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Biomedical subjects

Takuji Oyama

Publications and source records attributed to Takuji Oyama.

10 recordsLinked to original sources
PubMed2005

Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis.

Ring-shaped sliding clamps and clamp loader ATPases are essential factors for rapid and accurate DNA replication. The clamp ring is opened and resealed at the primer-template junctions by the ATP-fueled clamp loader function. The processivity of the DNA polymerase is conferred by its attachment to the clamp loaded onto the DNA. In eukarya and archaea, the replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) play crucial roles as the clamp loader and the clamp, respectively. Here, we report the electron microscopic structure of an archaeal RFC-PCNA-DNA complex at 12-A resolution. This complex exhibits excellent fitting of each atomic structure of RFC, PCNA, and the primed DNA. The PCNA ring retains an open conformation by extensive interactions with RFC, with a distorted spring washer-like conformation. The complex appears to represent the intermediate, where the PCNA ring is kept open before ATP hydrolysis by RFC.

DNA Replication↗
PubMed2005

Detection of subunit interfacial modifications by tracing the evolution of clamp-loader complex.

The archaeal and eukaryal clamp-loader and clamp proteins were investigated with the evolutionary trace method. The molecular phylogeny of the proteins suggested that the hetero-pentameric complex of the archaeal clamp-loader with two subunits (RFCL and RFCS) was not a preserved ancestral type, but a degenerated version of the eukaryal complex of five subunits (RFC1-5). The evolutionary trace of amino acid replacements during the course of subunit differentiation revealed that the replacements had accumulated preferentially at the subunit interface regions. Some of the interfacial modifications that might be responsible for the specific interaction between the subunits were conserved in the current complex.

Archaeal Proteins↗
PubMed2005

Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to OxLDL.

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.

Amino Acid Sequence↗
PubMed2004

Domain architectures and characterization of an RNA-binding protein, TLS.

Translocated in liposarcoma (TLS) is an important protein component of the heterogeneous nuclear ribonucleoprotein complex involved in the splicing of pre-mRNA and the export of fully processed mRNA to the cytoplasm. We examined the domain organization of human TLS by a combined approach using limited proteolysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, circular dichroism, inductively coupled plasma atomic emission spectroscopy, and NMR spectroscopy. We found that the RNA recognition motif (RRM) and zinc finger-like domains exclusively form protease-resistant core structures within the isolated TLS protein fragments, while the remaining regions, including the Arg-Gly-Gly repeats, appear to be completely unstructured. Thus, TLS contains the unstructured N-terminal half followed by the RRM and zinc finger-like domains, which are connected to each other by a flexible linker. We also carried out NMR analyses to obtain more detailed insights into the individual RRM and zinc finger-like domains. The 113Cd NMR analysis of the zinc finger-like domain verified that zinc is coordinated with four cysteines in the C4 type scheme. We also investigated the interaction of each domain with an oligo-RNA containing the GGUG sequence, which appears to be critical for the TLS function in splicing. The backbone amide NMR chemical shift perturbation analyses indicated that the zinc finger domain binds GGUG-containing RNA with a dissociation constant of about 1.0 x 10(-5) m, whereas the RRM domain showed no observable interaction with this RNA. This surprising result implies that the zinc finger domain plays a more predominant role in RNA recognition than the RRM domain.

Amino Acid Sequence↗
PubMed2004

Structural basis for channelling mechanism of a fatty acid beta-oxidation multienzyme complex.

The atomic view of the active site coupling termed channelling is a major subject in molecular biology. We have determined two distinct crystal structures of the bacterial multienzyme complex that catalyzes the last three sequential reactions in the fatty acid beta-oxidation cycle. The alpha2beta2 heterotetrameric structure shows the uneven ring architecture, where all the catalytic centers of 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT) face a large inner solvent region. The substrate, anchored through the 3'-phosphate ADP moiety, allows the fatty acid tail to pivot from the ECH to HACD active sites, and finally to the KACT active site. Coupling with striking domain rearrangements, the incorporation of the tail into the KACT cavity and the relocation of 3'-phosphate ADP bring the reactive C2-C3 bond to the correct position for cleavage. The alpha-helical linker specific for the multienzyme contributes to the pivoting center formation and the substrate transfer through its deformation. This channelling mechanism could be applied to other beta-oxidation multienzymes, as revealed from the homology model of the human mitochondrial trifunctional enzyme complex.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2004

The clamp-loading complex for processive DNA replication.

DNA polymerase requires two processing factors, sliding clamps and clamp loaders, to direct rapid and accurate duplication of genomic DNA. In eukaryotes, proliferating cell nuclear antigen (PCNA), the ring-shaped sliding clamp, encircles double-stranded DNA within its central hole and tethers the DNA polymerases onto DNA. Replication factor C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands in an ATP-dependent manner. Here we report the three-dimensional structure of an archaeal clamp-loading complex (RFC-PCNA-DNA) determined by single-particle EM. The three-dimensional structure of the complex, reconstituted in vitro using a nonhydrolyzable ATP analog, reveals two components, a closed ring and a horseshoe-shaped element, which correspond to PCNA and RFC, respectively. The atomic structure of PCNA fits well into the closed ring, suggesting that this ternary complex represents a state just after the PCNA ring has closed to encircle the DNA duplex.

Adenosine Triphosphate↗
PubMed2003

Mutational analysis of Pyrococcus furiosus replication factor C based on the three-dimensional structure.

In eukaryotic DNA replication, replication factor C (RFC) acts as a "clamp loader" that loads PCNA onto a primed DNA template in an ATP-dependent manner. Proteins with functions essentially identical to that of RFC exist in Archaea. We have determined the crystal structure of the small subunit (RFCS) of Pyrococcus furiosus RFC at 2.8-A resolution. Using the information from the determined tertiary structure, we prepared several mutations in RFCS and biochemically characterized them. Truncation of the C-terminal alpha-helix (alpha16) causes a failure in RFCS oligomerization and a loss of the stimulating activity for the PCNA-dependent DNA synthesis by DNA polymerases. The site-directed reduction of the negative charges at the center part of the RFCS complex affected the stability of the RFC-PCNA interaction and reduced the clamp-loading activity. These results contribute to our general understanding of the structure-function relationship of the RFC molecule for the clamp-loading event.

Adenosine Triphosphatases↗
PubMed2003

Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents.

The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.

Affinity Labels↗
PubMed2002

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery.

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.

Adenosine Triphosphatases↗