Tomoko Sunami

Structures of d(GCGAAGC) and d(GCGAAAGC) (tetragonal form): a switching of partners of the sheared G·A pairs to form a functional G·A×A·G crossing

The DNA fragments d(GCGAAGC) and d(GCGAAAGC) are known to exhibit several extraordinary properties. Their crystal structures have been determined at 1.6 and 1.65 A resolution, respectively. Two heptamers aligned in an antiparallel fashion associate to form a duplex having molecular twofold symmetry. In the crystallographic asymmetric unit, there are three structurally identical duplexes. At both ends of each duplex, two Watson-Crick G.C pairs constitute the stem regions. In the central part, two sheared G.A pairs are crossed and stacked on each other, so that the stacked two guanine bases of the G.AxA.G crossing expose their Watson-Crick and major-groove sites into solvent, suggesting a functional role. The adenine moieties of the A(5) residues are inside the duplex, wedged between the A(4) and G(6) residues, but there are no partners for interactions. To close the open space on the counter strand, the duplex is strongly bent. In the asymmetric unit of the d(GCGAAAGC) crystal (tetragonal form), there is only one octamer chain. However, the two chains related by the crystallographic twofold symmetry associate to form an antiparallel duplex, similar to the base-intercalated duplex found in the hexagonal crystal form of the octamer. It is interesting to note that the significant difference between the present bulge-in structure of d(GCGAAGC) and the base-intercalated duplex of d(GCGAAAGC) can be ascribed to a switching of partners of the sheared G.A pairs.

Insights into the Structures of DNA Damaged by Hydroxyl Radical: Crystal Structures of DNA Duplexes Containing 5-Formyluracil

Hydroxyl radicals are potent mutagens that attack DNA to form various base and ribose derivatives. One of the major damaged thymine derivatives is 5-formyluracil (fU), which induces pyrimidine transition during replication. In order to establish the structural basis for such mutagenesis, the crystal structures of two kinds of DNA d(CGCGRATfUCGCG) with R = A/G have been determined by X-ray crystallography. The fU residues form a Watson-Crick-type pair with A and two types of pairs (wobble and reversed wobble) with G, the latter being a new type of base pair between ionized thymine base and guanine base. In silico structural modeling suggests that the DNA polymerase can accept the reversed wobble pair with G, as well as the Watson-Crick pair with A.

High-resolution crystal structure of copper amine oxidase from Arthrobacter globiformis: assignment of bound diatomic molecules as O2

The crystal structure of a copper amine oxidase from Arthrobacter globiformis was determined at 1.08 Å resolution with the use of low-molecular-weight polyethylene glycol (LMW PEG; average molecular weight ∼200) as a cryoprotectant. The final crystallographic R factor and Rfree were 13.0 and 15.0%, respectively. Several molecules of LMW PEG were found to occupy cavities in the protein interior, including the active site, which resulted in a marked reduction in the overall B factor and consequently led to a subatomic resolution structure for a relatively large protein with a monomer molecular weight of ∼70,000. About 40% of the presumed H atoms were observed as clear electron densities in the Fo - Fc difference map. Multiple minor conformers were also identified for many residues. Anisotropic displacement fluctuations were evaluated in the active site, which contains a post-translationally derived quinone cofactor and a Cu atom. Furthermore, diatomic molecules, most likely to be molecular oxygen, are bound to the protein, one of which is located in a region that had previously been proposed as an entry route for the dioxygen substrate from the central cavity of the dimer interface to the active site.

X-ray analyses of d(GCGAXAGC) containing G and T at X: the base-intercalated duplex is still stable even in point mutants at the fifth residue.

DNA fragments containing the sequence d(GCGAAAGC) prefer to adopt a base-intercalated (zipper-like) duplex in the crystalline state. To investigate effects of point mutation at the 5th residue on the structure, two crystal structures of d(GCGAGAGC) and d(GCGATAGC) have been determined by X-ray crystallography. In the respective crystals, the two octamers related by a crystallographic two-fold symmetry are aligned in an anti-parallel fashion and associated to each other to form a duplex, suggesting that the base-intercalated duplex is stable even when the 5th residue is mutated with other bases. The sheared G3:A6 pair formation makes the two phosphate backbones closer and facilitates formation of the A-X*-X-A* base-intercalated motif. The three duplexes are assembled around the three-fold axis, and their 3rd and 4th residues are bound to the hexamine cobalt chloride. The central 5th residues are bound to another cation.

Crystallization and preliminary X-ray analyses of the active and the inactive forms of family GH-8 chitosanase with subclass II specificity from Bacillus sp. strain K17

Chitosanase from Bacillus sp. strain K17 (ChoK) belongs to glycoside hydrolase family 8 and exhibits subclass II specificity. The purified protein is structurally stable over a wide pH range (3-10), but is active in a much narrower pH range (4.5-7.5), with optimal activity around pH 6.0. The protein has been successfully crystallized at two different pH values corresponding to the active and inactive states. The crystals diffract to 1.5 and 2.0 A resolution, respectively.

Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-Ray Crystallography

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%-75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.

DNA conformational transitions inferred from re-evaluation of m|Fo| − D|Fc| electron-density maps

A re-evaluation of m|F o| − D|F c| electron-density maps revealed that potential conformational transitions of 27% of DNA phosphates are found in previous crystallographic data. The analysis suggests that some of these unassigned densities correspond to ZI ↔ ZII or A/B → BI transitions.

The structure of a d(gcGAACgc) duplex containing two consecutive bulged A residues in both strands suggests a molecular switch

In previous studies, it was reported that DNA fragments with the sequence d(gcGXYAgc) (where X = A or G and Y = A, T or G) form a stable base-intercalated duplex (Bi-duplex) in which the central X and Y residues are not involved in any base-pair interactions but are alternately stacked on each other between the two strands. To investigate the structural stability of the Bi-duplex, the crystal structure of d(gcGAACgc) with a point mutation at the sixth residue of the sequence, d(gcGAAAgc), has been determined. The two strands are associated in an antiparallel fashion to form two types of bulge-containing duplexes (Bc-duplexes), I and II, both of which are quite different from the Bi-duplex of the parent sequence. In both Bc-duplexes, three Watson-Crick G.C base pairs constitute the stem regions at the two ends. The A(4) residues are bulged in to form a pair with the corresponding A(4) residue of the opposite strand in either duplex. The A(4).A(4)* pair formation is correlated to the orientations of the adjacent A(5) residues. A remarkable difference between the two Bc-duplexes is seen at the A(5) residue. In Bc-duplex I, it is flipped out and comes back to interact with the G(3) residue. In Bc-duplex II, the A(5) residue extends outwards to interact with the G(7) residue of the neighbouring Bc-duplex I. These results indicate that trans sugar-edge/Hoogsteen (sheared-type) G(3).A(6)* base pairs are essential in the formation of a Bi-duplex of d(gcGXYAgc). On the other hand, the alternative conformations of the internal loops containing two consecutive bulged A residues suggest molecular switching.

Crystal structures of d(CGAA) and d(CGAAGC): parallel-stranded duplexes and their dimer

We found the first example of CGAA forming a parallelstranded right-handed double helix in the X-ray structure of the DNA d(GCGAAAGCT) (N6.5) [1]. The remaining AGCT forms a normal anti-parallel duplex. The parallel part is formed through homo base-pair formations, C:C, G:G, A:A and A:A ( indicates hemi-protonation). To examine whether the sequence CGAA can form such a parallel duplex without the anti-parallel part, and to survey a possibility of longer parallel duplex formation, the crystal structures of d(CGAA) (T5.5 at pH5.5, T6 at pH6, T7.5 at pH7.5) and d(CGAAGC) (H7 at pH7) have been determined at 1.45, 1.2, 1.05 and 2.5 Å resolutions, respectively. In the T5.5 crystal, the asymmetric unit contains two parallel duplexes, which are formed through homo base-pair formations similar to those in the N6.5 crystal, and the two duplexes are stacked on each other to form an infinitely long column. These structural features are retained in the T6 crystal, though the unit cell dimensions are quite different. Surprisingly, the same type of parallel duplexes is also formed in the T7.5 crystal. This means that the parallel duplex is stabilized by accepting a proton in the central hole of the C:C pair even at neutral pH. Furthermore, the most unexpected finding in the H7 crystal is that the two parallel strands form a duplex between the d(CGAA) parts while the remaining d(CG) residues participate in forming an anti-parallel duplex so that the two parallel duplexes are associated to form a tetramer. In any of the parallel duplexes, the C’...C’ atomic distance between the paired nucleotides is shortest at G2:G2 and longest at A4:A4. Between them, A3:A3 has an intermediate distance. Assuming that G5:G5 in d(CGAAGC) also has the same as C ’...C’ distance as that of G2:G2, a distortion in the backbone conformation between the fourth and the fifth residues might arise. This distortion may have to be released in order to form a long parallel duplex that is comprised of homo-base pairs. For this, it may be necessary to insert one or two A residues as spacers.

X-ray structure of D(GCGAAAGCT), parallel-stranded DNA duplex with homo base pairs

X-RAY STRUCTURE OF D(GCGAAAGCT), PARALLEL-STRANDED DNA DUPLEX WITH HOMO BASE PAIRS T. Sunami 1 J. Kondo I. Hirao K. Watanabe 3 K. Miura A. Takenaka Tokyo Institute of Technology Graduate School of Bioscience and Biotechnology 4259 Nagatsuta, Midori-Ku YOKOHAMA 226-8501 JAPAN RIKEN GSC, Wako-shi, Saitama 351-0198, Japan Graduate School of Engineering, University of Tokyo, Tokyo 113-8656, Japan Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan