Biomedical subjects

Mikako Shirouzu

Publications and source records attributed to Mikako Shirouzu.

100 records · Page 6Linked to original sources

PubMed2003

Crystal structure of the conserved protein TT1542 from Thermus thermophilus HB8.

The TT1542 protein from Thermus thermophilus HB8 is annotated as a conserved hypothetical protein, and belongs to the DUF158 family in the Pfam database. A BLAST search revealed that homologs of TT1542 are present in a wide range of organisms. The TT1542 homologs in eukaryotes, PIG-L in mammals, and GPI12 in yeast and protozoa, have N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-N-acetylase activity. Although most of the homologs in prokaryotes are hypothetical and have no known function, Rv1082 and Rv1170 from Mycobacterium tuberculosis are enzymes involved in the mycothiol detoxification pathway. Here we report the crystal structure of the TT1542 protein at 2.0 A resolution, which represents the first structure for this superfamily of proteins. The structure of the TT1542 monomer consists of a twisted beta-sheet composed of six parallel beta-strands and one antiparallel beta-strand (with the strand order 3-2-1-4-5-7-6) sandwiched between six alpha-helices. The N-terminal five beta-strands and four alpha-helices form an incomplete Rossmann fold-like structure. The structure shares some similarity to the sugar-processing enzymes with Rossmann fold-like domains, especially those of the GPGTF (glycogen phosphorylase/glycosyl transferase) superfamily, and also to the NAD(P)-binding Rossmann fold domains. TT1542 is a homohexamer in the crystal and in solution, the six monomers forming a cylindrical structure. Putative active sites are suggested by the structure and conserved amino acid residues.

Amino Acid Sequence↗

PubMed2003

Crystal structure of the Pyrococcus horikoshii DNA primase-UTP complex: implications for the mechanism of primer synthesis.

BACKGROUND: In chromosomal DNA replication, DNA primase initiates the synthesis of a dinucleotide on a single-stranded template DNA, and elongates it to form a primer RNA for the replicative DNA polymerase. Although the apo-structure of an archaeal primase has been reported, the mechanism of primer synthesis by the eukaryotic-type primase still remains to be elucidated. RESULTS: In this study, we present the crystal structure of the eukaryotic-type DNA primase from the hyperthermophilic archaeon (Pyrococcus horikoshii) with the uridine 5'-triphosphate (UTP). In the present primase-UTP complex, the primase binds the triphosphate moiety of the UTP at the active site, which includes Asp95, Asp97, and Asp280, the essential residues for the nucleotidyl transfer reaction. CONCLUSION: The nucleotide binding geometry in this complex explains the previous biochemical analyses of the eukaryotic primase. Based on the complex structure, we constructed a model between the DNA primase and a primer/template DNA for the primer synthesis. This model facilitates the comprehension of the reported features of DNA primase.

Amino Acid Sequence↗

PubMed2002

Structure and catalytic mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase, an enzyme in the non-mevalonate pathway of isoprenoid synthesis.

Precursors for isoprenoid synthesis are essential in all organisms. These compounds are synthesized by one of two known routes: the well characterized mevalonate pathway or a recently discovered non-mevalonate route which is used in many bacteria and human pathogens. Since the second pathway is both vital and unlike any found in humans, enzymes catalysing reactions along this synthetic route are possible drug targets. The structure of one such enzyme from the thermophilic bacterium Thermus thermophilus has been solved to high resolution in the presence of substrate and with a substrate analogue. Enzyme co-crystallized with substrate shows only one product, cytosine monophosphate (CMP), in the active site. At the high resolution of the refinement (1.6 A) the positions and coordination of the magnesium ions in the active site are clearly seen.

Amino Acid Sequence↗

PubMed2002

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells.

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.

Animals↗

PubMed2002

The Cdc42 binding and scaffolding activities of the fission yeast adaptor protein Scd2.

The small GTP-binding protein Cdc42, the guanine nucleotide exchange factor Scd1, the p21-activated kinase Shk1, and the adaptor protein Scd2 are involved in the Cdc42-dependent signaling cascade in fission yeast. In the present study, we analyzed the Cdc42 binding and scaffolding activities of Scd2 by co-precipitation assays. We found that two SH3-containing regions, amino acid residues 1-87 (CB1 (Cdc42-binding region 1)) and 110-266 (CB2), of Scd2 can bind to the GTP-bound form of Cdc42. CB2 is cryptic because of the intramolecular binding between the SH3 domain in CB2 (SH3(C)) and the PX domain and binds to Cdc42 only when the Scd2 PB1 domain binds to the PC motif-containing region (residues 760-872) of Scd1. This CB2.Cdc42 association, which would stabilize the open configuration of Scd2, enables the SH3(C) domain to bind to the polyproline motif of Shk1. We also found that the GTP-bound form of Cdc42 binds to the CRIB motif of Shk1 more strongly than to Scd2. Thus, Scd2 functions as a scaffold to form a protein complex, and the GTP-bound Cdc42 might be transferred effectively from the upstream activator Scd1 to the downstream effector Shk1 via Scd2.

Binding Sites↗

PubMed2002

Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains.

Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extracellular region, comprising domains I-IV, with the resultant dimerization of the receptor tyrosine kinase. In this study, the crystal structure of a 2:2 complex of human EGF and the EGFR extracellular region has been determined at 3.3 A resolution. EGFR domains I-III are arranged in a C shape, and EGF is docked between domains I and III. The 1:1 EGF*EGFR complex dimerizes through a direct receptor*receptor interaction, in which a protruding beta-hairpin arm of each domain II holds the body of the other. The unique "receptor-mediated dimerization" was verified by EGFR mutagenesis.

Amino Acid Sequence↗

PubMed2002

An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system.

Tyrosyl-tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-L-tyrosine rather than L-tyrosine for the site-specific incorporation of 3-iodo-L-tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-L-tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the L-tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo-L-tyrosine 10-fold more efficiently than L-tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNA(Tyr) was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo-l-tyrosine. After the use of the 3-iodotyrosyl-tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of L-tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-L-tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.

Amino Acid Substitution↗

PubMed2002

An enzyme with a deep trefoil knot for the active-site architecture.

Knots in polypeptide chains have been found in very few proteins. Only two proteins are considered to have a shallow 'trefoil' knot, which tucks a few residues at one end of the chain through a loop exposed on the protein surface. Recently, another protein was found by a mathematical algorithm to have a deep 'figure-of-eight' knot which had not been visually identified. In the present study, the crystal structure of a hypothetical RNA 2'-O-ribose methyltransferase from Thermus thermophilus (RrmA) was determined at 2.4 A resolution and a deep trefoil knot was found for the first time. The present knot is formed by the threading of a 44-residue polypeptide chain through a 41-residue loop and is better defined than the previously reported knots. Two of the three catalytic residues conserved in the 2'-O-ribose methyltransferase family are located in the knotting loop and in the knotted carboxy-terminal chain, which is the first observation that the enzyme active site is constructed right on the knot. On the other hand, the amino-terminal domain exhibits a geometrical similarity to the ribosomal proteins which recognize an internal loop of RNA.

Algorithms↗

PubMed2002

Increased Ras expression and caspase-independent neuroblastoma cell death: possible mechanism of spontaneous neuroblastoma regression.

BACKGROUND: Neuroblastoma undergoes spontaneous regression frequently during its natural course. Although programmed cell death (PCD) has been implicated in this process, accumulating evidence suggests that apoptosis, a form of PCD that is regulated by caspases, may not play a major role. We examined the mechanism(s) of spontaneous regression of neuroblastoma, focusing on the role of Ras, a favorable prognostic marker of neuroblastoma. METHODS: Tumor tissues were analyzed by light microscopy, electron microscopy, and immunohistochemistry to examine cell degeneration and expression of Ras and several indicators of PCD. Cell degeneration was also studied in vitro in neuroblastoma cells transfected with the H-ras gene. All statistical tests were two-sided. RESULTS: Immunohistochemical analyses revealed that Ras expression was increased in areas of cellular degeneration lacking apoptotic characteristics. The degenerating cells were fragmented without nuclear condensation and, essentially, lacked caspase-3 activation and apoptotic DNA fragmentation. These cells had ultrastructural features of autophagic degeneration, another form of PCD that is distinct from apoptosis. Focal areas of degeneration associated with Ras expression were seen more frequently in tumors from patients detected in a mass-screening program (53 [60.9%] of 87) than in tumors from clinically detected, advanced-stage patients over 1 year of age (7 [29.2%] of 24) (P =.006; chi-square test), suggesting a positive relationship between Ras-associated degeneration and probability of spontaneous regression/favorable prognosis. The characteristic features of Ras-associated nonapoptotic degeneration observed in tumor samples were recapitulated in vitro by transfection-mediated Ras expression, and Ras-mediated degeneration was augmented by TrkA, another favorable prognostic marker. CONCLUSIONS: High-level expression of H-Ras in neuroblastoma cells is associated with caspase cascade-independent, nonapoptotic PCD. This Ras-mediated nonapoptotic tumor cell death may play a key role in spontaneous regression of neuroblastoma.

Apoptosis↗

PubMed2002

Anti-(Raf-1) RNA aptamers that inhibit Ras-induced Raf-1 activation.

RNA aptamers with affinity for the Ras-binding domain (RBD) of Raf-1 were isolated from a degenerate pool by in vitro selection. These aptamers efficiently inhibited the Ras interaction with the Raf-1 RBD, and also inhibited Ras-induced Raf-1 activation in a cell-free system. The RNA aptamer with the most potent inhibitory effect specifically inhibited the Ras-Raf-1 interaction and had no affinity for the RBD of the RGL protein, a homolog of the Ral GDP dissociation stimulator. Although the aptamer was capable of binding to the B-Raf RBD, the RNA did not inhibit the interaction between Ras and the B-Raf RBD. Enzymatic and chemical probing experiments indicated that the aptamer was folded into a pseudoknot structure, and some loop regions of the pseudoknot were located at the binding interface for the Raf-1 RBD.

Base Sequence↗