Packing features of the all-AT oligonucleotide d(AAATTT).

We describe the packing features of the oligonucleotide duplex d(AAATTT)2, as determined by X-ray diffraction. There is little information on sequences that only contain A and T bases. The present structure confirms that these sequences tend to pack as a helical arrangement of stacked oligonucleotides in a B conformation with Watson-Crick hydrogen bonding. Our results demonstrate that the virtual TA base step between stacked duplexes has a negative twist that improves base stacking. This observation is consistent with the low stability of TA base steps in B-form DNA.

DNA coiled coils.

We report the coiled-coil structure of DNA, which is generated by the dodecanucleotide d(ATATATATATAT). The structure has been determined by single-crystal x-ray crystallography. The molecules form duplexes with single-stranded overhangs, which associate with neighbor molecules and give rise to infinite double helices in a coiled-coil conformation, with staggered nicks in both strands. The coiled coils have six dodecamer duplexes per turn. Despite the presence of nicks, the structure is very rigid. Statistical disorder is present, which gives rise to continuous scattering along the layer lines. This observation can be interpreted by the classical theory of coiled coils developed for proteins, which we apply here to a DNA structure. A clear splitting of the original layer lines of the DNA double helix is detected. The structure we have found adds a previously unrecognized element to the architectures that can be built from DNA oligonucleotides. Any duplex with complementary single-strand overhangs should be expected to give rise to regular coiled-coil structures.

Extra-helical guanine interactions in DNA.

We review the extra-helical guanine interactions present in many oligonucleotide crystals. Very often terminal guanines interact with other guanines in the minor groove of neighboring oligonucleotides through N2 x N3 hydrogen bonds. In other cases the interaction occurs with the help of Ni2+ ions. Guanine/netropsin stacking in the minor groove has also been found. From these studies we conclude that guanine may have multiple extra-helical interactions. In particular it may be considered a very effective minor groove binder, which could be used in the design of sequence selective binding drugs. Interactions through the major groove are seldom encountered, but might be present when DNA is stretched. Such interactions are also analyzed, since they might be important for homologous chromosome pairing during meiosis.

Structure of a cyclic dimer of amino undecanoic acid as a model of folding and hydration in polyamides, polypeptides, and related substances.

Aliphatic amides are often used in the synthesis of peptidomimetic compounds. Here we present the structure of two cyclic dimers of aminoundecanoic acid as determined by x-ray diffraction. Each dimer contains two peptide groups and twenty methylene units. In one of the crystal structures, water is associated with the peptide groups, forming a chain of hexagons similar to those found in crambin, and in other protein and nucleic acid crystals. The aminoundecanoic rings show a fold at the peptide group, similar to either beta-turn type III or V found in proteins. Such folds are an adequate model for the peptide bond structure in nylon crystallites and peptidomimetic compounds.

Solvent organization in an oligonucleotide crystal. The structure of d(GCGAATTCG)2 at atomic resolution.

We describe the crystal structure of d(GCGAATTCG) determined by x-ray diffraction at atomic resolution level (0.89 A). The duplex structure is practically identical to that described at 2.05 A resolution (Van Meervelt, L., Vlieghe, D., Dautant, A., Gallois, B., Précigoux, G., and Kennard, O. (1995) Nature 374, 742-744), however about half of the phosphate groups show multiple conformations. The crystal has three regions with different solvent structure. One of them contains several ordered Mg(+2) ions and can be considered as an ionic crystal. A second region is formed by a network of ordered water molecules with a polygonal organization that binds three duplexes. The third region is formed by channels of solvent in which very few ordered solvent molecules are visible. The less ordered phosphates are found facing this channel. The latter region provides a view of DNA with highly movable charges, both negative phosphates and counterions, without a precise location.

Alternation of DNA and solvent layers in the A form of d(GGCGCC) obtained by ethanol crystallization.

We have determined by X-ray crystallography the structure of the hexamer duplex d(GGCGCC)2 in the A-form using ethanol as a precipitant. The same sequence had previously been crystallized in the B-form, but with 2-methyl-2,4-pentanediol as a precipitant. It appears that ethanol precipitation is a useful method to induce the formation of A-form crystals of DNA. Packing of the molecules in the crystal has unique features: the known interaction of A-DNA duplexes between terminal base-pairs and the minor groove of neighbor molecules is combined with a superstructure consisting in an alternation of DNA layers and solvent layers (water/ions). This organization in layers has been observed before, also with hexamers in the A conformation which crystallize in the same space group (C2221). The solvent layer has a precise thickness, although very few ordered water molecules can be detected. Another feature of this crystal is its large unit cell, which gives rise to an asymmetric unit with three hexamer duplexes. One of the three duplexes is quite different from the other two in several aspects: the number of base pairs per turn, the twist pattern, the mean value of the twist angle and the fact that one terminal base-pair is not stacked as part of the duplex and appears to be disordered. So the variability in conformation of this sequence is remarkable.

Stacking interaction of guanine with netropsin in the minor groove of d(CGTATATACG)2.

We present the structure of the decanucleotide d(CGTATATACG) determined by single crystal X-ray diffraction at 1.58 A resolution. A netropsin drug is found in the minor groove with guanine stacked on a pyrrole ring of the drug, a feature described here for the first time. The stacked guanine is an extra-helical base coming from the end of a neighbour oligonucleotide. This observation may open the way to the development of minor groove binding drugs with a higher sequence selectivity. The oligonucleotide is in the B-conformation, but the terminal base-pairs are disrupted: the cytosine residues are disordered while the guanine residues penetrate into the minor groove of neighbouring duplexes. Four hydrated Ni ions with octahedral co-ordination are found associated with the N7 atoms of each guanine. The high affinity of these ions with guanine suggests that they may be used as probes for specific guanine residues.

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.

Water and ions in a high resolution structure of B-DNA.

A detailed picture of hydration and counterion location in the B-DNA duplex d(GCGAATTCG) is presented. Detailed data have been obtained by single crystal x-ray diffraction at atomic resolution (0.89 A) in the presence of Mg(2+). The latter is the highest resolution ever obtained for a B-DNA oligonucleotide. Minor groove hydration is compared with that found in the Na(+) and Ca(2+) crystal forms of the related dodecamer d(CGCGAATTCGCG). High resolution data (1.45 A) of the Ca(2+) form obtained in our laboratory are used for that purpose. The central GAATTC has a very stable hydration spine identical in all cases, independent of duplex length and crystallization conditions (counterions, space group). However, the organization of the water molecules (tertiary and quaternary layers) associated with the central spine vary in each case.

Structural features of single amino acid repeats in proteins.

Single amino acid repeats are found in different kinds of proteins. Some of these repeats are pathogenic. It is striking that some amino acids are able to form such repeats, but other amino acids are not. We suggest an explanation for this fact based on the different tendency of each amino acid to form aggregates. Aggregation may be due to the formation of incipient lamellar crystals as they have been described in poly-alpha-amino acids and in most synthetic polymers.

Influence of packing interactions on the average conformation of B-DNA in crystalline structures.

The molecular interactions in crystals of oligonucleotides in the B form have been analysed and in particular the end-to-end interactions. Phosphate-phosphate interactions in dodecamers are also reviewed. A strong influence of packing constraints on the average conformation of the double helix is found. There is a strong relationship between the space group, the end-to-end interactions and the average conformation of DNA. Dodecamers must have a B-form average conformation with 10 +/- 0.1 base pairs per turn in order to crystallize in the P212121 and related space groups usually found. Decamers show a wider range of conformational variation, with 9.7-10. 6 base pairs per turn, depending on the terminal sequence and the space group. The influence of the space group in decamers is quite striking and remains unexplained. Only small variations are allowed in each case. Thus, crystal packing is strongly related to the average DNA conformation in the crystals and deviations from the average are rather limited. The constraints imposed by the crystal lattice explain why the average twist of the DNA in solution (10.6 base pairs per turn) is seldom found in oligonucleotides crystallized in the B form.

Structure of the oligonucleotide d(CGTATATACG) as a site-specific complex with nickel ions.

In this paper we explore the application of Ni2+to the crystallization of oligonucleotides. We have determined in this way the structure of a fully alternating (Y-R) decanucleotide d(CGTATATACG) by single crystal X-ray diffraction. This is the first oligonucleotide crystal structure with an alternating 5'-(TA)3-3' central part. Alternating oligonucleotides have a particular interest since they often have a unique structure. In this case the general conformation is B-like with an alternating twist and an end-to-end interaction which involves terminal guanines. The crystal belongs to space group P41212 with a = b = 52.46, c = 101.49 A. This packing imposes a 90 degrees crossing of the symmetry related helices. This is a new way of packing for decamers. The oligonucleotide structure is characterized by the specific association with seven nickel ions, involving the N7 atom of every guanine. One of the Ni2+ions is shared between two guanines of symmetry related molecules. Until now no oligonucleotide has been crystallized in the presence of this metal ion. A novel C.A.T triplet structure has also been tentatively identified.

DNA-interacting proteins in the spermiogenesis of the mollusc Murex brandaris.

Sperm chromatin of Murex brandaris (a neogastropod mollusc) undergoes a series of structural transitions during spermiogenesis. The DNA-interacting proteins responsible for these changes as well as the mature protamines present in the ripe sperm nucleus have been characterized. The results reveal that spermiogenic nuclear proteins are protamine precursors that are subjected to a substantial number of small N-terminal deletions that gradually modify their overall charge. The composition of mature protamines is remarkably simple in turn, promoting an efficient and extremely tight packaging of DNA. The pattern of spermiogenic chromatin condensation in M. brandaris clearly departs from that corresponding to vertebrate chromatin.

Structure of the d(CGCCCGCGGGCG) dodecamer: a kinked A-DNA molecule showing some B-DNA features.

We have determined the structure of the dodecamer duplex d(CGCCCGCGGGCG)2. A careful use of the molecular replacement programme AMoRe has been essential in order to solve the structure. This dodecamer shows a unique conformation, quite different from all the previously studied oligonucleotide duplexes: the central octamer has an A conformation, but with a sharp 65 degrees kink in the centre; the terminal base-steps have a B-like conformation; the major groove is completely closed in the centre, a hollow molecule is thus found. The results obtained confirm the high degree of variability of DNA structure. A new type of kink and an intermediate A/B double-helical conformation have been found. Such intermediate conformation differs from those described in DNA polymerase complexes.

Physicochemical and functional comparison of Xenopus laevis nucleoplasmin obtained from oocytes and from overexpression in bacteria.

We compare the physicochemical and functional characteristics of nucleoplasmin obtained from Xenopus laevis oocytes and by bacterial overexpression of a plasmid containing the nucleoplasmin gene. The comparison shows that, while the secondary structure of the protein is not affected by the method used to obtain this protein, the bacterial expressed form exhibits a marked tendency to form large aggregates and an impaired ability to displace protamines from sperm nuclei. These results add a word of caution to the indiscriminate use, in functional or structural (crystallographic) studies, of bacterially overproduced proteins that have been end-terminally tagged with polyhistidine.

The structure of the most studied DNA fragment changes under the influence of ions: a new packing of d(CGCGAATTCGCG).

The title oligonucleotide and many related dodecamers have been extensively studied alone and as DNA-drug complexes. In practically all cases they were found to crystallize in the same space group, stabilized by interactions among the terminal guanine bases. Here we report new packing interactions (R3) in the presence of Ca2+. The oligonucleotides interact by placing their terminal guanines in the narrow groove of a neighbor molecule, an interaction which had never been found in dodecamers.

Structural variability and new intermolecular interactions of Z-DNA in crystals of d(pCpGpCpGpCpG).

We have determined the single crystal x-ray structure of the synthetic DNA hexamer d(pCpGpCpGpCpG) in two different crystal forms. The hexamer pCGCGCG has the Z-DNA conformation and in both cases the asymmetric unit contains more than one Z-DNA duplex. Crystals belong to the space group C222(1) with a = 69.73, b = 52.63, and c = 26.21 A, and to the space group P2(1) with a = 49.87, b = 41.26, c = 21.91 A, and gamma = 97.12 degrees. Both crystals show new crystal packing modes. The molecules also show striking new features when compared with previously determined Z-DNA structures: 1) the bases in one duplex have a large inclination with respect to the helical axis, which alters the overall shape of the molecule. 2) Some cytosine nitrogens interact by hydrogen bonding with phosphates in neighbor molecules. Similar base-phosphate interactions had been previously detected in some B-DNA crystals. 3) Basepair stacking between the ends of neighbor molecules is variable and no helical continuity is maintained between contiguous hexamer duplexes.

Structure and supramolecular packing features of the dipeptide Arg-Val acetate.

The title compound crystallizes in the zwitterionic form. The crystal forms a supramolecular structure with the peptide molecules organized in head-to-tail columns in the b direction. The arginine side-chains and acetate ions interact with neighbor peptides in the c direction. Infinite hydrophobic columns are present in the a direction; they involve the valine side-chains, the acetate methyl groups and the methylene groups of the arginine side-chains. This three-dimensional organization is similar to that found in Lys-Val hydrochloride.

A crystallographic approach to DNA bending: prediction of nucleosome formation by DNA triple repeats and other repetitive sequences.

DNA bending is due to two main factors: the inherent curvature of the sequence and its flexibility. Most methods of analysis do not allow a differentiation between these two factors. In this paper I show that the flexibility of DNA sequences can be estimated from the standard deviation of roll values determined by X-ray crystallography for each base step. As an application of this approach, the nucleosome formation ability of triple repeat sequences has been determined and shown to be in agreement with the experimental results. Local variations in twist do not appear to have any influence on nucleosome formation.

Structural variability of A-DNA in crystals of the octamer d(pCpCpCpGpCpGpGpG)

We have determined the structure of the synthetic DNA octamer d(pCpCpCpGpCpGpGpG) in five different crystal forms by single crystal X-ray diffraction. One crystal belongs to the space group P4(3)2(1)2 with a = b = 41.77, c = 25.15 A, whereas all others have the space group P2(1)2(1)2(1) with progressively decreasing unit cell volumes. In all crystals the octamer forms duplexes of A-DNA and all crystals display a similar packing mode, typical for A-DNA. The structure of the duplex varies from loose to very compact when going from one crystal form to another. The most compact form exhibits a volume of 995 A3 per base pair. Such a high density has never been found in A-DNA, being more characteristic of Z-DNA crystals. A comparison of the most with the least compact forms gives a RMS value of 1.7 A, with the distance between the phosphate centers through the major groove being almost twice shorter in the compact form. The phosphate-phosphate separation across the major groove in the compact form is extremely small, 0.7 A. The helical parameters also vary significantly in the various crystal forms. Differences in the helical twist can reach 13 degrees in the same step of the octamer in different crystal forms. The results prove that A-DNA is structurally very variable and demonstrate that the local structure of the same DNA fragment can strongly depend on the crystal environment.