Local and overall conformations of DNA double helices with the A - T base pairs.

Raman spectra have been observed of two different poly[d(A-T)].poly[d(A-T)] fibers, whose X-ray diffractions were confirmed to be purely of A and B forms. A number of spectral differences were found between the A and B forms of this DNA duplex, and they were ascribed to local conformational differences in the adenosine, thymidine and phosphodiester portions. The ascription was made on the basis of a separate series of Raman examinations on six crystals involving adenosine or thymidine, and fifteen other nucleotide crystals, whose structures are all known by previous crystallographic works. By taking these structure-spectrum correlations thus obtained into account, a Raman spectroscopic investigation was made of a few double-helical DNAs in aqueous solutions. It has been concluded that both poly[d(A-T)].poly[d(A-T)] and poly(dA).poly(dT) have a C2'endo-anti adenosine, C2'endo-anti thymidine, a b-type mainchain (beta = 160 +/- 15 degrees, gamma = 45 +/- 15 degrees, delta = 140 +/- 10 degrees) and an a2-type mainchain (beta = 210 +/- 10 degrees, gamma = 45 +/- 15 degrees, delta = 140 +/- 10 degrees) not only in low-salt medium but also in 6.6 M CsF solutions, where beta, gamma and delta are the torsion angles around O5'-C5', C5'-C4' and C4'-C3' axes, respectively. Poly(rA).poly(dT), on the other hand, was considered to have a heteronomous duplex structure, in which the poly(rA) strand has a C3'endo-anti adenosine and a1-type mainchain (beta = 175 +/- 25 degrees, gamma = 45 +/- 15 degrees, delta = 80 +/- 10 degrees) whereas the poly(dT) strand has a C2'endo-anti thymidine and b-type mainchain.

Crystal structure at 2.6 A resolution of the complex of subtilisin BPN' with streptomyces subtilisin inhibitor.

The crystal structure of the complex of a bacterial alkaline serine proteinase, subtilisin BPN', with its proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was solved at 2.6 A resolution. Compared with other similar complexes involving serine proteinases of the trypsin family, the present structure is unique in several respects. (1) In addition to the usual antiparallel beta-sheet involving the P1, P2 and P3 residues of the inhibitor, the P4, P5 and P6 residues form an antiparallel beta-sheet with a previously unnoticed chain segment (residues 102 through 104, which was named the S4-6 site) of subtilisin BPN'. (2) The S4-6 site does not exist in serine proteinases of the trypsin family, whether of mammalian or microbial origin. (3) Global induced-fit movement seems to occur on SSI: a channel-like structure in SSI where hydrophobic side-chains are sandwiched between two lobes becomes about 2 A wider upon complexing with subtilisin. (4) The complex is most probably a Michaelis complex, as in most of the other complexes. (5) The main role of the "secondary contact region" of SSI seems to be to support the reactive site loop ("primary contact region"). Steric homology of the two contact regions between the inhibitors of the SSI family and the pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family is so high that it seems to indicate divergent evolutionary processes and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forward by Doolittle (1978).

X-ray crystallographic and chromatographic characterization of the crystals of Ca2+-calmodulin complexed with bee venom melittin.

Crystals of calmodulin complexed with both Ca2+ and melittin, a peptide from bee venom, have been grown from 2-methyl-2,4-pentanediol solution by using the hanging drop method of vapour diffusion. The crystals belong to space group P2(1)2(1)2(1) with a = 97.3(9) A, b = 56.5(0) A, c = 33.4(9) A and Z = 4. Analyses of the dissolved crystals by high performance liquid chromatography show that the crystals contain a 1:1 complex of calmodulin and melittin. An asymmetric unit contains one such complex and the solvent content of the crystals is 47.5% (v/v).

Crystal structure of a protein proteinase inhibitor, SSI (Streptomyces subtilisin inhibitor), at 4 A resolution.

The crystal structure of a protein proteinase inhibitor, SSI (Streptomyces subtilisin inhibitor), which strongly inhibits bacterial alkaline proteinases specifically, was determined at 4 A resolution using four heavy-atom derivatives. The SSI molecule can be described as an ellipsoid of about 30 X 40 X 65 A composed of two identical subunits each having dimensions of about 35 X 25 X 40 A and a molecular weight of 11,483. The subunit has an extensive beta-sheet structure, but no long alpha-helices are present. Based on the binding sites of platinum reagents known to form coordination complexes with methionine, it is speculated that the P1 residue, Met 73, of the reactive site is at the protruding edge of the subunit. At the subunit-subunit interface, a beta-sheet of one subunit is stacked on top of the corresponding beta-sheet of the other subunit.

Crystal structure of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor, at 2.3 angstrom resolution.

The crystal structure of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor which strongly inhibits bacterial alkaline proteinases, was determined at 2.3 angstrom resolution. The subunit (molecular weight, 11,485) of this dimeric molecule has a unique fold of polypeptide chain with a five-fold anti-parallel beta-sheet structure (about 21% of the 113 amino acid residues) and two small segments of alpha-helices (about 16%). The region around the apparent reactive site, Met(73)-Val(74), is held tight by a combination of various structural features. The conformation of this region seems to have close similarity to that found in substrate analogues of low molecular weight bound to subtilisin BPN'.

Preliminary chemical and X-ray studies on the interactions of E. coli DNA with putrescine.

The interactions of putrescine, the major diamine in E. coli, with E. coli DNA as a model of phage DNA were studied by melting temperature analysis, equilibrium dialysis and X-ray diffraction with the aid of molecular model building. The chemical analysis of the DNA-putrescine complex shows that the molar binding ratio of putrescine to DNA (phosphate) is nearly 1 to 2. The equilibrium (or reversible) binding of putrescine to DNA was suggested by the fact that the melting temperature increased according to the concentration of added putrescine, and its elevation was not saturated even at the molar ratio of 6 to 1. The equilibrium dialysis experiments indicate that the association constant for the complex is a little smaller than, but in the same order (10(3) liter/mol) as, that of DNA-spermine complex. The binding of putrescine stabilizes the B-form of DNA fiber, which is well preserved even at 66% relative humidity. The distance between the neighboring DNA helices in the wet fiber increased with the increasing degree of hydration, as in the case of native DNA. Unlike spermine, putrescine does not form precipitate upon mixing with DNA in the concentration range for UV measurement, suggesting that the cross-bridge formed by putrescine is intra-double helical. The equilibrium binding of putrescine to DNA, seems to be important for the life cycle of lambda-phage.

Crystal structure of monoclinic ribonuclease-S at 4 A resolution. The mode of binding of 4-thiouridylic acid and a fragment of folic acid, p-aminobenzoylglutamic acid.

A four-A electron density map was calculated for the monoclinic crystal of ribonuclease-S (RNase-S) based on two heavy-atom derivatives. Close geometrical similarity was found between the two crystallographically independent RNase-S molecules (called molecules ZA and ZB) in this crystal and that (called molecule Y) in the trigonal crystal. Using the rotational and translational parameters relating these three molecules, it was established that the crystallographic two-fold symmetry between the two molecules ZA in the monoclinic crystal was exactly identical to that between the two molecules Y in the trigonal crystal, suggesting the tendency of RNase-S molecules to associate in this way although the interaction is weak. The 4-A difference Fourier maps calculated for the monoclinic crystal established the following conclusions. (1) 4-Thiouridine-2'(3')-monophosphates binds to the B1 and R1 sites like other pyrimidine nucleoside-2'(3')-monophosphates as expected from previous spectrophotometric studies, but not to the B2 site even at the concentration of 20 mM. An attempt to visualize the photoproduct generated by irradiation of near-ultraviolet light in this complex failed. (2) p-Aminobenzoylglutamic acid, a fragment of folic acid, seems to bind to RNase-S with its benzene ring close to the B2 site and the alpha-carboxylate group close to the p1 site. The model is compatible with most of the chemical results obtained by Sawada et al. ((1977) Biochim. Biophys. Acta 479, 188-197).

Interactions of Streptomyces subtilisin inhibitor with Streptomyces griseus proteases A and B. Enzyme kinetic and computer simulation studies.

Streptomyces subtilisin inhibitor (SSI), a dimeric protein that strongly inhibits subtilisins, was shown to form tight inhibitory complexes with Streptomyces griseus proteases A and B (SGPA and SGPB). The apparent dissociation constants of the SGPA-SSI and SGPB-SSI complexes were found to be orders of magnitude less than those of subtilisin-SSI complexes. Using the known atomic coordinates for SGPA and SSI, the highly complementary nature of the surface geometries of the two proteins was confirmed by a computer graphics study, which led to a proposed structure for the SGPA-SSI complex. Kinetic studies further suggested that the SSI dimer can bind two molecules of either SGPA or SGPB, and the 2:1-complexes (consisting of one inhibitor dimer and one enzyme molecule) apparently possess lower intrinsic dissociation constants than the 2:2-complexes. It was also shown that both of SGPA and SGPB are inhibited by both soybean trypsin inhibitor (Kunitz) and bovine pancreatic trypsin inhibitor (Kunitz), but far less strongly than by SSI.

Crystallization of calcium-calmodulin-trifluoperazine complex and an attempt at crystallizing calcium-free calmodulin.

Crystals suitable for X-ray structure analysis were obtained for CaM complexed with both calcium ions and a phenothiazine drug, trifluoperazine (TFP). The TFP/CaM binding ratio in crystals was experimentally determined to be nearly 1. An attempt at crystallizing calcium-free calmodulin (CaM) resulted in rigid but non-birefringent solids which exhibited no X-ray reflections.

Crystallization of arginine-, formylmethionine-tyrosine-, and glycine-transfer RNAs from Escherichia coli.

Crystals as large as 0.5 X 0.3 X 0.2 mm of purified arginine-transfer RNA from Escherichia coli have been prepared by a vapour diffusion method. X-ray diffraction photographs showed that the crystals gave reflections up to 3.7 A spacing. They have a trigonal space group P31 2 1 (OR P32 2 1) and cell-dimensions a=97.2, b=97.2, c=94.8A. Crystals of a mercury derivative of this transfer RNA have also been obtained, and an X-ray diffusion photography of one of them is presented. Formylmethionine-transfer RNA from E. coli was crystallized in various forms, and the appearance of the polymorphs was found to depend upon the amount of spermine in the solution from which the crystallization took place. Crystals of tyrosine-transfer RNA and glycine-transfer RNA have also been obtained.

Structure of aplasmomycin.

A new antibiotic, aplasmomycin, was isolated from a broth cultivated with a marine isolate of actinomycete, and inhibits Gram-positive bacteria in vitro and Plasmodium berghei in vivo. It is a natural ionophore and the structure of the Ag-salt was solved by an X-ray crystallographic analysis. It has symmetric structure having boron in the centre of the molecule.

Production, isolation and structure determination of a novel beta-glucosidase inhibitor, cyclophellitol, from Phellinus sp.

In the course of our screening of beta-glucosidase inhibitor, a culture filtrate of a mushroom, Phellinus sp. strongly inhibited the enzyme activity. The active substance was isolated through charcoal separation, column chromatography and crystallization. Spectroscopic and crystallographic analysis revealed that it had a novel cyclitol structure, (1S,2R,3S,4R,5R,6R)-5-hydroxymethyl-7-oxabicyclo[4,1,0]heptane-2,3,4-tri ol, and we named it cyclophellitol. It inhibited almond-derived beta-glucosidase with an IC50 of 0.8 micrograms/ml.

Structures of new antibiotics napyradiomycins.

Structures of novel antibiotics, napyradiomycins A, B1, B2, B3, C1 and C2 were determined. By X-ray crystallography, napyradiomycin B2 was determined to be (3R,10aR)-3-chloro-10a-[[(1R,3S)-3-chloro-2, 2-dimethyl-6-methylenecyclohexyl]methyl]-3,10a-dihydro-6,8-dihydro xy-2, 2-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione. The structures of other napyradiomycins were elucidated by NMR studies. Napyradiomycins C1 and C2 have unique structures which contain 14-membered ring cyclized by carbon-carbon bond.

Benastatins A and B, new inhibitors of glutathione S-transferase, produced by Streptomyces sp. MI384-DF12. II. Structure determination of benastatins A and B.

Benastatins A and B, new inhibitors of glutathione S-transferase, have been isolated from the culture broth of Streptomyces sp. MI384-DF12. By X-ray crystallography, benastatin A was determined to be 8,13-dihydro-1,7,9,11-tetrahydroxy-13-dimethyl-8-oxo-3-pentyl- benzo[a]naphthacene-2-carboxylic acid. The structure of benastatin B was elucidated by NMR studies.

Pironetin, a novel plant growth regulator produced by Streptomyces sp. NK10958. II. Structural elucidation.

A novel plant growth regulator, pironetin was isolated from the culture broth of Streptomyces sp. NK10958. The structure of pironetin was determined to be (5R,6R)-5-ethyl- 5,6-dihydro-6-[(E)-(2R,3S,4R,5S)-2-hydroxy-4-methoxy-3,5-dimethyl-7- nonenyl]-2H-pyran-2-one by FAB-MS, 1H and 13C NMR, COSY, COLOC, DEPT, IR, X-ray crystallographic analyses and adapted Mosher's method.

New antibiotic napyradiomycins A2 and B4 and stereochemistry of napyradiomycins.

Napyradiomycins A2 and B4, new members of the napyradiomycins, have been isolated from the culture broth of Chainia rubra MG802-AF1. The structure of napyradiomycin A2 was elucidated as 16-hydroxy-17-methylenenapyradiomycin A1 by NMR studies. The absolute structure of napyradiomycin B4 was determined as 13-hydroxy-13-methylnapyradiomycin B1 by X-ray crystallography and therefore the configuration of C(4a) in other napyradiomycins is assumed as the R configuration. The geometrical isomerism of napyradiomycin C1 was estimated as 12E and 16E by nuclear Overhauser effect experiments.

Studies on macrocyclic lactone antibiotics. VIII. Absolute structures of rhizoxin and a related compound.

The absolute structure of rhizoxin, a potent antifungal and antitumor antibiotic, was determined by interrelation with compound 2 whose structure was established by X-ray analysis. Since a 18OH group was introduced at C-3 on a hydrolytic cleavage of C-2, C-3 epoxy group with alkaline H2(18)O, the original epoxy oxygen should be retained at C-2. The stereo-chemistry at C-2 and C-3 positions in rhizoxin was, therefore, determined as 2R,3S.

Gualamycin, a novel acaricide produced by Streptomyces sp. NK11687. II. Structural elucidation.

A novel acaricide, gualamycin was isolated from the culture broth of Streptomyces sp. NK11687. The structure of gualamycin was determined to be (2R,3S,4S)-2-O-[4-O-(2-amino-2-deoxy-beta-D-gulopyranosyl)-alpha-D - galactopyranosyl]-2,3,4-trihydroxy-4-[(2S,3S,4S,5S)-3,4-dihydroxy-5-hydr oxy - methylpyrrolidin-2-yl] butanoic acid by FAB-MS, 1H and 13C NMR, COSY, HMQC, HMBC, IR, X-ray crystallographic analyses and synthetic studies.

Thrazarine, a new antitumor antibiotic. II. Physico-chemical properties and structure determination.

A new antitumor antibiotic thrazarine was soluble in water and positive to anisaldehyde-sulfuric acid and ninhydrin color reactions. The absolute structure of thrazarine was determined to be O-[3R)-2-diazo-3-hydroxybutyryl)-L-serine by acid hydrolysis, spectroscopic analysis and X-ray crystallographic analysis. Structurally, thrazarine was a new member of azaserine group antibiotics.

Bisucaberin, a new siderophore, sensitizing tumor cells to macrophage-mediated cytolysis. II. Physico-chemical properties and structure determination.

The structure of bisucaberin, a new siderophore, was determined to be 1,12-dihydroxy-1,6,12,17-tetraazacyclodocosane-2,5,13,16-tetron e by spectroscopic analysis and X-ray crystallographic analysis. The molecule of bisucaberin consists of a cyclic dimer of 1-hydroxy-1,6-diazaundecane-2,5-dione moiety and is closely related to nocardamine, the trimer of the same moiety.