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D Kohda

Publications and source records attributed to D Kohda.

At least 19 recordsLinked to original sources
PubMed2001

The archaeal DNA primase: biochemical characterization of the p41-p46 complex from Pyrococcus furiosus.

We characterized the primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus. The two proteins, Pfup41 and Pfup46, have similar sequences to the p48 and p58 subunits, respectively, of the eukaryotic DNA polymerase alpha-primase complex. Unlike previously reported primases, the Pfup41 preferentially utilizes deoxyribonucleotides for its de novo synthesis, and moreover, it synthesizes up to several kilobases in length in a template-dependent manner (Bocquier, A., Liu, L., Cann, I., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol. 11, 452-456). The p41-p46 complex showed higher DNA binding activity than the catalytic p41 subunit alone. In addition, the amount of DNA synthesized by the p41-p46 complex was much more abundant and shorter in length than that by Pfup41 alone. The activity for RNA primer synthesis, which was not detected with Pfup41, was observed from the reaction using the p41-p46 complex in vitro. The in vitro replication of M13 single-stranded DNA by the P. furiosus proteins was stimulated by ATP. Observation of the labeled primers by using [gamma-(32)P]ATP in the substrates suggests ATP as the preferable initiating nucleotide for the p41-p46 complex. These results show that the primer synthesis activity of Pfup41 is regulated by Pfup46, and the p41-p46 complex may function as the primase in the DNA replication machinery of P. furiosus, in a similar fashion to the eukaryotic polymerase alpha-primase complex.

Adenosine Triphosphate↗
PubMed2001

The PX domain as a novel phosphoinositide- binding module.

The phox (phagocyte oxidase) homology (PX) domain occurs in the mammalian phox proteins p40(phox) and p47(phox), the polarity establishment protein Bem1p in budding yeast, and a variety of proteins involved in membrane trafficking. Here we show that the PX domains of p40(phox) and p47(phox) directly bind to phosphoinositides: p40(phox) prefers Ptdlns(3)P, while p47(phox) does Ptdlns(4)P and Ptdlns(3,4)P(2). In addition, the Bem1p PX domain also interacts with Ptdlns(4)P. When the p40(phox) PX domain is expressed as a fusion to green fluorescent protein in HeLa cells, it exists at early endosomes where Ptdlns(3)P is enriched. Furthermore, a mutant p40(phox) PX carrying the substitution of Lys for Arg105 only weakly binds to phosphoinositides in vitro, and fails to locate to early endosomes. Thus the PX domain functions as a novel phosphoinositide-binding module and likely participates in targeting of proteins to membranes.

Amino Acid Sequence↗
PubMed2001

Archaeal primase: bridging the gap between RNA and DNA polymerases.

In the evolution of life, DNA replication is a fundamental process, by which species transfer their genetic information to their offspring. DNA polymerases, including bacterial and eukaryotic replicases, are incapable of de novo DNA synthesis. DNA primases are required for this function, which is sine qua non to DNA replication. In Escherichia coli, the DNA primase (DnaG) exists as a monomer and synthesizes a short RNA primer. In Eukarya, however, the primase activity resides within the DNA polymerase alpha-primase complex (Pol alpha-pri) on the p48 subunit, which synthesizes the short RNA segment of a hybrid RNA-DNA primer. To date, very little information is available regarding the priming of DNA replication in organisms in Archaea. Available sequenced genomes indicate that the archaeal DNA primase is a homolog of the eukaryotic p48 subunit. Here, we report investigations of a p48-like DNA primase from Pyrococcus furiosus, a hyperthermophilic euryarchaeote. P. furiosus p48-like protein (Pfup41), unlike hitherto-reported primases, does not catalyze by itself the synthesis of short RNA primers but preferentially utilizes deoxynucleotides to synthesize DNA fragments up to several kilobases in length. Pfup41 is the first DNA polymerase that does not require primers for the synthesis of long DNA strands.

DNA↗
PubMed2001

NMR identification of the Tom20 binding segment in mitochondrial presequences.

Many mitochondrial proteins are synthesized in the cytosol as precursors with N-terminal presequences, and are imported into mitochondria with the aid of translocator protein complexes containing presequence-binding proteins. Tom20, a receptor protein which functions in an early step of the mitochondrial protein import, recognizes presequences with divergent amino acid sequences. Here, we report the identification of the segments involved in binding to Tom20 in mitochondrial presequences. We monitored the chemical shift perturbation of the NMR signals of five different 15N-labeled presequence peptides by the addition of the cytosolic receptor domain of rat or yeast Tom20. The perturbed segments occupy different positions, either near the N terminus or at the C terminus, in the presequences. Spin label experiments revealed that this is not due to different orientations of the presequence peptides bound to Tom20. The results presented here will offer a starting point to perform detailed analyses of Tom20-binding elements by systematic amino acid replacements.

Amino Acid Sequence↗
PubMed2001

Solution structure of the PX domain, a target of the SH3 domain.

The phox homology (PX) domain is a novel protein module containing a conserved proline-rich motif. We have shown that the PX domain isolated from the human p47phox protein, a soluble subunit of phagocyte NADPH oxidase, binds specifically to the C-terminal SH3 domain derived from the same protein. The solution structure of p47 PX has an alpha + beta structure with a novel folding motif topology and reveals that the proline-rich motif is presented on the molecular surface for easy recognition by the SH3 domain. The proline-rich motif of p47 PX in the free state adopts a distorted left-handed polyproline type II helix conformation.

Amino Acid Motifs↗
PubMed2000

Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with a cleavable N-terminal presequence and are imported into mitochondria. We report here the NMR structure of a general import receptor, rat Tom20, in a complex with a presequence peptide derived from rat aldehyde dehydrogenase. The cytosolic domain of Tom20 forms an all alpha-helical structure with a groove to accommodate the presequence peptide. The bound presequence forms an amphiphilic helical structure with hydrophobic leucines aligned on one side to interact with a hydrophobic patch in the Tom20 groove. Although the positive charges of the presequence are essential for import ability, presequence binding to Tom20 is mediated mainly by hydrophobic rather than ionic interactions.

Aldehyde Dehydrogenase↗
PubMed2000

Characterization of the N-oligosaccharides attached to the atypical Asn-X-Cys sequence of recombinant human epidermal growth factor receptor.

The extracellular domain of human EGF receptor (sEGFR) produced by CHO cells has been used in various biophysical studies to elucidate the molecular mechanism of EGF-induced receptor activation. We have found that the CHO sEGFR contains one oligosaccharide chain attached to an atypical N-glycosylation consensus sequence, Asn(32 )-X( 33 )-Cys(34 ). The oligosaccharide structure at Asn(32 ) is a mixture of the monosialo and asialo forms of a core fucosylated biantennary complex-type oligosaccharide. Deletion of this atypical glycosylation site by replacement of Asn(32 ) with lysine changed neither the expression nor function of the full length EGFR in CHO cells. The glycosylation at Asn(32 ) in CHO sEGFR was incomplete: 20% of Asn(32 ) remained unmodified. Thus, CHO sEGFR itself is heterogeneous with respect to the glycosylation at Asn(32 ), which may cause problems in biophysical studies. An attempt to remove the oligosaccharide at Asn(32 ) enzymatically did not succeed under nondenaturing conditions. Therefore, sEGFR with the mutation of Asn(32) -> Lys(32 )is useful for biophysical and biochemical studies, and, particularly, for X-ray crystallography.

Amino Acid Sequence↗
PubMed2000

Soft metal ions, Cd(II) and Hg(II), induce triple-stranded alpha-helical assembly and folding of a de novo designed peptide in their trigonal geometries.

We previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded alpha-helix structure. The addition of Cd(II), however, resulted in the reconstitution of the triple-stranded alpha-helix bundle, as revealed by circular dichroism (CD) spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant Kd was determined to be 1.5 +/- 0.8 microM for Cd(II). The triple-stranded complex formed by the 113Cd(II) ion showed a single 113Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl- ions. The 113Cd NMR resonance was connected with the betaH protons of the cysteine residue by 1H-113Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg(II) also induced the assembly of the peptide into a triple-stranded alpha-helical bundle below the Hg(II)/peptide ratio of 1/3. With excess Hg(II), however, the alpha-helicity of the peptide was decreased, with the change of the Hg(II) coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.

Amino Acid Sequence↗
PubMed1999

Crystallographic and functional studies of very short patch repair endonuclease.

Vsr endonuclease plays a crucial role in the repair of TG mismatched base pairs, which are generated by the spontaneous degradation of methylated cytidines; Vsr recognizes the mismatched base pair and cleaves the phosphate backbone 5' to the thymidine. We have determined the crystal structure of a truncated form of this endonuclease at 1.8 A resolution. The protein contains one structural zinc-binding module. Unexpectedly, its overall topology resembles members of the type II restriction endonuclease family. Subsequent mutational and biochemical analyses showed that certain elements in the catalytic site are also conserved. However, the identification of a critical histidine and evidence of an active site metal-binding coordination that is novel to endonucleases indicate a distinct catalytic mechanism.

Alanine↗
PubMed1998

Disulfide bond structure of human epidermal growth factor receptor.

The extracellular domain of the human epidermal growth factor receptor (sEGFR) consists of 621 amino acid residues, including 50 cysteines. The connections of the 25 disulfide bonds in the recombinant sEGFR protein, obtained from Chinese hamster ovary cells, have been determined using N-terminal sequencing and matrix-assisted laser desorption/ionization mass spectroscopy. We identified a basic repeat of eight cysteines with a 1-3, 2-4, 5-6, and 7-8 disulfide pairing pattern in the two cysteine-rich regions of sEGFR. By comparison to other cysteine-rich motifs, it was concluded that the cysteine-rich repeat of sEGFR belongs to the laminin-type EGR-like (LE) structural motif. Three-dimensional structure models of the two cysteine-rich regions have been built, based on the three-dimensional structures of the LE domains from the laminin gamma1 chain and secondary structure predictions for the EGF receptor.

Amino Acid Sequence↗
PubMed1998

An isoleucine zipper peptide forms a native-like triple stranded coiled coil in solution.

Recent studies in the field of de novo protein design have focused on the construction of native-like structures. Here we describe the design and characterization of an isoleucine zipper peptide intended to form a parallel triple-stranded coiled coil. To obtain the native-like structural uniqueness, the hydrophobic interface of the peptide consists of beta-branched Ile residues for complementary side chain packing. The peptide forms a stable triple-stranded coiled coil, as determined by circular dichroism and sedimentation equilibrium analyses. A fluorescence quenching assay after the incorporation of acridine revealed a parallel orientation of the peptides. The structural uniqueness of the coiled coil was confirmed by proton-deuterium amide hydrogen exchange and hydrophobic dye binding. The peptide contains amide protons with hydrogen exchange rates that are approximately an order of magnitude slower than those expected if the exchange occurred via global unfolding. A hydrophobic dye does not bind to the peptide. These results strongly suggest that the peptide folds into a well-packed structure that is very similar to the native state of a natural protein. Thus, Ile residues in the hydrophobic interface can improve the side chain packing, which can impart native-like structural uniqueness to the designed coiled coil.

Circular Dichroism↗
PubMed1997

Ligand-binding enhances the affinity of dimerization of the extracellular domain of the epidermal growth factor receptor.

We studied the dimerization of the recombinant soluble extracellular domain of the epidermal growth factor receptor (sEGFR) in response to EGF-binding using multi-angle laser light scattering with size exclusion chromatography (SEC-MALLS). In the absence of EGF, sEGFR behaved as a monomer. However, upon EGF-binding, sEGFR formed a dimer with the stoichiometry of two EGF molecules bound to two sEGFR molecules [(EGF)2-(sEGFR)2]. We analyzed the chemical equilibrium of the dimer formation by SEC-MALLS using a dissociation constant of 0.25 microM for the binding of EGF to sEGFR. The calculated dissociation constant for EGF-induced sEGFR dimerization was found to be 2.4 +/- 0.9 microM. These experiments demonstrated that EGF induces receptor dimerization and that two EGF molecules are bound to an EGF-receptor dimer.

Binding Sites↗
PubMed1996

Solution structure of the link module: a hyaluronan-binding domain involved in extracellular matrix stability and cell migration.

Link modules are hyaluronan-binding domains found in proteins involved in the assembly of extracellular matrix, cell adhesion, and migration. The solution structure of the Link module from human TSG-6 was determined and found to consist of two alpha helices and two antiparallel beta sheets arranged around a large hydrophobic core. This defines the consensus fold for the Link module superfamily, which includes CD44, cartilage link protein, and aggrecan. The TSG-6 Link module was shown to interact with hyaluronan, and a putative binding surface was identified on the structure. A structural database search revealed close similarity between the Link module and the C-type lectin domain, with the predicted hyaluronan-binding site at an analogous position to the carbohydrate-binding pocket in E-selectin.

Amino Acid Sequence↗
PubMed1995

Three-dimensional solution structure of bombyxin-II an insulin-like peptide of the silkmoth Bombyx mori: structural comparison with insulin and relaxin.

The three-dimensional solution structure of bombyxin-II, an insulin-like two-chain peptide produced by the brain of the silkworm Bombyx mori, has been determined by simulated annealing calculations based on 535 distance constraints and 24 torsion-angle constraints derived from NMR data and three distance constraints of the disulfide bonds. To our knowledge, this is the first three-dimensional structure determined for an invertebrate insulin-related peptide. The root-mean-square deviations between the best 10 structures and the mean structure are 0.58(+/- 0.15) A for the backbone heavy atoms (N, C alpha, C) and 1.03(+/- 0.18) A for all non-hydrogen atom if less well-defined N and C termini (A1, A20, B(-2) to B4 and B23 to B25) are excluded. The overall main-chain structure of bombyxin-II is similar to that of insulin. However, there are significant conformational and functional differences in their B-chain C-terminal parts. The B-chain C-terminal part of bombyxin-II adopts an extension of the B-chain central helix like that of relaxin and is not required for bombyxin activity, while the corresponding part of insulin adopts a sharp turn and a beta-strand and is essential for insulin activity. This structure demonstrates that bombyxin-II is more closely related to relaxin than to insulin, and suggests that insulin might have evolved the additional receptor-recognition site in the B-chain C-terminal beta-strand to distinguish itself from bombyxin and relaxin. The structure of bombyxin-II thus provides novel insights into the receptor recognition and divergent molecular evolution of insulin-superfamily peptides.

Amino Acid Sequence↗
PubMed1995

Identification of the receptor-recognition surface of bombyxin-II, an insulin-like peptide of the silkmoth Bombyx mori: critical importance of the B-chain central part.

Bombyxin-II, a brain-secretory peptide of the silkmoth Bombyx mori, shares 40% sequence identify and the characteristics core structure with human insulin. In spite of the structural similarity, no cross-activity is observed between them. To localize the active region of bombyxin-II, we have synthesized chimeric molecules of bombyxin-II and human insulin, and examined their bombyxin activity. Two chimeric molecules, which were sequentially identical except for the B-chain central part, showed significantly different potencies in bombyxin activity. Solution structure determination of these chimeric molecules revealed that their B-chain central parts took similar main-chain conformation, but formed dissimilar patches on their molecular surfaces. Therefore, the surface patch formed by the central part of the bombyxin-II B-chain is of critical importance for recognition of the bombyxin receptor. The above results, together with other data on the structure-activity relationships of bombyxin, indicate that the receptor-recognition surface of bombyxin-II includes the A-chain N and C, termini in addition to the B-chain central part. Though bombyxin-II, human insulin and human relaxin 2 use the common surface as their receptor-recognition sites, each of the surface patches is characterized by the variety of involved side-chains. Insulin and relaxin involve additional parts for receptor recognition, particularly the B-chain C-terminal part and the extended A-chain N-terminal helix, respectively. In conclusion, these ligands have evolved their own specific mechanisms for receptor recognition while retaining the major recognition surface.

Amino Acid Sequence↗