Examination of the platelet membrane glycoprotein IIb-IIIa complex and its interaction with fibrinogen and other ligands by electron microscopy.

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), is a calcium-dependent heterodimer that binds fibrinogen, von Willebrand factor, and fibronectin after platelet activation. We examined GPIIb-IIIa alone and bound to these ligands by electron microscopy after rotary shadowing with platinum/tungsten. We found, as observed previously, that in the presence of detergent and 2 mM Ca2+, GPIIb-IIIa consists of an 8 x 12-nm globular head with two 18-nm flexible tails extending from one side. We also found that in the presence of EDTA, GPIIb-IIIa dissociates into two similar comma-shaped subunits, each containing a portion of the globular head and a single tail. Using monoclonal antibodies to GPIIb, GPIIIa, and the GPIIb-IIIa heterodimer, we found that the tails contained the carboxyl termini of each subunit, while the nodular head was composed of amino-terminal segments of both subunits. Electron microscopy of GPIIb-IIIa bound to fibrinogen revealed a highly specific interaction of the nodular head of GPIIb-IIIa with the distal end of the trinodular fibrinogen molecule and with the tails of GPIIb-IIIa extended laterally at an angle of approximately 98 degrees with respect to the long axis of fibrinogen. When a GPIIb-IIIa was bound to each end of a single fibrinogen, the tails were oriented to opposite sides of fibrinogen, enabling fibrinogen to bridge two adjacent platelets. Electron microscopy of GPIIb-IIIa bound to fibronectin revealed GPIIb/IIIa-binding sites approximately two-thirds of the distance from the amino terminus of each end of the fibronectin molecule, while GPIIb-IIIa was found to bind to von Willebrand factor protomers along a rod-like region near the central nodule of the molecule.     

Two ubiquitin-long-tail fusion genes arranged as closely spaced direct repeats in barley.

Ubiquitin (Ubi) genes encode two types of fusion proteins: polyUbi with a varying number of direct repeats of Ubi, and Ubi-tail fusions with long or short basic C-terminal extensions. A barley (Hordeum vulgare) genomic clone has been isolated with two very similar, intronless genes encoding monoUbi-long-tail fusion peptides. The genes are arranged as direct repeats separated by 3 kb of DNA and account for two of the probable three long-tail genes in the haploid barley genome. Both genes are active and give rise to messengers about 800 nt long. The sequence of the encoded Ubi moieties is identical to the sequence of Ubi repeats of polyUbi precursors from barley and other plants. The basic tails of the peptides are 79 aa long and 71-72% homologous to corresponding sequences from yeast and man. Recently, it was found that the long and short tails are ribosomal proteins in yeast [Finley et al., Nature 338 (1989) 394-401] and the evolutionary conservation of the structure of the Ubi-tail fusion genes suggests that they serve the same function in plants. The similarity between yeast and barley Ubi-long-tail fusion genes may extend to the regulatory regions, since upstream activating sites characteristic of ribosomal protein-encoding genes in yeast (UASrpg) were found in the barley genes.     

Structure and mass analysis of 14S dynein obtained from Tetrahymena cilia

Scanning transmission electron microscopic analysis revealed that the 14S fraction of Tetrahymena dynein was of a mixture of two types of particles in approximately equal proportions. The 14S dynein molecules were roughly ellipsoid in shape with approximate axes of 9.5 and 14.5 nm. About half of the particles had tails 20-24-nm long. By the integration of electron scattering intensities, particles with tails had an average mass of 510 kD with a SD of 90 kD. The globular heads of both types of particles had an average mass of 330 kD with a SD of 60 kD. The mass of the tail structure was about 180 kD. By SDS-PAGE, the 14S dynein consisted of two high molecular mass polypeptides above 300 kD that could be distinguished by immunoblot analysis.     

Structural analysis of homologous repeated domains in alpha-actinin and spectrin

The amino acid sequences of chick and slime mould alpha-actinin each contain four repeats of approximately 122 residues. These repeats are homologous to the 18-22 repeats, each of approximately 106 residues, found in the alpha and beta subunits of spectrin and fodrin, and to the multiple repeats of approximately 110 residues found in the Duchenne muscular dystrophy protein (dystrophin). The repeats correspond to the elongated rod-like portion of these molecules. We present a multiple sequence alignment of 21 repeats from this superfamily (8 alpha-actinin and 13 spectrin/fodrin), based on optimal pairwise alignments, from which a characteristic consensus pattern of amino acid types is deduced. Trp 46 is invariant in all but one repeat, and physicochemical classes of amino acids are conserved at 25 other positions. Secondary structure prediction on both the alpha-actinin and spectrin repeats taken together with the distribution of proline residues in the sequences, strongly suggest that each repeated domain consists of a four-helix structure. Our predictions differ significantly from previous three-helix models based on analyses of fewer sequences. To determine possible interdomain regions, sites of limited proteolysis of the native chick alpha-actinin dimer were determined and located in the amino acid sequence. The majority of these sites were in corresponding positions in different repeats within a segment predicted as a long helix. We propose a model, consistent with the overall dimensions of the rod-like portions of the molecules, in which these long, probably interrupted helices, link adjacent domains.     

Low resolution crystal structure of Arenicola erythrocruorin: influence of coiled coils on the architecture of a megadalton respiratory protein

Annelid erythrocruorins are extracellular respiratory complexes assembled from 180 subunits into hexagonal bilayers. Cryo-electron microscopic experiments have identified two different architectural classes. In one, designated type I, the vertices of the two hexagonal layers are partially staggered, with one hexagonal layer rotated by about 16 degrees relative to the other layer, whereas in the other class, termed type II, the vertices are essentially eclipsed. We report here the first crystal structure of a type II erythrocruorin, that from Arenicola marina, at 6.2 A resolution. The structure reveals the presence of long continuous triple-stranded coiled-coil "spokes" projecting towards the molecular center from each one-twelfth unit; interdigitation of these spokes provides the only contacts between the two hexagonal layers of the complex. This arrangement contrasts with that of a type I erythrocruorin from Lumbricus terrestris in which the spokes are broken into two triple-stranded coiled coils with a disjointed connection. The disjointed connection allows formation of a more compact structure in the type I architecture, with the two hexagonal layers closer together and additional extensive contacts between the layers. Comparison of sequences of the coiled-coil regions of various linker subunits shows that the linker subunits from type II erythrocruorins possess continuous heptad repeats, whereas a sequence gap places these repeats out of register in the type I linker subunits, consistent with a disjointed coiled-coil arrangement.     

Bacteriophage SPO1 structure and morphogenesis. I. Tail structure and length regulation.

Bacteriophage SPO1, a structually complex phage with hydroxymethyl uracil replacing thymine, has been studied by structural and chemical methods with the aim of defining the virion organization. The contractile tail of SPO1 consists of a complex baseplate, a tail tube, and a 140-nm-long sheath composed of stacked disks (4.1 nm repeat), each containing six subunits of molecular weight 60,300. The subunits are arranged in six parallel helices, each with a helical screw angle (omega 0) of 22.5 degrees. The baseplate was shown to undergo a structural rearrangement during tail contraction into a hexameric pinwheel. A mutation in gene 8 which produced unattached heads and tails also produced tails of different lengths. The tail length distribution suggests that the smallest integral length increment is a single disk of subunits. The structural arrangement of subunits in long tails is identical to that of normal tails, and the tails can contract. Many of the long tails showed partial stain penetration within the tail tube to a point which coincides with the top of a unit-length tail. The implications of these findings with respect to tail length regulation are discussed.     

Ultrastructure of C4b-binding protein fragments formed by limited proteolysis using chymotrypsin

C4b-binding protein is a regulator of the classical pathway of the complement system, acting as a cofactor to the serine protease factor I in the degradation of C4b. Its molecular weight is approximately 570,000 and it is composed of multiple, disulfide-linked 70-kDa subunits. Visualized by electron microscopy (Dahlb?ck, B., Smith, C. A., and Muller-Eberhard, H. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3641-3645), it has an unusual spider-like structure with multiple thin (30 A), elongated (330 A) tentacles. The number of tentacles was estimated to be seven. Limited proteolysis by chymotrypsin produces fragments of approximately 50- and 160-kDa, the latter composed of multiple, disulfide-linked, 25-kDa polypeptides. We now have isolated the undenatured C4b-binding protein fragments formed by treatment of the protein with chymotrypsin and have visualized them by electron microscopy. The 160-kDa fragment comprises the central portion of the C4b-binding protein, which appears as a ringlike structure with an inner diameter of 13 A and an outer diameter of 60 A and having attached an approximately 40-A long piece of each tentacle. The liberated 50-kDa fragment constitutes the major part (290-A long) of the tentacles. Chymotrypsin digestion of C4b-binding protein was also monitored as a function of time by polyacrylamide gel electrophoresis and the number of subunits cleaved was found to be seven, supporting our previous ultrastructural data which suggested that C4b-binding protein contains seven identical tentacle-like subunits.     

Evidence for a repeating cross-beta sheet structure in the adenovirus fibre

The amino acid sequence of the adenovirus fibre protein reveals an approximately repeating motif of 15 residues. A diagonal comparison matrix established that these repeats extended from residue 43 to residue 400 of the 581 residue sequence. Assignment of secondary structure combined with model building showed that each 15-residue segment contained two short beta-strands and two beta-bends, one of which incorporated an extra residue in a beta-bulge of the Gx type. The 44 strands together gave a long (210 A) narrow, amphipathic beta-sheet, which could be stabilised by dimer formation to give the shaft of the fibre. The knob could arise from a dimer of the C-terminal 180 residue segment, predicted to be an 8-10 stranded beta-sandwich. This model is consistent with the electron micrographs of the fibre and it was supported by measurements of c.d. and of electron diffraction from microcrystals. The latter gave a pair of wide angle arcs, corresponding to a repeat of 4.7 A, oriented appropriately for a cross-beta structure. The relation of this structure to globular structures is discussed and a folding pathway is proposed. In its general features the structure resembles that proposed for the tail fibre of bacteriophage T4.     

3D reconstruction of mammalian septin filaments

Mammalian septins are a family of guanosine triphosphate-binding proteins thought to play a role in a number of key cellular processes, such as cytokinesis, protein scaffolding and vesicle trafficking. Although their precise functions remain to be determined, electron microscopy has shown septin filament formation in vitro and a role as a cytoskeletal polymer has been proposed. Here, we present a 3D reconstruction of septin filaments determined using electron microscopy of negatively stained specimens and single-particle image processing. Septin was isolated from rat brain as an approximately 240-kDa complex, from which immunoblotting and N-terminal sequencing identified the major components as septins 3, 5 and 7. Electron microscopy and single-particle analysis indicated that the majority of the septin filaments were ∼ 27 nm long. A comparison of 3D volumes obtained using two independent starting models (a row of spheres or a helix) and projection matching techniques revealed no major differences at the final resolution of 27 Å, and this structure was highly reproducible when the entire procedure was repeated several times. The reconstruction revealed three apparent subunits, each separated by a cleft; these subunits were similar, but not identical, possibly indicating multiple isoforms within each filament. In some views a smaller cleft appeared to separate the subunits into two smaller regions, perhaps reflecting the presence of septin dimers. This is the first 3D reconstruction of the native septin assembly, and appears compatible with the hypothesis that the septin complex is a hexamer consisting of dimers or heterotrimers. Further investigations are necessary to confirm how the structure of the filaments determined in the present study correlates with the roles of septins in vivo.     

Structure of quinoprotein methylamine dehydrogenase at 2.25 A resolution.

The three-dimensional structure of quinoprotein methylamine dehydrogenase from Thiobacillus versutus has been determined at 2.25 A resolution by a combination of multiple isomorphous replacement, phase extension by solvent flattening and partial structure phasing using molecular dynamics refinement. In the resulting map, the polypeptide chain for both subunits could be followed and an X-ray sequence was established. The tetrameric enzyme, made up of two heavy (H) and two light (L) subunits, is a flat parallellepiped with overall dimensions of approximately 76 x 61 x 45 A. The H subunit, comprising 370 residues, is made up of two distinct segments: the first 31 residues form an extension which embraces one of the L subunits; the remaining residues are found in a disc-shaped domain. This domain is formed by a circular arrangement of seven topologically identical four-stranded antiparallel beta-sheets, with approximately 7-fold symmetry. In spite of distinct differences, this arrangement is reminiscent of the structure found in influenza virus neuraminidase. The L subunit consists of 121 residues, out of which 53 form a beta-sheet scaffold of a central three-stranded antiparallel sheet flanked by two shorter two-stranded antiparallel sheets. The remaining residues are found in segments of irregular structure. This subunit is stabilized by six disulphide bridges, plus two covalent bridges involving the quinone co-factor and residues 57 and 107 of this subunit. The active site is located in a channel at the interface region between the H and L subunits, and the electron density in this part of the molecule suggests that the co-factor of this enzyme is not pyrrolo quinoline quinone (PQQ) itself, but might be instead a precursor of PQQ.     

A pre-ribosome with a tadpole-like structure functions in ATP-dependent maturation of 60S subunits

Analyses of isolated pre-ribosomes yielded biochemical "snapshots" of the dynamic, nascent 60S and 40S subunits during their path from the nucleolus to the cytoplasm. Here, we present the structure of a pre-60S ribosomal intermediate located in the nucleoplasm. A huge dynein-related AAA-type ATPase (Rea1) and the Rix1 complex (Rix1-Ipi1-Ipi3) are components of an extended (approximately 45 nm long) pre-60S particle. Antibody crosslinking in combination with electron microscopy revealed that the Rea1 localizes to the "tail" region and ribosomal proteins to the "head" region of the elongated "tadpole-like" structure. Furthermore, in vitro treatment with ATP induces dissociation of Rea1 from the pre-60S subunits. Rea1 and the Rix1 complex could mediate ATP-dependent remodeling of 60S subunits and subsequent export from the nucleoplasm to the cytoplasm.     

Quaternary structure of pig liver phosphofructokinase

The quaternary structure of phosphofructokinase from pig liver has been studied by electron microscopy. Particles ranging in size from tetramers to long flexible chains of tetramers were commonly observed. Phosphofructokinase tetramers are square planar and approximately 110 A on a side; individual subunits are roughly spherical, with a mean radius of 28 A. Chains are formed by end-to-end association of tetramers rather than by tetramer stacking. The geometry of association implies that phosphofructokinase tetramers possess D2 symmetry, with distinct isologous bonding domains for dimer, tetramer, and chain formation.     

Cytochrome oxidase: structural insights from electron microscopy and from secondary structure prediction

Electron microscopic images of selectively contrasted cytochrome oxidase dimer crystals are interpreted in a manner consistent with the structure of monomers determined by Fuller et al. (J. Molec. Biol. 134, 305-327). The arms of the y-shaped monomers lie within and perpendicular to the lipid bilayer protruding approximately 25 A on the matrix side of the membrane. The cytoplasmic-side tails of two monomers spread apart in a dimer forming a large cleft. Decoration of the exposed matrix side of vesicle crystals with antisubunit IV antibody fragments indicates that subunit IV lies along the a-crystal axis roughly 20 A from the center of the dimer. A membrane propensity algorithm applied to the sequences of cytochrome oxidase subunits predicts a total of 19 transmembrane alpha-helices per monomer.     

T cell receptor engagement triggers its CD3epsilon and CD3zeta subunits to adopt a compact, locked conformation

How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3epsilon subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3epsilon and CD3zeta subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3epsilon and CD3zeta, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.     

The quaternary structure and activity of newly purified fatty acid synthetase from the Harderian gland of guinea-pig

Fatty acid synthetase was isolated from the Harderian gland of guinea-pig. The fatty acids synthesized by the purified enzyme were analyzed by mass fragmentography. The purified enzyme had an inherent capacity to utilize methylmalonyl-CoA and synthesize methyl-branched fatty acids. Physicochemical studies indicated that an active enzyme was a dimer, consisted of two subunits of Mr = 2.5 X 10(5). The negatively stained enzyme had an electron micrographic image of an ellipsoidal contour with a continuous middle cleft along the major axis. The major and minor axes were approximately equal to 220 and 150 A, respectively. In a dimer, the subunit had a rod-like structure about 220 A long and 50 A wide. The enzyme was inactivated and dissociated into subunits by incubation at 0 degree C. The inactivated enzyme was fully reactivated by raising the temperature of the solution. The relationship between the quaternary structure of the enzyme and the occurrence of enzymatic activity was studied by high-performance liquid chromatography. Neither active monomers nor inactive dimers were found in inactivation and reactivation processes. The initial velocity of reactivation was proportional to the enzyme concentration over a concentration range of 160-800 micrograms/ml, indicating that the rate-determining step in the reactivation reaction was unimolecular.     

Two distant regions of the Epstein-Barr virus genome with sequence homologies have the same orientation and involve small tandem repeats.

The two regions of the Epstein-Barr virus genome (DSL and DSR) carrying homologous sequences at distant parts of the long unique region are described. Cleavage of cloned DNA containing the DSR region with restriction endonucleases revealed a so far unrecognized small tandem repeat of approximately 120 base pairs present in approximately 20 copies. Heteroduplexes of the DNA of two clones containing DSL and DSR respectively, visualized in the electron microscope by cytochrome c spreading, revealed that the region of homology is approximately 2.5 kb long, involves small tandem repeats, and has the same orientation in the viral genome. Mica adsorption of the heteroduplex showed, that the homologous region consists of approximately 1.5 kb with only partial homology including the small internal repeats and 0.9 kb with well-matched duplexes. When DNA containing the DSL region reanneals, it can give rise to two single-stranded loops of the same size at different positions suggesting the presence of a row of tandem repeats also in this region.     

Distributions of dimeric tandem repeats in non-coding and coding DNA sequences

We study the length distribution functions for the 16 possible distinct dimeric tandem repeats in DNA sequences of diverse taxonomic partitions of GenBank (known human and mouse genomes, and complete genomes of Caenorhabditis elegans and yeast). For coding DNA, we find that all 16 distribution functions are exponential. For non-coding DNA, the distribution functions for most of the dimeric repeats have surprisingly long tails, that fit a power-law function. We hypothesize that: (i) the exponential distributions of dimeric repeats in protein coding sequences indicate strong evolutionary pressure against tandem repeat expansion in coding DNA sequences; and (ii) long tails in the distributions of dimers in non-coding DNA may be a result of various mutational mechanisms. These long, non-exponential tails in the distribution of dimeric repeats in non-coding DNA are hypothesized to be due to the higher tolerance of non-coding DNA to mutations. By comparing genomes of various phylogenetic types of organisms, we find that the shapes of the distributions are not universal, but rather depend on the specific class of species and the type of a dimer.     

Expansion of polyglutamine induces the formation of quasi-aggregate in the early stage of protein fibrillization

We examined the effects of the expansion of glutamine repeats on the early stage of protein fibrillization. Small-angle x-ray scattering (SAXS) and electron microscopic studies revealed that the elongation of polyglutamine from 35 to 50 repeats in protein induced a large assembly of the protein upon incubation at 37 degrees C and that its formation was completed in approximately 3 h. A bead modeling procedure based on SAXS spectra indicated that the largely assembled species of the protein, quasi-aggregate, is composed of 80 to approximately 90 monomers and a bowl-like structure with long and short axes of 400 and 190 A, respectively. Contrary to fibril, the quasi-aggregate did not show a peak at S = 0.21 A-1 corresponding to the 4.8-A spacing of beta-pleated sheets in SAXS spectra, and reacted with a monoclonal antibody specific to expanded polyglutamine. These results imply that beta-sheets of expanded polyglutamines in the quasi-aggregate are not orderly aligned and are partially exposed, in contrast to regularly oriented and buried beta-pleated sheets in fibril. The formation of non-fibrillary quasi-aggregate in the early phase of fibril formation would be one of the major characteristics of the protein containing an expanded polyglutamine.     

Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes

A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes is specific and saturable, with a Kd of 1.3 nM. [3H]Ryanodine binding is enriched in membranes from the hippocampus but is significantly lower in membranes from the brain stem and spinal cord. Approximately 60% of [3H]ryanodine-labeled receptor is solubilized from brain membranes using 2.5% CHAPS and 10 mg/ml phosphatidylcholine containing 1 M NaCl. The solubilized brain [3H]ryanodine receptor sediments through sucrose gradients like the skeletal receptor as a large (approximately 30 S) complex. Solubilized receptor is specifically immunoprecipitated by sheep polyclonal antibodies against purified skeletal muscle ryanodine receptor coupled to protein A-Sepharose. [3H]Ryanodine-labeled receptor binds to heparin-agarose, and a protein of approximately 400,000 Da, which is cross-reactive with two polyclonal antibodies raised against the skeletal muscle ryanodine receptor, elutes from the column and is enriched in peak [3H]ryanodine binding fractions. These results suggest that the approximately 400,000-Da protein is the brain form of the high affinity ryanodine receptor and that it shares several properties with the skeletal ryanodine receptor including a large oligomeric structure composed of approximately 400,000-Da subunits.