CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs.

Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 A resolution. Each subunit within the homodimeric protein displays a mushroom-like structure, composed of five alpha-helices arranged in 'stalk' and 'head' subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so-called tetraspan-web.

Crystallization and preliminary crystallographic studies on the large extracellular domain of human CD81, a tetraspanin receptor for hepatitis C virus.

The large extracellular domain of CD81, a member of the tetraspanin family and a receptor protein for hepatitis C virus envelope E2 glycoprotein, has been expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data to 1.6 A resolution were obtained at the ID14 beamline of the European Synchrotron Radiation Facility from a flash-frozen crystal at 100 K. The crystals belong to space group P2(1), with unit-cell parameters a = 31.5, b = 77.2, c = 38.5 A, beta = 107.4 degrees, and are likely to contain two extracellular domains (2 x 99 residues) per asymmetric unit.

Crystal structure of human secretory phospholipase A2-IIA complex with the potent indolizine inhibitor 120-1032.

Phospholipase A2 is a key enzyme in a number of physiologically important cellular processes including inflammation and transmembrane signaling. Human secretory phospholipase A2-IIA is present at high concentrations in synovial fluid of patients with rheumatoid arthritis and in the plasma of patients with septic shock. Inhibitors of this enzyme have been suggested to be therapeutically useful non-steroidal anti-inflammatory drugs. The crystal structure of human secretory phospholipase A2-IIA bound to a novel potent indolizine inhibitor (120-1032) has been determined. The complex crystallizes in the space group P3121, with cell dimensions of a = b = 75.8 A and c = 51.3 A. The model was refined to an R-factor of 0. 183 for the intensity data collected to a resolution of 2.2 A. It was revealed that the inhibitor is located near the active site and bound to the calcium ion. Although the binding mode of the 120-1032 inhibitor to human secretory phospholipase A2-IIA is similar to that previously determined for an indole inhibitor LY311299, the specific interactions between the enzyme and the inhibitor in the present complex include the oxycarboxylate group which was introduced in this inhibitor. The oxycarboxylate group in 120-1032 is coordinated to the calcium ion and included in the water-mediated hydrogen bonding to the catalytic Asp49. In addition, the ethyl group in 120-1032 gains hydrophobic contacts with the cavity wall of the hydrophobic channel of the enzyme.

Crystal structure of DNA photolyase from Anacystis nidulans.

The crystal structure at 1.8 A resolution of 8-HDF type photolyase from A. nidulans shows a backbone structure similar to that of MTHF type E. coli photolyase but reveals a completely different binding site for the light-harvesting cofactor.

Crystallization and preliminary X-ray crystallographic studies of glutamic acid-specific proteinase from Bacillus licheniformis complex with Z-Leu-Glu-CH2Cl.

A glutamic acid specific proteinase from Bacillus licheniformis has been crystallized as a complex with the inhibitor Z-Leu-Glu-CH(2)Cl. Crystals were grown by the vapor-diffusion method using sodium formate as a precipitant. The crystals diffracted to about 2.0 A resolution and belonged to the trigonal space group P3(1)21 (P3(2)21) with unit-cell parameters a = b = 134.3, c = 109.7 A. A total of 26 964 independent reflections were obtained up to 2.2 A resolution, the merging R-factor being 0.05 for 42 614 measurements.

Crystal structure analysis of a serine proteinase from Streptomyces fradiae at 0.16-nm resolution and molecular modeling of an acidic-amino-acid-specific proteinase.

We have determined the three-dimensional structure of a proteinase from Streptomyces fradiae ATCC 14544 (SFase-2) at 0.16-nm resolution. SFase-2, a typical serine proteinase, has broad substrate specificity. The characterization and crystallographic analysis of this enzyme have been reported previously [Kitadokoro, K., Tsuzuki, H., Nakamura, E., Sato, T. & Teraoka, H. (1994) Eur. J. Biochem. 220, 55-61]. In the present study, data were collected to approximately 0.16-nm resolution on a Rigaku R-AXIS IIC imaging plate detector system. Preliminary phases were obtained by molecular replacement methods with a search model derived from the previously determined structure of Streptomyces griseus protease A [Sielecki, A. R., Hendrickson, W. A., Broughton, C. G., Delbaere, L. T., Brayer, G. D. & James, M. N. (1979) J. Mol. Biol. 134, 781-804]. The starting model gave an initial crystallographic R factor of 0.443. Refinement with restrained least-squares converged at a final R factor of 0.182 for 16128 observed reflections. The final model includes 86 water molecules. The crystal structure showed that the enzyme consists of two domains, each of which is comprised of a beta barrel with six-stranded beta sheets and two alpha helices. The overall tertiary structure of SFase-2 is similar to the structures of other chymotrypsin-like proteinases from S. griseus, namely proteinase A and proteinase B. The essential residues of the catalytic triad are located on the cleft between the two domains. These two domains have different sequences, but possess similar three-dimensional structures, indicating that a gene duplication event has occurred to produce these two domains. We predicted the tertiary structure of an acidic-amino-acid-specific proteinase on the basis of the crystal structure of SFase-2, and compared the active-site conformations of these two enzymes. We found a characteristic histidine cluster of three histidine residues in the active site of the acidic-amino-acid-specific proteinase. The substrate recognition mechanism of SFase-2 may be mediated through the hydrophobic amino acid residues. However, in the acidic-amino-acid-specific proteinase, the positive charge of this histidine cluster would attract the negative charges of glutamic acid and aspartic acid.

Purification, characterization, primary structure, crystallization and preliminary crystallographic study of a serine proteinase from Streptomyces fradiae ATCC 14544.

A proteinase having wide substrate specificity was isolated from Streptomyces fradiae ATCC 14544. This proteinase, which we propose to call SFase-2, was purified from the culture filtrate by S-Sepharose chromatography. The purified enzyme showed an apparent molecular mass of 19 kDa on SDS/PAGE. When synthetic peptides were used as substrates, SFase-2 showed broad substrate specificity. It also hydrolyzed keratin, elastin and collagen as proteinaceous substrates. It was completely inhibited by diisopropylfluorophosphate and chymostatin, but not by tosylphenylalaninechloromethane, tosyllysinechloromethane or EDTA, indicating that it can be classified as a serine proteinase. The matured protein sequence of SFase-2 was determined by a combination of amino acid sequencing and the DNA sequencing of the gene. SFase-2, consisting of 191 amino acids, is a novel proteinase. It showed 76% similarity in the amino acid sequence with Streptomyces griseus proteinase A [Johnson P. and Smillie L. B. (1974) FEBS Lett. 47, 1-6]. For insight into the three-dimensional structure of SFase-2, we obtained single crystals by the vapor diffusion method using sodium phosphate as a precipitant. These crystals belonged to the orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 6.92 nm, b = 7.28 nm, c = 2.99 nm; one molecule was present in the asymmetric unit.

Synthesis of manool-related labdane diterpenes as platelet aggregation inhibitors.

Enantioselective total synthesis of the labdane diterpene (-)-1, was achieved starting from the R-(-)-enantiomer of the Wieland-Miescher ketone. The enantiomer (+)-1 was obtained by partial synthesis via microbial transformation of sclareol. These results established that the natural compound (+)-1, a platelet aggregation inhibitor, has a normal absolute stereochemistry like that of manool. The B-norlabdane-related compound 44 was also synthesized using a novel ring contraction reaction.

Purification, characterization and molecular cloning of an acidic amino acid-specific proteinase from Streptomyces fradiae ATCC 14544.

We have isolated a novel acidic amino-acid-specific proteinase from Streptomyces fradiae ATCC 14544, using benzyloxycarbonyl-L-Phe-L-Leu-L-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. A proteinase, which we propose to call SFase, was purified from the culture filtrate by salting out, repeated S-Sepharose chromatography, and affinity chromatography (CH-Sepharose-Phe-Leu-D-Glu-OMe). The purified enzyme showed a single band having an apparent molecular weight of 19,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. When synthetic peptides were used as substrates, SFase showed high specificity for Z-Phe-Leu-Glu-pNA. Comparison with nitroanilides of glutamic acid and aspartic acid as substrates revealed that the reactivity was about 10-fold higher for a glutamyl bond than an aspartyl bond. SFase selectively hydrolyzed the -Glu-Ala-bond of two glutamyl bonds in the oxidized insulin B-chain within the initial reaction time until the starting material was completely digested. Diisopropylfluorophosphate and benzyloxycarbonyl-Phe-Leu-Glu chloromethylketone completely inhibited SFase, while metalloproteinase inhibitors, such as EDTA and o-phenanthrolin, did not inhibit the enzyme. The findings indicate that SFase can be classified as a serine proteinase, and is highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure and precursor of SFase, its gene was cloned from genomic DNA of the producing strain, and the nucleotide sequence was determined. Consideration of the N- and C-terminal amino-acid sequences of the mature protein of SFase indicates that it consists of 187 amino acids, which follows a prepropeptide of 170 residues. In comparison with the acidic amino-acid-specific proteinase from Streptomyces griseus (Svendsen, I., Jensen, M.R. and Breddam, K. (1991) FEBS Lett. 292, 165-167), SFase had 82% homology in the amino acid sequence. The processing site for maturation of SFase was a unique sequence (-Glu-Val-), so that the propeptide could be released by cleavage of the peptide bond between Glu and Val.

Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580.

A glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized. The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis. This protease, which we propose to call BLase (glutamic acid-specific protease from B. licheniformis ATCC 14580), was characterized enzymatically. Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds. Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme. The findings clearly indicate that BLase can be classified as a serine protease. To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B. licheniformis ATCC 14580, and the nucleotide sequence was determined. Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues. The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity. Its key physical and chemical characteristics were the same as those of the wild-type enzyme. BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K. (1992) Eur. J. Biochem. 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.

Crystallization and main-chain structure of neutral protease from Streptomyces caespitosus.

A neutral protease, i.e., a zinc-containing metalloendoprotease from Streptomyces caespitosus, has been crystallized using acetone as a precipitating agent. The crystals diffract to better than 1.5 A resolution when a rotating anode X-ray generator is used as an X-ray source. Protein phase angles were calculated by the multiple isomorphous replacement method using two heavy-atom derivatives (HgCl2 and CH3HgCl). A 6 A resolution electron density map clearly showed molecular boundaries. Although its amino acid sequence is not known, the folding pattern of the polypeptide chain could be traced on a 2.5 A resolution electron density map. A large cleft, which is located on the molecular surface, was proved to be the active site of the enzyme by structure analyses of inhibitor-complex crystals. The highest electron density peak, which corresponds to the cleft, was assigned to a catalytically essential zinc atom on difference Fourier synthesis between native and EDTA-soaked crystals.

Preliminary X-ray crystallographic study of lysozyme produced by Streptomyces globisporus.

Lysozyme from Streptomyces globisporus has been crystallized in a form suitable for X-ray structure analysis using ammonium sulfate as a precipitant. The crystals are hexagonal, space group P6(1)22 (P6(5)22) with unit cell dimensions: a = b = 129 A, c = 143 A. There are three or four molecules per asymmetric unit. The crystals diffract X-rays to at least 3.0 A resolution.