Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.

N-terminal activation function is dominant in ligand-dependent transactivation of medaka estrogen receptor alpha in human cells.

Two independent transcriptional activation functions have been mapped to the N- and C-terminal domains of estrogen receptors (ERs), and are named activation function-1 (AF-1) and AF-2, respectively. Due to the lower activity of AF-1 and difficulties in producing AF-1 recombinant protein, little information is available regarding the biochemical properties of ER AF-1 and its coactivators compared to AF-2. In this study, we characterized the AF domains from medaka fish ERalpha (meERalpha) using a transient expression assay in cultured mammalian cells. While both meERalpha AF-1 and AF-2 were functional and gave similar results to human ERalpha AFs, meERalpha AF-1 displayed significant activity even in HeLa cells that exhibit little human ERalpha (hERalpha) AF-1 activity. Evidence of transcriptional squelching between hERalpha and meERalpha AF-1 molecules suggested that the molecules utilized common coactivators in mammalian cells. We also showed that large amounts of the meERalpha A/B domain could be expressed in Escherichia coli cells as a soluble protein, in contrast to hERalpha A/B domain protein which was not observed. Taken together, our results suggested that meERalpha AF-1 may have a more significant role in estrogen-induced function of meERalpha than AF-2 in medaka fish.

Caged and clustered structures of endothelin inhibitor BQ123, cyclo(-D-Trp-D-Asp--Pro-D-Val-Leu-)-Na+, forming five and six coordination bonds between sodium ions and peptides.

BQ123 is a cyclic pentapeptide and a potent endothelin-1 inhibitor. The crystal structure of the BQ123 sodium salt was determined as the first example of an endothelin inhibitor. Four independent molecules and many solvent molecules were found in the asymmetric unit; the total weight was about 3000 Da. The precise structure including the solvent molecules was determined using high-resolution data collected on a synchrotron source. Sodium ions formed unique structures with five and six coordination bonds and their forms were distinguished into three classes. An ion was sandwiched by two BQ123 molecules. This peptide-sodium (2:1) complex showed a cage-like structure and octahedral coordination was observed. Sodium ions also formed a cluster composed of hydrated water molecules and peptides. Two sodium ions were contained in this cluster, making five coordination bonds. Despite having the same coordination numbers, these ions were distinguishable by differences in the polyhedra. One was trigonal bipyramidal (having six planes) and the other was square pyramidal (having five planes). Both shapes were very similar to each other, although the synchrotron data clearly revealed slight geometrical differences.

Crystallization and structural analysis of intact maltotetraose-forming exo-amylase from Pseudomonas stutzeri.

The intact maltotetraose-forming exo-amylase from Pseudomonas stutzeri (G4-1), which has a raw starch binding domain, has been crystallized. The structure was identified (PDB entry 1GCY) by the molecular replacement method using the structure of its catalytic domain (G4-2). The result showed that the raw starch binding domain is in a disordered state, the corresponding electron densities being almost invisible. Superposition of these two enzyme forms showed evidence for the possible location of the raw starch binding domain (SBD). This crystal is a novel case, in that it forms a regular lattice incorporating flexibly bound SBD in the channel of crystal packing of the catalytic domains.

Effects of amino acids and chirality for molecular folding of desoxazoline-ascidiacyclamide derivatives: X-ray crystal structures of four cyclic octapeptides including unusual amino acids, cyclo(-Ile-aThr-D-Val-Thz-)(2), cyclo(-Ala-aThr-D-Val-Thz-Ile-aThr-D-Val-Thz-), cyclo(-Val-aThr-D-Val-Thz-Ile-aThr-D-Val-Thz-), and cyclo(-Ile-aThr-Val-Thz-Ile-aThr-D-Val-Thz-).

Desoxazoline derivative of ascidiacyclamide (1), cyclo(-L-Ile-L-allo-threonine-D-Val-thiazole-)(2), was modified to disturb the C(2)-symmetry. An Ile(1) residue of 1 was replaced for Ala (2) or Val (3), and the D-Val(3) residue was replaced for Val (4). The crystal structures of 1-4 were analyzed by x-ray diffraction methods. The molecules of all compounds were folded and this type of structure was not observed in x-ray structures of ascidiacyclamide derivatives so far except for patellamide D. The folding patterns of 1-4 were similar to each other and resembled that of patellamide. The asymmetric modifications at position 1 caused the conformational changes at local area, and these were related with the peptide-peptide and peptide-solvent interactions. Despite the diverse backbone conformation by the epimeric modification at position 3, the entire molecule of 4 was folded. These results mean that (1) the desoxazoline-ascidiacyclamides favored the folded structures and (2) the modifications of the side chain size at position 1 and the chirality at position 3 brought the local conformational changes to derivatives, suggesting that (3) the lack of the oxazoline block leads to conformational flexibility of 1-4, which accepts the conformational change with no drastic change on the entire structure.

Amphipathic structure of theonellapeptolide-Id, a hydrophobic tridecapeptide lactone from the Okinawa marine sponge Theonella swinhoei.

Theonellapeptolide-Id (TNLP), a cyclic tridecapeptide lactone, was crystallized from dimethylformamide-water solution. In the asymmetric unit, two peptide molecules were combined with solvent molecules, and the total molecular weight was over 3000 Dalton. The crystal structure including solvent molecules was finally determined at 0.80 A resolution using synchrotron radiation. The conformations of two independent molecules were similar to each other and were also similar to the previously reported structure (Doi, Ishida, Kobayashi, Deschamps and Flippen-Anderson, 1999, Acta Crystallogr Sect C, 55, 796-798). About 13 hydrated water molecules were found at disordered 19 sites; they were located at a certain region to avoid contact with aliphatic side-chains of peptolide in the crystal. The spatial disposition of the solvent molecules and peptides subsequently caused the formation of the amphipathic layer.

Crystallization and preliminary X-ray crystallographic analysis of alginate lyase A1-II from Sphingomonas species A1.

Alginate lyase A1-II of Sphingomonas species A1 was purified and crystallized using the hanging drop vapor-diffusion method in 0.1 M Tris-HCl buffer containing 43% saturated ammonium sulfate, 8% polyethylene glycol 4000 and 0.2 M Li(2)SO(4) at pH 8.5 and 20 degrees C. The crystals are tetragonal and belong to the space group P4(3)2(1)2 or P4(1)2(1)2 with unit cell dimensions of a=b=144.07 and c=296.38 A. The diffraction data up to 2.8 A were collected by a synchrotron radiation source at SPring-8 in Japan.

Three-dimensional structure of Pseudomonas isoamylase at 2.2 A resolution.

The three-dimensional structure of isoamylase from Pseudomonas amyloderamosa, which hydrolyzes alpha-1,6-glucosidic linkages of amylopectin and glycogen, has been determined by X-ray structure analysis. The enzyme has 750 amino acid residues and a molecular mass of 80 kDa, and it can be crystallized from ammonium sulfate solution. The structure was elucidated by the multiple isomorphous replacement method and refined at 2.2 A resolution, resulting in a final R-factor of 0.161 for significant reflections with a root-mean-square deviation from ideality in bond lengths of 0.009 A. The analysis revealed that in the N-terminal region, isoamylase has a novel extra domain that we call domain N, whose three-dimensional structure has not so far been reported. It has a (beta/alpha)8-barrel-type supersecondary structure in the catalytic domain common to the alpha-amylase family enzymes, though the barrel is incomplete, with a deletion of an alpha-helix between the fifth and sixth beta-strands. A long excursed region is present between the third beta-strand and the third alpha-helix of the barrel but, in contrast to the so-called domain B that has been identified in the other enzymes of alpha-amylase family, it cannot be considered to be an independent domain, because this loop forms a globular cluster together with the loop between the fourth beta-strand and the fourth alpha-helix. Isoamylase contains a bound calcium ion, but this is not in the same position as the conserved calcium ion that has been reported in other alpha-amylase family enzymes.

Scintigraphic and MRI studies in a patient with granulocytic sarcoma accompanied with CML and myelofibrosis.

Indium bone marrow, bone, and gallium scans and magnetic resonance imaging (MRI) were performed in a patient with granulocytic sarcoma accompanied with chronic myelocytic leukemia (CML) and myelofibrosis. The significance and implications of the findings of these studies are discussed.