Structural organization of the fibrinogen molecule

A brief review of the chemical structure of the fibrinogen molecule, its proteolytic fragmentation, physicochemical and functional properties of proteolytic fragments is presented. The previous notions on the structure of the fibrinogen molecule are considered as well as new data obtained by the methods of scanning microcalorimetry, electron microscopy and by other methods which permit suggesting some models for structural organization of the fibrinogen molecule.     

Two contrary functions of tenascin: dissection of the active sites by recombinant tenascin fragments

A structural and functional model of tenascin was elaborated using recombinant parts of three alternatively spliced tenascin variants and anti-tenascin monoclonal antibodies. The fusion proteins were compared with intact tenascin for their functions and by electron microscopy. A strong cell binding site was localized within 104 amino acids. This fragment also contains the epitope of the monoclonal antibody anti-Tn68, which inhibits cell attachment to tenascin and binds near the tips of the six arms of tenascin. In contrast, constructs containing the 13 1/2 EGF-like repeats of tenascin showed an antiadhesive effect. The coexistence of the two contrary signals on the same molecule might be responsible for the versatile features of tenascin.     

Isolation and Characterization of a Bacteriophage Lytic for Desulfovibrio salexigens, a Salt-Requiring, Sulfate-Reducing Bacterium

A bacteriophage that lysed Desulfovibrio salexigens cells was isolated from marine sediments and preliminarily characterized by electron microscopy and electrophoretic analysis of structural proteins and genomic nucleic acid. The bacteriophage had an icosahedral head and a long flexible tail, and the buoyant density of the bacteriophage particles was 1.468 g/ml in cesium chloride. The particles consisted of a double-stranded DNA molecule about 33 kilobase pairs long and at least 11 structural proteins.     

The unique hydrogen bonded water in the reduced form of<Emphasis Type="Italic"> Clostridium pasteurianum</Emphasis> rubredoxin and its possible role in electron transfer

Rubredoxin is a small iron-sulfur (FeS₄) protein involved in oxidation–reduction reactions. The side chain of Leu41 near the iron-sulfur center has two conformations, which we suggested previously serve as a gate for a water molecule during the electron transfer process. To establish the role of residue 41 in electron transfer, an [L41A] mutant of Clostridium pasteurianum rubredoxin was constructed and crystallized in both oxidation states. Despite the lack of the gating side chain in this protein, the structure of the reduced [L41A] rubredoxin reveals a specific water molecule in the same position as observed in the reduced wild-type rubredoxin. In contrast, both the wild-type and [L41A] rubredoxins in the oxidized state do not have water molecules in this location. The reduction potential of the [L41A] variant was ~50 mV more positive than wild-type. Based on these observations, it is proposed that the site around the S of Cys9 serves as a port for an electron acceptor. Lastly, the Fe–S distances of the reduced rubredoxin are expanded, while the hydrogen bonds between S of the cysteines and the backbone amide nitrogens are shortened compared to its oxidized counterpart. This small structural perturbation in the Fe(II)/Fe(III) transition is closely related to the small energy difference which is important in an effective electron transfer agent.     

Structure and biological activity of hagfish insulin

An isomorphously phased electron density map of hagfish (Myxine glutinosa) insulin has been calculated at a resolution of 3·1 Å spacing. The molecule crystallises with one molecule per asymmetric unit but is organised as a symmetric dimer lying on a 2-fold crystal axis. The structure of the hagfish insulin monomer is much more similar to that of pig insulin molecule 2 than molecule 1 of the dimer that constitutes one third of the 2 Zn insulin hexamer. There are different conformations however at the N and C termini of the B-chain. At the C terminus the two final residues on hagfish insulin partially obscure the A1 glycine residue, which in pig insulin is exposed. This structural difference has been shown, however, not to be responsible for the reduced activity of the hagfish insulin.     

Structure of calmodulin refined at 2.2 A resolution

The crystal structure of mammalian calmodulin has been refined at 2.2 A (1 A = 0.1 nm) resolution using a restrained least-squares method. The final crystallographic R-factor, based on 6685 reflections in the range 2.2 A less than or equal to d less than or equal to 5.0 A with intensities exceeding 2.5 sigma, is 0.175. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.7 degrees, respectively. The refined model includes residues 5 to 147, four Ca2+ and 69 water molecules per molecule of calmodulin. The electron density for residues 1 to 4 and 148 is poorly defined, and they are not included in the model. The molecule is shaped somewhat like a dumbbell, with an overall length of 65 A; the two lobes are connected by a seven-turn alpha-helix. Prominent secondary structural features include seven alpha-helices, four Ca2+-binding loops, and two short, double-stranded antiparallel beta-sheets between pairs of adjacent Ca2+-binding loops. The four Ca2+-binding domains in calmodulin have a typical EF hand conformation (helix-loop-helix) and are similar to those described in other Ca2+-binding proteins. The X-ray structure determination of calmodulin shows a large hydrophobic cleft in each half of the molecule. These hydrophobic regions probably represent the sites of interaction with many of the pharmacological agents known to bind to calmodulin.     

Mechanism of photosynthetic water oxidation: combining biophysical studies of photosystem II with inorganic model chemistry

A mechanism for photosynthetic water oxidation is proposed based on a structural model of the oxygen-evolving complex (OEC) and its placement into the modeled structure of the D1/D2 core of photosystem II. The structural model of the OEC satisfies many of the geometrical constraints imposed by spectroscopic and biophysical results. The model includes the tetranuclear manganese cluster, calcium, chloride, tyrosine Z, H190, D170, H332 and H337 of the D1 polypeptide and is patterned after the reversible O2-binding diferric site in oxyhemerythrin. The mechanism for water oxidation readily follows from the structural model. Concerted proton-coupled electron transfer in the S2-->S3 and S3-->S4 transitions forms a terminal Mn(V)=O moiety. Nucleophilic attack on this electron-deficient Mn(V)=O by a calcium-bound water molecule results in a Mn(III)-OOH species, similar to the ferric hydroperoxide in oxyhemerythrin. Dioxygen is released in a manner analogous to that in oxyhemerythrin, concomitant with reduction of manganese and protonation of a mu-oxo bridge.     

Comparison of backbone structures of glucose isomerase from Streptomyces and Arthrobacter

The C alpha backbones of the glucose isomerase molecules of Streptomyces rubiginosus and Arthrobacter have been determined by X-ray crystallography and compared. Each molecule is a tetramer of eight-stranded alpha/beta barrels, and the mode of association of the tetramers is identical in each case. The Arthrobacter electron density shows four additional amino acids at the carboxyl terminus. There is also an insertion of six amino acids at position 277, and two individual insertions at about positions 348 and 357 (numbering according to the Streptomyces structure). There is a close structural homology throughout the whole molecule, which is most accurate up to position 325. The r.m.s. displacement for 315 homologous C alpha positions up to this position is 0.92 A.     

Structure of Streptomyces erythraeus lysozyme at 6 A resolution

A 6 A resolution electron density map has been calculated for a bacterial lysozyme produced by Streptomyces erythraeus. This lysozyme differs from the vertebrate lysozyme in its size, amino acid composition, and specificity. The structure was determined by the method of isomorphous replacement. Three heavy atom derivatives were obtained by soaking crystals of the lysozyme in HgCl2, K2PtCl4, and UO2(NO3)26H2O. The resulting electron density map clearly shows the molecular boundary. The molecule is ellipsoidal in shape with average dimensions 50 A X 35 A X 35 A. High resolution analysis and sequence analysis of the molecule are in progress.     

Secondary structure of coliphage Q beta RNA. Analysis by electron microscopy

The secondary structure of genomic RNA from the coliphage Q beta has been examined by electron microscopy in the presence of varying concentrations of spermidine using the Kleinschmidt spreading technique. The size and position of structural features that cover 70% of the viral genome have been mapped. The structural features that are visualized by electron microscopy in Q beta RNA are large. They range in size from 170 to 1600 nucleotides. A loop containing approximately 450 nucleotides is located at the 5' end of the RNA. It includes the initiation region for the viral maturation protein. A large hairpin containing approximately 1600 nucleotides is located in the center of the molecule. It is multibranched and includes most of the viral coat gene, the readthrough region of the A1 gene, and approximately one third of the viral replicase gene. Within the central hairpin, the initiation region for the viral replicase gene pairs with a region within the distal third of the viral coat gene. This structure may participate in the regulation of translational initiation of the viral replicase gene. Two structural variants of the central hairpin were observed. One of them brings the internal S and M viral replicase binding regions into juxtaposition. These observations suggest that the central hairpin may also participate in the regulation of translation of the viral coat gene. The secondary structures that are observed in Q beta RNA differ significantly from structures that we described previously in the genomic RNA of coliphage MS2 but are similar to structures we observed by electron microscopy in the related group B coliphage SP.     

An open conformation of switch I revealed by Sar1-GDP crystal structure at low Mg2+

Mg2+ is essential for guanosine triphosphatase activity and plays key roles in guanine nucleotide binding and preserving the structural integrity of GTP-binding proteins. To understand the structural basis for Mg2+ function during the GDP/GTP exchange process, we determined the crystal structure of Delta9-Sar1-GDP at low Mg2+ concentration at 1.8A. Two Sar1-GDP molecules in the crystal form a dimer with Mg2+ presenting only in molecule B but not in molecule A. The absence of Mg2+ induces significant conformational changes in the switch I region in molecule A that shows similarities with those of Ha-Ras bound to Sos. The current structure reveals an important regulatory role for Mg2+. We suggest that guanine nucleotide exchange factor may utilize this feature to generate an open conformation for GDP/GTP exchange. Furthermore, we propose a mechanism for COPII assembly and disassembly in which dimerization of Sar1 plays an important role.     

The reaction of Rhodospirillum rubrum cytochrome c2 with iron hexacyanides.

The reaction of Rhodospirillum rubrum cytochrome c2 with the nonphysiological reactants, ferrocyanide and ferricyanide has been investigated as a function of ionic strength, temperature and pH, using both stopped-flow and temperature-jump kinetic methods. The results are consistent with a complex reaction mechanism involving the formation of two intermediate complexes. The site of electron transfer appears to be at the front of the cytochrome c2 molecule near the hem e crevice with interacton of both ferri and ferrocyanide with a positively charged region of the molecule. Comparison of the proposed electron transfer mechanism of cytochrome c2 with ferro-ferricyanide is made with the mechanism proposed based upon structural considerations.     

Crystals of crotoxin suitable for high resolution x-ray diffraction analysis

The phospholipasic presynaptic neurotoxin, crotoxin, has been crystallized in a morphology suitable for single crystal x-ray diffraction analysis. The conditions for growth and the unit cell parameters (P4(1)22 or P4(3)22, a = b = 38.5 A, c = 256.9 A, 1 molecule/asymmetric unit) are similar to the very thin plate-like crystals which have been studied with electron diffraction and electron microscopy by Chiu and his colleagues (Jeng, T.-W., Chiu, W., Zemlin, F., and Zeitler, E. (1984) J. Mol. Biol. 175, 93 - 97). These two macroscopic crystal morphologies of what is likely to be a very similar, if not identical, lattice structure will permit the complementary application of electron diffraction/microscopy and x-ray diffraction to understanding the structural basis of the interactions between a phospholipasic neurotoxin and its membrane target.     

Study of the structure of immunoglobins by small-angle x-ray diffraction. I. The structure of IgMCep in solution

The data of small-angle X-ray scattering from monoclonal immunoglobulin MCep (IgM) enabled the shape and geometrical parameters of the molecule in solution at 23 degrees C to be established. The molecule is a flat, strongly anisometric particle with radius of gyration 115 A, volume 1,8 X 10(6) A3, maximum size 380 A, thickness 35-40 A. The most probable molecular model in the approximation of homogeneous electron density in the molecule was suggested, its geometry fitting the experimental parameters. The five IgM subunits are located in the equatorial plane, low-electronic-density regions are located in the centre and at the periphery of the macromolecule. In addition, the absence of fixed angle values between Fab-regions in each subunit is indicative of rather high structural mobility at the periphery of the IgM molecule.     

Interaction of bromophenol blue with serum albumin: criteria for using dyes for assessing the state and quantity of protein

The optical properties of the complexes of the pH-dependent dye bromophenol blue (BPB) with human serum albumin were investigated by the spectrophotometric method. The solvatochromic longwave displacement of bound BPB-2 absorption and BPB-1/BPB-2 redistribution were shown to form the optical signal of complexes. Because of the distortion of the bound BPB-2 signal its quantity was determined as delta A630 = A630 - A660 and the use of lambda max as structural parameter was limited to low pH less than or equal to 3. The conclusion was made that BPB is inapplicable as a structural probe on account of low structural dependence of delta A630 and pH-limitation of lambda max used. The maximal absorption delta Amax = Amax - A660 and its structural independence were obtained in the region of 70-100% occupation of the dye-binding centers of the protein. It is the optimal conditions for the quantitative determination of protein. After maximal dye binding (15-16 molecules of BPB per 1 molecule of albumin) the aggregation and precipitation of the complexes occurred.     

Tricolorin A, a potent natural uncoupler and inhibitor of photosystem II acceptor side of spinach chloroplasts

Tricolorin A, (11 S )-11-hydroxyhexadecanoic acid 11- O - α - l - rhamnopyranosyl-(1↠3)- O - α - l -{2- O -(2 S -methylbutanoyl)-4- O -(2 S -methylbutanoyl)}-rhamnopyranosil-(1↠2)- O - β - d -glucopyranosil-(1↠2)- β -fucopyranoside-(1,3'-lactone), the major phytogrowth inhibitor isolated from Ipomoea tricolor Cav. (Convolvulaceae) was found to be a potent uncoupler (U₅₀=0.33 μ M ) of photophosphorylation in spinach chloroplasts. Tricolorin A inhibited H⁺-uptake and adenosine 5'-triphosphate (ATP) synthesis, and stimulated basal and phosphorylating electron flows. Using a combination of two well-known fluorescent ΔpH probes, 9-aminoacridine and 9-amino-6-chloro-2-methoxyacridine, the uncoupling behavior of tricolorin A was also demonstrated for submitochondrial particles. Polarographic data showed that high concentrations (20 μ M ) of tricolorin A inhibited photosystem II (PSII) electron flow at the level of plastoquinone B (Q_B). Chlorophyll (Chl) a fluorescence analysis showed that tricolorin A induced accumulation of Q_A⁻ and strongly decreased the electron transport capacity, suggesting that the target of this molecule was located at the Q_B level. The macrocyclic lactone-type structure of this allelopathic agent proved to be an important structural requirement for uncoupling activity since its hydrolysis caused loss of the inhibitory potential.     

Purification and analysis of authentic CLIP-170 and recombinant fragments.

We have purified authentic CLIP-170 (cytoplasmic linker protein of 170 kDa) and fragments comprising functional domains of the protein to characterize the structural basis of the function of CLIP-170. Analysis of authentic CLIP-170 and the recombinant fragments by electron microscopy after glycerol spraying/low angle rotary metal shadowing reveals CLIP-170 as a thin, 135-nm-long molecule with two kinks in its central rod domain, which are approximately equally spaced from the two ends of the protein. The central domain consisting of heptad repeats, which is alpha-helical in nature and forms a 2-stranded coiled-coil, mediates dimerization of CLIP-170. The rod domain harbors two kinks, each spaced approximately 37 nm from the corresponding end of the molecule, thus providing mechanical flexibility to the highly elongated molecule. The N-terminal domain of CLIP-170 binds to microtubules in vitro with a stoichiometry of one dimeric head domain per four tubulin heterodimers. Authentic CLIP-170 binds to microtubules with lower stoichiometry, indicating that the rod and tail domains affect microtubule binding of CLIP-170. These results document that CLIP-170 is a highly elongated polar molecule with the microtubule-binding domain and the organelle-interacting domains at opposite ends of the homodimer, thus providing a structural basis for the function of CLIP-170 as a microtubule-organelle linker protein.     

Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Ogamma. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.     

Visualization of beta-sheets and side-chain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography.

The biotin-binding protein streptavidin was crystallized as two-dimensional periodic arrays on biotinylated phospholipid monolayers. Electron diffraction patterns and images of the arrays embedded in vitreous ice were recorded to near-atomic resolution. Amplitudes and phases of structure factors were computed and combined to produce a 3 A projection density map. The reliability of the map was verified by comparing it to the available x-ray atomic model of the molecule. Projection densities from beta-strands and some amino acid side chains were identified from the electron cryomicroscopy map. These results demonstrate the first near-atomic image of this type of protein periodic array by electron crystallography, which has a great potential to aid in the structural characterization of molecular arrays engineered on a monolayer for various basic or biotechnological applications.     

Three-dimensional structure of transporter associated with antigen processing (TAP) obtained by single Particle image analysis

The transporter associated with antigen processing (TAP) is an ATP binding cassette transporter responsible for peptide translocation into the lumen of the endoplasmic reticulum for assembly with major histocompatibility complex class I molecules. Immunoaffinity-purified TAP particles comprising TAP1 and TAP2 polypeptides, and TAP2 particles alone were characterized after detergent solubilization and studied by electron microscopy. Projection structures of TAP1+2 particles reveal a molecule approximately 10 nm across with a deeply staining central region, whereas TAP2 molecules are smaller in projection. A three-dimensional structure of TAP reveals it is isolated as a single heterodimeric complex, with the TAP1 and TAP2 subunits combining to create a central 3-nm-diameter pocket on the predicted endoplasmic reticulum-lumenal side. Its structural similarity to other ABC transporters demonstrates a common tertiary structure for this diverse family of membrane proteins.