High-resolution crystal structure of Z-DNA in complex with Cr3+ cations

This work is part of our project aimed at characterizing metal-binding properties of left-handed Z-DNA helices. The three Cr3+ cations found in the asymmetric unit of the d(CGCGCG)2–Cr3+ crystal structure do not form direct coordination bonds with atoms of the Z-DNA molecule. Instead, the hydrated Cr3+ ions are engaged in outer-sphere interactions with phosphate groups and O6 and N7 guanine atoms of the DNA. The Cr3+(1) and Cr3+(2) ions have disordered coordination spheres occupied by six water molecules each. These partial-occupancy chromium cations are 2.354(15) Å apart and are bridged by three water molecules from their hydration spheres. The Cr3+(3) cation has distorted square pyramidal geometry. In addition to the high degree of disorder of the DNA backbone, alternate conformations are also observed for the deoxyribose and base moieties of the G2 nucleotide. Our work illuminates the question of conformational flexibility of Z-DNA and its interaction mode with transition-metal cations. Electronic supplementary material The online version of this article (doi:10.1007/s00775-015-1247-5) contains supplementary material, which is available to authorized users.


Introduction
Trivalent chromium, a d 3 cation, is poorly taken up by living cells. The Cr 3+ ions are the final product of in vivo Cr 6+ metabolism. However, Cr 3+ in contrast to Cr 6+ can form coordination complexes with macromolecules in the cells [1][2][3]. In vitro biochemical experiments have shown that exposure of cells to Cr 6+ yields binary (DNA-Cr 3+ ) and ternary (DNA-Cr 3+ -ligand) adducts, DNA crosslinks, as well as oxidative DNA lesions [2,3]. The DNA-Cr 3+ complexes are most abundant during in vitro Cr 6+ reduction and are less mutagenic than the ternary adducts. It was also found that the Cr 3+ ions decrease the fidelity of DNA polymerase [4]. Several attempts have been made to explain the nature of the DNA-Cr 3+ interactions. Phosphate groups and/or the N7 guanine atoms have been indicated as the interaction sites in DNA-Cr 3+ adducts [2,[5][6][7][8]. Some nucleotide-Cr 3+ and DNA-[Cr(Schiff base)] complexes were characterized by different techniques, such as 31 P NMR, EPR, UV-Vis and IR spectroscopy; however, those studies provided little, if any, precise structural data [9,10]. Structures of phosphate-based DNA-Cr 3+ adducts have also been proposed [11]. Despite the interest in DNA-Cr 3+ interactions in biological systems, the existing literature provides detailed crystallographic structural data for only two, low-resolution DNA-Cr 3+ :DNA polymerase-β complexes, PDB 1zqe (3.7 Å) [12] and 1huz (2.6 Å) [13]. For these two structures, direct interactions of Cr 3+ with the DNA phosphates were described. In particular, no 1 3 structures of Z-DNA-Cr 3+ complexes have been deposited in the PDB [14] or the NDB [15] databases.
Left-handed duplexes of Z-DNA with the self-complementary d(CGCGCG) sequence are remarkable for their potential to form crystals that diffract X-rays to very high resolution. We have used those crystals to obtain accurate information about the geometrical parameters characterizing the coordination of Cr 3+ ions by left-handed Z-DNA. In the present study, using high-resolution crystallographic data, we describe the coordination geometry of three hydrated Cr 3+ ions and their outer-sphere interactions with a highly disordered Z-DNA chain in a d(CGCGCG) 2 -Cr 3+ complex. To the best of our knowledge, a double-conformation guanine base ring is also reported for the first time in any Z-DNA structure.

Materials and method
Oligonucleotide synthesis, purification and crystallization A DNA hexamer with the d(CGCGCG) sequence was synthesized on an Applied Biosystems DNA/RNA synthesizer using phosphoramidite chemistry. The methods of deprotection and purification of the oligodeoxynucleotides have been described previously [16]. A 1.5 mM water solution of the DNA oligonucleotide was annealed at 338 K for 12 min to form a DNA duplex. Single crystals of the DNA were grown at 292 K by the hanging-drop vapor diffusion method by mixing 2 μl of nucleic acid solution and 2 μl of precipitating solution containing 10 % (v/v) (±)2-methyl-2,4-pentanediol (MPD), 40 mM sodium cacodylate, pH 6.0, 80 mM KCl, 12 mM NaCl and 12 mM sperminium tetrachloride. The drops were equilibrated against 0.5 ml of 35 % (v/v) MPD. The crystals appeared within 1 week and grew to the dimensions of 0.2 × 0.1 × 0.1 mm. The best crystals were used for metal ion soaking. For metal soaking, the crystals were placed for several days in 2 μl of the reservoir solution mixed with 2 μl of 5 mM [Cr(H 2 O) 6 ]Cl 3 .
Data collection, structure solution and refinement X-Ray diffraction data for Cr 3+ -soaked crystals were collected to the resolution of 0.97 Å at the BESSY synchrotron beamline 14.2 in Berlin ( Table 1). The crystal was vitrified in a stream of cold nitrogen gas at 100 K. The mother liquor served as a cryoprotectant solution. The data were recorded in four passes, using different crystal-to-detector distances and exposure times to ensure reliable measurement of the high-resolution data and of the strong low-resolution reflections, and to avoid oversaturation of the MAR225 detector. The crystal-to-detector distances, the corresponding maximum resolution, oscillation and the numbers of images in  Supplementary Table  S1. The diffraction data were indexed, integrated and scaled using the XDS package [17]. The X-ray data statistics are summarized in Table 1. The structure was solved by molecular replacement using PHASER [18]. The DNA part of the PDB structure 4hig, corresponding to our earlier model of a d(CGCGCG) 2 -Spk-Mn 2+ complex [16], was used as a molecular probe. At the initial stages of the refinement, the model was refined using REFMAC5 [19] from the CCP4 program suite [20]. Later, anisotropic refinement with SHELXL [21] was performed using the full resolution of the diffraction data. The anomalous signal, clearly visible  [22]. Therefore, the structure-factor refinement was carried out with Bijvoet pairs unmerged. The Flack parameter is −0.08 (9), confirming the obvious fact that the absolute structure has been determined correctly. After each round of 50 cycles of CGLS (conjugategradient least-squares) minimization, the COOT [23] program was used for visualization of electron density maps and manual rebuilding of the atomic model. The occupancies of the DNA moieties in alternative conformations were refined with their sum constrained to unity. Water molecules were added to the model based on mFo-DFc electron density and stereochemical considerations. There are four close pairs of water molecules with combined occupancy of 1.0. The remaining water sites were classified as fully (11) or partially (52) occupied. Ordered water molecules in the coordination sphere of a given Cr 3+ ion have the same occupancy as the central atom. Hydrogen atoms were added at their expected positions only to DNA atoms and refined isotropically using the "riding" option.
The model was validated using the free R test [24] and the NuCheck program [25]. ~7 % of all reflections were selected at random and set aside for R free calculations. The test reflections were included in the work set during the final rounds of refinement. Most of the stereochemical restrains for the DNA moieties were taken from Clowney et al. [26], Gelbin et al. [27] and Parkinson et al. [28]. However, the target values for the C4-N9-C1′ [129.0(7)°] and C8-N9-C1′ [124.5(7)°] exocyclic angles in syn purines, which seem to be inadequate in the above dictionaries, were taken from the ultrahigh-resolution Z-DNA-Spm structure (0.55 Å; PDB 3p4j) reported by Brzezinski et al. [29]. The 3p4j structure was also used for restraining the O-P-O valence angles in a conformation-dependent manner [30]. At the conclusion of the refinement, one cycle of full-matrix least-squares minimization was calculated with all reflections, but no restraints included to estimate the standard uncertainties (e.s.u.) of the structural and all refined parameters.
The coordinates of the Cr 3+ (1), Cr 3+ (2) and Cr 3+ (3) ions coincide with the most prominent peaks in the anomalous difference Fourier map at 15.0, 10.0 and 16.1σ, respectively (Fig. 1). The pseudorotation amplitude (τ m ) and phase angle (P) of the DNA furanose rings as well as their deviations from the ideal (cyclopentane) model were calculated by the method of Jaskolski [31] (Supplementary Table  S2). The Z-DNA helical parameters were calculated using 3DNA [32] and the figures were generated with PyMol [33].
Overall structure and helical parameters The asymmetric unit of the complex contains one d(CGCGCG) 2 Z-DNA duplex. The d(5′-CGCGCG-3′) nucleotides of chain A are numbered 1-6, and the The disordered phosphate groups have Z I and Z II conformation for the alternative positions. Z II conformation of the nucleotide fragments can be attributed to G2(I) (ζ = 59.2°), G4(I) (61.9°), G8(II) (73.3°) and G10(I) (74.7°). The Z II conformations are stabilized by hydrogen bonds between a phosphate oxygen atom and water molecules form the coordination spheres of the Cr 3+ cations, as well as by contacts with symmetry-related Z-DNA helices in the crystal lattice.
The presence of the double-conformation G2 nucleotide causes significant distortions of the helical parameters for the two dinucleotide pairs, C1G2-C11G12 and G2C3-G10C11, as well as of the average values for the entire duplex (tip and inclination; Supplementary Table S2) [32]. The helical parameters shift/tilt/x-displacement have the following values: −0.04 and 0.23 Å/0.84 and 2.39°/−48.89 and −31.99 Å, for the pairs of dinucleotides C1G2(I)-C11G12 and C1G2(II)-C11G12, respectively. For the dinucleotide pair C3G4-C9G10, there is a difference in the width of the minor groove (after subtraction of 5.8 Å for the combined radii of the phosphorus atoms) between conformations Z I (7.2 Å) and Z II (6.0 Å).

Coordination of the Cr 3+ cations and crystal packing
The Cr 3+ (1), Cr 3+ (2) and Cr 3+ (3) ions have partial occupancies of 35, 26 and 40 %, respectively (Fig. 1). The Cr 3+ (1) and Cr 3+ (2) cations, which are unexpectedly only 2.354(15) Å apart, were treated as separate individual ions during the refinement. It was not possible to unambiguously correlate their partial populations as alternatives within the networks of partially occupied water and disordered DNA fragments. In conclusion, despite their partial occupancies, the possibility of a direct interaction between the Cr 3+ (1) and Cr 3+ (2) ions in the crystal lattice cannot be excluded, especially in view of the bridging water molecules. A similarly short contact [2.394(1) Å] between Cr 3+ ions was found in a μ-methylene complex, as reported by Noh et al. [35].
The crystals of the Z-DNA-Cr 3+ complex belong to space group P2 1 2 1 2 1 and have the less common B type of crystal packing of Z-DNA structures (also called purespermine type), as reported for the first time by Egli et al. [d(CGCGCG) 2 -Spm; PDB 1d48] [36]. As described in detail by Brzezinski et al. [29], d(CGCGCG) 2 Z-DNA crystallizes into two polymorphic forms (A and B) of the same space group and cell parameters, but with different crystal packing. A detailed discussion of the influence of metal and/or polyamine cations, and disorder of Z-DNA on crystal packing can be found in Brzezinski et al. [29], Schuerman et al. [37] and Harper et al. [38].

Hydration
The d(CGCGCG) 2 -Cr 3+ complex is similar to other highresolution structures of Z-DNA with regard to the architecture of the hydration shell, which comprises a complicated network of hydrogen bonds that stabilize the molecular packing in the crystal. The asymmetric unit contains 67 water sites. The characteristic features of hydration geometry noted in other Z-DNA crystals, such as the spine of hydration [40], water molecules between N2_G and phosphate O atoms, two water molecules H-bonded to each O6_G group, or absence of water molecules H-bonded to the N3_G atoms, are also found in the present Z-DNA-Cr 3+ complex. The positions of two disordered water molecules, Wat2 and Wat53, are correlated with the alternate conformations of the DNA chain between the G2-C3 and G8-C9 nucleotides, respectively. The G2(I)·C11 and G2(II)·C11 base pairs have different schemes of hydration. There are no N2···Wat interactions for the G2(I) conformation. Atom O6_G2(I) forms hydrogen bonds with Wat10

Discussion
The high-resolution crystal structure of the d(CGCGCG) 2 -Cr 3+ complex presented in this work shows the presence of three Cr 3+ ion sites per one Z-DNA duplex, which do not form any direct coordination bonds with either the guanine N/O atoms or the phosphate groups of the DNA. Only water-mediated contacts between the nucleic acid and the Cr 3+ cations are observed. The degree of disorder of the DNA strands in the Z-DNA-Cr 3+ complex is comparable with that in the recently presented metaland polyamine-cation free Z-DNA hexamer (PDB 3wbo) [41] and dodecamer (4ocb) [42] structures, and even higher than in the Z-DNA complexes with Mn 2+ (4hig) and Zn 2+ (4hif) reported by Drozdzal et al. [16] or in d(CGCGCA):d(TGCGCG)-[Ru(NH 3 ) 6 ] 3+ (2hto) [43]. The high degree of disorder in this and similar Z-DNA structures is in stark contradiction to the rigidity of the same chemical molecule (crystallized without metal cations) reported by Brzezinski et al. [29]. In view of the accumulated facts, it seems that the rigidity observed in that ultrahigh-resolution structure (PDB 3p4j) was a fortuitous exception rather than a rule. In addition to alternative positions of the backbone phosphate groups, the present electron density maps also clearly show a double conformation of the G2 nucleoside. The alternate conformations of G2 in the Z-DNA-Cr 3+ structure resulted in deviations from typical hydrogen bonding and stacking interaction observed for the G2C3-G10C11 base pairs in similar d(CGCGCG) 2 structures. Analysis of the positions of the base rings of G2(I) and G2(II) indicates a tendency to keep the DNA strands in favorable contacts with water molecules. The alternative positions of the G2 nucleotide do not affect the orientation of the complementary C11 base. The mFo-DFc map for the C11 nucleotide does not indicate any peaks (even at the 2.5σ level) to suggest possible alternate positions of the cytosine base, which could be correlated with the G2(I)/(II) conformations. This observation is in contrast to findings based on a high-resolution (0.96 Å) B-DNA-Mg 2+ structure with full base-pair positional heterogeneity (PDB 3u89) [44]. The present structure of the d(CGCGCG) 2 -Cr 3+ complex confirms the inherent polymorphism in the positions of the DNA atoms and suggests that complementary bases can move independently within the DNA scaffold. Thus, the tendency to maintain the exact Watson-Crick base-pairing geometry with both G2 conformations is not the predominant factor for the C11 nucleotide and many other factors, such as stacking interactions, water and/or ligand interactions, as well as crystal packing, should be taken into account when considering the energy balance of a single nucleotide conformation. Moreover, the stable position of the base ring of C11 within the G2(I)/(II)·C11 base pair may confirm the crucial role played by water molecules in DNA stabilization [45]. The crystal structure of the d(CGCGCG) 2 -Cr 3+ complex analyzed in this study has the same packing mode of the Z-DNA helices as the d(CACGIUG) 2 -[Co(NH 3 ) 6 ] 3+ (PDB 1omk) [37] and d(TGCGCA) 2 -[Co(NH 3 ) 6 ] 3+ (362d) structures [38]. Although sperminium tetrachloride was present in the crystallization conditions, the spermine 4+ tetracation has not been identified in the electron density maps of the d(CGCGCG) 2 -Cr 3+ crystal. This suggests either a complete disorder of the entire spermine 4+ molecule, or its total absence in the crystal structure.

Conclusions
Our studies of the d(CGCGCG) 2 -Cr 3+ complex indicate that Z-DNA, especially in complexes with metal cations, has a large potential for conformational flexibility. The present structure, as well as other examples deposited in the PDB, strongly supports the notion that Z-DNA helices should not be regarded as having extremely regular and rigid stereochemistry, in contradiction to the suggestion made by Brzezinski et al. [29]. The d(CGCGCG) 2 -Cr 3+ structure is an excellent illustration of the flexibility of the Z-DNA molecule, visible in the adoption of multiple conformations (by the phosphate groups and the G2 nucleotide), in response to changes in its electrostatic and hydration environment, caused by the introduction of hydrated metal complexes.