Domains in human plasminogen

Calorimetric studies of intramolecular melting of human plasminogen and of its fragments under various solvent conditions show that the intact plasminogen molecule consists of seven compact co-operative subunits, which can be regarded as structural domains. Five of these domains are formed by the homologous regions, the kringles, two domains are formed by the C-terminal part of the polypeptide chain that is split at activation, forming the light chain in plasmin, while the initial 76 amino acid residue peptide does not form any compact co-operative structure. The specific influence of epsilon-aminocaproic acid on the stability of the first, the fourth and, to a lesser extent, on the second kringle domain, provides evidence that these three domains in plasminogen possess lysine-binding ability. The first four kringle domains are almost independent in the molecule, while the fifth interacts with that part of the light chain not included in either of the two domains of this chain. These two domains are of different size and co-operate strongly in plasminogen, but at its activation into plasmin they decooperate and the stability of the smaller domain, which is formed by the N-terminal part of the light chain, decreases significantly. Since the light chain is responsible for the proteolytic activity of plasmin, it becomes clear that the active site of this protein is composed of two domains, as is the case for other serine proteases.     

Domainal organization of the molecules of Lys-plasminogen

The intramolecular melting of the human Lys-plasminogen and its different fragments were studied by the differential scanning microcalorimetry method. Thermodynamical analysis of melting curves showed that the Lys-plasminogen molecule consists of 7 domains. Five of them are formed by five homologeus regions of the polypeptide chain (kringle), while two domains are formed by the part of the polypeptide chain corresponding to the plasmin light chain. The domains included in the fragments seem to be rather independent, since fragmentation does not lead to noticeable changes of their stability in comparison to that of the intact molecule. It has been shown also that plasminogen-plasmin conversion is accompanied by structural transformation of the molecule which results in the destabilization of one of the light chain domains.     

Domains in the fibrinogen molecule

Intramolecular melting of fibrinogen and its degradation products has been studied by a scanning microcalorimetric method in various solution environments (especially variations in pH), and inferences are made about the features that seemed to be independently folding segments (“domains”), as evidenced by their independent resistance to thermal denaturation. It was shown that there are 12 more or less independent co-operative regions of ordered compact structure in fibrinogen, which can be considered as structural domains of this macromolecule. Of these 12 domains, two are in the central part of the molecule, corresponding to the E fragment, four are in each terminal part, corresponding to the D fragments, and two are formed by the carboxy-terminal portions of the α-chains. All fibrinogen domains can be divided into two groups according to their thermodynamic properties: (1) thermolabile domains, to which belong three domains from each terminal part of the molecule and the domains formed by the carboxy-terminal portions of the α-chains; (2) thermostable domains, to which belong both domains from the central part and one domain from each terminal part of the molecule. This division seems to reflect the structural differences between the domains.     

Structural and functional comparisons of chicken and human cellular fibronectins

We have investigated the structural and functional differences between chicken and human cellular fibronectin by comparing the tryptic peptide patterns using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analyzing the binding properties of isolated trypsin-resistant polypeptide fragments. Although the overall functional organization of chicken and human cellular fibronectins was similar, the tryptic patterns obtained from these two molecules were strikingly different. For example, the tryptic digest of chicken cellular fibronectin contained two unique peptide fragments having molecular sizes of 45 and 70 kilodaltons. The previously unidentified carboxyl-terminal 45-kDa fragment is an intermediate that appears between 15 to 120 s of digestion. The 70-kDa fragment binds to gelatin, to fibrin (with unusually high apparent affinity), to heparin (at low ionic strength), and to fixed Staphylococcus aureus cells; it also contains an acceptor site for factor XIIIa (plasma transglutaminase). These results suggest that the functional domains of chicken and human fibronectins remain constant and that structural variations occur in the protease-susceptible regions of the molecule. The present findings are discussed in terms of the previously existing discrepancies in the literature on fibronectin.     

Domain structure of fibronectin and its relation to function. Disulfides and sulfhydryl groups.

Hamster cell fibronectin is a glycoprotein consisting of two 230,000-dalton subunits in a disulfide-bonded dimer. The molecule is composed of domains which can be separated by partial proteolytic cleavage. The carbohydrates, disulfide bonds, and a single free sulfhydryl group per chain are distributed nonuniformly among these regions. All the interchain disulfides are within 10,000 daltons of the end of the molecule and are removed by mild proteolysis which also generates 200,000- and 25,000-dalton fragments which do not contain interchain disulfides. The 200,000-dalton fragment contains all or most of the carbohydrate side chains, and the free sulfhydryl group, but is relatively poor in cystine. The 25,000-dalton fragment is carbohydrate-free and cystine-rich but has no free sulfhydryl groups. There is heterogeneity in carbohydrate content among the monomeric chains of intact fibronectin and the 200,000-dalton fragments. The gelatin binding site of fibronectin is in the 200,000 fragment. Intact disulfide bonds are required for binding of fibronectin to cells and to gelatin and blockage of the free sulfhydryl groups prevents binding of fibronectin to cells, suggesting that intermolecular disulfide bonding may be important.     

The wac gene product of bacteriophage T4 contains coiled-coil structural patterns

The bacteriophage T4 late gene wac (whisker antigen control) encodes the protein which forms the fibrous structure on the neck of the virion called whiskers. Amino acid sequence analysis of wac gene product, as deduced from the nucleotide sequence, indicate ten alpha-helical domains (19-40 residues long) with coiled-coil structural patterns. These regions comprise about 70% of the entire 486 amino acid sequence. The alpha-helices are separated by short stretches of polypeptide chain which are similar to the loop regions of the globular protein sequences. We propose a structural model for the dimer of wac gene product molecule, that we call fibritin in which two polypeptide chains associate in a parallel fashion and form a segmented alpha-helical coiled-coil rod similar to epidermal keratins.     

Laminins and other strange proteins.

Laminins are large multidomain proteins of the extracellular matrix (ECM) with important functions in the development and maintenance of cellular organization and supramolecular structure, in particular in basement membranes. Each molecule is composed of three polypeptide chains, A (300-400 kDa) and B1 and B2 (180-200 kDa), which together form the characteristic cross-shaped laminin structure with three short arms and one long arm. Many different domains have been identified in laminin by sequence analysis, structural investigations, and functional studies. Each short arm is formed by homologous N-terminal portions of one of the three chains. Structurally, each short arm contains two or three globular domains which are connected by rows of manyfold-repeated Cys-rich "EGF-like" domains. In all three chains this region is followed by a long heptad repeat region similar to those found in many alpha-helical coiled-coil proteins. These parts of the three laminin chains constitute a triple-stranded coiled-coil domain, which forms the extended rodlike structure of the long arm. This is the only domain in the protein which is made up of more than one chain and consequently serves the function of chain assembly. The two B chains are terminated by the coiled-coil domain, but the A chain contains an additional C-terminal segment which accounts for five globular domains located at the tip of the long arm. Several important functions of laminin have been assigned to individual domains in either the short arms or terminal regions of the long arm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Structural and functional effects of selective chemical modifications of Scapharca inaequivalvis haemoglobins in relation to their unique assembly.

The structural and functional roles of lysyl and thiol groups in the dimeric (HbI) and tetrameric (HbII) haemoglobins from the mollusc Scapharca inaequivalvis have been assessed. In these haemoglobins a unique mode of assembly (the haem-carrying E and F helices form the intersubunit contact of the dimeric unit) is associated with co-operative oxygen binding. Extensive acylation is accompanied by significant haem oxidation. Modification of one or two lysyl residues per chain (corresponding to approximately 20% of the total residues) does not affect the structural and functional properties of both haemoglobins, in line with the proposal that the intersubunit contacts are rich in hydrophobic residues. The modification of the thiol groups does not influence the state of association in both HbI and HbII, despite the location of the cysteine residue common to all polypeptide chains in the vicinity of the major intersubunit contact. The effect on the functional properties depends on the size of the thiol reagent: p-chloromercuribenzoate and phenylmercuric acetate increase the oxygen affinity about 20-fold, but iodoacetamide and mercuric chloride have no effect. Moreover, electrophoresis experiments indicate that p-chloromercuribenzoate is bound in a co-operative fashion, the degree of co-operativity being much higher in the dimeric HbI. Thus, only in HbII are intermediates containing substoichiometric amounts of p-chloromercuribenzoate formed in significant amounts. Their oxygen binding properties show that reaction of only one thiol group/tetramer suffices to alter the oxygen affinity of the molecule.     

Similarity between protein-protein and protein-carbohydrate interactions, revealed by two crystal structures of lectins from the roots of pokeweed

The roots of pokeweed (Phytolacca americana) are known to contain the lectins designated PL-A, PL-B, PL-C, PL-D1, and PL-D2. Of these lectins, the crystal structures of two PLs, the ligand-free PL-C and the complex of PL-D2 with tri-N-acetylchitotriose, have been determined at 1.8A resolution. The polypeptide chains of PL-C and PL-D2 form three and two repetitive chitin-binding domains, respectively. In the crystal structure of the PL-D2 complex, one trisaccharide molecule is shared mainly between two neighboring molecules related to each other by a crystallographic 2(1)-screw axis, and infinite helical chains of complexed molecules are generated by the sharing of ligand molecules. The crystal structure of PL-C reveals that the molecule is a dimer of two identical subunits, whose polypeptide chains are located in a head-to-tail fashion by a molecular 2-fold axis. Three putative carbohydrate-binding sites in each subunit are located in the dimer interface. The dimerization of PL-C is performed through the hydrophobic interactions between the carbohydrate-binding sites of the opposite domains in the dimer, leading to a distinct dimerization mode from that of wheat-germ agglutinin. Three aromatic residues in each carbohydrate-binding site of PL-C are involved in the dimerization. These residues correspond to the residues that interact mainly with the trisaccharide in the PL-D2 complex and appear to mimic the saccharide residues in the complex. Consequently, the present structure of the PL-C dimer has no room for accommodating carbohydrate. The quaternary structure of PL-C formed through these putative carbohydrate-binding residues may lead to the lack of hemagglutinating activity.     

Mapping protein family interactions: intramolecular and intermolecular protein family interaction repertoires in the PDB and yeast

In the postgenomic era, one of the most interesting and important challenges is to understand protein interactions on a large scale. The physical interactions between protein domains are fundamental to the workings of a cell: in multi-domain polypeptide chains, in multi-subunit proteins and in transient complexes between proteins that also exist independently. To study the large-scale patterns and evolution of interactions between protein domains, we view interactions between protein domains in terms of the interactions between structural families of evolutionarily related domains. This allows us to classify 8151 interactions between individual domains in the Protein Data Bank and the yeast Saccharomyces cerevisiae in terms of 664 types of interactions, between protein families. At least 51 interactions do not occur in the Protein Data Bank and can only be derived from the yeast data. The map of interactions between protein families has the form of a scale-free network, meaning that most protein families only interact with one or two other families, while a few families are extremely versatile in their interactions and are connected to many families. We observe that almost half of all known families engage in interactions with domains from their own family. We also see that the repertoires of interactions of domains within and between polypeptide chains overlap mostly for two specific types of protein families: enzymes and same-family interactions. This suggests that different types of protein interaction repertoires exist for structural, functional and regulatory reasons. Copyright 12001 Academic Press.     

Fibronectin molecule visualized in electron microscopy: a long, thin, flexible strand

We have determined the structure of plasma fibronectin by electron microscopy of shadowed specimens. the 440,000 molecular weight, dimeric molecule appears to be a long, thin, highly flexible strand. The contour length of the most extended molecules is 160 nm, but a distribution of lengths down to 120 nm was observed, indicating flexibility in extension as well as in bending. The average diameter of the strand is 2 nm and there are no large globular domains. the large fragments produced by limited digestion with plasmin are not globular domains but are segments of the strand, whose length corresponds to the molecular weight of the polypeptide chain. We conclude that each polypeptide chain of the dimeric molecule spans half the length of the strand, with their carboxyl termini joined at the center of the strand and their amino termini at the ends. This model is supported by images of fibronectin-fibrinogen complexes, in which the fibrinogen is always attached to an end of the fibronectin strand.     

Two Lyt-2 polypeptides arise from a single gene by alternative splicing patterns of mRNA

The Lyt-2/3 molecule is a glycoprotein expressed on T lymphocytes and has classically been considered a marker for the cytotoxic/suppressor T cell subset. It has been postulated to be a receptor for class I major histocompatibility complex proteins. We have used a cDNA clone encoding the analogous human protein, Leu-2/T8, to isolate mouse cDNA clones, which were used as probes to isolate mouse genomic clones. By transfection we have shown that the mouse homologue of Leu-2/T8 is Lyt-2 and not Lyt-3. We have further demonstrated that two Lyt-2 polypeptide chains are encoded by a single gene and result from alternative modes of mRNA splicing. The nucleotide sequence of cDNA clones encoding each of these polypeptide chains has been determined and shows the difference between the two Lyt-2 polypeptide chains to be in the lengths of their cytoplasmic tails.     

Cyclic conformation of parallel polypeptide chains closed by cross bridges

The method of possible conformations calculations for cyclic structures, formed by two (or more) identical parallel polypeptide chains, closed by cross bridges has been elaborated. The algorithm is necessary for rigorous conformational analysis of cyclic regions in immunoglobulin, fibronectin and myosin.     

The three-dimensional structure of canavalin from jack bean (Canavalia ensiformis).

The three-dimensional structure of the vicilin storage protein canavalin, from Canavalia ensiformis, has been determined in a hexagonal crystal by x-ray diffraction methods. The model has been refined at 2.6 A resolution to an R factor of 0.197 with acceptable geometry. Because of proteolysis, 58 of 419 amino acids of the canavalin polypeptide are not visible in the electron density map. The canavalin subunit is composed of two extremely similar structural domains that reflect the tandem duplication observed in the cDNA and in the amino acid sequence. Each domain consists of two elements, a compact, eight-stranded beta-barrel having the "Swiss roll" topology and an extended loop containing several short alpha-helices. The root mean square deviation between 84 pairs of corresponding C alpha atoms making up the strands of the two beta-barrels in a subunit is 0.78 A, and for 112 pairs of structurally equivalent C alpha atoms of the two domains the deviation is 1.37 A. The interface between domains arises from the apposition of broad hydrophobic surfaces formed by side chains originating from one side of the beta-barrels, supplemented by at least four salt bridges. The interfaces between subunits in the trimer are supplied by the extended loop elements. These interfaces are also composed primarily of hydrophobic residues supplemented by six salt bridges. The canavalin subunits have dimensions about 40 x 40 x 86 A, and the oligomer is a disk-shaped molecule about 88 A in diameter with a thickness of about 40 A. The distribution of domains lends a high degree of pseudo-32-point group symmetry to the molecule. There is a large channel of 18 A diameter, lined predominantly by hydrophilic and charged amino acids, running through the molecule along the 3-fold axis. The majority of residues conserved between domains and among vicilins occur at the interface between subunits but appear otherwise arbitrarily distributed within the subunit, although predominantly on its exterior.     

Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily

The neuron-glia cell adhesion molecule (Ng-CAM) mediates both neuron-neuron and neuron-glia adhesion; it is detected on SDS-PAGE as a predominant 135-kD glycoprotein, with minor components of 80, 190, and 210 kD. We have isolated cDNA clones encoding the entire sequence of chicken Ng-CAM. The predicted extracellular region includes six immunoglobulin-like domains followed by five fibronectin-type III repeats, structural features that are characteristic of several neural CAMs of the N-CAM superfamily. The amino acid sequence of chicken Ng-CAM is most similar to that of mouse L1 but the overall identity is only 40% and Ng-CAM contains a short fibronectin-like segment with an RGD sequence that has no counterpart in L1. These findings suggest that Ng-CAM and L1 may not be equivalent molecules in chicken and mouse. The amino-terminal sequences of the 210-, 190-, and 135-kD components of Ng-CAM are all the same as the predicted amino terminus of the molecule, whereas the 80-kD component begins within the third fibronectin repeat. The cDNA sequence is continuous across the junction between the 135- and 80-kD components, and a single 170-kD Ng-CAM polypeptide was isolated from tunicamycin-treated cells. In addition, all cDNA probes hybridized on Northern blots to a 6-kb RNA, and most hybridized to single bands on Southern blots. These results indicate that the Ng-CAM components are derived from a single polypeptide encoded by a single gene, and that the 135- and 80-kD components are generated from the 210/190-kD species by proteolytic cleavage. The 135-kD component contains most of the extracellular region including all of the immunoglobulin-like domains. It has no transmembrane segment, but it is tightly associated with the membrane. The 80-kD component contains two and a half type III repeats plus the RGD-containing segment, as well as the single transmembrane and cytoplasmic domains. These structural features of Ng-CAM provide a framework for understanding its multiple functions in neuron-neuron interactions, neurite fasciculation, and neuron-glia interactions.     

Expression and characterization of human lamin C

We have expressed human lamin C cDNA in E. coli using a modification of the pLcII vector system. Protein produced in this way had seven additional amino acids at its N-terminus, but retained key lamin structural and assembly properties. The modified vector we produced may prove useful when difficulties are encountered in removal of the cII fusion peptide by factor X cleavage in the pLcII system. Shadowed preparations of expressed lamin C showed the presence of 50-nm rod-like particles that closely resembled those observed for native material. Isolated molecules had two globular domains at one end, indicating that they were constructed from two parallel polypeptide chains. The expressed material also formed paracrystals with a characteristic 22.5 nm axial repeat, indicating that its assembly properties had also been retained. We also used site-specific mutagenesis to engineer a lamin fragment that lacked the C-terminal non-helical domain of the molecule. This material formed paracrystals similar to those obtained with the intact molecule, indicating that the large C-terminal non-helical domain did not contain information vital for lamin assembly.     

What distinguishes tenascin from fibronectin?

Tenascin and fibronectin are two major extracellular matrix glycoproteins. They both consist of large disulfide-linked subunits composed of multiple structural domains. More than half of each molecule consists of so-called fibronectin type III repeats, but the other domains differ. Fibronectin is a dimer, whereas tenascin is a hexamer. Often fibronectin and tenascin are colocalized in tissues, but the occurrence of tenascin is much more restricted when compared with fibronectin. Tenascin is transiently expressed in many developing organs such as connective tissues, the mesenchyme of epithelial organs, and also the central and peripheral nervous systems, and it reappears in the stroma of many tumors. The distinctive and highly regulated expression of tenascin has provoked interest in trying to identify possible functions of tenascin in cell-cell and cell-substratum adhesion, cell migration, growth, and cell differentiation during morphogenesis.