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.

Thermostable xylanase from Thermoascus aurantiacus at ultrahigh resolution (0.89 A) at 100 K and atomic resolution (1.11 A) at 293 K refined anisotropically to small-molecule accuracy.

Thermoascus aurantiacus xylanase is a thermostable enzyme which hydrolyses xylan, a major hemicellulose component of the biosphere. The crystal structure of this F/10 family xylanase, which has a triosephosphate isomerase (TIM) barrel (beta/alpha)(8) fold, has been solved to small-molecule accuracy at atomic resolution (1.11 A) at 293 K (RTUX) and at ultrahigh resolution (0.89 A) at 100 K (CTUX) using X-ray diffraction data sets collected on a synchrotron light source, resulting in R/R(free) values of 9.94/12.36 and 9.00/10.61% (for all data), respectively. Both structures were refined with anisotropic atomic displacement parameters. The 0.89 A structure, with 177 476 observed unique reflections, was refined without any stereochemical restraints during the final stages. The salt bridge between Arg124 and Glu232, which is bidentate in RTUX, is water-mediated in CTUX, suggesting the possibility of plasticity of ion pairs in proteins, with water molecules mediating some of the alternate arrangements. Two buried waters present inside the barrel form hydrogen-bond interactions with residues in strands beta2, beta3, beta4 and beta7 and presumably contribute to structural stability. The availability of accurate structural information at two different temperatures enabled the study of the temperature-dependent deformations of the TIM-barrel fold of the xylanase. Analysis of the deviation of corresponding C(alpha) atoms between RTUX and CTUX suggests that the interior beta-strands are less susceptible to changes as a function of temperature than are the alpha-helices, which are on the outside of the barrel. betaalpha-loops, which are longer and contribute residues to the active-site region, are more flexible than alphabeta-loops. The 0.89 A structure represents one of the highest resolution structures of a protein of such size with one monomer molecule in the asymmetric unit and also represents the highest resolution TIM-barrel fold structure to date. It may provide a useful template for theoretical modelling studies of the structure and dynamics of the ubiquitous TIM-barrel fold.

First evidence to show the topological change of DNA from B-dNA to Z-DNA conformation in the hippocampus of Alzheimer's brain.

Alzheimer's disease (AD) is a complex neurodegenerative disorder. Our studies for the first time showed evidence for altered DNA conformation in the hippocampus of Alzheimer's disease affected brain. The Circular dichroism spectra of severely affected AD DNA showed a typical left-handed Z-DNA conformation, whereas normal, young, and aged brain DNA have the usual B-DNA conformation. Moderately affected AD DNA has modified B-DNA conformation (probable B-Z intermediate form). The ELISA, ethidium bromide binding pattern to DNA and melting temperature (Tm) profiles also revealed the conformational transition from B to Z DNA in AD brain DNA. The altered conformation of DNA will have tremendous implications in gene expressions.

Crystallization and preliminary investigations on a telomeric repeat sequence C4A2C4A2.

Repeat units based on the telomeric sequence of the ciliated protozoan Tetrahymena, d(C(4)A(2))(2), have been crystallized. Cytosine-rich DNA stretches are known to reside in telomeres and centromeres of eukaryotic chromosomes, playing crucial roles in the structural stability of the chromosome in addition to their connection with cancer and aging. Preliminary investigations on the telomeric repeat sequence C(4)A(2)C(4)A(2) from CD studies and X-ray crystal data suggest it to be a right-handed interdigitated tetraplex structure with hemiprotonated C.C(+) base pairs. The molecules appear to be packed one on top of another forming a discontinuous helix along c simulating a poly-C fibre, an arrangement which maximizes the number of cytosines stacked.

The tertiary structure at 1.59 A resolution and the proposed amino acid sequence of a family-11 xylanase from the thermophilic fungus Paecilomyces varioti bainier.

We report the crystal structure at 1.59 A and the proposed amino acid sequence of an endo-1,4-beta-xylanase (PVX) from the thermophilic fungus Paecilomyces varioti Bainier (PvB), stable up to 75 degrees C. This fungus is attracting clinical attention as a pathogen causing post-surgical infections. Its xylanase, known as a skin-contact allergen, is the first protein from this fungus whose three-dimensional structure has been elucidated. The crystals of PVX conform to the space group P2(1)2(1)2(1 )with a=38.76 A, b=54.06 A and c=90.06 A. The structure was solved by molecular replacement techniques using polyalanine coordinates of the Thermomyces lanuginosus xylanase (PDB code 1YNA) and a careful model building based on the amino acid sequence known for two trypsin-digested peptide fragments (17 residues), the sequence and structural alignment of family-11 xylanases and electron density maps. The final refined model has 194 amino acid residues and 128 water molecules, with a crystallographic R-factor of 19.07 % and a free R-factor of 21.94 %. The structure belongs to an all-beta fold, with two curved beta-sheets, forming the cylindrical active-site cleft, and a lone alpha-helix, as present in other family-11 xylanases. We have carried out a quantitative comparison of the structure and sequence of the present thermophilic xylanase (PVX) with other available native structures of mesophiles and thermophiles, the first such detailed analysis to be carried out on family-11 xylanases. The analysis provides a basis for the rationalisation of the idea that the "hinge" region is made more compact in thermophiles by the addition of a disulphide bridge between Cys110 and Cys154 and a N-H.O hydrogen bond between Trp159 near the extremity of the lone alpha-helix and Trp138 on beta-strand B8. This work brings out explicitly the presence of the C-H.O and the C-H.pi type interactions in these enzymes. A complete description of structural stability of these enzymes needs to take account of these weaker interactions.

Crystal structure at 1.8 A resolution and proposed amino acid sequence of a thermostable xylanase from Thermoascus aurantiacus.

Thermoascus aurantiacus xylanase is a thermostable enzyme which hydrolyses xylan, a major hemicellulose component in the biosphere. Crystals belonging to P21 space group with a=41.7 A, b=68.1 A, c=51. 4 A and beta=113.6 degrees, Z=2 were grown that could diffract to better than 1.8 A resolution. The structure was solved by molecular replacement method using the Streptomyces lividans xylanase model. The amino acid sequence was determined from the electron density map aided by multiple alignment of related xylanase sequences. The sequence thus obtained provides a correction to the sequence reported earlier based on biochemical methods. The final refined protein model at 1.8 A resolution with 301 amino acid residues and 266 water molecules has an R-factor of 16.0 % and free R of 21.1 % with good stereochemistry. The single polypeptide chain assumes (alpha/beta)8 TIM-barrel fold and belongs to F/10 family of glycoside hydrolases. The active site consists of two glutamate residues located at the C terminus end of the beta-barrel, conforming to the double displacement mechanism for the enzyme action. A disulphide bond and more than ten salt bridges have been identified. In particular, the salt bridge Arg124-Glu232 which is almost buried, bridges the beta-strands beta4 and beta7 where the catalytic glutamate residues reside, and it may play a key role in the stability and activity at elevated temperature. To our knowledge, for the first time in the F/10 family xylanases, we observe a proline residue in the middle of the alpha-helix alpha6 which may be contributing to better packing. Earlier studies show that the enzyme retains its activity even at 70 degrees C. The refined protein model has allowed a detailed comparison with the other known structures in the F/10 family of enzymes. The possible causative factors for thermostability are discussed.

An A-DNA structure with two independent duplexes in the asymmetric unit.

The crystal and molecular structure of the self-complementary A-DNA decamer sequence d(G4CGC4) was solved at 1.9 A resolution. The decamer crystallizes in space group P21 with two independent duplexes in the asymmetric unit. Duplex 1 has interactions which are distributed symmetrically about its length compared with duplex 2. The two end base pairs of duplex 1 have a similar NH.O hydrogen-bond pattern involving GGC segments of duplex 2 and a symmetry-related neighbour, while the end base pairs of duplex 2 interact with the GCC and GGG segments of its symmetry-related neighbours through NH.O and NH.N hydrogen bonds and a water-mediated hydrogen bond between the carboxyl groups of C40 and C8. In addition to the C4'-C5' torsion angle gamma assuming the trans conformation in certain steps, this angle also adopts the gauche- conformation at C37 as opposed to the preferred gauche+ conformation, with a concomitant change in phosphodiester P-O5' (alpha) in the opposite sense. This facilitates stacking between adjacent bases. The study suggests that the structural alterations in the two molecules in the asymmetric unit originate from an inherent propensity of the d(G4CGC4) base sequence for varied intermolecular interactions and malleability.

Interaction of Co, Mn, Mg and Al with d(GCGTACGC): a spectroscopic study.

Spectroscopic study on the interactions of trace elements Co., Mn, Mg and Al with d(GCGTACGC) indicated the following: Al and Mg did not alter Tm values. Mn enhanced Tm at lower concentration and decreased it at higher concentrations. Interestingly Co at higher concentration elevated the Tm. These studies also showed lower concentrations of Mn displaced EtBr, whereas Al could displace it at higher ionic strength. Mg and Co displaced EtBr fluorescence at moderate concentrations. The binding constant values and CD spectra clearly indicated strong binding of these elements to DNA.

Crystallization and preliminary X-ray crystallographic studies of thermostable xylanase crystals isolated from Paecilomyces varioti.

A highly thermostable xylanase isolated from the thermophilic fungus Paecilomyces varioti has been crystallized by the vapour diffusion method. The isolation of this enzyme by crystallization directly from the culture filtrate projects this fungus as an important source for large-scale production of pure xylanase. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with the unit cell dimensions a = 38.48 A, b = 53.87 A and c = 90.23 A. Four molecules occupy a volume of 187,039.4 A3 along with 34% of solvent. The data collected with an area detector to the resolution of 2.7 A were used to calculate the unit cell parameters and Matthews' constant. The optical behaviour of the crystal was studied at different temperatures to understand its thermal stability.

Crystallization and preliminary X-ray diffraction analysis of crystals of Thermoascus aurantiacus xylanase.

Crystals suitable for high resolution X-ray diffraction analysis have been grown of the 29,774-Da protein, xylanase (1,-4-beta-xylan xylanohydrolase EC 3.2.1.8) from the thermophilic fungus Thermoascus aurantiacus. This protein, an endoxylanase demonstrates the hydrolysis of beta-(1-4)-D-xylose linkage in xylans and crystallizes as monoclinic pinacoids in the presence of ammonium sulphate buffered at pH 6.5, and also with neutral polyethylene glycol 6000. The crystals belong to space group P2(1) and have cell dimensions, a = 41.2 A, b = 67.76 A, c = 51.8 A; beta = 113.2 degrees.

Crystal and molecular structure of L-valyl-L-lysine hydrochloride.

L-Valyl-L-lysine hydrochloride, C11N3O3H23 HCl, crystallizes in the monoclinic space group P2(1) with a = 5.438(5), b = 14.188(5), c = 9.521(5) A, beta = 95.38(2) degrees and Z = 2. The crystal structure, solved by direct methods, refined to R = 0.036, using full matrix least-squares method. The peptide exists in a zwitterionic form, with the N atom of the lysine side-chain protonated. The two gamma-carbons of the valine side-chain have positional disorder, giving rise to two conformations, chi 1(11) = -67.3 and 65.9 degrees, one of which (65.9 degrees) is sterically less favourable and has been found to be less popular amongst residues branching at beta-C. The lysine side-chain has the geometry of g- tgt, not seen in crystal structures of the dipeptides reported so far. Interestingly, chi 2(3) (63.6 degrees) of lysine side-chain has a gauche+ conformation unlike in most of the other structures, where it is trans. The neighbouring peptide molecules are hydrogen bonded in a head-to-tail fashion, a rather uncommon interaction in lysine peptide structures. The structure shows considerable similarity with that of L-Lys-L-Val HCl in conformational angles and H-bond interactions [4].

The identification of 14 alpha,17 beta-dihydroxyandrost-4-en-3-one monohydrate and 14 alpha,17 beta-dihydroxyandrosta-1,4-dien-3-one monohydrate, metabolites of androstenedione in Mucor piriformis.

The microorganism Mucor piriformis transforms androst-4-ene-3,17-dione into a major and several minor metabolites. X-ray crystallographic analysis of two of these metabolites was undertaken to determine unambiguously their composition and chirality. Crystals belong to the orthorhombic space-group P2(1)2(1)2(1), with a = 7.199(4) A and a = 6.023(3) A, b = 11.719(3) A and b = 13.455(4) A, c = 20.409(3) A and c = 20.702(4) A for the two title compounds, respectively. The structures have been refined to final R values of 0.060 and 0.040, respectively.

Visualisation of a 2'-5' parallel stranded double helix at atomic resolution: crystal structure of cytidylyl-2',5'-adenosine.

X-ray crystallographic studies on 3'-5' oligomers have provided a great deal of information on the stereochemistry and conformational flexibility of nucleic acids and polynucleotides. In contrast, there is very little information available on 2'-5' polynucleotides. We have now obtained the crystal structure of Cytidylyl-2',5'-Adenosine (C2'p5'A) at atomic resolution to establish the conformational differences between these two classes of polymers. The dinucleoside phosphate crystallises in the monoclinic space group C2, with a = 33.912(4)A, b = 16.824(4)A, c = 12.898(2)A and beta = 112.35(1) with two molecules in the asymmetric unit. Spectacularly, the two independent C2'p5'A molecules in the asymmetric unit form right handed miniature parallel stranded double helices with their respective crystallographic two fold (b axis) symmetry mates. Remarkably, the two mini duplexes are almost indistinguishable. The cytosines and adenines form self-pairs with three and two hydrogen bonds respectively. The conformation of the C and A residues about the glycosyl bond is anti same as in the 3'-5' analog but contrasts the anti and syn geometry of C and A residues in A2'p5'C. The furanose ring conformation is C3' endo, C2' endo mixed puckering as in the C3'p5'A-proflavine complex. A comparison of the backbone torsion angles with other 2'-5' dinucleoside structures reveals that the major deviations occur in the torsion angles about the C3'-C2' and C4'-C3' bonds. A right-handed 2'-5' parallel stranded double helix having eight base pairs per turn and 45 degrees turn angle between them has been constructed using this dinucleoside phosphate as repeat unit. A discussion on 2'-5' parallel stranded double helix and its relevance to biological systems is presented.

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.

A proposal for a specific double-helical structure in which the polynucleotide strands intercalate instead of forming base-pairs.

Double helices, since the discovery of the DNA structure by Watson and Crick, represent the single most important secondary structural form of nucleic acids. The secondary structures of a variety of polynucleotide helices have now been well characterised with hydrogen-bonded base-pairs as building blocks. We wish to propose here the possibility, in a specific case, of a double stranded helical structure without any base-pair, but having a repeat unit of two nucleotides with their bases stacked through intercalation. The proposal comes from the initial models we have built for poly(dC) using the stacking patterns found in the crystal structures of 5'-dCMPNa2 which crystallises in two forms depending on the degree of hydration. These structures have pairs of nucleotides with the cytosine rings partially overlapping and separated by 3.3A. Using these as repeat units one could generate a model for poly(dC) with parallel strands, having a turn angle of 30 degrees and a base separation of 6.6A along each strand. Both right and left handed models with these parameters can be built in a smooth fashion without any obviously unreasonable stereochemical contacts. The helix diameter is about 13.5A, much smaller than that of normal helices with base-pair repeats. The changes in the sugar-phosphate backbone conformation in the present models compared to normal duplexes only reflect the torsional flexibility available for extension of polynucleotide chains as manifested by the crystal structures of drug-inserted oligonucleotide complexes. Intercalation proposed here could have some structural relevance elsewhere, for instance to the base-mismatched regions on the double helix and the packing of noncomplementary single strands as found in the filamentous bacteriophage Pf1.

Sequence-dependent conformation of an A-DNA double helix. The crystal structure of the octamer d(G-G-T-A-T-A-C-C).

The crystal structures of the synthetic self-complementary octamer d(G-G-T-A-T-A-C-C) and its 5-bromouracil-containing analogue have been refined to R values of 20% and 14% at resolutions of 1.8 and 2.25 A, respectively. The molecules adopt and A-DNA type double-helical conformation, which is minimally affected by crystal forces. A detailed analysis of the structure shows a considerable influence of the nucleotide sequence on the base-pair stacking patterns. In particular, the electrostatic stacking interactions between adjacent guanine and thymine bases produce symmetric bending of the double helix and a major-groove widening. The sugar-phosphate backbone appears to be only slightly affected by the base sequence. The local variations in the base-pair orientation are brought about by correlated adjustments in the backbone torsion angles and the glycosidic orientation. Sequence-dependent conformational variations of the type observed here may contribute to the specificity of certain protein-DNA interactions.

Self base pairing in a complementary deoxydinucleoside monophosphate duplex: crystal and molecular structure of deoxycytidylyl-(3'-5')-deoxyguanosine.

The molecular structure of ammonium deoxycytidylyl-(3'-5')-deoxyguanosine, crystallized from aqueous acetone near pH 4, was determined for X-ray diffraction data. The crystals were tetragonal, space group P43212 with a = b = 11.078 (1) A and c = 45.826 (4) A. The structure was solved by tangent expansion of phases based on a derived phosphorus position and refined to R = 0.060 by full matrix least squares. Molecules related by a 2-fold symmetry axis are connected by hydrogen bonds between the bases and form parallel right-handed duplexes. Pairs of cytosines share a proton at N(3) and are joined by three hydrogen bonds: N(4)-H...O(2)...H-N(4), and N(3)-H...N(3). Guanines are joined by two hydrogen bonds: N(2)-H...N(3) and N(3)...H-N(2). Base-stacking interactions within the duplex are weak with the cytosine and guanine ring planes inclined at 24 degrees to each other in each monomer. Despite the unusual arrangement of the molecules, the sugar phosphate backbone has the g-g- conformation normally associated with right-handed double helical structures. Conformational parameters of the nucleosides are also typical with both sugars C(2')-endo and glycosidic torsion angles 55 degrees for cytidine and 94 degrees for guanosine. The bonding geometry of the bases is influenced by hydrogen bonding and charge-transfer networks in the crystal lattice. The solvent molecules interact with the dimer in three fused circular hydrogen bonding domains with a single disordered ammonium cation per d(CpG) dimer. Parallels with the formation of self base pairs and their implications in molecular biology are discussed.