol. Chem. 2001; 276: 38774-38780Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and platinum complexes with different spectator ligands inhibit transcription to varying degrees (37Sandman K.E. Marla S.S. Zlokarnik G. Lippard S.J. Chem. Biol. 1999; 6: 541-551Abstract Full Text PDF PubMed Scopus (32) Google Scholar). This result suggests that hydrogen bonding, hydrophobic, and other major groove interactions between protein, DNA, and the ligands on the platinum may affect drug activity. The bulky dach ligand in oxaliplatin and the cyclohexylamine ligand in {Pt(NH3)(NH2Cy)}2+ adducts fill and increase the hydrophobicity of the major groove in the vicinity of the adduct. These major groove effects may fine tune platinum drug activity by interfering with transcription. We thank Dr. Chuan He for assistance with data collection and Dr. Bernhard Spingler, Matthew Sazinsky, and the Drennan laboratory for helpful discussions.

Referência(s)