Crystal and molecular structure of the ammonium salt of the dinucleoside monophosphate d(CpG)

The crystal and molecular structure of the ammonium salt of deoxycytidylyl-(3'-5')-deoxyguanosine has been determined from 0.85 A resolution single crystal X-ray diffraction data. The crystals obtained by acetone diffusion technique at -20 degrees C, are orthorhombic, P212121, a = 12.880(2), b = 17444(2) and c = 27.642(2) A. The structure was solved by high resolution Patterson and Fourier methods and refined to R = 0.136. There are two d(CpG) molecules in the asymmetric unit forming a mini left handed Z-DNA helix. This is in contrast to the earlier reported forms of d(CpG) where the molecules form self base paired duplexes. There are two ammonium ions in the asymmetric unit. The major groove NH+4 ion interacts with N7 of guanines through water bridges besides making H-bonded interactions directly with the phosphate oxygen atoms. A second NH+4 ion is found in the minor groove interacting directly with the phosphate oxygen atoms. Symmetry related molecules pack in such a way that the cytosine base stacks on cytosine and guanine base on guanine. Our structure demonstrates that alternating d(CpG) sequences have the ability to adopt the left handed Z-DNA structure even at the dimer level i.e., in a sequence which is only two base pairs long.     

Crystal and molecular structure of a DNA fragment: d(CGTGAATTCACG)

The crystal structure of the dodecanucleotide d(CGTGAATTCACG) has been determined to a resolution of 2.7 A and refined to an R factor of 17.0% for 1532 reflections. The sequence crystallizes as a B-form double helix, with Watson-Crick base pairing. This sequence contains the EcoRI restriction endonuclease recognition site, GAATTC, and is flanked by CGT on the 5'-end and ACG on the 3'-end, in contrast to the CGC on the 5'-end and GCG on the 3'-end in the parent dodecamer d(CGCGAATTCGCG). A comparison with the isomorphous parent compound shows that any changes in the structure induced by the change in the sequence in the flanking region are highly localized. The global conformation of the duplex is conserved. The overall bend in the helix is 10 degrees. The average helical twist values for the present and the parent structures are 36.5 degrees and 36.4 degrees, respectively, corresponding to 10 base pairs per turn. The buckle at the substituted sites are significantly different from those seen at the corresponding positions in the parent dodecamer. Step 2 (GpT) is underwound with respect to the parent structure (27 degrees vs 36 degrees) and step 3 (TpG) is overwound (34 degrees vs 27 degrees). There is a spine of hydration in the narrow minor groove. The N3 atom of adenine on the substituted A10 and A22 bases are involved in the formation of hydrogen bonds with other duplexes or with water; the N3 atom of guanine on G10 and G22 bases in the parent structure does not form hydrogen bonds.     

Crystal structure of d(GCGAAAGCT) containing a parallel-stranded duplex with homo base pairs and an anti-parallel duplex with Watson-Crick base pairs

A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized in the space group I4₁22 with the unit-cell dimensions a = b = 53.4 Å and c = 54.0 Å, and the crystal structure has been determined at 2.5 Å resolution. The four nucleotide residues CGAA of the first half of the oligomer form a parallel duplex with another half through the homo base pairs, C₂:C₂⁺ (singly-protonated between the Watson– Crick sites), G₃:G₃ (between the minor groove sites), A₄:A₄ (between the major groove sites) and A₅:A₅ (between the Watson–Crick sites). The two strands remaining in the half of the parallel duplex are split away in different directions, and they pair in an anti-parallel B-form duplex with the second half extending from a neighboring parallel duplex, so that an infinite column is formed in a head-to-tail fashion along the c-axis. It seems that a hexa-ammine cobalt cation supports such a branched and bent conformation of the oligomer. One end of the parallel duplex is stacked on the corresponding end of the adjacent parallel duplex; between them, the guanine base of the first residue is stacked on the fourth ribose of another duplex.     

Crystal and Molecular Structure of the Sodium Salt of the Dinucleotide Duplex d(CpG)

Abstract

The crystal and molecular structure of the sodium salt of deoxycytidylyl-{3′ ?5′)-deoxyguanosine has been determined from X-ray diffraction data. The crystals, obtained from an aqueous y- butyrolactone solution at pH = 5.3, are orthorhombic, P2₁2₁2₁, a= 10.640(2), b= 11.184(2) and c=44.618(4) A. The structure was refined to an R = 0.041. The d(CpG) structure is similar to the ammonium salt solved by Cruse et al.(1). Both structures form a parallel self base paired mini-double helix. In d(CpG).Na⁺, one of the two paired cytosines is protonated on N(3). The cytosines form 3 hydrogen bonds while the guanines form only 2. The Na⁺ ion is coordinated with five groups: two water molecules, 0(6) of guanine A, N(7) of guanine B and 0(5′) of cytosine B, forming a square pyramid. The hydration shell around the mini-helix is analysed and compared with that of the ammonium salt. d(CpG).Na⁺ is the second d(CpG) oligonucleotide found with a self base pairing arrangement despite of the fact that the crystallization conditions and counterion were different in both cases. The hypothesis that self base pairing is not only a crystallization artifact but may play a role under physiological conditions as a source of transversion mutations is discussed.     

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.     

A duplex of the oligonucleotides d(GGGGGTTTTT) and d(AAAAACCCCC) forms an A to B conformational junction in concentrated salt solutions

The coexistence of both A form and B form tracts and formation of an A-B junction in the oligomer d(GGGGGTTTTT).d(AAAAACCCCC) in saturated sodium chloride solution have been detected by Raman spectroscopy. The entire duplex adopts the familiar B-form conformation in aqueous solution at low salt concentrations (0.1M NaCl). In 6M NaCl the adoption of an A form is observed within the G,C tract while a B-form is maintained in the A.T tract. The experimental results indicate that two different helical forms can co-exist in a rather short oligonucleotide and that formation of an A-B junction can occur over a fairly small span of bases. This is in agreement with recent rules governing the relation between base sequence and secondary structure of DNA published from this laboratory. The conformational preferences of each of the individual oligomers d(AAAAACCCCC) and d(GGGGGTTTTT) have also been investigated. The oligomer d(AAAAACCCCC) is single stranded but some evidence for base stacking is observed at 2 degrees C. In contrast, a double stranded B-form structure characterized by wobble G-T base pairing is observed for d(GGGGGTTTTT) in 0.1M and 6M NaCl.     

Study of the spatial structure of the duplex d(pTGTTTGGC) d(pCCAAAC)A in aqueous solution by methods of uni- and two-dimensional (1)H-NMR spectroscopy and organic molecular mechanics

Structure of the complementary complex d(pTGTTTGGC) d(pCCAAAC)A in the aqueous solution has been investigated by one- and two-dimensional 1H NMR spectroscopy. The resonances of nonexchangeable protons of bases as well as methyl and deoxyribose 1', 2'a, 2'b, 3' and 4' protons have been assigned by means of two-dimensional J-correlated spectroscopy (COSY) and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY). Using one-dimensional NOE measurements, 62 interproton distances (intranucleotide: (H6/H8)i--(H1')t, (H6/H8)i--(H2'a)i, (H1')i--(H2'a)i, (H1')i--(H2'b)i; internucleotide: (H6/H8)i--(H1')i-1, (H6/H8)i--(H2'a)i-1, (H6/H8)i--(H2'b)i-1, (H5/CH3)i--(H6/H8)i-1, (H5/CH3)i--(H2'a/H2'b)i-1) have been determined for nearest-neighbour protons. Spin-coupling constant values for some sugar protons have been obtained from COSY spectra. The restrained molecular mechanics calculations have yielded the possible solution structures of duplex fitting the experimental set of interproton distances and coupling constants.     

Crystal and molecular structure of the alternating dodecamer d(GCGTACGTACGC) in the A-DNA form: comparison with the isomorphous non-alternating dodecamer d(CCGTACGTACGG).

The crystal structure of the alternating dodecamer d(GCGTACGTACGC) (5'-GC) has been determined to a resolution of 2.55A using oscillation film data. The crystals belong to space group P6(1) 22, a = b = 46.2A, c = 71.5A with one strand in the asymmetric unit, and are isomorphous with a previously described non-alternating dodecamer, d(CCGTACGTACGG) (5'-CC). Refinement by X-PLOR/NUCLSQ gave a final R factor of 14.2% for 1089 observations. The molecule adopts the A-DNA form. The interchange of the terminal base pairs in the two dodecamers results in differences in the intermolecular contacts and may account for the differences in the bending. This dodecamer shows an axial deflection of 30 degrees, in the direction of the major groove compared to 20 degrees in 5'-CC and may be a consequence of additional contacts generated in 5'-GC by the interchange of end base pairs. The high helical axis deflection appreciably influences the local helical parameters. The molecule exhibits relatively high inclination angles, and has a narrow major groove. The helical parameters when described relative to the dyad-related hexamer halves of the molecule give more reasonable values. The crystal packing, local helical parameters, torsion angles, and hydration are described and also compared with the non-alternating 5'-CC dodecamer.     

Crystal and molecular structure of the A-DNA dodecamer d(CCGTACGTACGG). Choice of fragment helical axis.

The crystal structure of the dodecamer d(CCGTACGTACGG) has been determined at 2.5 A resolution. The crystals grow in the hexagonal space group P6(1)22, a = b = 46.2 A, c = 71.5 A with one strand as the asymmetric unit. Diffraction data were collected by the oscillation film method yielding 1664 unique reflections with an Rmerge of 0.04. The structure was solved by real-space rotational translational searches with idealized helical models of A, B and Z-DNA. The best agreement was given by an A-DNA model with its dyad axis along the diagonal crystallographic dyad axis, with an R-factor 0.43 and correlation coefficient of 0.59 for data between 10 and 5 A. Iterative map fitting and restrained least-squares refinement and addition of 40 solvent molecules brought the R-factor to 0.15 and the correlation coefficient to 0.97 for all data between 8.0 and 2.5 A. The stereochemistry of the atomic model is good, with a root-mean-square deviation in bond distances of 0.006 A. This is the first example of an A-DNA containing a full helical turn. The dodecamer displays a novel packing motif. In addition to the characteristic contacts between the terminal base-pairs and the minor grooves of symmetry-related molecules, there are also minor groove to minor groove interactions not previously observed. The packing leaves an approximately 25 A diameter solvent channel around the origin, along the c-axis. The presence of a prominent 3.4 A meridional reflection and other diffuse features in the diffraction pattern provided evidence for the presence of disordered B-DNA along the c-axis, which can be accommodated in these solvent channels. The molecular conformation of the dodecamer also displays novel features. The dyad-related halves of the molecule are bent at an angle of 20 degrees, and the helical parameters are affected by this bend. Unlike the shorter A-DNA octamers, the dimensions of the major groove can be directly measured. Novel correlations between local helical parameters and global conformational features are presented. Most of the solvent molecules are associated with the major groove and the sugar-phosphate backbone.     

Crystal and molecular structure of a collagen-like polypeptide (Pro-Pro-Gly)10

(Pro-Pro-Gly)₁₀ forms single crystals, providing X-ray diffraction data to 0.22 nm resolution. In the crystals, the polypeptides form triplexes that aggregate end-to-end in quasi-infinite helices with axial translation per tripeptide h = 0.287 nm and the corresponding rotation t = ?102.9 °. The structure, which may be an allomorph of collagen, has been refined by the linked-atom least-squares procedure. In addition, three water molecules per tripeptide have been detected by Fourier difference syntheses. One of them forms an intrachain hydrogen-bonded bridge O(Pro₂) - - - W - - - O(Gly). There are also interchain hydrogen bonds (Gly)NH - - - O(Pro₁) within the triplex.     

Crystal structure of RNase A complexed with d(pA)4

Co-crystals of pancreatic RNase A complexed with oligomers of d(pA)4 were grown from polyethylene glycol 4000 at low ionic strength and the X-ray diffraction data were collected to 2.5 A resolution. From a series of heavy-atom derivatives a multiple isomorphous replacement-phased electron density map of the RNase-d(pA)4 complex was calculated to 3.5 A. By inspection, the disposition of the known structure of RNase in the unit cell was determined and this was confirmed by calculation of a standard crystallographic residual, R. Refinement of the protein alone in the unit cell as a strictly rigid body yielded an R factor of 0.32 at 2.8 A resolution. From difference Fourier syntheses DNA fragments were elucidated and incorporated into a model of the complex. The entire asymmetric unit was refined using a restrained-constrained least-squares procedure (CORELS) interspersed with difference Fourier syntheses. At the present time the crystal structure has been refined to an overall R value of 0.215 at 2.5 A resolution. The asymmetric unit of the complex crystals contains four oligomers of d(pA)4 associated with each molecule of RNase. In addition, there may also be partially ordered fragments of DNA at low occupancy present in the unit cell, but these have not, at this time, been incorporated into the model. One tetramer of d(pA)4 is entirely bound by a single protein molecule and occupies a portion of the active site cleft, filling the purine binding site and the phosphate site at the catalytic center with its 5' nucleotide. Two other tetramers are partly intermolecular. One passes from near the pyrimidine binding site over the surface of the protein toward arginine 39 and into a solvent region. A third tetramer is anchored at its 5' terminus by a salt link to lysine 98, passes near arginine and then through a solvent region to terminate with its 3' end near the surface of another protein molecule in the lattice. The fourth tetramer of d(pA)4 is bound at its 5' end on the opposite side of the protein from the active site in an electropositive anion trap that includes lysines 31 and 91 as well as arginine 33. There may be a DNA-DNA interaction involving the 5' phosphate of one tetramer and the 3' bases of two other tetramers and this may help to stabilize the crystalline complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure and properties of the copper(II) complex of sodium monensin A

The preparation and structural characterization of a new copper(II) complex of the polyether ionophorous antibiotic sodium monensin A (MonNa) are described. Sodium monensin A binds Cu(II) to produce a heterometallic complex of composition [Cu(MonNa)₂Cl₂]·H₂O, 1. The crystallographic data of 1 show that the complex crystallizes in monoclinic space group C2 with Cu(II) ion adopting a distorted square-planar geometry. Copper(II) coordinates two anionic sodium monensin ligands and two chloride anions producing a neutral compound. The sodium ion remains in the inner cavity of the ligand retaining its sixfold coordination with oxygen atoms. Replacement of crystallization water by acetonitrile is observed in the crystal structure of the complex 1. Copper(I) salt of the methyl ester of MonNa, 2, was identified by X-ray crystallography as a side product of the reaction of MonNa with Cu(II). Compound 2, [Me-MonNa][H-MonNa][CuCl₂]Cl, crystallizes in monoclinic space group C2 with the same coordination pattern of the sodium cation but contains a chlorocuprate(I) counter [CuCl₂]⁻, which is linear and not coordinated by sodium monensin A. The antibacterial and antioxidant properties as two independent activities of 1 were studied. Compound 1 is effective against aerobic Gram(+)-microorganisms Bacillus subtilis, Bacillus mycoides and Sarcina lutea. Complex 1 shows SOD-like activity comparable with that of the copper(II) ion.     

Crystal structure and stability of a DNA duplex containing A(anti).G(syn) base-pairs

The synthetic dodecanucleotide d(CGCAAATTGGCG) has been analysed by single-crystal X-ray diffraction techniques and the structure refined to R = 0.16 and 2.25 A resolution, with the location of 94 solvent molecules. The sequence crystallizes as a full turn of a B-DNA helix with ten Watson-Crick base-pairs and two adenine-guanine mispairs. The analysis clearly shows that the mismatches are of the form A(anti).G(syn). Thermal denaturation studies indicate that the stability of the duplex is strongly pH dependent, with a maximum at pH 5.0, suggesting that the base-pair is stabilized by protonation. Three different arrangements have been observed for base-pairs between guanine and adenine and it is likely that A.G mismatch conformation is strongly influenced by dipole-dipole interactions with adjacent base-pairs.     

Structures of the mismatched duplex d(GGGTGCCC) and one of its Watson-Crick analogues d(GGGCGCCC)

The mismatched duplex d(GGGTGCCC) (I) and its two Watson-Crick analogues (dGGGCGCCC) (II) and d(GGGTACCC) (III) were synthesized. The X-ray crystal structures of (I) and (II) were determined at resolutions of 2.5 and 1.7 A (1 A = 0.1 nm) and refined to R factors of 15 and 16%, respectively. (I) and (II) crystallize as A-DNA doublehelical octamers in space groups P61 and P4(3)2(1)2, respectively, and are stable at room temperature. The central two G.T mispairs of (I) adopt the wobble geometry as observed in other G.T mismatches. The two structures differ significantly in their local conformational features at the central helical regions as well as in some global ones. In particular, T-G adopts a large helical twist (44 degrees) whereas C-G adopts a small one (24 degrees). This difference can be rationalized on the basis of simple geometrical considerations. Base-pair stacking energies which were calculated for the two duplexes indicate that (I) is destabilized with respect to (II). Helix-coil transition measurements were performed for each of the three oligomers by means of ultraviolet absorbance spectrophotometry. The results indicate that the stability of the duplexes and the co-operativity of the transition are in the following order: (I) less than (III) less than (II). Such studies may help in understanding why certain regions of DNA are more likely to undergo spontaneous mutations than others.     

Crystal and molecular structure of d(GTGCGCAC): investigation of the effects of base sequence on the conformation of octamer duplexes.

The structure of the self-complementary deoxyoctanucleotide d(GTGCGCAC), which crystallized as an A-type helix in the space group P4(3)2(1)2, with one strand in the crystallographic asymmetric unit has been determined and refined to a final R-value of 0.154 using 1.64-A diffraction data collected on an area detector. In contrast to the closely related sequence d(GTGTACAC)tet, there was no evidence for an ordered spermine molecule in the major groove of this octamer. Ordered water is found associated with almost all the exposed hydrogen bonding groups of the octamer. A pentagonal ring of water molecules is hydrogen bonded to O6 and N7 of G3 and the N4 and O6 of the C4.G13 base pair. A detailed comparison of the local helical parameters of d(GTGCGCAC) and d(GTGTACAC)tet is presented. The base sequence change at the center of the octamers affects several of the local helical parameters, via both intra- and interduplex interactions within the crystal.     

Crystal and molecular structure of methyl O-alpha-D-manno-pyranosyl-(1----2)-alpha-D-mannopyranoside

The crystal and molecular structure of a synthetic mannosyl disaccharide, methyl O-alpha-D-mannopyranosyl-(1----2)-alpha-D-mannopyranoside, has been determined from X-ray diffractometer data by direct methods by use of the Multan programs. The crystals are monoclinic, space group P2 with unit cell dimensions, a 8.086(1), b 9.775(1), c 9.975(2) A, beta 104.58(1) degrees, Z 2, and Dm 1.54 g/cm3. The structure was refined to an R-value of 0.033 for 1359 reflections measured with CuK alpha radiation. The mannopyranose units have the chair conformations 4C(D) with C-5' and C-2' deviating from the best plane through the other four atoms of the ring by -0.68 and +0.53 A in the nonreducing group, and C-3 and O-5 deviating from the mean plane through the other four atoms by +0.57 and -0.66 A, respectively, in the "potentially" reducing residue. The ring-to-ring conformation can be described as (phi, psi) = (-64.5, 105.5 degrees). The conformation across the C-5--C-6 bond is gauche-gauche in both the sugars. The crystal structure is stabilized by a network of intermolecular O-H...O hydrogen bonds.     

The molecular structure of Okazaki fragment r(CGCA)d(AAAAAGCG):d(CGCTTTTTTGCG) in solution

The hetero duplex molecule, r(CGCA)d(AAAAAGCG):d(CGCTTTTTTGCG) which corresponds to Okazaki fragment was synthesized and its molecular structure has been analyzed by NMR study. The RNA strand of RNA-DNA hybrid region adopts A-form and DNA strand of the same region deviates from the standard B-form. The conformation of DNA-DNA duplex segment belongs to B-form. The hybrid-DNA duplex junction shows a structural discontinuities, A-B junction. The same conformational characteristic of oligo(dA): oligo(dT) tract as that of DNA oligomer which has same base sequence has been observed.     

Crystal structure of an RNA duplex r(gugucgcac)(2) with uridine bulges.

The crystal structure of a nonamer RNA duplex with a uridine bulge in each strand, r(gugucgcac)(2), was determined at 1.4 A resolution. The structure was solved by multiple anomalous diffraction phasing method using a three-wavelength data set collected at the Advanced Protein Source and refined to a final R(work)/R(free) of 21.2 %/23.4 % with 33,271 independent reflections (Friedel pairs unmerged). The RNA duplex crystallized in the tetragonal space group P4(1)22 with two independent molecules in the asymmetric unit. The unit cell dimensions are a=b=47.18 A and c=80.04 A. The helical region of the nonamer adopts the A-form conformation. The uridine bulges assume similar conformations, with uracils flipping out and protruding into the minor groove. The presence of the bulge induces very large twist angles (approximately +50 degrees) between the base-pairs flanking the bulges while causing profound kinks in the helix axis at the bulges. This severe twist and the large kink in turn produces a very narrow major groove at the middle of the molecule. The ribose sugars of the guanosines before the bulges adopt the C2'-endo conformation while the rest, including the bulges, are in the C3'-endo conformation. The intrastrand phosphate-phosphate (P-P) distance of the phosphate groups flanking the bulges (approximately 4.4 A) are significantly shorter than the average P-P distance in the duplex (6.0 A). This short distance between the two phosphate groups brings the non-bridging oxygen atoms close to each other where a calcium ion is bound to each strand. The calcium ions in molecule 1 are well defined while the calcium ions in molecule 2 are disordered.