The crystal structure of Z-DNA with untypically coordinated Ca²⁺ ions

Abstract

DNA oligomer duplexes with alternating cytosines and guanines in their sequence tend to form helices of the Z-DNA type, where the sugar and phosphate backbone forms a left-handed helix in a zigzag fashion with a repeat of two successive Wat-son–Crick pairs of nucleotides. Z-DNA duplexes often crystallize in complexes with diverse metal ions interacting with polar DNA atoms in various ways. This work describes the high-resolution crystal structure of a Z-DNA d(CGC GCG )₂ duplex in complex with Ca²⁺ ions, unusually coordinated as an approximate pentagonal bipyramid by two neighboring guanines through their O6 and N7 atoms and a water molecule in the equatorial plane and a phosphate oxygen atom and another water molecule in the apical positions.

Introduction

The first crystal structures of the left-handed DNA oligonucleotides were determined almost four decades ago [1], and since then a large number of crystal structures of Z-DNA in various crystal forms are available in the Protein Data Bank (PDB, [2]). Their features and characteristics have been thoroughly analyzed and reviewed (e.g., [3, 4]). A vast majority of these crystal structures contain hexamer duplexes, consisting of alternating purines and pyrimidines, usually cytosines and guanines, in many cases with nucleobases chemically modified. The glycosidic bond between the nucleobase and sugar always has syn conformation in purines and anti conformation in pyrimidines. This results in the Z-DNA phosphate–sugar backbone having a left-handed zigzag conformation, not the monotonous thread observed in the right-handed A- and B-DNA forms. The Z-DNA double helix is therefore formed by a repeat of two pairs of nucleotides, not a single one as in the right-handed DNA. The helical tube in Z-DNA is straight and narrower than in the other forms and the average distance between consecutive bases is 3.7 Å, larger than in B-DNA (3.4 Å) and A-DNA (2.5 Å). The twist of Z-DNA double helix is 60° per two pairs of nucleotides, and in consequence two hexamer duplexes positioned one after another along a twofold screw axis form the full turn of the double helix of about 44 Å length.

Indeed, in crystals, Z-DNA molecules almost universally form parallel straight tubes along twofold screw axes packed in a hexagonal fashion, which in various crystal forms differ in their mutual orientational and translational relation. The direct interaction between neighboring tubes is limited by the lack of hydrogen bond donors, since the outer surface of the Z-DNA duplexes is populated by the phosphate oxygen atoms that can serve as acceptors, but not donors of H bonds. Potentially available donors are only the terminal 3′ and 5′ hydroxyl groups at each end of a duplex. In effect, these hydroxyl groups interact indirectly through the solvent of water and other molecules.

The space between the Z-DNA tubes and the interior of the minor groove of duplexes is filled by solvent, i.e., molecules of water and other components of the crystallization buffer. Z-DNA is usually crystalized in a slightly acidic pH, when this form is most stable, in the presence of cationic counterions, such as polyamines (e.g., spermine, spermidine) and metal ions. However, these non-water molecules and ions are often not identifiable in crystal structures due to the inherent disorder within the bulk solvent regions in crystals. Moreover, cations such as NH₄⁺, Na⁺ and Mg²⁺ are isoelectronic with water molecules and their identification in the electron density maps is difficult. The metal ions can be recognized from their characteristic coordination, their stronger electron density (if they are heavier than Na or Mg) and possibly from their anomalous diffraction signal. However, if their sites are not fully occupied and shared with water molecules, the identification may not be possible at all, especially in the X-ray analysis at lower than atomic resolution.

The inspection of all Z-DNA crystal structures in the PDB [2] (Table 1) shows that out of 95 available crystal structures, 51 contain no metal ions, and in the remaining structures 20 contain Mg²⁺ ions. The majority of identified metal ions are coordinated by oxygen atoms of water and phosphate oxygen atoms. In 14 complexes, metal ions are also coordinated by the N7 atoms of the guanine bases, available in the major groove of Z-DNA (Supplementary Table). Only in six structures containing Cu²⁺ ions, the metal is coordinated by two N7 atoms of guanines in two neighboring duplexes.

Table 1.

Statistics of metal ions in all 95 Z-DNA structures identified in the PDB current on 7 Aug. 2017, separately for ions coordinating and non-coordinating the aromatic nucleotide nitrogen atoms

Without Me–N(nucl) coordination		With Me–N(nucl) coordination
Mg2+	15	Mg2+	5
$\text{Co}{\left({\text{NH}}_3\right)}_6^{3+}$	7	Cu2+	6
$\text{Ru}{\left({\text{NH}}_3\right)}_6^{3+}$	2	Zn2+	1
Ba2+	2	Mn2+	1
Na+	2	$\text{Pt}{\left({\text{NH}}_3\right)}_3^{2+}$	1
Cr3+	1
Ca2+	1
All	30		14

51 Z-DNA structures do not contain any metal ions

Only one structure contains Ca²⁺ ions, which are coordinated by two phosphate oxygen atoms and four or five water molecules (and for one of five identified Ca²⁺ ions also by the sugar O3′ atom). In this work, we present the structure of Z-DNA containing the Ca²⁺ ion untypically coordinated by N7 and O6 atoms of two guanine molecules from two adjacent duplexes, in addition to three other oxygen ligands.

Materials and methods

Crystallization and diffraction data collection

The salt-free hexamer d(CGC GCG )₂ was purchased from Eurofins MWG Operon (Huntsville, USA) and was used without further purification. This oligomer has no terminal phosphate groups, only ten –O–PO₂–O– moieties between pairs of nucleotides. The oligomer was dissolved in 1 mM Tris–HCl, pH 7.5, at a concentration of 2 mM, kept at 90 °C for 1 min, annealed at 60 °C for 10 min, and then slowly cooled down to room temperature. Crystals were grown using the sitting-drop vapor-diffusion method at room temperature by mixing 0.6 μl of the 0.4 mM DNA oligomer solution and 1.2 μl of the precipitant solution consisting of 10 mM CaCl₂, 40 mM Na cacodylate, pH 6.0, 6 mM spermidine and 10% v/v MPD, equilibrated against the well solution of 35% MPD. The initial crystals appeared after 2 days and were used for micro-seeding by streaking a new crystallization drop pre-equilibrated for 2 h. Small, well-developed crystals appeared after 2 days and were dipped in a cryoprotecting solution containing 50 mM Tris–HCl, pH 7.0, and 40% MPD for a few seconds and then vitrified in liquid nitrogen.

High-resolution diffraction data set was collected at the SER-CAT beamline 22ID at the Advanced Photon Source (Argonne National Laboratory, USA). The data were processed using HKL2000 [5] with and without merging the Bijvoet pairs. The anomalous data were used only to confirm the presence of calcium ion by the anomalous difference map and all diffraction data and refinement statistics were based on the merged “native” data. The data collection statistics are presented in Table 2.

Table 2.

Data collection and refinement statistics

PDB code	6B01
Data collection
Beam line	SER-CAT 22ID
Detector	Rayonix MX300HS
Wavelength (Å)	0.9920
Space group	P212121
a (Å)	17.76
b (Å)	28.82
c (Å)	42.36
Resolution (Å)	24–1.45 (1.50–1.45)a
Reflections total	22,556 (1051)
Reflections unique	4114 (357)
Completeness (%)	98.3 (87.9)
Multiplicity	5.5 (2.9)
Mosaicity range (°)	0.95–1.15
Rmerge	0.086 (0.427)
Rmeas	0.096 (0.577)
<I/σ (I)>	33.5 (1.6)
${\text{CC}}_{\text{1/2}}^{\text{b}}$	0.79
Wilson B factor (Å2)	14.0
Refinement
No. of nucleotides	12
No. of all reflections	4083
R factor	0.121
No. of free reflections	209
Rfree	0.172
Rmsd bonds (Å)	0.010
Rmsd angles (°)	1.61
<B> (Å2)
Overall	19.3 (289)c
DNA	16.3 (243)
Solvent water	33.3 (35)
Spermidinium3+	42.7 (10)
Ca2+	17.7 (1)

^aValues in parentheses correspond to the highest resolution shell

^bCorrelation between intensities from random half-sets in the highest resolution shell as defined by Karplus and Diederichs (2012)

^cThe number of atom sites is given in parentheses

Structure determination and refinement

The structure was solved by molecular replacement using PHASER [6], with the Z-DNA hexamer duplex from the ultrahigh-resolution crystal structure 3P4J [7] serving as a search model. Using MR_AUTO mode, one copy of the hexamer duplex was located in the asymmetric unit of the cell. The initial model was refined with REFMAC5 [8], initially in the isotropic mode and then anisotropically. The hydrogen atoms were introduced as “riding” on their parent atoms. Manual corrections to the atomic model and selection of solvent water atoms were executed with COOT [9]. The fully occupied Ca²⁺ ion site was identified in the structure according to the coordination environment and confirmed by the anomalous difference map.

There is no restraint file for the spermidine tri-cation available in the current CCP4 library and the existing restraints for glycosidic bonds and phosphate groups in the DNA library are not appropriate for Z-DNA. The restraints for spermidinium tri-cation were therefore created on the basis of the existing library for the spermine tetra-cation (SPK). The restraint targets for the glycosidic bonds and phosphate groups in Z-DNA were used as suggested in the recent work of Kowiel et al. [10]. The phosphate group restraints for Z_I and Z_II conformations were taken from the RestraintLib option of the ACHESYM server (http://achesym.ibch.poznan.pl/) [11]. All these additional restraints were introduced using the appropriate external restraints option of REFMAC5.

On introduction of anisotropic ADPs, the R factor diminished from 0.162 to 0.122 and R_free dropped from 0.223 to 0.172. In spite of the scarcity of the “free” reflections (209, constituting 5% of the total number), it was decided to accept the anisotropic ADP model in the refinement procedure. All reflections, including those previously used as “free”, were used in the last cycles of refinement and in the calculation of the final electron density maps. The structure factors and atomic coordinates of d(CGC GCG )₂:Ca²⁺ were deposited into PDB with entry ID 6BST.

Results and discussion

Quality of the structure

The atomic model of d(CGC GCG )₂ was refined to an R value of 0.125 and R_free of 0.172. All atoms of DNA are well defined in the electron density (Fig. 1), and the geometry of the duplex agrees well with the expected stereochemistry of Z-DNA. One well-defined Ca²⁺ was identified in the solvent region as well as 35 water sites and one spermidinium tri-cation. The definition of spermidinium in the electron density is satisfactory, but not perfect, suggesting its substantial flexibility in the crystal. This syndrome is often observed in other Z-DNA crystal structures. All (except one) water molecules were refined with full occupancies, although some of them acquired elevated B factors, again suggesting some degree of disorder.

Fig. 1.

A portion of the 2F_o–F_c electron density map, as a blue mesh, around the Ca²⁺ ion and its ligands at the 1.0 σ contour level. The anomalous difference density at the ion site is shown in magenta at the 3.0 σ contour level

Overall structure and crystal packing

The crystal of the Z-DNA:Ca²⁺ complex is orthorhombic and its lattice corresponds to the P2₁2₁2₁A type, isomorphous with the highest resolution structure of Z-DNA (PDB Id 3P4J, [7]) and a number of other structures. This form has one d(CGC GCG )₂ duplex in the asymmetric unit and its local (non-crystallographic) twofold axis, perpendicular to the helix and passing through the center of the duplex, is inclined by 25° with respect to the x-axis direction of the crystal. The duplex in one helical tube is surrounded by six symmetry equivalent tubes with two of them related by x ± 1 translations and four by the crystal 2₁ axes parallel to the y-direction. All base pairs are therefore at the same z-level in two neighboring duplex tubes, whereas the nucleobase pair 1–12 is approximately on the same level as pair 3–10 and pair 2–11 as the equivalent pair 2–11 in the other four adjacent tubes.

A comparison with the 3P4J structure shows a high degree of similarity of the duplex conformation, except the terminal base pairs 1–12 and 6–7 and the presence of double conformation of one of the phosphate groups. As evidenced in Fig. 2, all base pairs in d(CGC GCG )₂:Ca²⁺ are relatively parallel to each other, whereas in the 3P4J structure the two terminal pairs are more inclined than the rest. The average deviation of the six base pair planes from perpendicularity to the helix axis is 7.2° in 3P4J and 4.4° in the current complex. This feature has a consequence in shortening of the helix and the c crystal cell dimension from 43.90 to 42.36 Å.

Fig. 2.

The d(CGC GCG )₂ duplex from the Ca²⁺ complex in yellow with Ca²⁺ ion as a green sphere overlapped on the highest resolution Z-DNA structure 3P4J [7] in blue color, with differences at the terminal residues marked

The phosphate groups in Z-DNA can adopt two main conformations, termed Z_I and Z_II [12, 13], and in many Z-DNA structures the phosphate groups are in either of them or are disordered in both conformations at once. The most indicative is the torsion angle ζ (C3′–O3′–P–O5′), which for the more frequent Z_I conformation lies in the + gauche region (or + synclinal, sc⁺) around + 60°, whereas for the Z_II conformation it is − gauche (or – synclinal, sc⁻) around − 60°.

Out of ten phosphate groups in the d(CGC GCG )₂:Ca²⁺ complex, the majority are in conformation corresponding to the Z_I type, except the C5 residue having the Z_II type; Table 3. Moreover, in the C3 residue, the phosphate group is disordered into two, half-occupied conformations, as illustrated in Fig. 3.

Table 3.

Conformational angles and pseudorotation parameters in the d(CGCGCG)₂:Ca²⁺ complex given in (°)

Angle	Contributing atoms	Type	C1	G2	C3	G4	C5	G6	C7	G8	C9	G10	C11	G12
α	O3′–P–O5′–C5′	ZI	–	66.9	162.1	71.0	− 177.6	80.1	–	69.5	171.2	71.4	169.4	62.6
α		ZII			− 154.8
β	P–O5′–C5′–C4′	ZI	–	− 174.8	169.5	− 174.0	− 129.3	179.9	–	− 174.3	171.6	− 170.2	147.0	− 172.2
β		ZII			− 118.7
γ	O5′–C5′–C4′–C3′	ZI	62.4	176.4	51.7	− 179.2	58.9	− 176.4	55.8	− 176.3	51.4	176.9	52.7	177.9
δ	C5′–C4′–C3′–O3′	ZI	140.4	90.3	145.6	107.1	136.1	131.7	142.2	90.5	144.3	95.1	140.7	87.6
ε	C4′–C3′–O3′–P	ZI	− 92.7	163.3	− 100.7		− 105.6	–	− 108.8	− 175.5	− 102.6	− 161.8	− 89.3	–
ε		ZII		− 125.0		− 120.5
ζ	C3′–O3′–P′O5′	ZI	75.2	61.7	73.8		72.7	–	75.6	52.1	70.7	61.8	75.1	–
ζ		ZII		− 65.4		− 58.4
χ (C)	O4′–C1′–N1–C2		− 142.9		− 155.2		− 166.6		− 160.1		− 154.5		− 151.0
χ (G)	O4′–C1′–N9–C4			62.7		61.7		66.2		53.9		55.7		64.1
ν0	C4′–O4′–C1′–C2′		− 25.0	− 6.3	− 29.7	− 12.7	− 36.3	− 16.9	− 33.9	− 4.7	− 29.6	− 4.8	− 29.9	− 6.5
ν1	O4′–C1′–C2′–C3′		35.1	− 10.0	41.4	0.9	42.1	22.1	41.5	− 11.1	40.0	− 13.3	36.8	− 13.4
ν2	C1′–C2′–C3′–C4′		− 31.5	20.8	− 36.3	10.3	− 31.5	− 19.0	− 32.8	21.3	− 34.9	25.1	− 29.3	26.9
ν3	C2′–C3′–C4′–O4′		17.8	− 24.7	20.4	− 17.6	11.5	9.3	13.7	− 24.7	19.4	− 28.5	12.5	− 31.0
ν4	C3′–C4′–O4′–C1′		4.5	20.1	5.7	19.2	15.7	4.7	12.7	18.7	6.3	20.9	11.0	23.7
τm			154.6	32.4	153.9	57.9	140.3	149.8	144.2	29.0	152.8	27.6	144.7	29.8
P			35.7	25.2	41.8	19.5	42.2	22.3	41.4	25.0	40.5	28.8	36.8	31.4

Pseudorotation parameters were calculated according to Jaskolski [16]

Fig. 3.

Two conformations of the disordered phosphate group of the C3 residue shown with the corresponding 2F_o–F_c electron density map (blue mesh) at the 1.0 σ contour level

Solvent region

Within the solvent region of the crystal, the electron density allowed us to identify 33 water sites, one spermidinium tri-cation and one Ca²⁺ ion. 11 water molecules are located within the minor groove of the duplex and all of them are hydrogen bonded to polar atoms of DNA and between themselves. Within the Watson–Crick base pairs, two H-bond-connected atoms C(O2) and G(N2) are positioned toward the inside of the groove. These atoms have a potential to form additional H bonds through the remaining lone pair of electrons at the C(O2) atom and the second hydrogen atom of the G(N2) amino group. All these potential H-bond capabilities are satisfied by the 11 water molecules located inside the groove, except for the C(O2) atom of residue C7, which is the terminal nucleotide in the duplex. The water molecules within the groove are well defined in the electron density and have on average somewhat lower B factors (29.9 Ų) than the remaining water molecules located between the duplexes (34.4 Ų).

The region of the crystal between the duplexes contains 22 modeled water sites. All of them are presented as fully occupied but, as mentioned earlier, some of these sites may represent other species (e.g., ions) partially occupying the same locations with water molecules. Since it is not possible to identify the content of these sites with certainly, their occupancy and positional uncertainty are represented by the refined B factors. Two water molecules with relatively low B factors (17.1 and 27.5 Ų) coordinate the Ca²⁺ ion.

It was possible to locate in the space between duplexes one moiety of spermidinium tri-cation, but the corresponding electron density is not even along the length of this linear molecule. Evidently, as in many other crystal structures of Z-DNA, the polyamine is highly flexible and some of its atoms may share their locations with partially occupied water molecules. Again, this effect is left to be represented by the refined B factors. However, the nitrogen atoms of spermidinium form several good hydrogen bonds. Its N1 atom forms H bonds with one water molecule (3.04 Å), N5 atom with phosphate oxygen atoms of the G4 residue (2.77 Å) and both disordered oxygen atoms of C3 in the neighboring duplex (2.75 and 3.01 Å), and N10 atom with phosphate oxygen atoms of C5 (2.95 Å) and of G2 in the other duplex (2.80 Å).

Metal identification and coordination

One metal ion was identified in a place between two neighboring duplexes. The crystallization medium contained about 7 mM Ca²⁺ and 30 mM Na⁺ ions, but the characteristics of this site fit well with all the properties of Ca²⁺ ions. The typical ion:ligand distances and coordination preferences are similar for Ca²⁺ and Na⁺ [14], but these ions differ in their number of electrons and their anomalous scattering properties, and these characteristics strongly suggest that this ion is properly identified as Ca²⁺. The ion refines with B factor (17.7 Ų) comparable with its ligands having B factors in the range 15.1–18.2 Ų (except the equatorial water with B = 27.4 Ų). A fully occupied Na⁺ ion with 10 electrons could not satisfactorily refine with B factor so similar to those of its ligands as the Ca²⁺ ion possessing 18 electrons. Additional support for the identity of the ion is provided by its anomalous scattering signal, which is small, but evident as a peak in the anomalous difference map presented in Fig. 1. At the X-ray wavelength used for data collection, 0.9920 Å, the fʹ value of Ca²⁺ is 0.58 e, as estimated by the program CROSSEC [15], while for Na⁺, another potential ion present in the crystallization buffer, it would be 0.05 e, a value that would not show any meaningful features in the anomalous difference map.

The ion is coordinated by seven ligands in an arrangement close to a pentagonal bipyramid; Fig. 4. In the equatorial plane there are two guanine molecules G10 and G12 from two neighboring duplexes and one water molecule; Fig. 5. The two apical positions are occupied by the OP1 atom of the phosphate group of the C11 residue and another molecule of water. The two coordinating guanines are parallel, but their planes are shifted by about 1.0 Å out of coplanarity. The Ca²⁺ ion lies between these planes, at a distance of 0.2 Å from the G10 plane and 0.8 Å from the G12 plane. The equatorial water molecule is most shifted from the average plane of the remaining four equatorial ligands by about 0.9 Å.

Fig. 4.

Details of the coordination of the Ca²⁺ ion with relevant distances and angles marked in Å and °

Fig. 5.

The location of the Ca²⁺ ion between two neighboring Z-DNA duplexes

The distances between Ca²⁺ and the three non-G oxygen ligands, in the range 2.21–2.39 Å, are around the values typical for Ca–O distances observed in macromolecules and small compounds, 2.3–2.4 Å [12]. The distances to both O6 atoms of G residues (2.47 and 2.57 Å) are somewhat longer. The distances to both N7 atoms of these residues (2.67 and 2.79 Å) are also somewhat longer than 2.50(± 10) Å, the average distance between Ca²⁺ and the nitrogen atoms within the aromatic rings of 2212 crystal structures in the Cambridge Structural Database. These distances to both G residues are elongated probably as a consequence of constraints resulting from the specific packing of duplexes in the crystal.

The position of the Ca²⁺ ion with respect to the N7 and O6 atoms of guanines is almost identical in relation to both bases; the only difference is that the ion lies in the plane of G10 and 0.9 Å from the plane of G12; Fig. 4. The ion is located 28° away from the direction bisecting the endocyclic angle C–N7–C of the imidazole rings of the guanines, representing the ideal position of the lone electron pair at the sp² hybridized nitrogen atom. With respect to the O6 atoms, the ion is located 10° from the angle of 120°, expected for the direction of the lone pair at the sp² hybridized oxygen atom.

Conclusions

The high-resolution crystal structure described here presents a Z-DNA:Ca²⁺ complex preserving the typical structure of a d(CGC GCG )₂ hexamer duplex, but containing an unusually coordinated Ca²⁺ ion. This ion is located between two neighboring duplexes and is coordinated by the N7 and O6 atoms of two guanine bases as well as three other oxygen ligands forming a distorted pentagonal bipyramid. The somewhat distorted stereochemistry of coordination results from constraints introduced by the specific pseudohexagonal packing geometry of Z-DNA duplexes in the crystal. Nevertheless, the ion is convincingly identified as a fully occupied Ca²⁺ cation.