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Toru Nakatsu

Publications and source records attributed to Toru Nakatsu.

12 recordsLinked to original sources
PubMed2005

Crystal structures of pyruvate phosphate dikinase from maize revealed an alternative conformation in the swiveling-domain motion.

Pyruvate phosphate dikinase (PPDK) reversibly catalyzes the conversion of ATP, phosphate, and pyruvate into AMP, pyrophosphate, and phosphoenolpyruvate (PEP), respectively. Since the nucleotide binding site (in the N-terminal domain) and the pyruvate/PEP binding site (in the C-terminal domain) are separated by approximately 45 A, it has been proposed that an intermediary domain, called the central domain, swivels between these remote domains to transfer the phosphate. However, no direct structural evidence for the swiveling central domain has been found. In this study, the crystal structures of maize PPDK with and without PEP have been determined at 2.3 A resolution. These structures revealed that the central domain is located near the pyruvate/PEP binding C-terminal domain, in contrast to the PPDK from Clostridium symbiosum, wherein the central domain is located near the nucleotide-binding N-terminal domain. Structural comparisons between the maize and C. symbiosum PPDKs demonstrated that the swiveling motion of the central domain consists of a rotation of at least 92 degrees and a translation of 0.5 A. By comparing the maize PPDK structures with and without PEP, we have elucidated the mode of binding of PEP to the C-terminal domain and the induced conformational changes in the central domain.

Amino Acid Sequence↗
PubMed2004

Crystal structure of gamma-glutamylcysteine synthetase: insights into the mechanism of catalysis by a key enzyme for glutathione homeostasis.

Gamma-glutamylcysteine synthetase (gammaGCS), a rate-limiting enzyme in glutathione biosynthesis, plays a central role in glutathione homeostasis and is a target for development of potential therapeutic agents against parasites and cancer. We have determined the crystal structures of Escherichia coli gammaGCS unliganded and complexed with a sulfoximine-based transition-state analog inhibitor at resolutions of 2.5 and 2.1 A, respectively. In the crystal structure of the complex, the bound inhibitor is phosphorylated at the sulfoximido nitrogen and is coordinated to three Mg2+ ions. The cysteine-binding site was identified; it is formed inductively at the transition state. In the unliganded structure, an open space exists around the representative cysteine-binding site and is probably responsible for the competitive binding of glutathione. Upon inhibitor binding, the side chains of Tyr-241 and Tyr-300 turn, forming a hydrogen-bonding triad with the carboxyl group of the inhibitor's cysteine moiety, allowing this moiety to fit tightly into the cysteine-binding site with concomitant accommodation of its side chain into a shallow pocket. This movement is caused by a conformational change of a switch loop (residues 240-249). Based on this crystal structure, the cysteine-binding sites of mammalian and parasitic gammaGCSs were predicted by multiple sequence alignment, although no significant sequence identity exists between the E. coli gammaGCS and its eukaryotic homologues. The identification of this cysteine-binding site provides important information for the rational design of novel gammaGCS inhibitors.

Amino Acid Sequence↗
PubMed2004

Crystal structure of the C-terminal clock-oscillator domain of the cyanobacterial KaiA protein.

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria, and KaiA activates kaiBC expression whereas KaiC represses it. Here we show that KaiA is composed of three functional domains, the N-terminal amplitude-amplifier domain, the central period-adjuster domain and the C-terminal clock-oscillator domain. The C-terminal domain is responsible for dimer formation, binding to KaiC, enhancing KaiC phosphorylation and generating the circadian oscillations. The X-ray crystal structure at a resolution of 1.8 A of the C-terminal clock-oscillator domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 shows that residue His270, located at the center of a KaiA dimer concavity, is essential to KaiA function. KaiA binding to KaiC probably occurs via the concave surface. On the basis of the structure, we predict the structural roles of the residues that affect circadian oscillations.

Amino Acid Sequence↗
PubMed2004

Crystal structure of the PsbP protein of photosystem II from Nicotiana tabacum.

PsbP is a membrane-extrinsic subunit of the water-oxidizing complex photosystem II (PS II). The evolutionary origin of PsbP has long been a mystery because it specifically exists in higher plants and green algae but not in cyanobacteria. We report here the crystal structure of PsbP from Nicotiana tabacum at a resolution of 1.6 A. Its structure is mainly composed of beta-sheet, and is not similar to any structures in cyanobacterial PS II. However, the electrostatic surface potential of PsbP is similar to that of cyanobacterial PsbV (cyt c(550)), which has a function similar to PsbP. A structural homology search with the DALI algorithm indicated that the folding of PsbP is very similar to that of Mog1p, a regulatory protein for the nuclear transport of Ran GTPase. The structure of PsbP provides insight into its novel function in GTP-regulated metabolism in PS II.

Amino Acid Sequence↗
PubMed2004

Crystal structure of a myristoylated CAP-23/NAP-22 N-terminal domain complexed with Ca2+/calmodulin.

A variety of viral and signal transduction proteins are known to be myristoylated. Although the role of myristoylation in protein-lipid interaction is well established, the involvement of myristoylation in protein-protein interactions is less well understood. CAP-23/NAP-22 is a brain-specific protein kinase C substrate protein that is involved in axon regeneration. Although the protein lacks any canonical calmodulin (CaM)-binding domain, it binds CaM with high affinity. The binding of CAP-23/NAP-22 to CaM is myristoylation dependent and the N-terminal myristoyl group is directly involved in the protein-protein interaction. Here we show the crystal structure of Ca2+-CaM bound to a myristoylated peptide corresponding to the N-terminal domain of CAP-23/NAP-22. The myristoyl moiety of the peptide goes through a hydrophobic tunnel created by the hydrophobic pockets in the N- and C-terminal domains of CaM. In addition to the myristoyl group, several amino-acid residues in the peptide are important for CaM binding. This is a novel mode of binding and is very different from the mechanism of binding in other CaM-target complexes.

Calcium↗
PubMed2003

Crystallization and preliminary crystallographic studies on the extrinsic 23 kDa protein in the oxygen-evolving complex of photosystem II.

The extrinsic 23 kDa protein in the oxygen-evolving complex (OEC23 or PsbP) of photosystem II (PS II) helps to retain calcium and chloride ions, which are essential cofactors for the photosynthetic evolution of oxygen. OEC23 is found in the PS II complex from higher plants and green algae, but not in that from cyanobacteria or other ancestral photosynthetic organisms. In order to elucidate the three-dimensional structure and its molecular evolution, OEC23 was crystallized by the hanging-drop vapour-diffusion technique with PEG 4000 as a precipitant. Diffraction data to a resolution of 2.0 A were collected from a native crystal belonging to space group P2(1)2(1)2, with unit-cell parameters a = 91.36, b = 74.15, c = 52.16 A.

Crystallization↗
PubMed2003

Capturing enzyme structure prior to reaction initiation: tropinone reductase-II-substrate complexes.

To understand the catalytic mechanism of an enzyme, it is crucial to determine the crystallographic structures corresponding to the individual reaction steps. Here, we report two crystal structures of enzyme-substrate complexes prior to reaction initiation: tropinone reductase-II (TR-II)-NADPH and TR-II-NADPH-tropinone complexes, determined from the identical crystals. A combination of two kinetic crystallographic techniques, a continuous flow of the substrates and Laue diffraction measurements, enabled us to capture the transit structures prior to the reaction proceeding. A structure comparison of the enzyme-substrate complex elucidated in this study with the enzyme-product complex in our previous study indicates that one of the substrates, tropinone, is rotated relative to the product so as to make the spatial organization in the active site favorable for the reaction to proceed. Side chains of the residues in the active site also alter their conformations to keep the complementarity of the space for the substrate or the product and to assist the rotational movement.

Alcohol Oxidoreductases↗
PubMed2003

Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+-calmodulin.

The calmodulin-binding domain of myristoylated alanine-rich C kinase substrate (MARCKS), which interacts with various targets including calmodulin, actin and membrane lipids, has been suggested to function as a crosstalk point among several signal transduction pathways. We present here the crystal structure at 2 A resolution of a peptide consisting of the MARCKS calmodulin (CaM)-binding domain in complex with Ca2+-CaM. The domain assumes a flexible conformation, and the hydrophobic pocket of the calmodulin N-lobe, which is a common CaM-binding site observed in previously resolved Ca2+-CaM-target peptide complexes, is not involved in the interaction. The present structure presents a novel target-recognition mode of calmodulin and provides insight into the structural basis of the flexible interaction module of MARCKS.

Amino Acid Sequence↗
PubMed2002

Active-site architecture of endopolygalacturonase I from Stereum purpureum revealed by crystal structures in native and ligand-bound forms at atomic resolution.

Crystal structures of endopolygalacturonase from Stereum purpureum were solved in native and two galacturonic acid complex states at atomic resolution. Endopolygalacturonase catalyzes the hydrolysis of alpha-1,4-glycosidic linkage of polygalacturonate in pectin. The native structure was determined by the multiple wavelength anomalous dispersion method and was refined anisotropically with SHELXL-97, with an R factor of 11.4% and an R(free) factor of 14.0% at 0.96 A resolution. The enzyme folds into a right-handed parallel beta-helix with 10 complete turns. The crystal structures of its binary complex with one D-galacturonate and its ternary complex with two D-galacturonates were also determined to identify the substrate binding site at 1.0 and 1.15 A resolutions, respectively. In the binary complex, one beta-D-galactopyranuronate was found in the +1 subsite, thus proving the strong affinity of the +1 subsite expected from the bond cleavage frequency on oligogalacturonates. In the ternary complex, an additional beta-D-galactofuranuronate was found in the -1 subsite. In both subsites, the recognition of the galacturonate carboxy group is important in galacturonate binding. In the +1 subsite, the carboxy group interacts with three basic residues, His195, Arg226, and Lys228, which were conserved in all endopolygalacturonases. In the -1 subsite, the unique nonprolyl cis-peptide bond is believed to be involved in binding the carboxy group of the substrate. The active site architecture of the complexes provides insight into the mechanism of inverting glycosyl hydrolases and also sheds light on the basis of the differences between the family 28 and the other inverting glycosyl hydrolases.

Amino Acid Sequence↗
PubMed2002

Crystal structure of the Homer 1 family conserved region reveals the interaction between the EVH1 domain and own proline-rich motif.

PSD-Zip45 (also named Homer 1c/Vesl-1L) is a synaptic scaffolding protein, which interacts with neurotransmitter receptors and other scaffolding proteins to target them into post-synaptic density (PSD), a specialized protein complex at the synaptic junction. Binding of the PSD-Zip45 to the receptors and scaffolding proteins results in colocalization and clustering of its binding partners in PSD. It has an Ena/VASP homology 1 (EVH1) domain in the N terminus for receptor binding, two leucine zipper motifs in the C terminus for clustering, and a linking region whose function is unclear despite the high level of conservation within the Homer 1 family. The X-ray crystallographic analysis of the largest fragment of residues 1-163, including an EVH1 domain reported here, demonstrates that the EVH1 domain contains an alpha-helix longer than that of the previous models, and that the linking part included in the conserved region of Homer 1 (CRH1) of the PSD-Zip45 interacts with the EVH1 domain of the neighbour CRH1 molecule in the crystal. The results suggest that the EVH1 domain recognizes the PPXXF motif found in the binding partners, and the SPLTP sequence (P-motif) in the linking region of the CRH1. The two types of binding are partly overlapped in the EVH1 domain, implying a mechanism to regulate multimerization of Homer 1 family proteins.

Amino Acid Motifs↗
PubMed2002

Escherichia coli B gamma-glutamylcysteine synthetase: modification, purification, crystallization and preliminary crystallographic analysis.

Escherichia coli B gamma-glutamylcysteine synthetase (gammaGCS) catalyzes the ATP-dependent coupling of L-Glu and L-Cys to form the glutathione precursor gamma-L-Glu-Cys and is a target for development of potential therapeutic agents. By introducing four point mutations of surface-exposed cysteine residues to serine, the gammaGCS was purified to homogeneity; single crystals have been obtained using the hanging-drop vapour-diffusion method with sodium formate. The gammaGCS crystal diffracted to 2.8 A and belongs to space group R3, with unit-cell parameters a = b = 326.7, c = 103.9 A.

Crystallization↗