Ella Czarina Magat Juan

Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(N-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules

Oligonucleotides containing 5-(N-aminohexyl)carbamoyl-modified uracils have promising features for applications as antigene and antisense therapies. Relative to unmodified DNA, oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2′-deoxyuridine (NU) or 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine (NUm), respectively exhibit increased binding affinity for DNA and RNA, and enhanced nuclease resistance. To understand the structural implications of NU and NUm substitutions, we have determined the X-ray crystal structures of DNA:DNA duplexes containing either NU or NUm and of DNA:RNA hybrid duplexes containing NUm. The aminohexyl chains are fixed in the major groove through hydrogen bonds between the carbamoyl amino groups and the uracil O4 atoms. The terminal ammonium cations on these chains could interact with the phosphate oxygen anions of the residues in the target strands. These interactions partly account for the increased target binding affinity and nuclease resistance. In contrast to NU, NUm decreases DNA binding affinity. This could be explained by the drastic changes in sugar puckering and in the minor groove widths and hydration structures seen in the NUm containing DNA:DNA duplex structure. The conformation of NUm, however, is compatible with the preferred conformation in DNA:RNA hybrid duplexes. Furthermore, the ability of NUm to render the duplexes with altered minor grooves may increase nuclease resistance and elicit RNase H activity.

Insights into the DNA stabilizing contributions of a bicyclic cytosine analogue: crystal structures of DNA duplexes containing 7,8-dihydropyrido [2,3-d]pyrimidin-2-one

The incorporation of the bicyclic cytosine analogue 7,8-dihydropyrido[2,3-d]pyrimidin-2-one (X) into DNA duplexes results in a significant enhancement of their stability (3–4 K per modification). To establish the effects of X on the local hydrogen-bonding and base stacking interactions and the overall DNA conformation, and to obtain insights into the correlation between the structure and stability of X-containing DNA duplexes, the crystal structures of [d(CGCGAATT-X-GCG)]2 and [d(CGCGAAT-X-CGCG)]2 have been determined at 1.9–2.9 Å resolutions. In all of the structures, the analogue X base pairs with the purine bases on the opposite strands through Watson–Crick and/or wobble type hydrogen bonds. The additional ring of the X base is stacked on the thymine bases at the 5′-side and overall exhibits greatly enhanced stacking interactions suggesting that this is a major contribution to duplex stabilization.

The structures of pyruvate oxidase from Aerococcus viridans with cofactors and with a reaction intermediate reveal the flexibility of the active-site tunnel for catalysis.

The crystal structures of pyruvate oxidase from Aerococcus viridans (AvPOX) complexed with flavin adenine dinucleotide (FAD), with FAD and thiamine diphosphate (ThDP) and with FAD and the 2-acetyl-ThDP intermediate (AcThDP) have been determined at 1.6, 1.8 and 1.9 A resolution, respectively. Each subunit of the homotetrameric AvPOX enzyme consists of three domains, as observed in other ThDP-dependent enzymes. FAD is bound within one subunit in the elongated conformation and with the flavin moiety being planar in the oxidized form, while ThDP is bound in a conserved V-conformation at the subunit-subunit interface. The structures reveal flexible regions in the active-site tunnel which may undergo conformational changes to allow the entrance of the substrates and the exit of the reaction products. Of particular interest is the role of Lys478, the side chain of which may be bent or extended depending on the stage of catalysis. The structures also provide insight into the routes for electron transfer to FAD and the involvement of active-site residues in the catalysis of pyruvate to its products.

Structure of d-3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate d-3-hydroxybutyrate and NAD+

D-3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between D-3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate D-3-hydroxybutyrate and the cofactor NAD(+) at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD(+) and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of D-3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure.

Crystallographic study of wild-type carbonic anhydrase αCA1 from Chlamydomonas reinhardtii

Carbonic anhydrases (CAs) are ubiquitously distributed and are grouped into three structurally independent classes (alphaCA, betaCA and gammaCA). Most alphaCA enzymes are monomeric, but alphaCA1 from Chlamydomonas reinhardtii is a dimer that is uniquely stabilized by disulfide bonds. In addition, during maturation an internal peptide of 35 residues is removed and three asparagine residues are glycosylated. In order to obtain insight into the effects of these structural features on CA function, wild-type C. reinhardtii alphaCA1 has been crystallized in space group P6(5), with unit-cell parameters a=b=134.3, c=120.2 A. The crystal diffracted to 1.88 A resolution and a preliminary solution of its crystal structure has been obtained by the MAD method.

Crystallization and preliminary crystallographic studies of putative threonyl-tRNA synthetases from Aeropyrum pernix and Sulfolobus tokodaii

Threonyl-tRNA synthetase (ThrRS) plays an essential role in protein synthesis by catalyzing the aminoacylation of tRNA(Thr) and editing misacylation. ThrRS generally contains an N-terminal editing domain, a catalytic domain and an anticodon-binding domain. The sequences of the editing domain in ThrRSs from archaea differ from those in bacteria and eukaryotes. Furthermore, several creanarchaea including Aeropyrum pernix K1 and Sulfolobus tokodaii strain 7 contain two genes encoding either the catalytic or the editing domain of ThrRS. To reveal the structural basis for this evolutionary divergence, the two types of ThrRS from the crenarchaea A. pernix and S. tokodaii have been overexpressed in Eschericha coli, purified and crystallized by the hanging-drop vapour-diffusion method. Diffraction data were collected and the structure of a selenomethionine-labelled A. pernix type-1 ThrRS crystal has been solved using the MAD method.

Crystal structure of d(gcGXGAgc) with X=G: a mutation at X is possible to occur in a base-intercalated duplex for multiplex formation.

DNA fragments with the sequences d(gcGX[Y]n Agc) (n = 1, X = A, and Y = A, T, or G) form base-intercalated duplexes, which is a basic unit for formation of multiplexes such as octaplex and hexaplex. To examine the stability of multiplexes, a DNA with X = Y = G and n = 1 was crystallized under conditions different from those of the previously determined sequences, and its crystal structure has been determined. The two strands are coupled in an anti-parallel fashion to form a base-intercalated duplex, in which the first and second residues form Watson-Crick type G:C pairs and the third and sixth residues form a sheared G:A pairs at both ends of the duplex. The G4 and G5 bases are stacked alternatively on those of the counter strand to form a long G column of G3-G4-G5*-G5-G4*-G3*, the central four Gs being protruded. In addition, the three duplexes are associated to form a hexaplex around a mixture of calcium and sodium cations on the crystallographic threefold axis. These structural features are similar to those of the previous crystals, though slightly different in detail. The present study indicates that mutation at the 4th position is possible to occur in a base-intercalated duplex for multiplex formations, suggesting that DNA fragments with any sequence sandwiched between the two triplets gcG and Agc can form a multiplex.

Duplex with non-WC pairings: crystal structure of d(gcGAGGGAgc)

DNA fragments with the sequence d(gcGAGAgc) (G1) easily form a base-intercalated duplex, which is the basic unit for further association to form a quadruplex and an octaplex depending on potassium concentration [1]. In the octaplex, the eight G5 residues form two G-quartets through the direct N-H ...O and N-H ...N hydrogen bonds. Between, above and below the two G-quartets, potassium ions are bound to the O atoms of the G5 residues. To examine the stability of longer octaplexes, several G residues were added in the central part of G1 for the present study. Electrophoresis experiments have shown that as the number of G residues increases at the center, octaplex formation becomesmore stable. The DNA fragment d(gcGAGGGAgc) was crystallized in two forms: P212121 and P21. In the latter form obtained at higher cobalt-hexamine concentration, the two fragments in the asymmetric unit form a duplex, in which the two strands are aligned in an anti-parallel fashion. At both ends of the duplex, two Watson-Crick (WC) type G1:C10 and C2:G9 pairs are followed by a sheared-type G3:A8 pair. These parts are the same as those of the base-intercalated duplexes [2,3,4]. In the remaining part, however, the associationmode of the two strands is quite different from that of the base-intercalated duplex. Surprisingly, it is found that the subsequent A4 residue also forms a sheared-type pair with the G7 residue and that the central two G residues form G5:G6 pairs through the N-H ...O andN-H ...N hydrogen bonds. These pairingmodes comprise just half of the G-quartet. However, the alignment of the two phosphate backbones is anti-parallel, different from that of the octaplex, which is parallel. Two A:GxG:A crossings occur at both side of the central two G:G pairs. It could be concluded that the major part of the present duplex is formed by non-WC pairings. It is interesting to examine whether the central sequence d(GAGGGA) can form such a non-WCduplex without two WC pairs at both ends. Electrophoresis patterns on a native gel containing 20mM potassium show that all of d(GAG[G]nGA) (where n=1-4) form not only duplexes, but also multiplexes such as quadruplexes, octaplexes, and so on. Crystallizations of those multiplexes are in progress.

Crystal structures of DNA duplexes stabilized by bicyclic-C residues.

Chemical modification of nucleic acids is being studied extensively as an approach for the development of nucleic acid-based therapies. We found that a nucleotide carrying 7,8-dihydropyrido[2,3-d]pyrimidin-2-one (bicyclic-C or X), which is a cytosine derivative with a propene attached at the N4 and C5 atoms, increases the stability of DNA duplexes. To establish the conformational effects of X on DNA and to obtain insight into the correlation between the structure and stability of X-containing DNA duplexes, the crystal structures of [d(CGCGAATT-X-GCG)](2) and [d(CGCGAAT-X-CGCG)](2) have been determined at 2.9 A resolutions. In both duplexes, the bicyclic-C bases form pairs with the counter bases through hydrogen bonds, and stabilize the duplex formation in part by stacking interactions between X and the subsequent thymine base of the same strand.

Structural basis for antigene and antisense duplexes with modified nucleotides

C60 signals of the snRNPs. As an import adaptor snurportin1 bridges the interaction between the m3G-cap bearing snRNPs and the nuclear import receptor importin, which mediates the interaction with and translocation through the nuclear pore complex. Snurportin1 contains a N-terminal importin-binding (IBB) domain and a m3G-capbinding region, which shows no similarity to other known nuclear import factors. We have solved the crystal structure of the m3G-cap binding domain of snurportin1 by means of MIRAS, and the structure was refined at 2.4 Å resolution. The crystal structure reveals an unexpected binding mode for the m3G-cap, that significantly differs from other cap-binding proteins such as eIF4E and CBP20. The structural basis for the discrimation of mG-cap bearing RNAs by snurportin1 will be discussed.