The 1.5-A crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii

The crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii has been determined at 1.5-A resolution with a crystallographic R factor of 16.8%. Plastocyanin is a small (98 amino acids), blue copper-binding protein that catalyzes the transfer of electrons in oxygenic photosynthesis from cytochrome f in the quinol oxidase complex to P700+ in photosystem I. Chlamydomonas reinhardtii plastocyanin is an eight-stranded, antiparallel beta-barrel with a single copper atom coordinated in quasitetrahedral geometry by two imidazole nitrogens (from His-37 and His-87), a cysteine sulfur (from Cys-84), and a methionine sulfur (from Met-92). The molecule contains a region of negative charge surrounding Tyr-83 (the putative distant site of electron transfer) and an exclusively hydrophobic region surrounding His-87; these regions are thought to be involved in the recognition of reaction partners for the purpose of directing electron transfer. Chlamydomonas reinhardtii plastocyanin is similar to the other plastocyanins of known structure, particularly the green algal plastocyanins from Enteromorpha prolifera and Scenedesmus obliquus. A potential "through-bond" path of electron transfer has been identified in the protein that involves the side chain of Tyr-83, the main-chain atoms between residues 83 and 84, the side chain of Cys-84, the copper atom, and the side chain of His-87.     

Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis

In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH. GS inactivation was completed within 30 min. Both the inactivated GS and the active enzyme were isolated. No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide. Mass spectrometry revealed that the molecular masses of active and inactive GS are equal. While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract. This result indicates that the active site was affected. From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration. GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen. In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2. The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen. Accordingly, the inactivating fraction was inhibited by catalase and EDTA. This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli. We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation.     

Solution structure of oxidized cytochrome c6 from the green alga Monoraphidium braunii

Cytochrome c6 from Monoraphidium braunii, an 89-amino acid electron transfer protein, has been investigated by NMR in solution, in its oxidized form, at pH 7 and 300 K. By using a combination of COSY, TOCSY, and NOESY experiments, 84% of the proton resonances have been assigned. A total of 1668 experimental NOE constraints, 1109 of which were meaningful, together with 288 pseudocontact shifts, have been used to determine the structure in solution. This is represented as a family of 40 structures which have been energy minimized. The rmsd values with respect to the mean structure are 0.57 +/- 0.08 and 0.94 +/- 0.09 A for the backbone and heavy atoms, respectively. The structure has been found to be very similar to that of the reduced form, except for a rearrangement in propionate 7, a feature which has been observed in all c-type cytochromes investigated so far. Such a feature could be relevant for the efficiency of the electron transfer pathway with either the oxidizing or the reducing partners. Other differences in the oxidation states have been noted in the region proposed to be involved in the interaction with the physiological partners.     

Lysogeny of blue-green alga Plectonema boryanum by long tailed virus

Lysogeny and its induction with mitomycin C by the long tailed virus is reported. The active virus was liberated in the medium during the growth period and ability of trichomes to produce virus was not reduced by treatment with EDTA.     

Solution structure of reduced microsomal rat cytochrome b5

The solution structure of the major form of the reduced soluble fragment of rat microsomal cytochrome b5 has been solved through 1H-NMR spectroscopy. The protein contains 98 amino acids. Proton assignment was available for residues 1-94, except 90 [Guiles, R. D., Basus, V. J., Kuntz, I. D. & Waskell, L. (1992) Biochemistry 31, 11,365-11,375] and has been confirmed. From 1722 NOEs, of which 1203 were found to be meaningful, a family of 40 energy-minimized structures has been obtained with average backbone rmsd (for residues 5-89) of 0.078 +/- 0.018 nm and average target function of 0.0045 nm2, no distance violations being larger than 0.029 nm. The structure has been compared with the X-ray structure of the oxidized rat mitochondrial isoenzyme and with that of the highly similar bovine microsomal isoenzyme in the oxidized form. The analysis of the elements of secondary structure is instructive in terms of their stability and of their occurrence in related structures, and of the capability of NMR and X-ray spectroscopy to observe them. Some detailed structural variations are noticed among the solved structures of the various isoenzymes and between solid and solution. The structural features in solution of the residues proposed to be involved in protein-protein recognition are found to be largely conserved with respect to the solid state.     

Cloning and correct expression in Escherichia coli of the petE and petJ genes respectively encoding plastocyanin and cytochrome c6 from the cyanobacterium Anabaena sp. PCC 7119

The genes coding for plastocyanin (petE) and cytochrome c6 (petJ) from Anabaena sp. PCC 7119 have been cloned and properly expressed in Escherichia coli. The recombinant proteins are identical to those purified from the cyanobacterial cells. The products of both the petE and petJ genes are correctly processed in E. coli, as deduced from their identical N-terminal amino acid sequences as compared with those of the metalloproteins isolated from the cyanobacterium. Physicochemical and functional properties of the native and recombinant protein preparations are also identical, thereby confirming that expression of petE and petJ genes in E. coli is an adequate tool to address the study of the structure/function relationships in plastocyanin and cytochrome c6 from Anabaena by site-directed mutagenesis.     

Sequence and mutational analysis of the devBCA gene cluster encoding a putative ABC transporter in the cyanobacterium Anabaena variabilis ATCC 29413

The devBCA gene cluster (dev for development), shown to be essential for envelope formation in heterocysts of Anabaena sp. strain PCC 7120, was identified in the gene bank of a second heterocyst-forming strain, Anabaena variabilis ATCC 29413. Sequence and structural organization of the three genes, encoding subunits of a presumptive ABC transporter, were nearly identical in both strains. Mutants of A. variabilis defective in the devA gene were constructed. As devA mutants of Anabaena 7120, A. variabilis mutants were unable to grow on N2 as sole nitrogen source due to incomplete differentiation of heterocysts.     

Solution structure of the SH3 domain from Bruton's tyrosine kinase

X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk). Btk belongs to the Tec family of tyrosine kinases. Each member of the family contains five regions and mutations causing XLA have been isolated in all five regions. We have determined the solution structure of the Src homology 3 (SH3) domain of Btk using two- and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on natural abundance and 15N-labeled protein material. The structure determination is complemented by investigation of backbone dynamics based on 15N NMR relaxation. The Btk SH3 forms a well-defined structure and shows the typical SH3 topology of two short antiparallel beta-sheets packed almost perpendicular to each other in a sandwich-like fold. The N- and C-termini are more flexible as are peptide fragments in the RT and n-Src loops. The studied Btk SH3 fragment adopts two slowly interconverting conformations with a relative concentration ratio of 7:1. The overall fold of the minor form is similar to that of the major form, as judged on the basis of observed NOE connectivities and small chemical shift differences. A tryptophan (W251) ring flip is the favored mechanism for interconversion, although other possibilities cannot be excluded. The side chain of Y223, which becomes autophosphorylated upon activation of Btk, is exposed within the potential SH3 ligand binding site. Finally, we compare the present Btk SH3 structure with other SH3 structures.     

Isolation and structure/activity features of halomon-related antitumor monoterpenes from the red alga Portieria hornemannii

Ten halogenated monoterpenes (2-6 and 8-12) related to the novel antitumor compound halomon (1) or to the carbocyclic analog 7 have been isolated from different geographic collections of the red alga, Portieria hornemannii. Structures were assigned to the basis of spectral analyses (primarily NMR and MS). The absolute configuration of isohalomon (2) was further established by X-ray crystallography. The compounds were comparatively evaluated alongside 1 and 7 in the U.S. National Cancer Institute's in vitro human tumor cell line screening panel. The results provide some interesting initial insights into the structure/activity relationships in this series.     

Ab initio determination of the crystal structure of cytochrome c6 and comparison with plastocyanin

BACKGROUND: Electron transfer between cytochrome f and photosystem I (PSI) can be accomplished by the heme-containing protein cytochrome c6 or by the copper-containing protein plastocyanin. Higher plants use plastocyanin as the only electron donor to PSI, whereas most green algae and cyanobacteria can use either, with similar kinetics, depending on the copper concentration in the culture medium. RESULTS: We report here the determination of the structure of cytochrome c6 from the green alga Monoraphidium braunii. Synchrotron X-ray data with an effective resolution of 1.2 A and the presence of one iron and three sulfur atoms enabled, possibly for the first time, the determination of an unknown protein structure by ab initio methods. Anisotropic refinement was accompanied by a decrease in the 'free' R value of over 7% the anisotropic motion is concentrated at the termini and between residues 38 and 53. The heme geometry is in very good agreement with a new set of heme distances derived from the structures of small molecules. This is probably the most precise structure of a heme protein to date. CONCLUSIONS: On the basis of this cytochrome c6 structure, we have calculated potential electron transfer pathways and made comparisons with similar analyses for plastocyanin. Electron transfer between the copper redox center of plastocyanin to PSI and from cytochrome f is believed to involve two sites on the protein. In contrast, cytochrome c6 may well use just one electron transfer site, close to the heme unit, in its corresponding reactions with the same two redox partners.     

Inhibition of tumor invasion and metastasis by calcium spirulan (Ca-SP), a novel sulfated polysaccharide derived from a blue-green alga, Spirulina platensis

We have investigated the effect of calcium spirulan (Ca-SP) isolated from a blue-green alga, Spirulina platensis, which is a sulfated polysaccharide chelating calcium and mainly composed of rhamnose, on invasion of B16-BL6 melanoma, Colon 26 M3.1 carcinoma and HT-1080 fibrosarcoma cells through reconstituted basement membrane (Matrigel). Ca-SP significantly inhibited the invasion of these tumor cells through Matrigel/fibronectin-coated filters. Ca-SP also inhibited the haptotactic migration of tumor cells to laminin, but it had no effect on that to fibronectin. Ca-SP prevented the adhesion of B16-BL6 cells to Matrigel and laminin substrates but did not affect the adhesion to fibronectin. The pretreatment of tumor cells with Ca-SP inhibited the adhesion to laminin, while the pretreatment of laminin substrates did not. Ca-SP had no effect on the production and activation of type IV collagenase in gelatin zymography. In contrast, Ca-SP significantly inhibited degradation of heparan sulfate by purified heparanase. The experimental lung metastasis was significantly reduced by co-injection of B16-BL6 cells with Ca-SP. Seven intermittent i.v. injections of 100 microg of Ca-SP caused a marked decrease of lung tumor colonization of B16-BL6 cells in a spontaneous lung metastasis model. These results suggest that Ca-SP, a novel sulfated polysaccharide, could reduce the lung metastasis of B16-BL6 melanoma cells, by inhibiting the tumor invasion of basement membrane probably through the prevention of the adhesion and migration of tumor cells to laminin substrate and of the heparanase activity.     

Sulfoquinovosyl diacylglycerols from the alga Heterosigma carterae

An extract of the chloromonad Heterosigma carterae (Raphidophyceae), cultivated in natural seawater, contained a complex mixture of sulfoquinovosyl diacylglycerols. Palmitoyl (16:0), three isomers of hexadecenoyl (16:1 cis delta 9, delta 11, delta 13), and eicosapentenoyl (20:5) were found to be the main fatty acyl substituents. Exact double-bond sites were determined by mass spectrometry analysis of the corresponding nicotinyl derivatives. Four major sulfoquinovosyl diacylglycerol components were partially purified and identified as 1-4 by interpretation of their nuclear magnetic resonance and mass spectral data. In addition, complete analysis of the H. carterae sulfoquinovosyl diacylglycerols was performed using high-performance liquid chromatography combined with electrospray tandem mass spectrometry.     

Solution structure of drosomycin, the first inducible antifungal protein from insects

Drosomycin is the first antifungal protein characterized recently among the broad family of inducible peptides and proteins produced by insects to respond to bacterial or septic injuries. It is a small protein of 44 amino acid residues extracted from Drosophila melanogaster that exhibits a potent activity against filamentous fungi. Its three-dimensional structure in aqueous solution was determined using 1H 2D NMR. This structure, involving an alpha-helix and a twisted three-stranded beta-sheet, is stabilized by three disulfide bridges. The corresponding Cysteine Stabilized alpha beta (CS alpha beta) motif, which was found in other defense proteins such as the antibacterial insect defensin A, short- and long-chain scorpion toxins, as well as in plant thionins and potent antifungal plant defensins, appears as remarkably persistent along evolution.     

Solution structure and peptide binding of the SH3 domain from human Fyn

BACKGROUND: The Src family of tyrosine kinases is involved in the propagation of intracellular signals from many transmembrane receptors. Each member of the family contains two domains that regulate interactions with other molecules, one of which is the Src homology 3 (SH3) domain. Although structures have previously been determined for SH3 domains, and ideas about peptide-binding modes have been proposed, their physiological role is still unclear. RESULTS: We have determined the solution structure of the SH3 domain from the Src family tyrosine kinase Fyn in two forms: unbound and complexed with a peptide corresponding to a putative ligand sequence from phosphatidylinositol 3' kinase. Fyn SH3 shows the typical SH3 topology of two perpendicular three-stranded beta sheets and a single turn of 3(10) helix. The interaction of SH3 with three potential ligand peptides was investigated, demonstrating that they all bind to the same site on the molecule. A previous model for ligand binding to SH3 domains predicts binding in one of two orientations (class I or II), each characterized by a consensus sequence. The ligand with the closest match to the class I consensus sequence bound with highest affinity and in the predicted orientation. CONCLUSIONS: The Fyn SH3 domain has a well-defined structure in solution. The relative binding affinities of the three ligand peptides and their orientation within the Fyn SH3 complex were consistent with recently proposed models for the binding of 'consensus' polyproline sequences. Although the affinities of consensus and non-consensus peptides are different, the degree of difference is not very large, suggesting that SH3 domains bind to polyproline peptides in a promiscuous manner.     

Solution structure of a beta-neurotoxin from the New World scorpion Centruroides sculpturatus Ewing

We report the detailed solution structure of the 7.2 kDa protein CsE-I, a beta-neurotoxin from the New World scorpion Centruroides sculpturatus Ewing. This toxin binds to sodium channels, but unlike the alpha-neurotoxins, shifts the voltage of activation toward more negative potentials causing the membrane to fire spontaneously. Sequence-specific proton NMR assignments were made using 600 MHz 2D-NMR data. Distance geometry and dynamical simulated annealing refinements were performed using experimental distance and torsion angle constraints from NOESY and pH-COSY data. A family of 40 structures without constraint violations was generated, and an energy-minimized average structure was computed. The backbone conformation of the CsE-I toxin shows similar secondary structural features as the prototypical alpha-neurotoxin, CsE-v3, and is characterized by a short 2(1/2)-turn alpha-helix and a 3-strand antiparallel beta-sheet, both held together by disulfide bridges. The RMSD for the backbone atoms between CsE-I and CsE-v3 is 1.48 A. Despite this similarity in the overall backbone folding, the these two proteins show some important differences in the primary structure (sequence) and electrostatic potential surfaces. Our studies provide a basis for unravelling the role of these differences in relation to the known differences in the receptor sites on the voltage sensitive sodium channel for the alpha- and beta-neurotoxins.     

Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus

The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, has been determined from 2D 1H NMR data. Robustoxin is a polypeptide of 42 residues cross-linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1-15, 8-20, 14-31 and 16-42 (a 1-4/2-6/3-7/5-8 pattern). The structure consists of a small three-stranded, anti-parallel beta-sheet and a series of interlocking gamma-turns at the C-terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the omega-conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non-polar residues. Both of these structural features may play a role in its binding to the voltage-gated sodium channel.     

Solution structure of the tobramycin-RNA aptamer complex

We have solved the solution structure of the aminoglycoside antibiotic tobramycin complexed with a stem-loop RNA aptamer. The 14 base loop of the RNA aptamer 'zippers up' alongside the attached stem through alignment of four mismatches and one Watson-Crick pair on complex formation. The tobramycin inserts into the deep groove centered about the mismatch pairs and is partially encapsulated between its floor and a looped out guanine base that flaps over the bound antibiotic. Several potential intermolecular hydrogen bonds between the charged NH3 groups of tobramycin and acceptor atoms on base pair edges and backbone phosphates anchor the aminoglycoside antibiotic within its sequence/structure specific RNA binding pocket.