Crystal structures of the catalytic domain of human protein kinase associated with apoptosis and tumor suppression.

We have determined X-ray crystal structures with up to 1.5 A resolution of the catalytic domain of death-associated protein kinase (DAPK), the first described member of a novel family of pro-apoptotic and tumor-suppressive serine/threonine kinases. The geometry of the active site was studied in the apo form, in a complex with nonhydrolyzable AMPPnP and in a ternary complex consisting of kinase, AMPPnP and either Mg2+ or Mn2+. The structures revealed a previously undescribed water-mediated stabilization of the interaction between the lysine that is conserved in protein kinases and the beta- and gamma-phosphates of ATP, as well as conformational changes at the active site upon ion binding. Comparison between these structures and nucleotide triphosphate complexes of several other kinases disclosed a number of unique features of the DAPK catalytic domain, among which is a highly ordered basic loop in the N-terminal domain that may participate in enzyme regulation.

X-ray crystallographic observation of "in-line" and "adjacent" conformations in a bulged self-cleaving RNA/DNA hybrid.

The RNA strand in an RNA/DNA duplex with unpaired ribonucleotides can undergo self-cleavage at bulge sites in the presence of a variety of divalent metal ions (Hüsken et al., Biochemistry, 1996, 35:16591-16600). Transesterification proceeds via an in-line mechanism, with the 2'-OH of the bulged nucleotide attacking the 3'-adjacent phosphate group. The site-specificity of the reaction is most likely a consequence of the greater local conformational freedom of the RNA backbone in the bulge region. A standard A-form backbone geometry prohibits formation of an in-line arrangement between 2'-oxygen and phosphate. However, the backbone in the region of an unpaired nucleotide appears to be conducive to an in-line approach. Therefore, the bulge-mediated phosphoryl transfer reaction represents one of the simplest RNA self-cleavage systems. Here we focus on the conformational features of the RNA that underlie site-specific cleavage. The structures of an RNA/DNA duplex with single ribo-adenosyl bulges were analyzed in two crystal forms, permitting observation of 10 individual conformations of the RNA bulge moiety. The bulge geometries cover a range of relative arrangements between the 2'-oxygen of the bulged nucleotide and the P-O5' bond (including adjacent and near in-line) and give a detailed picture of the conformational changes necessary to line up the 2'-OH nucleophile and scissile bond. Although metal ions are of crucial importance in the catalysis of analogous cleavage reactions by ribozymes, it is clear that local strain or conformational flexibility in the RNA also affect cleavage selectivity and rate (Soukup & Breaker, RNA, 1999, 5:1308-1325). The geometries of the RNA bulges frozen out in the crystals provide snapshots along the reaction pathway prior to the transition state of the phosphoryl transfer reaction.

Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments.

The observation of light metal ions in nucleic acids crystals is generally a fortuitous event. Sodium ions in particular are notoriously difficult to detect because their X-ray scattering contributions are virtually identical to those of water and Na(+.)O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules. We demonstrate here that replacement of Na(+) by K(+), Rb(+) or Cs(+) and precise measurements of anomalous differences in intensities provide a particularly sensitive method for detecting alkali metal ion-binding sites in nucleic acid crystals. Not only can alkali metal ions be readily located in such structures, but the presence of Rb(+) or Cs(+) also allows structure determination by the single wavelength anomalous diffraction technique. Besides allowing identification of high occupancy binding sites, the combination of high resolution and anomalous diffraction data established here can also pinpoint binding sites that feature only partial occupancy. Conversely, high resolution of the data alone does not necessarily allow differentiation between water and partially ordered metal ions, as demonstrated with the crystal structure of a DNA duplex determined to a resolution of 0.6 A.

Crystal structure of a conformation-selective casein kinase-1 inhibitor.

Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.

The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding.

The yrdC family of genes codes for proteins that occur both independently and as a domain in proteins that have been implicated in regulation. An example for the latter case is the sua5 gene from yeast. SuaS was identified as a suppressor of a translation initiation defect in cytochrome c and is required for normal growth in yeast (Na JG, Pinto I, Hampsey M, 1992, Genetics 11:791-801). However, the function of the Sua5 protein remains unknown; Sua5 could act either at the transcriptional or the posttranscriptional levels to compensate for an aberrant translation start codon in the cyc gene. To potentially learn more about the function of YrdC and proteins featuring this domain, the crystal structure of the YrdC protein from Escherichia coli was determined at a resolution of 2.0 A. YrdC adopts a new fold with no obvious similarity to those of other proteins with known three-dimensional (3D) structure. The protein features a large concave surface on one side that exhibits a positive electrostatic potential. The dimensions of this depression, its curvature, and the fact that conserved basic amino acids are located at its floor suggest that YrdC may be a nucleic acid binding protein. An investigation of YrdC's binding affinities for single- and double-stranded RNA and DNA fragments as well as tRNAs demonstrates that YrdC binds preferentially to double-stranded RNA. Our work provides evidence that 3D structures of functionally uncharacterized gene products with unique sequences can yield novel folds and functional insights.

Structural origins of the exonuclease resistance of a zwitterionic RNA.

Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2'-O-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1 degrees C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3' ends competitively inhibit the degradation of single-stranded DNA by the Escherichia coli Klenow fragment (KF) 3'-5' exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-A resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 A and compared with the structure of the complex between oligo(dT) and the D355A/E357A KF mutant. The structural models suggest that interference of the positively charged 2'-O-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.

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.

Crystal structure and improved antisense properties of 2'-O-(2-methoxyethyl)-RNA.

2'-O-(2-Methoxyethyl)-RNA (MOE-RNA) is a nucleic acid analog with promising features for antisense applications. Compared with phosphorothioate DNA (PS-DNA), the MOE modification offers improved nuclease resistance, enhanced RNA affinity, improved cellular uptake and intestinal absorption, reduced toxicity and immune stimulation. The crystal structure of a fully modified MOE-RNA dodecamer duplex (CGCGAAUUCGCG) was determined at 1.7 A resolution. In the majority of the MOE substituents, the torsion angle around the ethylene alkyl chain assumes a gauche conformation. The conformational preorganization of the MOE groups is consistent with the improved RNA affinity and the extensive hydration of the substituents could play a role in the improved cellular uptake of MOE-RNA. A specific hydration pattern that bridges substituent and phosphate oxygen atoms in the minor groove of MOE-RNA may explain its high nuclease resistance.

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.

Correlating structure and stability of DNA duplexes with incorporated 2'-O-modified RNA analogues.

Chemically modified nucleic acids are currently being evaluated as potential antisense compounds for therapeutic applications. 2'-O-Ethylene glycol substituted oligoribonucleotides are second-generation antisense inhibitors of gene expression with promising features for in vivo use. Relative to DNA, they display improved RNA affinity and higher nuclease resistance. Moreover, chimeric oligonucleotides with 2'-O-methoxyethyl ribonucleoside wings and a central DNA phosphorothioate window have been shown to effectively reduce the growth of tumors in animal models at low doses. Using X-ray crystallography, we have determined the structures of three A-form DNA duplexes containing the following 2'-O-modified ribothymidine building blocks: 2'-O-methoxyethyl ribo-T, 2'-O-methyl[tri(oxyethyl)] ribo-T, and 2'-O-ethoxymethylene ribo-T. In contrast to 2'-O-ethylene glycol substituents, the presence of a 2'-O-ethoxymethylene group leads to slightly reduced RNA affinity of the corresponding oligonucleotides. The three structures allow a qualitative rationalization of the differing stabilities of duplexes between oligonucleotides comprising these types of 2'-O-modified ribonucleotides and complementary RNAs. The stabilizing 2'-O-ethylene glycol substituents are conformationally preorganized for the duplex state. Thus, the presence of one or several ethylene glycol moieties may reduce the conformational space of the substituents in an oligonucleotide single strand. In addition, most of these preferred conformations appear to be compatible with the minor groove topology in an A-type duplex. Factors that contribute to the conformational rigidity of the 2'-O-substituents are anomeric and gauche effects, electrostatic interactions between backbone and substituent, and bound water molecules.

Crystal structures of B-DNA with incorporated 2'-deoxy-2'-fluoro-arabino-furanosyl thymines: implications of conformational preorganization for duplex stability.

The fundamental conformational states of right-handed double helical DNA, the A- and B-forms, are associated with distinct puckers of the sugar moieties. The furanose conformation itself is affected by the steric and electronic nature of the ring substituents. For example, a strongly electronegative substituent at the C2' position, such as in the 2'-deoxy-2'-fluoro ribo furanosyl analogue, will drive the conformational equilibrium towards the C3'- endo type (north). Conversely, the 2'-deoxy-2'-fluoro arabino furanosyl modification with opposite stereochemistry at C2' appears to have a preference for a C2'- endo type pucker (south). Incorporation of 2'-fluoroarabinofuranosyl thymines was previously shown to enhance the thermodynamic stability of B-DNA duplexes. We have determined the crystal structures of the B-DNA dodecamer duplexes [d(CGCGAASSCGCG)]2and [d(CGCGAASTCGCG)]2with incorporated 2'-deoxy-2'-fluoroarabinofuranosyl thymines S (south) at 1.55 A resolution. In the crystal structures, all S residues adopt an O4'- endo conformation (east), well compatible with an overall B-form duplex geometry. In addition to the increased rigidity of S nucleosides, a clathrate-like ordered water structure around the 2'-fluorines may account for the observed larger thermodynamic stability of DNA duplexes containing 2'-deoxy-2'-fluoroarabino thymidines.

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.

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.

Structure of the B-DNA oligomers d(CGCTAGCG) and d(CGCTCTAGAGCG) in new crystal forms.

We present the structure of the dodecamer CGCTCTAGAGCG and the related octamer CGCTAGCG, both in the B form, determined by single crystal X-ray diffraction. Two different crystal forms of the octamer have been obtained, with either three or four duplexes in the asymmetric unit. The dodecamer crystallizes in the P2(1) space group with two duplexes in the asymmetric unit. Very few such structures have been previously reported, while the octamer structure is the first one determined with three duplexes in the asymmetric unit. It is also the first octamer with standard Watson-Crick base pairs to be crystallized in the B form. The crystal structure is stabilized in both cases by interactions between the guanines in the two terminal base pairs of each duplex. This interaction is similar to that found in most dodecamers which have been previously studied, but here it is found in a new unit cell (for the dodecamer) and in one octamer. In the dodecamer cytosine-stacking interactions between neighbor duplexes are also present. The two dodecamer duplexes in the asymmetric unit show different patterns of bending, while the octamer molecule has a rather straight helical axis. The results presented confirm the strong conformational variability of the TA pyrimidine-purine step and demonstrate a clear alternating structure for the (CT/GA)n sequence.

Incorporation of diacids into the polyglycine II structure: model studies.

Aliphatic diacids are often incorporated into polypeptide structures in order to obtain model compounds for hormones, protein turns, etc. They are also fundamental components of many commercial polyamides. On the other hand glycine, the simplest amino acid, shows unique conformational features. In order to better understand the structure of such compounds, we have synthetized and determined the molecular structure of three models represented by the general formula CH3-CH2-CH2-NH-CO-CH2-NH-CO-(CH2)n-2-CO-NH-CH2-CO-NH-CH2-CH2-CH3, with n = 3, 4, or 6. Conformational differences have been found in the dicarboxylic moiety, whereas glycine always has the polyglycine II conformation. The -CO-(CH2)n-2-CO-segment adopts a folded conformation: SS, TGT, and SGTGS for n = 3, 4, and 6, respectively. Molecular packing is always pseudohexagonal and a network of hydrogen bonds oriented in three directions at 120 degrees is formed. The results are of interest in order to provide information about polyamides in which glycine residues are incorporated. Our results confirm the tendency of glycine residues to adopt the polyglycine II conformation in its copolymers with aliphatic compounds.

[New packing of B-DNA in crystals]. / Novaia upakovka B-DNK v kristallakh.

Two crystal forms of the self-complementary tetramer GpGpCpC have been obtained by phase diagram technique: P6(2)22/P6(4)22. a = b = 67.7 A, c = 105.6 A and P3(2)12/P3(1)12, a = b = 116.9 A. c = 116.4 A. Both crystals form diffract at least up to 3.2 A. Diffraction patterns of both crystal forms have strongest base-stacking reflections corresponding to the Bragg spacing 3.38 A which is typical for B-DNA. Moreover the self-rotation function of the first crystal form shows regular located two-fold pseudo-axes periodicity of which also indicates that this is B-conformation. The same conclusion can be reached on the basis of the crystal packing of the duplexes in the unit cell. It should be emphasized that this is a new example of B-DNA crystal packing.

Different forms of the double helix in the pCpGpCpGpCpG crystals.

Four different crystal forms of the self-complementary oligonucleotide pCpGpCpGpCpG have been obtained. The space groups and the unit cell parameters of these crystals, as well as the location of the base-stacking reflections, indicate that the oligonucleotide is able to assume different double helical conformations that are dependent on the crystallization conditions.

[Use of phase diagrams in crystallization of oligonucleotide duplexes. II. Setting of the crystallized samples]. / Ispol'zovanie fazovykh diagramm pri kristallizatsii oligonukleotidnykhdupleksov. II. Postanovka kristallizatsionnykh prob.

Oligonucleotide crystallization technique based on the method of phase diagrams is described in detail with (pGpT)3.(pApC)3 hexamer as an example. The key point of the technique consists of dividing the multiparameter crystallization space into a set of regions, each of which corresponds to the precipitation of a duplex in complex with a certain number of counterions.

Phase diagrams for DNA crystallization systems.

Phase diagrams for several oligonucleotide duplex-spermine systems have been constructed. These diagrams characterize the duplex and spermine concentrations ranges in which crystalline precipitates are formed. All of them are wedge-like form. The slope of the upper branch of the diagram is determined by the oligonucleotide length. The position of the lower branch depends on both the nucleotide sequence and its length. The position of the lower branch depends on both the nucleotide sequence and its length. It has been shown that the addition to the system of MgCl2 and NaCl salts and MPD results in specific changes in the diagrams. A model for oligonucleotide duplex-spermine system has been suggested which explains the main characteristic features of the obtained phase diagrams. The experimental phase diagrams for the (pGpT)n (pApC)n-spermine system (n = 2,3,4) have been analyzed ion terms of this model and the values of the binding constants of spermine and Mg2+ ions binding to duplexes have been determined. It permitted to identify the complexes that precipitated in different regions of the phase diagrams under various conditions. The diagram obtained in the presence of a cobalt hexammine counterion is also considered. It has been shown that this phase diagram, in general, is similar to those obtained for the oligonucleotide duplex-spermine system.