Search PubMed⌕ Search

Biomedical subjects

T Hakoshima

Publications and source records attributed to T Hakoshima.

At least 55 records · Page 3Linked to original sources
PubMed1996

Interaction between left-handed Z-DNA and polyamine - 3. The crystal structure of the d(CG)3 and thermospermine complex.

The DNA fragment, d(CG)3, was co-crystallized with N-(3-amino-propyl)-N-(5-aminopropyl)-l,4 -diaminobutane (thermospermine; PA(334)), a polyamine metabolized from the nucleic acid. By using a good crystal with dimensions of 0.5 x 0.5 x 0.5 mm3, X-ray intensity data were collected up to 1.0 A resolution. Two thermospermine molecules and a magnesium cation were bound to the left-handed double-helical d(CG)3 molecule. The d(CG)3 molecule adopted the left-handed Z-conformation and two thermospermine molecules and a magnesium cation neutralized the negative charges of the phosphate groups of the d(CG)3 molecule. Furthermore, the binding modes between d(CG)3 and thermospermine were different from those of d(CG)3 complexes with PA(24), spermidine and spermine. This is the first case in which it was determined by X-ray crystallographic analysis that one of two thermospermine molecules bound three d(CG)3 duplexes which were symmetrically related to each other, and the other formed two hydrogen bonds at the N(5) and N(9) atoms with two adjacent nucleotide phosphate groups of a single d(CG)3 strand at the minor groove. Furthermore, no direct coordination bond was found between the d(CG)3 molecule and the magnesium cation.

Crystallization↗
PubMed1996

Crystallization and preliminary X-ray analysis of a histidine kinase domain of the anaerobic sensor protein ArcB from Escherichia coli.

Crystals of a novel histidine protein kinase domain of the anaerobic sensor protein ArcB from Escherichia coli have been obtained by a hanging-drop vapor-diffusion method with micro- and macroseeding techniques. Preliminary X-ray crystallographic analysis revealed that they belong to space group P2(1)2(1)2(1) with dimensions a = 30.56, b = 34.93 and c = 110.78 A, having one molecule in the crystallographic asymmetric unit. The crystals diffract to at least 2.0 A resolution.

Journal Article↗
PubMed1996

Interaction between the left-handed Z-DNA and polyamine-2. The crystal structure of the d(CG)3 and spermidine complex.

This paper deals with the crystal structure of d(CG)3-spermidine complex. The DNA fragment, d(CG)3, was crystallized with N-(2-amino-propyl)-1,4-diamino-butane, PA(34), spermidine. The results of its X-ray crystallographic analysis showed many intermolecular contacts between d(CG)3 and spermidine, but the binding mode of spermidine to the d(CG)3 molecule is different from that of the d(CG)3 and N-(2-amino-ethyl)-1,4-diamino-butane [PA(24)] complex: a spermidine molecule bound to the d(CG)3 and its symmetrically related neighboring d(CG)3 molecules through the water molecules with hydrogen bonds, while one PA(24) molecule connected directly to one d(CG)3 molecule, but not to its neighboring d(CG)3 molecule. In the crystal, the d(CG)3 molecule was the left-handed Z-form, and three magnesium cations and a sodium cation were observed around the d(CG)3 moiety with different binding modes from the case of the d(CG)3-PA(24) complex.

Base Sequence↗
PubMed1994

Crystal structure of RNase T1 complexed with the product nucleotide 3'-GMP. Structural evidence for direct interaction of histidine 40 and glutamic acid 58 with the 2'-hydroxyl group of the ribose.

The crystal structure of RNase T1 complexed with 3'-GMP has been determined. The glycosyl conformation of 3'-GMP is in the syn conformation, and the ribose adopts the O4'-endo pucker. This observed pucker is different from that in any complex structures of RNase T1. In the present complex, this energetically unfavorable conformation is stabilized by the water molecule with the bridged hydrogen bonds between the O2' and the O3' atoms of the ribose. The guanine base is recognized in the same manner as observed in the complex of 2'-GMP. The 2'-hydroxyl group of the ribose shows a tight hydrogen bond to both His-40 and Glu-58 with the suitable geometry for the proton transfer. These hydrogen bonds suggest that the two residues can participate directly in the proton transfer. His-92 is hydrogen bonded to two the proton transfer. His-92 is hydrogen bonded to two oxygen atoms of the phosphate group. Based on the geometry in the active site, the O1P atom may correspond to the O5' atom of the leaving nucleotide in the phosphoryl transfer or a water molecule as a nucleophile in the hydrolysis reaction. In the present complex, the conformations of the 3'-GMP molecule and the side chains of the catalytic residues would be represented as the conformation before the phosphoryl transfer reaction and/or after the hydrolysis reaction.

Binding Sites↗
PubMed1993

Crystallographic characterization of a PHO4-DNA complex.

Crystals have been obtained of the DNA-binding domain of the yeast transcription factor PHO4 in complexes with several synthetic fragments of DNA with appropriate cognate sequences. Crystals suitable for X-ray diffraction studies were produced in the case of a complex of the protein with a 17 base-pair fragment of DNA from a solution in polyethylene glycol and calcium chloride. The crystals have the space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 56.7 A, c = 447.8 A. The diffraction data at 3 A resolution were collected using synchrotron radiation with a Weissenberg camera for macromolecular crystallography.

Base Sequence↗
PubMed1993

Crystal structure of RNase T1(Y45W) complexed with 3'AMP and GflpA.

We have previously reported the crystallization of a mutant RNase T1(Y45W) with a synthetic modified trinucleotide ApGflpA [Hakoshima, T. et al. (1990) J. Biochem. 108, 695-698]. In the present report, we describe the crystal structure refined at 2.4 A resolution. During the refinement process, we found that the ApGflpA molecule was cleaved at the phosphodiester bond between the 5'-terminal adenosine and the subsequent 2'-fluoroguanosine. At the end of the refinement (R = 17.1%), it was supposed that the resulting molecules, i.e., 3'AMP and GflpA, were separately bound to the enzyme. In the complex structure, the binding-site of the enzyme was occupied by the guanine base of GflpA via a similar interaction to that of the enzyme complexed with 2'GMP, while the phosphate group of GflpA was not bound to the active site. The guanosine adopted the anti orientation on the glycosyl torsion angle with a C2'-endo-C3'-exo sugar pucker. This conformation resulted in the phosphate group protruding from the active site. The phosphate group of 3'AMP was bound to the active site of the enzyme and oriented itself toward the solvent region. This orientation was different from that of 2'AMP bound to the RNase T1(Y45W).

Adenosine↗
PubMed1992

Phosphorescence and optically detected magnetic resonance measurements of the 2'AMP and 2'GMP complexes of a mutant ribonuclease T1 (Y45W) in solution: correlation with X-ray crystal structures.

Phosphorescence and ODMR measurements have been made on ribonuclease T1 (RNase T1), the mutated enzyme RNase T1 (Y45W), and their complexes with 2'GMP and 2'AMP. It is not possible to observe the phosphorescence of Trp45 in RNase T1 (Y45W). Only that of the naturally occurring Trp59 is seen. The binding of 2'GMP to wild-type RNase T1 produces only a minor red shift in the phosphorescence and no change in the ODMR spectrum of Trp59. However, a new tryptophan 0,0-band is found 8.2 nm to the red of the Trp59 0,0-band in the 2'GMP complex of the mutated RNase T1 (Y45W). Wavelength-selected ODMR measurements reveal that the red-shifted emission induced by 2'GMP binding, assigned to Trp45, occurs from a residue with significantly different zero-field splittings than those of Trp59, a buried residue subject to local polar interactions. The phosphorescence red shift and the zero-field splitting parameters demonstrate that Trp45 is located in a polarizable environment in the 2'GMP complex. In contrast with 2'GMP, binding of 2'AMP to RNase T1 (Y45W) induces no observable phosphorescence emission from Trp45, but leads only to a minor red shift in the phosphorescence origin of Trp59 in both the mutated and wild-type enzyme. The lack of resolved phosphorescence emission from Trp45 in RNase T1 (Y45W) implies that the emission of this residue is quenched in the uncomplexed enzyme. We conclude that local conformational changes that occur upon binding 2'GMP remove quenching residues from the vicinity of Trp45, restoring its luminescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1992

Three-dimensional structure of a mutant ribonuclease T1 (Y45W) complexed with non-cognizable ribonucleotide, 2'AMP, and its comparison with a specific complex with 2'GMP.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2'GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2'GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2'AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.

Adenosine Monophosphate↗
PubMed1991

Non-cognizable ribonucleotide, 2'AMP, binds to a mutant ribonuclease T1 (Y45W) at a new base-binding site but not at the guanine-recognition site.

Complex of a mutant ribonuclease T1 (Y45W) with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined by X-ray diffraction at 1.7 A resolution. The 2'AMP molecule locates at a new base-binding site which is remote from the guanine-recognition site, where 2'GMP was found to be bound. The nucleotide adopts the anti conformation of the glycosidic bond and C3'-exo sugar pucker. There exists a single hydrogen bond between the adenine base and the enzyme, and, therefore, the site found is apparently a non-specific binding site. The results indicate that the binding of 2'AMP to the guanine-recognition site is weaker than that to the new binding site.

Adenosine Monophosphate↗
PubMed1991

Molecular cloning, nucleotide sequence and expression of the tufB gene encoding elongation factor Tu from Thermus thermophilus HB8.

The tufB gene encoding elongation factor Tu (EF-Tu) of Thermus thermophilus HB8 was cloned and expressed. Compared with the known tufA gene of T. thermophilus, nucleotide differences were found at 10 positions out of 1221 nucleotides, and amino acid substitutions were found at 4 positions out of 406 amino acids. The tufB product was 70.9% homologous to the corresponding sequence of the tufB product of E. coli. The G+C content of the third base of the codon in the tufB gene was 84.8% and G was especially preferred in this position.

Amino Acid Sequence↗
PubMed1991

Interaction between the left-handed Z-DNA and polyamine. The crystal structure of the d(CG)3 and N-(2-aminoethyl)-1,4-diamino-butane complex.

The DNA fragment d(CG)3 was co-crystallized with N-(2-aminoethyl)-1,4-diaminobutane (PA(24], a chemically synthesized polyamine. The complex crystal contained one polyamine, 3 magnesium cations and one sodium cation per duplex of d(CG)3, and well diffracted the X-ray intensities up to 1.0 A resolution. The d(CG)3 took a left-handed Z-DNA conformation, and the PA(24) molecule electrostatically interacted with the phosphate groups of the d(CG)3 duplex.

Crystallization↗
PubMed1991

Conformation of 2'GMP bound to a mutant ribonuclease T1 (Y45W) determined by X-ray diffraction and NMR methods.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a specific inhibitor, 2'GMP, has been determined by X-ray diffraction and refined at 1.9 A resolution to a conventional R-factor of 0.164. The mode of recognition of the guanine base by the enzyme is similar to that found for the wild-type ribonuclease T1 complexed with 2'GMP. The binding of the guanine base is clearly enhanced by maximum overlapping of the indole ring of Trp45 and the base. The glycosyl torsion angle of the inhibitor is in the syn conformation and the sugar exhibits a C3'-endo type pucker, which differs from that observed in the crystal of the complex between the wild-type ribonuclease T1 and 2'GMP. Analysis of 500-MHZ NMR spectra has also indicated that the 2'GMP molecule as bound to the mutant enzyme in solution exhibits a C3'-endo type pucker, similar to that bound to the wild-type enzyme in solution [Inagaki, Shimada, & Miyazawa (1985) Biochemistry 24, 1013-1020].

Carbohydrate Conformation↗
PubMed1991

Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease T1.

Hydrophobic effects on binding of ribonuclease T1 to guanine bases of several ribonucleotides have been proved by mutating a hydrophobic residue at the recognition site and by measuring the effect on binding. Mutation of a hydrophobic surface residue to a more hydrophobic residue (Tyr45----Trp) enhances the binding to ribonucleotides, including mononucleotide inhibitor and product, and a synthetic substrate-analog trinucleotide as well as the binding to dinucleotide substrates and RNA. Enhancements on binding to non-substrate ribonucleotides by the mutation have been observed with free energy changes ranging from -2.2 to -3.9 kJ/mol. These changes are in good agreement with that of substrate binding, -2.3 kJ/mol, which is calculated from Michaelis constants obtained from kinetic studies. It is shown, by comparing the observed and calculated changes in binding free energy with differences in the observed transfer free energy changes of the amino acid side chains from organic solvents to water, that the enhancement observed on guanine binding comes from the difference in the hydrophobic effects of the side chains of tyrosine and tryptophan. Furthermore, a linear relationship between nucleolytic activities and hydrophobicity of the residues (Ala, Phe, Tyr, Trp) at position 45 is observed. The mutation could not change substantially the base specificity of RNase T1, which exhibits a prime requirement for guanine bases of substrates.

Binding Sites↗
PubMed1990

Crystallization and preliminary X-ray investigation of non-specific complexes of a mutant ribonuclease T1 (Y45W) with 2'AMP and 2'UMP.

We have succeeded in crystallizing complexes of a mutant ribonuclease T1 (Y45W) with the non-cognizable ribonucleotides 2'AMP and 2'UMP by macroscopic seeding of microcrystals of the mutant enzyme complexed with 2'GMP, which is the cognizable nucleotide inhibitor. The mutant enzyme has a tryptophan residue instead of Tyr45 of the wild-type enzyme and thus this mutation enhances the binding of ribonucleotides to the enzyme. The space group is P212121 with unit cell dimensions a = 49.40 A, b = 46.71 A, c = 41.02 A for the complex with 2'AMP and a = 48.97, b = 46.58 A, c = 40.97 A for the complex with 2'UMP, both of which are poorly isomorphous to the mother crystals. Diffraction data for the complexes with 2'AMP and 2'UMP were collected on a diffractometer at 1.7 A and 2.4 A resolution, respectively. The present studies show that crystallization of non-specific complexes of other protein-ligand systems with the dissociation constants around 10(-3) M, or even larger, could be feasible by application of the seeding technique. A comparison of the crystal structures of the complexes with that with 2'GMP may serve as a structural basis for the determination of differences between the specific and non-specific interactions of the enzyme.

Adenosine Monophosphate↗
PubMed1990

Crystallographic characterization of wild-type and mutant ribonuclease T1 complexes with several ribonucleotides.

Ribonuclease T1 and the mutant enzymes were cocrystallized with several ribonucleotides, including non-hydrolyzable substrate analogs of di- and triribonucleotides, which have a novel guanylate in which the 2'-hydroxyl group of the ribose is replaced by a fluorine atom. One of the mutant enzymes has a tryptophan residue, instead of Tyr45 of the wild-type enzyme, to enhance the binding of ribonucleotides to the enzyme and the other mutant enzyme has histidine and aspartate residues, instead of Asn43 and Asn44, respectively, to reproduce the natural substitutions found in ribonuclease Ms. Polymorphism of the crystals was observed for wild-type and mutant enzymes. However, orthorhombic crystals, which are virtually all isomorphous to each other, were successfully obtained from wild-type and mutant (Y45W) enzymes by the macroscopic seeding technique using mother crystals of the wild-type ribonuclease T1 complexed with 2'GMP or 3'GMP. The diffraction patterns of these crystals extend beyond 2.5 A resolution and the diffraction data were collected from some of the crystals on a diffractometer up to a range of 2.5 to 1.8 A resolution.

Aspergillus oryzae↗
PubMed1990

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds.

Molecular-dynamic calculations predict that, if Tyr24 and Asn84 are each replaced by a Cys residue, it should be possible to form a third disulfide bond in ribonuclease T1 (RNase T1) between these residues, with only minimal conformational changes at the catalytic site. The gene encoding such a mutant variant of RNase T1 (Tyr24----Cys24, Asn84----Cys84) was constructed by the cassette mutagenesis method using a chemically synthesized gene. In order to reduce the toxic effect of the mutant enzyme (RNase T1S) on an Escherichia coli host, we arranged for the protein to be secreted into the periplasmic space by using a vector that harbors a gene for an alkaline phosphatase signal peptide under the control of the trp promoter. The nucleolytic activity of RNase T1S toward pGpC was approximately the same as that of RNase T1 at 37 degrees C (pH 7.5). Moreover, at 55 degrees C, RNase T1S retained nearly 70% of its activity while the activity of the wild-type enzyme was reduced to less than 10%. RNase T1S was also more resistant to denaturation by urea than the wild-type enzyme. However, unlike RNase T1, RNase T1S was irreversibly and almost totally inactivated by boiling at 100 degrees C for 15 min.

Base Sequence↗
PubMed1989

Polyamine interaction with Z-DNA.

In order to elucidate the detailed Z-DNA interaction with polyamines and also to clarify the mutual molecular recognition between the left-handed helix and the biologically important polyamine molecule, several polyamine-Z-DNA hexamer complexes were crystallized and their crystal structures were determined by X-ray diffraction. The general interaction modes found in these crystal structures were discussed in comparison with those in the complexes between polyamine and the right-handed DNA or RNA.

Crystallization↗