M. Tsunoda

Water mediated Dickerson-Drew-type crystal of DNA dodecamer containing 2′-deoxy-5-formyluridine

To investigate the role of divalent cations in crystal packing, a Dickerson-Drew-type dodecamer with the sequence d(CGCGAATXCGCG), containing 2-deoxy-5-formyluridine at X, was crystallized under several conditions with Ba(2+) ion instead of Mg(2+) ion. The crystal structure is isomorphous with the original Dickerson-type crystal containing Mg(2+) ion. In the Mg(2+)-free crystals, however, a five-membered ring of water molecules occupies the same position as the magnesium site found in the Mg(2+)-containing crystals, and connects the two duplexes similarly to the hydrated Mg(2+) ion. It has been concluded that the five-membered water molecules can take the place of the hydrated magnesium cation in crystallization. The 5-formyluracil residues form the canonical Watson-Crick pair with the opposite adenine residues.

Crystallization and preliminary X‐ray analysis of a DNA dodecamer containing 2′‐deoxy‐5‐formyluridine; what is the role of magnesium cation in crystallization of Dickerson‐type DNA dodecamers?

To investigate the role of divalent cations in crystal packing, four different crystals of a Dickerson-type dodecamer with the sequence d(CGCGAATXCGCG), containing 2-deoxy-5-formyluridine at X, were obtained under several conditions with and without divalent cations. The crystal structures are all isomorphous. The octahedrally hydrated magnesium cations found in the major groove cement the two neighbouring duplexes along the b axis. In the Mg(2+)-free crystals, a five-membered ring of water molecules occupies the same position as the magnesium site and connects the two duplexes similarly to the hydrated Mg(2+) ion. It has been concluded that water molecules can take the place of the hydrated magnesium cation in crystallization, but the magnesium cation is more effective and gives X-ray diffraction at slightly higher resolution. In all four crystals, the 5-formyluracil residues form the canonical Watson-Crick pair with adenine residues.

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 (3–4u2009K 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.

Structures of DNA duplexes containing O6-carboxymethylguanine, a lesion associated with gastrointestinal cancer, reveal a mechanism for inducing pyrimidine transition mutations

N-nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O6-carboxymethylguanine (O6-CMG) in DNA. O6-CMG has been identified in DNA derived from human colon tissue, and its occurrence has been linked to diets high in red and processed meats. By analogy to O6-methylguanine, O6-CMG is expected to be highly mutagenic, inducing G to A mutations during DNA replication that can increase the risk of gastrointestinal and other cancers. Two crystal structures of DNA dodecamers d(CGCG[O6-CMG]ATTCGCG) and d(CGC[O6-CMG]AATTCGCG) in complex with Hoechst33258 reveal that each can form a self-complementary duplex to retain the B-form conformation. Electron density maps clearly show that O6-CMG forms a Watson–Crick–type pair with thymine similar to the canonical A:T pair, and it forms a reversed wobble pair with cytosine. In situ structural modeling suggests that a DNA polymerase can accept the Watson–Crick–type pair of O6-CMG with thymine, but might also accept the reversed wobble pair of O6-CMG with cytosine. Thus, O6-CMG would permit the mis-incorporation of dTTP during DNA replication. Alternatively, the triphosphate that would be formed by carboxymethylation of the nucleotide triphosphate pool d[O6-CMG]TP might compete with dATP incorporation opposite thymine in a DNA template.

Crystallization and preliminary X-ray analysis of the full-size cubic core of pig 2-oxoglutarate dehydrogenase complex

The full-length (untruncated) dihydrolipoamide succinyltransferase from pig heart was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction patterns indicate that the crystal belongs to space group I432, with unit-cell parameter a = 189.9 A. The crystal structure has been preliminarily solved at 7 A resolution by the molecular-replacement method. The unit cell contains two cubic cores, in each of which 24 subunits of E2 are associated according to crystallographic 432 symmetry. At the corners of each cubic core, the catalytic domains of E2s form a trimer through tight interactions around the crystallographic threefold axes. In the electron-density maps, many small broad peaks are observed in regions expected to contain the remaining N-terminal domains (the E1/E3-binding domain and the lipoyl domain), suggesting flexibility of these domains relative to the core. The architecture of the cubic core is similar to that of the other truncated E2s. In the unit cell, however, the core-core contact occurs in a different direction from that found for the truncated proteins.

Crystal structures of RNA 3'-terminal phosphate cyclase and its complexes with Mg2+ +ATP, ATP or Mn2+.

RNA 3-terminal phosphate cyclase (Rtc) is an enzyme related to RNA splicing, in which the 3-terminal hydroxyl group of a truncated RNA is converted to the 2,3-cyclic phosphate that is required prior to RNA ligation. This reaction may occur in the following two steps: (i) Rtc + ATP --> Rtc-AMP + Ppi and (ii) RNA-N3 + Rtc-AMP --> RNA-N>p + Rtc + AMP. In order to establish the reaction mechanism, Rtc of Sulfolobus tokodaii, overexpressed in E. coli, was crystallized in the following states, Rtc, Rtc-AMP, Rtc:AMP, Rtc:ATP and Rtc:Mn, and their crystal structures have been determined at 2.25, 2.25, 2.9, 2.4 and 3.2 A resolutions, respectively. Based on these structures, a possible reaction mechanism has been proposed.

The substrate recognition and the catalytic reaction mechanisms of D-3-hydroxybutyrate dehydrogenase

is grown on acetate, IDH is in its inactive phosphorylated form, thus inhibiting Krebs’ cycle. Alternatively, a change of carbon source to glucose or pyruvate results in the activation of IDH by dephosphorylation, and the initiation of Krebs’ cycle. The function of AceK and its involvement in the regulation of Krebs’ cycle and the glyoxylate bypass is well-characterized, but its structural and mechanistic qualities have remained relatively unknown. The determination of the crystal structure could provide confirmation of the function of AceK by identifying the various kinase, phosphatase and ATPase domains and insights into the coordination of the kinase and phosphatase activity of AceK. Of note, it is currently unknown how, or if at all, the active site changes conformation as it switches between kinase and phosphatase activity. Three Acek crystal forms were obtained and SAD datasets were collected at CHESS and BNL synchrotron source. The AceK structure is determined at 2.6 Å. The overall AceK structure displays a typical eukaryotic kinase folding.

Molecular basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms

The spliceosome is a highly dynamic macromolecular machinery that undergoes several structural and compositional rearrangements during its assembly and disassembly, respectively, its activation, as well as during the pre-mRNA splicing reaction. All these steps proceed in a highly ordered and strictly controlled manner with most of them driven by ATP. At least eight different ATPases, which all belong to the family of DExD/H-box helicases, are specifically involved in defined steps of the splicing cycle. These DExD/H-box proteins are thought not only to unwind dsRNA, but they might also act as RNPase disrupting protein-RNA complexes, which is an important process to achieve splicing and regeneration of the sliceosome. Besides the conserved helicase (DExD/H) core domain, the spliceosomal DExD/H-box proteins contain additional Nor C-terminal domains, which are important for the assembly and interaction with other proteins of the spliceosome. However, molecular details regarding their exact function and specificity are mostly unknown. We have recently determined the crystal structure of the catalytic domain of hPrp28 showing that the two halves of the catalytic domain are displaced with respect to each other and therefore no productive catalytic centre is formed. In contrast to other DExD/H-proteins the N-terminal half of the catalytic domain doesn’t bind ATP, raising the question on interacting and activating partners in the spliceosome. Additionaly we have solved the first structure of a helicase-associated C-terminal domain that is found in the spliceosomal DEAD/H-box proteins Prp22, Prp2, Prp16, and Prp43. Interestingly, this domain was predicted to consist of two domains, but the crystal structure clearly demonstrates that it folds into one functional domain.