Crystal structures of Scone: pseudosymmetric folding of a symmetric designer protein.

Recent years have seen an increase in the development of computational proteins, including symmetric ones. A ninefold-symmetric ß-propeller protein named Cake has recently been developed. Here, attempts were made to further engineer this protein into a threefold-symmetric nine-bladed propeller using computational design. Two nine-bladed propeller proteins were designed, named Scone-E and Scone-R. Crystallography, however, revealed the structure of both designs to adopt an eightfold conformation with distorted termini, leading to a pseudo-symmetric protein. One of the proteins could only be crystallized upon the addition of a polyoxometalate, highlighting the usefulness of these molecules as crystallization additives.

Parallel and antiparallel (G.GC)2 triple helix fragments in a crystal structure.

Nucleic acid triplexes are formed by sequence-specific interactions between single-stranded polynucleotides and the double helix. These triplexes are implicated in genetic recombination in vivo and have application to areas that include genome analysis and antigene therapy. Despite the importance of the triple helix, only limited high-resolution structural information is available. The x-ray crystal structure of the oligonucleotide d(GGCCAATTGG) is described; it was designed to contain the d(G middle dotGC)2 fragment and thus provide the basic repeat unit of a DNA triple helix. Parameters derived from this crystal structure have made it possible to construct models of both parallel and antiparallel triple helices.

DNA-drug interactions. The crystal structures of d(TGTACA) and d(TGATCA) complexed with daunomycin.

The anticancer drug daunomycin has been co-crystallized with the hexanucleotide duplex sequences d(TGTACA) and d(TGATCA) and single crystal X-ray diffraction studies of these two complexes have been carried out. Structure solution of the d(TGTACA) and d(TGATCA) complexes to 1.6 and 1.7 Angstrom resolution, respectively, shows two daunomycin molecules bound to the DNA hexamer. Binding occurs via intercalation of the drug chromophore at the d(TpG) step, and hydrogen bonding interactions involving the drug, DNA and solvent molecules. The daunomycin sugar is located in the minor groove of the DNA hexamer and is stabilized by hydrogen bonds between the amino group of the sugar and functional groups on the floor of the groove. The amino sugar of the d(TGATCA) duplex interacts directly with the DNA sequence, while in the d(TGTACA) duplex, the interaction is via solvent molecules. Two other complexes d(CGTACG)-daunomycin and d(CGATCG)-daunomycin have previously been structurally characterized. Comparison of the four structures with daunomycin bound to the triplet sequences 5'TGT, 5'TGA, 5'CGT and 5'CGA reveals changes in the conformation of both the DNA hexamer and the daunomycin upon complexation, as well as the hydrogen bonding and van der Waals' interactions.

Direct observation of two base-pairing modes of a cytosine-thymine analogue with guanine in a DNA Z-form duplex: significance for base analogue mutagenesis.

The pyrimidine nucleobase analogue 6H,8H-3,4-dihydropyrimido[4,5-c]- [1,2]oxazin-7-one (P) is a mimic both of cytosine and thymine, since it can form stable hydrogen-bonded base-pairs with either guanine or adenine. To investigate the geometric properties of pairing with guanine in a DNA double helix, the structure of d(CGCGPG)2 has been determined by single crystal X-ray analysis. The oligonucleotide crystallised as a left-handed Z-DNA duplex in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 18.23 A, b = 30.63 A, c = 43.78 A. Refinement using NUCLSQ with 51 water molecules included in the final model converged at R = 0.179 (Rw = 0.159) for 2798 reflections (F > 2 sigma (F)) in the range 8 A to 1.7 A. Remarkably, the two P.G pairs in the hexamer duplex are different: Watson-Crick and wobble types separately illustrate both cytosine-like and thymine-like behaviour. The result suggests that mutagenesis experiments involving P and other analogues which display pronounced base-pairing ambivalence can be used to examine the structural basis of substrate discrimination by polymerases that is essential to accurate genetic replication.

Molecular and crystal structure of d(CGCGmo4CG): N4-methoxycytosine.guanine base-pairs in Z-DNA.

The base analogue N4-methoxycytosine (mo4C) is ambivalent in its hydrogen-bonding potential, since it forms stable base-pairs with both adenine and guanine in oligomer duplexes. To investigate the base-pair geometry, the structure of d(CGCGmo4CG) has been determined by single-crystal X-ray diffraction techniques. The d(CGCGmo4CG)2 crystallized in a left-handed double helical structure (Z-type). Refinement using 2559 reflections between 10 and 1.7 A converged with a final R = 0.181 (Rw = 0.130) including 68 solvent molecules. The orthorhombic crystals are in the space group P2(1)2(1)2(1), with cell dimensions a = 18.17 A, b = 30.36 A, c = 43.93 A. The mo4C.G base-pair is of the wobble type, with mo4C in the imino form, and the methoxy group in the syn configuration.

A thymine-like base analogue forms wobble pairs with adenine in a Z-DNA duplex.

The DNA hexamer d(CACGPG), in which dP is the ambivalent pyrimidine nucleoside analogue 2'-deoxy-beta-d-ribofuranosyl-(6H,8H-3, 4-dihydropyrimido[4,5-c][1,2]oxazin-7-one), crystallises as a left-handed Z-DNA duplex. X-ray analysis at 1.5 A shows that both P. A base-pairs are of the wobble type. This result appears inconsistent with other evidence from hybridisation and NMR studies of P-containing oligonucleotides, which suggests that, while P can form stable base-pairs with either A or G, thymine-like properties are more pronounced. Thermal denaturation experiments over a range of solution pH values indicate that protonation of the P.A base-pairs is unlikely to be responsible for the anomalous behaviour. No specific crystal packing effects can be identfied as an explanation, and it is concluded that base stacking and other interactions between nucleotide residues in Z-DNA are responsible.

Synthesis, biological evaluation, and structure analysis of a series of new 1,5-anhydrohexitol nucleosides.

In view of the selective anti-HSV activity of 1,5-anhydro-2,3-dideoxy-2- (5-iodouracil-1-yl)-D-arabino-hexitol, a series of novel 1,5-anhydrohexitol nucleosides were synthesized and evaluated for their inhibitory activity against several viruses. The 5-iodouracil 3 and the 5-ethyluracil 4 derivatives are highly selective TK-dependent inhibitors of HSV-1 and HSV-2. Broad anti-herpes virus activity was noticed for 5-fluorocytosine 6 and 2,6-diaminopurine 10 analogues. From a transport study of 3, using the thymidine influx competition method, one can conclude that intracellular uptake of this compound most probably occurs by passive diffusion. X-ray analysis of compounds 3 and 9 showed that the heterocyclic base of 1,5-anhydrohexitol pyrimidine and purine is placed in the axial position and that the sugar ring adopts a slightly distorted chair conformation.

Asymmetric synthesis of alpha,beta-dialkyl-alpha-phenylalanines via direct alkylation of a chiral alanine derivative with racemic alpha-alkylbenzyl bromides. A case of high enantiomer differentiation at room temperature.

[figure: see text] This study demonstrates that the direct alkylation of a Ni(II)-complex of the chiral Schiff base of alanine with (S)-o-[N-(N-benzylprolyl)amino]- benzophenone, with racemic alpha-alkylbenzyl bromides, is a synthetically feasible and methodologically advantageous approach to the target alpha,beta-dialkylphenylalanines over previously reported methods. For the first time we report and rationalize a case of a high enantiomer differentiation process at room temperature.

Experimental and computational growth morphology of two polymorphs of a yellow isoxazolone dye.

We report on the crystal structures and the experimentally found and the computationally predicted growth morphologies of two polymorphs of a yellow isoxazolone dye. The stable polymorph I has a blocklike habit, and the metastable polymorph II grows as fine needles, nucleating only by heterogeneous or contact nucleation. The habits of both polymorphs depend on the supersaturation during growth. The experimental observations are compared with predictions of the attachment energy model and kinetic Monte Carlo lattice simulations in which the growth is modeled as an "atomistic process", governed by surface energetics. These Monte Carlo simulations correctly predict the shape and the dependence on supersaturation of the crystal morphology for both polymorphs: for polymorph I, a strong dependence on supersaturation is found from the simulations. For polymorph II, the order of morphological importance is reproduced correctly, as well as the needlelike morphology.

B-DNA at atomic resolution reveals extended hydration patterns.

Despite the importance of hydration around DNA in the understanding of its conformation and interactions with other molecules in many biological processes, only limited atomic resolution information is available. Crystal-engineering techniques, which were originally developed to mimic DNA base triplets in a crystal lattice, also eliminate the rotational disorder of oligonucleotides around their helical axis and thereby enhance the resolution of the structure analysis. We have determined the low-temperature crystal structure of the synthetic DNA decamer d(GGCCAATTGG) at atomic resolution (1. 15 A) using 17700 reflections and have characterized the highly organized hydration patterns in both grooves. The narrow d(AATT) minor groove is occupied by an 'extended hydration spine' alternately bridging base pairs and phosphate O1P atoms of opposite strands, while a distinctive pattern of parallel water ribbons is observed in the major groove. This analysis provides structural insight into the correlation found between narrow minor-groove width and occurrence of the B(I) conformation and can be used to design new minor-groove binders. By their location between adjacent helices, two fully hydrated magnesium ions further stabilize the crystal packing. The structure also provides details of the hydration and conformation of G.GC triple helices.

Crystal structure of d(GGCCAATTGG) complexed with DAPI reveals novel binding mode.

The single-crystal X-ray structure of the complex between the minor groove binder 4',6-diamidino-2-phenylindole (DAPI) and d(GGCCAATTGG) reveals a novel way of off-centered binding, with an unique hydrogen bond between the minor groove binder and a CG base pair. Application of crystal engineering and cryocooling techniques helped to extend the resolution to 1.9 A, resulting in an unambiguous determination of drug conformation and orientation. The structure was refined to completion using SHELXL-93, resulting in a residual factor R of 18. 0% for 3562 reflections with F(o) > 4sigma(F(o)) including 81 water molecules. As the bulky NH(2)-group on guanine is believed to prevent drug binding in the minor groove, the nature and stability of the CG-DAPI contact was further addressed in full detail using ab initio quantum chemical methods. The amino groups involved in the guanine-drug interaction are substantially nonplanar, resulting in an energy gain of about 5 kcal/mol. The combined structural and theoretical data suggest that the guanine NH(2)-group does not destabilize the drug binding to an extent that it prevents complexation.

Rational design of highly diastereoselective, organic base-catalyzed, room-temperature Michael addition reactions.

Via the rational design of a single-preferred transition state, stabilized by electron donor-acceptor-type attractive interactions, structural and geometric requirements for the corresponding starting compounds have been determined. The Ni(II) complex of the Schiff base of glycine with o-[N-alpha-picolylamino]acetophenone, as a nucleophilic glycine equivalent, and N-(trans-enoyl)oxazolidin-2-ones, as derivatives of an alpha,beta-unsaturated carboxylic acid, were found to be the substrates of choice featuring geometric/conformational homogeneity and high reactivity. The corresponding Michael addition reactions were found to proceed at room temperature in the presence of catalytic amounts of DBU to afford quantitatively the addition products with virtually complete diastereoselectivity. Acidic decomposition of the products followed by treatment of the reaction mixture with NH4OH gave rise to the diastereomerically pure 3-substituted pyroglutamic acids.

Formation of (C.G)*G triplets in a B-DNA duplex with overhanging bases.

Crystallization of a DNA double helix with overhanging bases at the 5'-ends of both strands, results in the formation of two crystallographically independent (C.G)*G triplets. In a previous report [Van Meervelt, Vlieghe, Dautant, Gallois, Précigoux & Kennard (1995). Nature (London), 374, 742-744] the unique molecular packing of the duplex and the Hoogsteen hydrogen-bond pattern and parallel backbone orientation of the guanine-containing strands in the triplets was described. The fine structural details and hydration of the d(GCGAATTCG) crystal structure refined to 2.05 A (R = 0.168, 86 water molecules, two Mg(2+) cations) are now presented. Helical parameters, stacking effects, the geometry at the duplex-triplex junction, and the hydration of the minor groove are discussed and compared with related theoretical and crystal structures.

High-resolution structure of a DNA helix forming (C.G)*G base triplets.

Triple helices result from interaction between single- and double-stranded nucleic acids. Their formation is a possible mechanism for recombination of homologous gene sequences in nature and provides, inter alia, a basis for artificial control of gene activity. Triple-helix motifs have been extensively studied by a variety of techniques, but few high-resolution structural data are available. The only triplet structures characterized so far by X-ray diffraction were in protein-DNA complexes studied at about 3 A resolution. We report here the X-ray analysis of a DNA nonamer, d(GCGAATTCG), to a resolution of 2.05 A, in which the extended crystal structure contains (C.G)*G triplets as a fragment of triple helix. The guanosine-containing chains are in a parallel orientation. This arrangement is a necessary feature of models for homologous recombination which results ultimately in replacement of one length of DNA by another of similar sequence. The present-structure agrees with many published predictions of triplex organization, and provides an accurate representation of an element that allows sequence-specific association between single- and double-stranded nucleic acids.

Oligonucleotides with 1,5-anhydrohexitol nucleoside building blocks: crystallization and preliminary X-ray studies of h(GTGTACAC).

Hexitol nucleic acids are oligonucleotides built up from natural nucleobases and a phosphorylated 1,5-anhydrohexitol backbone. The anhydrohexitol oligonucleotide h(GTGTACAC) was synthesized using phosphoramidite chemistry and standard protecting groups. Crystals of h(GTGTACAC) were obtained at either 279 or 289 K by the hanging-drop vapour-diffusion technique using a 24-matrix screen for nucleic acid fragments. The crystals diffract beyond 2.0 A resolution and belong to the hexagonal space group P6222 (or P6422) with unit-cell parameters a = 36.42 and c = 63.33 A.

DNA-drug refinement: a comparison of the programs NUCLSQ, PROLSQ, SHELXL93 and X-PLOR, using the low-temperature d(TGATCA)-nogalamycin structure.

In an earlier study [Smith, Davies, Dodson & Moore (1995). Biochemistry, 34, 415-425] the crystal structure of the d(TGATCA)-nogalamycin complex was determined to 1.8 A and refined with PROLSQ to R = 19.5% against 4767 reflections with F> 1sigma(F). A low-temperature crystallographic study on this complex has now been performed. Native data collection at liquid-nitrogen temperature (120 K) improved the resolution to 1.4 A. The structure has now been refined against these new diffraction data in the resolution range 8-1.4 A using NUCLSQ, PROLSQ, SHELXL93 and X-PLOR, in order to determine to what extent the resulting DNA conformation and associated solvent structure would differ and to examine the suitability of these programs for the refinement of oligonucleotide structures. With the advent of more DNA-protein structure determinations, it is of interest to see how well the protein-refinement packages, PROLSQ and X-PLOR, and the small-molecule program, SHELXL93, are able to accommodate DNA. Comparisons are made between the dictionaries, weights and restraints used and the final models obtained from each program. Although the final R values, using all data in the resolution range 8.0-1.4 A, from PROLSQ (22.8%), SHELXL93 (R1 =21.7% after isotropic refinement) and X-PLOR (24.4%) are higher than the R value from the NUCLSQ refinement (21.2%), the root-mean-square deviations between the four final models are very small. Using this high-quality 8.0-1.4 A data set neither the dictionary nor the refinement program leave an imprint on the final fully refined complex. Likewise, the helical parameters and backbone conformation including sugar-puckering modes are not influenced by the refinement procedure used. Although a different number of water molecules is found in each refinement, varying from 62 (X-PLOR) to 86 (NUCLSQ), the first hydration sphere is well conserved in all four models.

1,1,3-Trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazine (TTD) derivatives: a new class of nonnucleoside human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitors with anti-HIV-1 activity.

We report the development of a new group of nonnucleoside reverse transcriptase inhibitors (NNRTIs). One of the most active congeners of this series of 1,1,3-trioxo-2H,4H-thieno[3,4-e] [1,2,4]thiadiazine (TTD) derivatives, i.e., 2-(3-fluorobenzyl)-4-cyanomethylen-l,1,3-trioxo-2H,4H- thieno [3,4-e] [1,2,4] thiadiazine) (QM96639) was found to inhibit human immunodeficiency virus (HIV) type 1 [HIV-1 (IIIB)] replication in MT-4 cells at a concentration of 0.09 microM. This compound was toxic for the host cells only at a 1,400-fold higher concentration. The TTD derivatives proved effective against a variety of HIV-1 strains, including those that are resistant to 3'-azido-3'-deoxythymidine (AZT), but not against HIV-2 (ROD) or simian immunodeficiency virus (SIV/ MAC251). HIV-1 strains containing the L100I, K103N, V106A, E138K, Y181C, or Y188H mutations in their reverse transcriptase (RT) displayed reduced sensitivity to the compounds. Their cross-resistance patterns correlated with that of nevirapine. 2-Benzyl-4-cyanomethylen-1,1,3-trioxo-2H,4H-thieno[3,4-e] [1,2,4]thiadiazine (QM96521) enhanced the anti-HIV-1 activity of AZT and didanosine in a subsynergistic manner. HIV-1-resistant virus containing the V179D mutation in the RT was selected after approximately six passages of HIV-1 (IIIB) in CEM cells in the presence of different concentrations of QM96521. From structure-activity relationship analysis of a wide variety of TTD derivatives, a number of restrictions appeared as to the chemical modifications that were compatible with anti-HIV activity. Modelling studies suggest that in contrast to most other NNRTIs, but akin to nevirapine, QM96521 does not act as a hydrogen bond donor in the RT-drug complex.