Biomedical subjects

Yuji Kobayashi

Publications and source records attributed to Yuji Kobayashi.

At least 19 recordsLinked to original sources

PubMed2007

Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase.

The imidazole (15)N signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with (15)N at ring-N(delta1) and N(epsilon2), (13)Cat ring-Cepsilon1, (13)C and (15)N at all carbon and nitrogen, or (15)N at the amide nitrogen and the labeled glycine with (13)C at the carbonyl carbon. Using the pH dependence of ring-(15)N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K(T)) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon2-nitrogen as (a) releases one proton into the bulk, whereas its delta1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.

Binding Sites↗

PubMed2006

NMR structure of a human homologous methuselah gene receptor peptide.

Human APG1 gene is homologous to Drosophila methuselah gene associated with extended life span. A peptide (APG1: RNGKRSNRTLREE) corresponding to a predicted region of the intracellular third loop of G protein-coupled receptor coded in human APG1 gene could activate Gi protein alpha subunit directly. The three-dimensional molecular structure of the peptide in SDS-d25 micelles was determined by 2D 1H NMR spectroscopy. APG1 formed an alpha-helical structure at the C-terminal site and a positive charge cluster at the N-terminal site. The cluster was also found in several other Gi protein-coupled receptor peptides. Therefore, the positive charge cluster on the helical structure might be engaged in G protein activation.

Amino Acid Sequence↗

PubMed2006

Apo- and holo-structures of 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831. Loop-helix transition induced by coenzyme binding.

Bacterial 3alpha-hydroxysteroid dehydrogenase, which belongs to a short-chain dehydrogenase/reductase family and forms a dimer composed of two 26-kDa subunits, catalyzes the oxidoreduction of hydroxysteroids in a coenzyme-dependent manner. This enzyme also catalyzes the oxidoreduction of nonsteroid compounds that play an important role in xenobiotic metabolism of bacteria. We performed an x-ray analysis on the crystal of Ps3alphaHSD, the enzyme from Pseudomonas sp. B-0831 complexed with NADH. The resulting crystal structure at 1.8A resolution showed that Ps3alphaHSD exists as a structural heterodimer composed of apo- and holo-subunits. A distinct structural difference between them was found in the 185-207-amino acid region, where the structure in the apo-subunit is disordered whereas that in the holo-subunit consists of two alpha-helices. This fact proved that the NADH binding allows the helical structures to form the substrate binding pocket even in the absence of the substrate, although the region corresponds to the so-called "substrate-binding loop." The induction of alpha-helices in solution by the coenzyme binding was also confirmed by the CD experiment. In addition, the CD experiment revealed that the helix-inducing ability of NADH is stronger than that of NAD. We discuss the negative cooperativity for the coenzyme binding, which is caused by the effect of the structural change transferred between the subunits of the heterodimer.

3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)↗

PubMed2006

Crystallization and preliminary X-ray analysis of the complex of NADH and 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831.

The NAD(P)(+)-dependent enzyme 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus. The complex of NADH and 3alpha-HSD from Pseudomonas sp. B-0831 was crystallized by the hanging-drop vapour-diffusion method. Refinement of crystallization conditions with microseeding improved the quality of the X-ray diffraction data to a resolution of 1.8 A. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.46, b = 82.25, c = 86.57 A, and contained two molecules, reflecting dimer formation of 3alpha-HSD, in the asymmetric unit.

3-Hydroxysteroid Dehydrogenases↗

PubMed2006

Structure of cytochrome c552 from a moderate thermophilic bacterium, Hydrogenophilus thermoluteolus: comparative study on the thermostability of cytochrome c.

We have studied the structure-thermostability relationship using cytochromes c from mesophilic and thermophilic bacteria; Pseudomonas aeruginosa (PAc(551)) growing at 37 degrees C and Hydrogenobacter thermophilus (HTc(552)) at 72 degrees C and showed that only five residues primarily differentiate their stabilities. For a more comprehensive study, we found Hydrogenophilus thermoluteolus (Pseudomonas hydrogenothermophila) growing at 52 degrees C and showed the moderate stability of the cytochrome c from this bacterium (PHc(552)). To explore the stabilization mechanisms, the crystal structure of PHc(552) was determined by X-ray analysis. The solution structure of HTc(552) elucidated previously by NMR was refined using distributed computational implementation. Furthermore, the recently reported crystal structure of HTc(552) has become available [Travaglini-Allocatelli, C. et al. (2005) J. Biol. Chem. 280, 25729-25734]. When the structures of these three cytochromes c were combined, this revealed that the five residues, corresponding to those mentioned above, determine the difference of stabilities among them as well. These facts suggested the stabilization mechanisms as follows: (1) improved van der Waals interactions by packing optimization at the N-terminal helix, (2) attractive electrostatic interactions with the heme propionate group, and (3) favorable van der Waals interaction with the heme. This comparative study, by supplementing the structural information of PHc(552) with its complementary feature, demonstrates that just a small number of amino acid residues determine the overall molecular stability by means of additivity of the effects of their substitutions. It is interesting that, in naturally occurring proteins, these adaptation strategies are accommodated by these bacteria to survive in the wide range of thermal conditions.

Amino Acid Sequence↗

PubMed2006

NMR structure of an intracellular loop peptide derived from prostaglandin EP3alpha receptor.

We found that a peptide (EP3a: TIKALVSRCRAKAAV) corresponding to the N-terminal site of the intracellular third loop of human prostaglandin EP3alpha receptor could activate G protein alpha-subunit directly. The activity was almost same as Mastoparan-X, a G protein activating peptide from wasp venom. The three-dimensional molecular structure of the peptide in SDS-d(25) micelles was determined by 2D (1)H NMR spectroscopy. The structure of EP3a consists of a positive charge cluster on the C-terminal helical site. The cluster was also found in several corresponding receptor peptides. Therefore, the positive charge cluster on the helical structure might play a crucial role in activation of G protein.

Guanosine 5'-O-(3-Thiotriphosphate)↗

PubMed2006

The solution structure of the methylated form of the N-terminal 16-kDa domain of Escherichia coli Ada protein.

The N-terminal 16-kDa domain of Escherichia coli Ada protein (N-Ada16k) repairs DNA methyl phosphotriester lesions by an irreversible methyl transfer to its cysteine residue. Upon the methylation, the sequence-specific DNA binding affinity for the promoter region of the alkylation resistance genes is enhanced by 10(3)-fold. Then, it acts as a transcriptional regulator for the methylation damage. In this paper, we identified the methyl acceptor residue of N-Ada16k and determined the solution structure of the methylated form of N-Ada16k by using NMR and mass spectrometry. The results of a 13C-filtered 1H-13C HMBC experiment and MALDI-TOF MS and MS/MS experiments clearly showed that the methyl acceptor residue is Cys38. The solution structure revealed that it has two distinct subdomains connected by a flexible linker loop: the methyltransferase (MTase) subdomain with the zinc-thiolate center, and the helical subdomain with a helix-turn-helix motif. Interestingly, there is no potential hydrogen bond donor around Cys38, whereas the other three cysteine residues coordinated to a zinc ion have potential donors. Hence, Cys38 could retain its inherent nucleophilicity and react with a methyl phosphotriester. Furthermore, the structure comparison shows that there is no indication of a remarkable conformational change occurring upon the methylation. This implies that the electrostatic repulsion between the negatively charged DNA and the zinc-thiolate center may avoid the contact between the MTase subdomain and the DNA in the nonmethylated form. Thus, after the Cys38 methylation, the MTase subdomain can bind the cognate DNA because the negative charge of the zinc-thiolate center is reduced.

Amino Acid Motifs↗

PubMed2006

Conformational analysis of human calcitonin in solution.

The solution conformation of human calcitonin in a mixture of 60% water and 40% trifluoroethanol has been determined by the combined use of 1H NMR spectroscopy and distance geometry calculations with a distributed computing technique. 1H NMR spectroscopy provided 195 distance constraints and 13 hydrogen bond constraints. The 20 best converged structures exhibit atomic rmsd of 0.43 A for the backbone atoms from the averaged coordinate position in the region of Asn3-Phe22. The conformation is characterized by a nearly amphiphilic alpha-helix domain that extends from Leu4 in the cyclic region to His20. There are no significant differences observed among the overall structures of a series of calcitonins obtained from ultimobranchial bodies, including those that possess 20- to 50-fold greater activity. Three aromatic amino acid residues, Tyr12, Phe16 and Phe19, form a hydrophobic surface of human calcitonin. Bulky side chains on the surface could interfere with the ligand-receptor interaction thereby causing its low activity, relative to those of other species.

Animals↗

PubMed2006

Measurement of circular dichroism and structural chemical research of d(CG)6 and d(TA)6.

It was known that d(CG)(6) became left-handed Z-DNA by a theoretical calculation. We carried out a CD measurement of d(CG)(6) at room temperature. B-Z transition was occurred when I raised the concentration of Mg salt. In addition, I confirmed that the Mg salt concentration caused B-Z transition by the lower concentration under the existence of cobalt hexamine. Structure of d(TA)(6) is not known. We performed the CD measurement of d(TA)(6) at low temperature. Structure changed from B type when I raised the Mg salt concentration in an existence of cobalt hexamine. I am examining structure now.

Circular Dichroism↗

PubMed2006

Regional and segmental flexibility of antibodies in interaction with antigens of different size.

The interaction of antibodies (Abs) with protein antigens (Ags) of different size, such as hen egg white lysozyme, ovalbumin, and bovine serum albumin, was examined using analytical ultracentrifugation, electrospray ionization time-of-flight mass spectrometry, and surface plasmon resonance in order to estimate regional and segmental Ab flexibility. When both Abs and Ags were free in solution, sedimentation equilibrium and surface plasmon resonance analyses showed the formation of an Ag(2)Ab(1) complexes regardless of Ag size, suggesting that the Fab arms were able to move to avoid interference between Ags bound to Ab combining sites. The Ag(2)Ab(1) complex, as well as the Ag(1)Ab(1) complex, was observed by MS. However, when Abs were immobilized on the surface of a sensor chip through the Fc region, the stoichiometry of the Ag-Ab complex was dependent on the Ag size; Ag(2)Ab(1) forming with hen egg white lysozyme and Ag(1)Ab(1) with ovalbumin and bovine serum albumin. These results indicated that immobilization of the Fc region reduces the dynamic range of the Fab arms and results in interference from the first Ag bound to either combining site, which in turn prevents the binding of the second Ag to the other combining site. Our results allow us to propose that the Fab arms of B-cell receptors whose Fc regions are immobilized on cell surface have a reduced dynamic range.

Animals↗

PubMed2005

Effect of hydration on the stability of the collagen-like triple-helical structure of [4(R)-hydroxyprolyl-4(R)-hydroxyprolylglycine]10.

X-ray analysis has been carried out on a crystal of the collagen model peptide (Hyp(R)-Hyp(R)-Gly)10 [where Hyp(R) is 4(R)-hydroxyproline] with 1.5 A resolution. The triple-helical structure of (Hyp(R)-Hyp(R)-Gly)10 has the same helical parameters and Rich and Crick II hydrogen bond patterns as those of other collagen model peptides. However, our full-length crystal structure revealed that almost all consecutive Hyp(R) residues take the up-up pucker in contrast to putative down-up puckering propensities of other collagen model peptides. The unique feature of thermodynamic parameters associated with the conformational transition of this peptide from triple helix to single coil is that both enthalpy and entropy changes of the transition are much smaller than those of other model peptides such as (Pro-Pro-Gly)10 and (Pro-Hyp(R)-Gly)10. To corroborate the precise structural information including main- and side-chain dihedral angles and intra- and interwater bridge networks, we estimated the degrees of hydration by comparing molecular volumes observed experimentally in solution to those calculated ones from the crystal structure. The results showed that the degree of hydration of (Hyp(R)-Hyp(R)-Gly)10 is comparable to that of (Pro-Hyp(R)-Gly)10 in the triple-helical state, but the former was more highly hydrated than (Pro-Hyp(R)-Gly)10 in the single-coil state. Because hydration reduces the enthalpy due to the formation of a hydrogen bond with a water molecule and diminishes the entropy due to the restriction of water molecules surrounding a peptide molecule, we concluded that the high thermal stability of (Hyp(R)-Hyp(R)-Gly)10 is able to be described by its high hydration in the single-coil state.

Circular Dichroism↗

PubMed2005

Structural and functional study of an Anemonia elastase inhibitor, a "nonclassical" Kazal-type inhibitor from Anemonia sulcata.

Anemonia elastase inhibitor (AEI) is a "nonclassical" Kazal-type elastase inhibitor from Anemonia sulcata. Unlike many nonclassical inhibitors, AEI does not have a cystine-stabilized alpha-helical (CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solution structure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by a central alpha-helix and a three-stranded antiparallel beta-sheet of a typical Kazal-type inhibitor such as silver pheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forming a disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3. Synthesized AEI exhibited unexpected strong inhibition toward Streptomyces griseus protease B (SGPB). Our previous study [Hemmi, H., et al. (2003) Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motif could produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI (AEI analogue) was chemically synthesized when a Cys(4)-Cys(34) bond was changed to a Cys(6)-Cys(31) bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibited almost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structural difference was detected between the two, but the N-terminal loop from Pro(5) to Ile(7) near the putative reactive site (Met(10)-Gln(11)) in the analogue moved by 3.7 A toward the central helix to form the introduced Cys(6)-Cys(31) bond. Such a conformational change in the restricted region correlates with the specificity change of the inhibitor.

Amino Acid Motifs↗

PubMed2005

Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix.

Differential scanning calorimetry (DSC) analyses of a series of collagen model peptides suggest that 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) have different effects on the stability of the collagen triple helices according to the sequence of amino acids and stereochemistry at the 4 positions of these imino acids. The thermodynamic parameters indicate that the enhanced stabilities are classified into two different types: the enthalpy term is primarily responsible for the enhanced stability of the triple helix of (Pro-Hyp(R)-Gly)(10), whereas the entropy term dominates the enhanced stability of (Pro-fPro(R)-Gly)(10). The difference between the molecular volumes observed in solution and intrinsic molecular volumes calculated from the crystal structure indicates the different hydration states of these peptides. (Pro-Hyp(R)-Gly)(10) is highly hydrated compared to (Pro-Pro-Gly)(10), which contributes to the larger enthalpy. In contrast, the volume of (Pro-fPro(R)-Gly)(10) shows a smaller degree of hydration than that of (Pro-Pro-Gly)(10). The entropic cost of forming the triple helix of the fPro-containing peptides is compensated by a decrease in an ordered structure of water molecules surrounding the peptide molecule, although the contribution of enthalpy originating from the hydration is reduced. These arguments about the different contribution of entropic and enthalpic terms were successfully applied to interpret the stability of the triple helix of (fPro(S)-Pro-Gly)(10) as well.

Amino Acid Sequence↗

PubMed2005

Crystallization and preliminary X-ray crystallographic analysis of a conserved domain in plants and prokaryotes from Pyrococcus horikoshii OT3.

A plant- and prokaryote-conserved domain (PPC) has previously been found in AT-hook motif nuclear localized protein 1 (AHL1) localized in the nuclear matrix of Arabidopsis thaliana (AtAHL1). AtAHL1 has a DNA-binding function. Mutation analyses of AtAHL1 has previously revealed that the hydrophobic region of the PPC domain is essential for its nuclear localization. In this study, the PPC of the hyperthermophilic archaebacterium Pyrococcus horikoshii (PhPPC) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 53.69, c = 159.2 A. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 A. A complete data set from crystals of the SeMet-substituted protein was also obtained.

Amino Acid Motifs↗

PubMed2005

Cloning, expression, crystallization and preliminary X-ray characterization of cytochrome c552 from a moderate thermophilic bacterium, Hydrogenophilus thermoluteolus.

The amino-acid sequence of cytochrome c552 (PH c552) from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus, was more than 50% identical to that of cytochrome c from an extreme thermophile, Hydrogenobacter thermophilus (HT c552), and from a mesophile, Pseudomonas aeruginosa (PA c551). The PH c552 gene was overexpressed as a correctly processed holoprotein in the Escherichia coli periplasm. The overexpressed PH c552 has been crystallized by vapour diffusion from polyethylene glycol 4000 pH 6.5. The crystals belong to space group C222(1), with unit-cell parameters a = 48.98, b = 57.99, c = 56.20 A. The crystals diffract X-rays to around 2.1 A resolution.

Cloning, Molecular↗

PubMed2005

Solution NMR structure investigation for releasing mechanism of neocarzinostatin chromophore from the holoprotein.

Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel beta-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the beta-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.

Crystallography, X-Ray↗

PubMed2005

Collagen-like triple helix formation of synthetic (Pro-Pro-Gly)10 analogues: (4(S)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10, (4(R)-hydroxyprolyl-4(R)-hydroxyprolyl-Gly)10 and (4(S)-fluoroprolyl-4(R)-fluoroprolyl-Gly)10.

For the rational design of a stable collagen triple helix according to the conventional rule that the pyrrolidine puckerings of Pro, 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) should be down at the X-position and up at the Y-position in the X-Y-Gly repeated sequence for enhancing the triple helix propensities of collagen model peptides, a series of peptides were prepared in which X- and Y-positions were altogether occupied by Hyp(R), Hyp(S), fPro(R) or fPro(S). Contrary to our presumption that inducing the X-Y residues to adopt a down-up conformation would result in an increase in the thermal stability of peptides, the triple helices of (Hyp(S)-Hyp(R)-Gly)(10) and (fPro(S)-fPro(R)-Gly)(10) were less stable than those of (Pro-Hyp(R)-Gly)(10) and (Pro-fPro(R)-Gly)(10), respectively. As reported by Bächinger's and Zagari's groups, (Hyp(R)-Hyp(R)-Gly)(10) which could have an up-up conformation unfavorable for the triple helix, formed a triple helix that has a high thermal stability close to that of (Pro-Hyp(R)-Gly)(10). These results clearly show that the empirical rule based on the conformational preference of pyrrolidine ring at each of X and Y residues should not be regarded as still valid, at least for predicting the stability of collagen models in which both X and Y residues have electronegative groups at the 4-position.

Collagen↗

PubMed2004

NMR structure of transcription factor Sp1 DNA binding domain.

To understand the DNA recognition mechanism of zinc finger motifs of transcription factor Sp1, we have determined the solution structure of DNA-binding domain of the Sp1 by solution NMR techniques. The DNA-binding domain of Sp1 consists of three Cys(2)His(2)-type zinc finger motifs. They have typical betabetaalpha zinc finger folds and relatively random orientations. From DNA-binding analysis performed by NMR and comparison between structures determined here and previously reported structures of other zinc fingers, it was assumed that DNA recognition modes of fingers 2 and 3 would be similar to those of fingers of Zif268, in which each finger recognizes four base pairs strictly by using residues at positions -1, 2, 3, and 6 of the recognition helix. On the contrary, finger 1 can use only two residues for DNA recognition, Lys550 and His553 at positions -1 and 3 of the helix, and has more relaxed sequence and site specificity than other Cys(2)His(2) zinc fingers. It is proposed that this relaxed property of finger 1 allows transcription factor Sp1 to bind various DNA sequences with high affinity.

Amino Acid Sequence↗