Atomic-resolution crystal structures of B-DNA reveal specific influences of divalent metal ions on conformation and packing.

Crystal structures of B-form DNA have provided insights into the global and local conformational properties of the double helix, the solvent environment, drug binding and DNA packing. For example, structures of the duplex with sequence CGCGAATTCGCG, the Dickerson-Drew dodecamer (DDD), established a unique geometry of the central A-tract and a hydration spine in the minor groove. However, our knowledge of the various interaction modes between metal ions and DNA is very limited and almost no information exists concerning the origins of the different effects on DNA conformation and packing exerted by individual metal ions.Crystallization of the DDD duplex in the presence of Mg(2+)and Ca(2+)yields different crystal forms. The structures of the new Ca(2+)-form and isomorphous structures of oligonucleotides with sequences GGCGAATTCGCG and GCGAATTCGCG were determined at a maximum resolution of 1.3 A. These and the 1.1 A structure of the DDD Mg(2+)-form have revealed the most detailed picture yet of the ionic environment of B-DNA. In the Mg(2+)and Ca(2+)-forms, duplexes in the crystal lattice are surrounded by 13 magnesium and 11 calcium ions, respectively.Mg(2+)and Ca(2+)generate different DNA crystal lattices and stabilize different end-to-end overlaps and lateral contacts between duplexes, thus using different strategies for reducing the effective repeat length of the helix to ten base-pairs. Mg(2+)crystals allow the two outermost base-pairs at either end to interact laterally via minor groove H-bonds, turning the 12-mer into an effective 10-mer. Ca(2+)crystals, in contrast, unpair the outermost base-pair at each end, converting the helix into a 10-mer that can stack along its axis. This reduction of a 12-mer into a functional 10-mer is followed no matter what the detailed nature of the 5'-end of the chain: C-G-C-G-A-ellipsis, G-G-C-G-A-ellipsis, or a truncated G-C-G-A-ellipsis Rather than merely mediating close contacts between phosphate groups, ions are at the origin of many well-known features of the DDD duplex structure. A Mg(2+)coordinates in the major groove, contributing to kinking of the duplex at one end. While Ca(2+)resides in the minor groove, coordinating to bases via its hydration shell, two magnesium ions are located at the periphery of the minor groove, bridging phosphate groups from opposite strands and contracting the groove at one border of the A-tract.     

1 A crystal structures of B-DNA reveal sequence-specific binding and groove-specific bending of DNA by magnesium and calcium

The 1 A resolution X-ray crystal structures of Mg(2+) and Ca(2+) salts of the B-DNA decamers CCAACGTTGG and CCAGCGCTGG reveal sequence-specific binding of Mg(2+) and Ca(2+) to the major and minor grooves of DNA, as well as non-specific binding to backbone phosphate oxygen atoms. Minor groove binding involves H-bond interactions between cross-strand DNA base atoms of adjacent base-pairs and the cations' water ligands. In the major groove the cations' water ligands can interact through H-bonds with O and N atoms from either one base or adjacent bases, and in addition the softer Ca(2+) can form polar covalent bonds bridging adjacent N7 and O6 atoms at GG bases. For reasons outlined earlier, localized monovalent cations are neither expected nor found.Ultra-high atomic resolution gives an unprecedented view of hydration in both grooves of DNA, permits an analysis of individual anisotropic displacement parameters, and reveals up to 22 divalent cations per DNA duplex. Each DNA helix is quite anisotropic, and alternate conformations, with motion in the direction of opening and closing the minor groove, are observed for the sugar-phosphate backbone. Taking into consideration the variability of experimental parameters and crystal packing environments among these four helices, and 24 other Mg(2+) and Ca(2+) bound B-DNA structures, we conclude that sequence-specific and strand-specific binding of Mg(2+) and Ca(2+) to the major groove causes DNA bending by base-roll compression towards the major groove, while sequence-specific binding of Mg(2+) and Ca(2+) in the minor groove has a negligible effect on helix curvature. The minor groove opens and closes to accommodate Mg(2+) and Ca(2+) without the necessity for significant bending of the overall helix.The program Shelxdna was written to facilitate refinement and analysis of X-ray crystal structures by Shelxl-97 and to plot and analyze one or more Curves and Freehelix output files.     

Crystal structure of the B-DNA hexamer d(CTCGAG): model for an A-to-B transition.

The crystal structure of the B-DNA hexamer d(CTCGAG) has been solved at 1.9 A resolution by iterative single isomorphous replacement, using the brominated derivative d(CG5BrCGAG), and refined to an R-factor of 18.6% for 120 nonhydrogen nucleic acid atoms and 32 water molecules. Although the central four base pairs form a typical B-form helix, several parameters suggest a transition to an A-like conformation at the termini. Based on this observation, a B-to-A transition was modeled, maintaining efficient base stacking across the junction. The wide minor groove (approximately 6.9 A) is reminiscent of that in the side-by-side double drug-DNA complexes and hosts a double spine of hydration. The global helix axes of the pseudo-continuous helices are at an acute angle of 60 degrees. The pseudocontinuous stacking is reinforced by the minor groove water structure extending between the two duplexes. The crossover point of two pairs of stacked duplexes is at the stacking junction, unlike that observed in the B-DNA decamers and dodecamers. This arrangement may have implications for the structure of a four-way DNA junction. The duplexes are arranged around a large (approximately 20 A diameter) channel centered on a 6(2) screw axis.     

Interaction between the Z-type DNA duplex and 1,3-propanediamine: crystal structure of d(CACGTG)2 at 1.2 A resolution

The crystal structure of a hexamer duplex d(CACGTG)(2) has been determined and refined to an R-factor of 18.3% using X-ray data up to 1.2 A resolution. The sequence crystallizes as a left-handed Z-form double helix with Watson-Crick base pairing. There is one hexamer duplex, a spermine molecule, 71 water molecules, and an unexpected diamine (Z-5, 1,3-propanediamine, C(3)H(10)N(2)) in the asymmetric unit. This is the high-resolution non-disordered structure of a Z-DNA hexamer containing two AT base pairs in the interior of a duplex with no modifications such as bromination or methylation on cytosine bases. This structure does not possess multivalent cations such as cobalt hexaammine that are known to stabilize Z-DNA. The overall duplex structure and its crystal interactions are similar to those of the pure-spermine form of the d(CGCGCG)(2) structure. The spine of hydration in the minor groove is intact except in the vicinity of the T5A8 base pair. The binding of the Z-5 molecule in the minor grove of the d(CACGTG)(2) duplex appears to have a profound effect in conferring stability to a Z-DNA conformation via electrostatic complementarity and hydrogen bonding interactions. The successive base stacking geometry in d(CACGTG)(2) is similar to the corresponding steps in d(CG)(3). These results suggest that specific polyamines such as Z-5 could serve as powerful inducers of Z-type conformation in unmodified DNA sequences with AT base pairs. This structure provides a molecular basis for stabilizing AT base pairs incorporated into an alternating d(CG) sequence.     

X-ray crystallographic analysis of the hydration of A- and B-form DNA at atomic resolution

We have determined single crystal structures of an A-DNA decamer and a B-DNA dodecamer at 0.83 and 0.95 A, respectively. The resolution of the former is the highest reported thus far for any right-handed nucleic acid duplex and the quality of the diffraction data allowed determination of the structure with direct methods. The structures reveal unprecedented details of DNA fine structure and hydration; in particular, we have reexamined the overall hydration of A- and B-form DNA, the distribution of water around phosphate groups, and features of the water structure that may underlie the B to A transition.     

The structure of DAPI bound to DNA

The structure of the DNA fluorochrome 4'-6-diamidine-2-phenyl indole (DAPI) bound to the synthetic B-DNA oligonucleotide C-G-C-G-A-A-T-T-C-G-C-G has been solved by single crystal x-ray diffraction methods, at a resolution of 2.4 A. The structure is nearly isomorphous with that of the native DNA molecule alone. With one DAPI and 25 waters per DNA double helix, the residual error is 21.5% for the 2428 reflections above the 2-sigma level. DAPI inserts itself edgewise into the narrow minor groove, displacing the ordered spine of hydration. DAPI and a single water molecule together span the four AT base pairs at the center of the duplex. The indole nitrogen forms a bifurcated hydrogen bond with the thymine O2 atoms of the two central base pairs, as with netropsin and Hoechst 33258. The preference of all three of these drugs for AT regions of B-DNA is a consequence of three factors: (1) The intrinsically narrower minor groove in AT regions than in GC regions of B-DNA, leading to a snug fit of the flat aromatic drug rings between the walls of the groove. (2) The more negative electrostatic potential within the minor groove in AT regions, attributable in part to the absence of electropositive-NH2 groups along the floor of the groove, and (3) The steric advantage of the absence of those same guanine-NH2 groups, thus permitting the drug molecule to sink deeper into the groove. Groove width and electrostatic factors are regional, and define the relative receptiveness of a section of DNA since they operate over several contiguous base pairs. The steric factor is local, varying from one base pair to the next, and hence is the means of fine-tuning sequence specificity.     

Molecular structure of the B-DNA dodecamer d(CGCAAATTTGCG)2. An examination of propeller twist and minor-groove water structure at 2.2 A resolution.

The crystal structure of the dodecanucleotide duplex d(CGCAAATTTGCG)2 has been solved to 2.2 A resolution and refined to an R-factor of 18.1% with the inclusion of 71 water molecules. The structure shows propeller twists of up to -20 degrees for the A.T base-pairs, although there is probably only one (weak) three-centre hydrogen bond in the six base-pair AT narrow minor-groove region. An extensive ribbon of hydration has been located in this groove that has features distinctive from the classic "spine of hydration". Solvation around phosphate groups is described, with several instances of water molecules bridging between phosphates.     

Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin I C2B domain

Synaptotagmin I has two tandem Ca(2+)-binding C(2) domains, which are essential for fast synchronous synaptic transmission in the central nervous system. We have solved four crystal structures of the C(2)B domain, one of them in the cation-free form at 1.50 A resolution, two in the Ca(2+)-bound form at 1.04 A (two bound Ca(2+) ions) and 1.65 A (three bound Ca(2+) ions) resolution and one in the Sr(2+)-bound form at 1.18 A (one bound Sr(2+) ion) resolution. The side chains of four highly conserved aspartic acids (D303, D309, D363, and D365) and two main chain oxygens (M302:O and Y364:O), together with water molecules, are in direct contact with two bound Ca(2+) ions (sites 1 and 2). At higher Ca(2+) concentrations, the side chain of N333 rotates and cooperates with D309 to generate a third Ca(2+) coordination site (site 3). Divalent cation binding sites 1 and 2 in the C(2)B domain were previously identified from NMR NOE patterns and titration studies, supplemented by site-directed mutation analysis. One difference between the crystal and NMR studies involves D371, which is not involved in coordination with any of the identified Ca(2+) sites in the crystal structures, while it is coordinated to Ca(2+) in site 2 in the NMR structure. In the presence of Sr(2+), which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.     

Structure of d(CGCGAATTCGCG) in the presence of Ca(2+) ions.

The dodecamer d(CGCGAATTCGCG) was the first oligonucleotide to be crystallized as a B-DNA duplex. Its structure was analyzed in detail in the early 1980s. Here we show that, in the presence of Ca(2+), it crystallizes in a different way (R3 space group). The dodecamers form parallel columns of straight duplexes with ten base pairs in the B form. The terminal cytosines in each molecule are disordered, whereas the terminal guanines are placed in the minor groove of neighbor duplexes. The central GAATTC region is practically identical to that found in the classic structure of the same dodecamer crystallized in the P2(1)2(1)2(1) space group in the presence of Mg(2+) and spermine. Its structure is thus independent of the crystallization conditions which have been used.     

Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements

It has long been suspected that the structure and function of a DNA duplex can be strongly dependent on its degree of hydration. By neutron diffraction experiments, we have succeeded in determining most of the hydrogen (H) and deuterium (D) atomic positions in the decameric d(CCATTAATGG)₂ duplex. Moreover, the D positions in 27 D₂O molecules have been determined. In particular, the complex water network in the minor groove has been observed in detail. By a combined structural analysis using 2.0 Å resolution X-ray and 3.0 Å resolution neutron data, it is clear that the spine of hydration is built up, not only by a simple hexagonal hydration pattern (as reported in earlier X-ray studies), but also by many other water bridges hydrogen-bonded to the DNA strands. The complexity of the hydration pattern in the minor groove is derived from an extraordinary variety of orientations displayed by the water molecules.     

Mediation of the A/B-DNA helix transition by G-tracts in the crystal structure of duplex CATGGGCCCATG

The crystal structure of the DNA dodecamer duplex CATGGGCCCATG lies on a structural continuum along the transition between A- and B-DNA. The dodecamer possesses the normal vector plot and inclination values typical of B-DNA, but has the crystal packing, helical twist, groove width, sugar pucker, slide and x-displacement values typical of A-DNA. The structure shows highly ordered water structures, such as a double spine of water molecules against each side of the major groove, stabilizing the GC base pairs in an A-like conformation. The different hydration of GC and AT base pairs provides a physical basis for solvent-dependent facilitation of the A↔B helix transition by GC base pairs. Crystal structures of CATGGGCCCATG and other A/B-DNA intermediates support a ‘slide first, roll later’ mechanism for the B→A helix transition. In the distribution of helical parameters in protein–DNA crystal structures, GpG base steps show A-like properties, reflecting their innate predisposition for the A conformation.     

Crystal and molecular structure of a DNA duplex containing the carcinogenic lesion O6-methylguanine

The crystal and molecular structure of the first DNA duplex containing the carcinogenic lesion O6MeG has been determined to a resolution of 1.9 A and refined to an R factor of 19%. (d[CGC-(O6Me)GCG])2 crystallizes in the left-handed Z DNA form and has crystal parameters and conformational features similar to those of the parent sequence [d(CG)3]2. The methyl groups on O6 of G4 and G10 have C5-C6-O6-O6Me torsion angles of 73 degrees and 56 degrees, respectively, and protrude onto the major groove surface. The base-pairing conformation for the methylated G.C base pairs is of the Watson-Crick type as opposed to a wobble-type conformation that had been proposed in a B DNA fragment. As in other Z DNA structures, a spine of hydration is seen in the minor groove.     

The solution structure of [d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA duplexes

The solution structure and hydration of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)]₂, in which the central hybrid segment is flanked by DNA duplexes at both ends, was determined using two-dimensional NMR, simulated annealing and restrained molecular dynamics. The solution structure of this chimeric duplex differs from the previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)]₂ as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)]₂. Long-lived water molecules with correlation time τ_c longer than 0.3 ns were found close to the RNA adenine H2 and H1′ protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA–DNA junction but not with the other two thymines (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA–DNA junction adopts an O4′-endo sugar conformation, while the other DNA residues including 3C in the DNA–RNA junction, adopt C1′-exo or C2′-endo conformations. The exchange rates for RNA C2′-OH were found to be ~5–20 s^–1. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)]₂, which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The minor groove width of [d(CGC)r(aaa)d(TTTGCG)]₂ is wider than its B-DNA analog but narrower than that of the A-RNA analog. It was further confirmed by its titration with the minor groove binding drug distamycin. A possible 2:1 binding mode was found by the titration experiments, suggesting that this chimeric duplex contains a wider minor groove than its B-DNA analog but still narrow enough to hold two distamycin molecules. These distinct structural features and hydration patterns of this chimeric duplex provide a molecular basis for further understanding the structure and recognition of DNA·RNA hybrid and chimeric duplexes.     

Crystal structure analysis of the B-DNA dodecamer CGTGAATTCACG.

The crystal structure of the DNA dodecamer C-G-T-G-A-A-T-T-C-A-C-G has been determined at a resolution of 2.5 A, with a final R factor of 15.8% for 1475 nonzero reflections measured at 0 degrees C. The structure is isomorphous with that of the Drew dodecamer, with the space group P2(1)2(1)2(1) and cell dimensions of a = 24.94 A, b = 40.78 A, and c = 66.13 A. The asymmetric unit contains all 12 base pairs of the B-DNA double helix and 36 water molecules. The structure of C-G-T-G-A-A-T-T-C-A-C-G is very similar to that of C-G-C-G-A-A-T-T-C-G-C-G, with no major alterations in helix parameters. Water peaks in the refined structure appear to represent a selection of peaks that were observed in the Drew dodecamer. The minor-groove spine of hydration at 2.5 A is fragmentary, but as Narendra et al. (1991) [Biochemistry (following paper in this issue)] have observed, lowering the temperature leads to a more complete representation of the spine.     

Hydration of the DNA bases is local.

Ordered hydration sites were determined for the nucleotide bases in B-type conformations using the crystal structure data on 14 B-DNA decamer structures. A method of density representation was extended so that positions, occupancies, and distributions of the hydration sites were predicted around a B-DNA double helix by a method analogous to crystallographic refinement. The predicted hydration sites correctly reproduce the main features of hydration around the B-DNA dodecamer. In contrast to the previous observations, the newly available crystal data show the same extent of hydration of guanine and adenine, and of cytosine and thymine.     

Structural insights into the effect of hydration and ions on A-tract DNA: a molecular dynamics study

DNA structure is known to be sensitive to hydration and ionic environment. To explore the dynamics, hydration, and ion binding features of A-tract sequences, a 7-ns Molecular dynamics (MD) study has been performed on the dodecamer d(CGCAAATTTGCG)(2). The results suggest that the intrusion of Na(+) ion into the minor groove is a rare event and the structure of this dodecamer is not very sensitive to the location of the sodium ions. The prolonged MD simulation successfully leads to the formation of sequence dependent hydration patterns in the minor groove, often called spine of hydration near the A-rich region and ribbon of hydration near the GC regions. Such sequence dependent differences in the hydration patterns have been seen earlier in the high resolution crystal structure of the Drew-Dickerson sequence, but not reported for the medium resolution structures (2.0 approximately 3.0 A). Several water molecules are also seen in the major groove of the MD simulated structure, though they are not highly ordered over the extended MD. The characteristic narrowing of the minor groove in the A-tract region is seen to precede the formation of the spine of hydration. Finally, the occurrence of cross-strand C2-H2.O2 hydrogen bonds in the minor groove of A-tract sequences is confirmed. These are found to occur even before the narrowing of the minor groove, indicating that such interactions are an intrinsic feature of A-tract sequences.     

Molecular dynamics simulation study of oriented polyamine- and Na-DNA: sequence specific interactions and effects on DNA structure

Molecular dynamics (MD) computer simulations have been carried out on four systems that correspond to an infinite array of parallel ordered B-DNA, mimicking the state in oriented DNA fibers and also being relevant for crystals of B-DNA oligonucleotides. The systems were all comprised of a periodical hexagonal cell with three identical DNA decamers, 15 water molecules per nucleotide, and counterions balancing the DNA charges. The sequence of the double helical DNA decamer was d(5'-ATGCAGTCAG)xd(5'-TGACTGCATC). The counterions were the two natural polyamines spermidine(3+) (Spd(3+)) and putrescine(2+) (Put(2+)), the synthetic polyamine diaminopropane(2+) (DAP(2+)), and the simple monovalent cation Na(+). This work compares the specific structures of the polyamine- and Na-DNA systems and how they are affected by counterion interactions. It also describes sequence-specific hydration and interaction of the cations with DNA. The local DNA structure is dependent on the nature of the counterion. Even the very similar polyamines, Put(2+) and DAP(2+), show clear differences in binding to DNA and in effect on hydration and local structure. Generally, the polyamines disorder the hydration of the DNA around their binding sites whereas Na(+) being bound to DNA attracts and organizes water in its vicinity. Cation binding at the selected sites in the minor and in the major groove is compared for the different polyamines and Na(+). We conclude that the synthetic polyamine (DAP(2+)) binds specifically to several structural and sequence-specific motifs on B-DNA, unlike the natural polyamines, Spd(3+) and Put(2+). This specificity of DAP(2+) compared to the more dynamic behavior of Spd(3+) and Put(2+) may explain why the latter polyamines are naturally occurring in cells.     

The crystal structure of metal-free human EF-hand protein S100A3 at 1.7-A resolution

S100A3 is a unique member of the EF-hand superfamily of Ca(2+)-binding proteins. It binds Ca(2+) with poor affinity (K(d) = 4-35 mm) but Zn(2+) with exceptionally high affinity (K(d) = 4 nm). This high affinity for Zn(2+) is attributed to the unusual high Cys content of S100A3. The protein is highly expressed in fast proliferating hair root cells and astrocytoma pointing toward a function in cell cycle control. We determined the crystal structure of the protein at 1.7 A. The high resolution structure revealed a large distortion of the C-terminal canonical EF-hand, which most likely abolishes Ca(2+) binding. The crystal structure of S100A3 allows the prediction of one putative Zn(2+) binding site in the C terminus of each subunit of S100A3 involving Cys and His residues in the coordination of the metal ion. Zn(2+) binding induces a large conformational change in S100A3 perturbing the hydrophobic interface between two S100A3 subunits, as shown by size exclusion chromatography and CD spectroscopy.     

Structural and thermodynamic studies on the adenine.guanine mismatch in B-DNA.

The structure of the synthetic dodecamer d(CGCAAATTGGCG) has been shown by single crystal X-ray diffraction methods to be that of a B-DNA helix containing two A(anti).G(syn) base pairs. The refinement, based on data to a resolution of 2.25 A shows that the mismatch base pairs are held together by two hydrogen bonds. The syn-conformation of the guanine base of the mismatch is stabilised by hydrogen bonding to a network of solvent molecules in both the major and minor grooves. A pH-dependent ultraviolet melting study indicates that the duplex is stabilised by protonation, suggesting that the bases of the A.G mispair are present in their most common tautomeric forms and that the N(1)-atom of adenine is protonated. The structure refinement shows that there is some disorder in the sugar-phosphate backbone.