A novel cell binding site in the coiled-coil domain of laminin involved in capillary morphogenesis

Recently, we reported the isolation and characterization of an anti-laminin antibody that modulates the extracellular matrix-dependent morphogenesis of endothelial cells. Here we use this antibody to precisely map the binding site responsible for mediating this biologically important interaction. By using a phage display-assisted mapping strategy to preserve protein structure, we demonstrate for the first time that the coiled-coil region of laminin contains a cell binding site. The adhesion motif is formed by residues contributed by both alpha and gamma chains, and is located in the middle part of the rod-like portion in a highly flexible area, which corresponds to a protease-susceptible site. Based on this information, a peptide mimotope was used to characterize the cognate receptor. Although we can not rule out the implication of other receptors, our results demonstrate that the laminin helical rod active site interacts with alpha2beta1 integrin on the surface of endothelial cells. These findings provide new insight into the complex mechanisms regulating capillary morphogenesis.     

The GRIP domain - a novel Golgi-targeting domain found in several coiled-coil proteins

Many large coiled-coil proteins are being found associated peripherally with the cytoplasmic face of the organelles of the secretory pathway. Various roles have been proposed for these proteins, including the docking of donor vesicles or organelles to an acceptor organelle prior to fusion, and, in the case of the Golgi apparatus, the stacking of the cisternae [1] [2] [3] [4] [5]. Such critical roles require accurate recruitment to the correct organelle. For the endosomal coiled-coil protein EEA1, targeting requires a carboxy-terminal FYVE domain, which interacts with Rab5 and phosphatidylinositol 3-phosphate (PI(3)P), whereas the Golgi protein GM130 interacts with Golgi membranes via the protein GRASP65 [3] [6] [7]. In this paper, we show that two other mammalian Golgi coiled-coil proteins, golgin-245/p230 and golgin-97, have a conserved domain of about 50 amino acids at their carboxyl termini. This 'GRIP' domain is also found at the carboxyl terminus of several other large coiled-coiled proteins of unknown function, including two human proteins and proteins in the genomes of Caenorhabditis elegans and yeasts. The GRIP domains from several of these proteins, including that from the yeast protein Imh1p, were sufficient to specify Golgi targeting in mammalian cells when fused to green fluorescent protein (GFP). This result suggests that this small domain functions to recruit specific coiled-coil proteins to the Golgi by recognising a determinant that has been well conserved in eukaryotic evolution.     

The neurite-promoting domain of human laminin promotes attachment and induces characteristic morphology in non-neuronal cells

The interaction of cells with laminin and laminin fragments was studied in short-term cell attachment assays. Neurite-promoting chymotrypsin fragments of laminin were isolated using a monoclonal antibody which blocks neurite outgrowth on laminin. The fragments were shown, by electron microscopy after rotary shadowing and by immunological reactivity with different monoclonal antibodies, to contain only the distal end of the long arm. These fragments promoted the attachment and spreading of glioma, sarcoma, carcinoma, muscle, and endodermal cells to the same extent as intact laminin. The attachment was unaffected by peptides containing the RGD sequence. The morphology of the cells on the chymotrypsin fragments was indistinguishable from that on intact laminin but different from the morphology of the same cells on fibronectin. Light microscopy and scanning electron microscopy showed extensive process formation on laminin but not on fibronectin suggestive of increased cell motility in response to laminin. We conclude that the neurite-promoting domain of laminin contains a major site of interaction for non-neuronal cells and that this site induces a cellular response in certain non-neuronal cells that is unique to laminin.     

The coiled-coil structure potential of the laminin LCC domain is very fragmented and does not differentiate between natural and non-detected isoforms

There are 15 known laminins, which differ in the isoforms of the three chains that assemble into the cross-shape molecules that are observed by electron microscopy. The amino acid sequences of the rod-like portion of the long arm have long been recognized as having a potential for coiled-coil structure formation; however, an experimental determination of its structure is hampered by the complexity of laminin, a multidomain, heterotrimeric, and glycosilated 800 kDa molecule. Here, we have investigated the coiled-coil structure potential of laminin to evaluate its distribution along the long arm, the presence of conserved patterns, and differences between natural and non-natural isoforms. With these aims, we have analysed the sequences of each laminin chain in the context of the three-chain assemblies to yield an overall score of coiled-coil potential for the 15 natural laminins and for the other 30 possible but non-detected ones. The potential has been calculated with two different existing methods to exclude algorithm specific biases and with different chain alignments to evaluate the dependency of the results on uncertainties in the specific alignment along the domain. The analysis shows that the distribution of the potential is discontinuous, highly fragmented along the arm, without a common pattern except for a higher potential at the C-terminus, and that natural and non-natural laminins cannot be distinguished based on their coiled-coil potential, indicating that other factors are responsible for the selection of chain assembly.     

Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration

The ampA gene has a role in cell migration in Dictyostelium discoideum. Cells overexpressing AmpA show an increase in cell migration, forming large plaques on bacterial lawns. A second-site suppressor of this ampA-overexpressing phenotype identified a previously uncharacterized gene, ndm, which is described here. The Ndm protein is predicted to contain a coiled-coil BAR-like domain-a domain involved in endocytosis and membrane bending. ndm-knockout and Ndm-monomeric red fluorescent protein-expressing cell lines were used to establish a role for ndm in suppressing endocytosis. An increase in the rate of endocytosis and in the number of endosomes was detected in ndm(-) cells. During migration ndm(-) cells formed numerous endocytic cups instead of the broad lamellipodia structure characteristic of moving cells. A second lamellipodia-based function-cell spreading-was also defective in the ndm(-) cells. The increase in endocytosis and the defect in lamellipodia formation were associated with reduced chemotaxis in ndm(-) cells. Immunofluorescence results and glutathione S-transferase pull-down assays revealed an association of Ndm with coronin and F-actin. The results establish ndm as a gene important in regulating the balance between formation of endocytic cups and lamellipodia structures.     

Functional and structural effects of seven-residue deletions on the coiled-coil cytoplasmic domain of a chemoreceptor

Chemoreceptors transmit signals from the environment to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors is strongly conserved and consists of a long alpha-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Changes in this domain during evolution are characterized by the presence of seven-residue insertions/deletions located symmetrically with respect to the hairpin turn, suggesting that specific interactions between the helices that form the hairpin are required for function. We assessed the impact of seven-residue deletions on the signalling ability and higher-order organization of the serine chemoreceptor from Escherichia coli. Our results indicate that symmetry alterations between the two branches of the cytoplasmic hairpin seriously compromise chemoreceptor function. Shorter functional versions of Tsr with symmetrical deletions form mixed trimers of dimers when coexpressed with Tar, the aspartate receptor of E. coli. However, Tar function in those cells is impaired, suggesting that the length difference between receptors introduces non-functional distortions into the chemoreceptor cluster. This observation is reinforced by the analysis of coexpression of Tar with chemoreceptors from Rhodobacter sphaeroides that naturally belong to a shorter-length class.     

Monoclonal anti-mouse laminin antibodies: AL-1 reacts with laminin alpha1 chain, AL-2 with laminin beta1 chain, and AL-4 with the coiled-coil domain of laminin beta1 chain.

We analyzed the reactivity of three different commercially available rat monoclonal antibodies raised against mouse laminin-alpha1beta1gamma1 (laminin-111), AL-1, AL-2, and AL-4. Using ELISA assays, Western blot analysis and immunostainings we present refined epitope maps for these three laminin monoclonals. AL-1 reacted, as predicted with laminin alpha1 chain. AL-4 has also been marketed as an alpha1 chain specific probe, but we show here that AL-4 detects mouse laminin beta1 chain, in the distal part of the coiled-coil region. AL-2 was predicted to react with all three chains near the cross-region, but seems to primarily react with laminin beta1 chain.     

Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma1 chain

Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the gamma1 subunit of laminin-1, whereas no binding to alpha1 and beta1 chains was detected. By using recombinant gamma1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant gamma1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding. Interestingly, no binding to a chimeric gamma2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.     

Synthesis and conformation of the trimeric coiled-coil segment of laminin.

A disulfide linked 95-mer parallel hetero-trimeric active site segment of laminin was designed and synthesised. The three subunits, A (32-mer), B1 (30-mer) and B2 (33-mer), were prepared by Boc-based solid-phase peptide synthesis involving a two-step trimethylsilyl bromide-thioanisole and HF deprotection procedure. The interlinking of the three subunits was accomplished by the stepwise selective formation of two disulfide bridges using air-oxidation and thallium (III) trifluoroacetate oxidation. The conformations of the synthetic peptides were studied by circular dichroism (CD) spectroscopy, showing that the hetero-dimer, B1-B2, one of the homo-dimers, B1-B1, and the trimer are 30 to 40% in the alpha-helical conformation in aqueous buffer. Variable temperature CD studies demonstrated that the trimer is considerably more stable (melting temperature (Tm) = 61 degrees) than the hetero-dimer, B1-B2 (Tm = 36 degrees).     

The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.     

Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype

Skeletal myoblasts from fetal muscle respond adversely to fibronectin and laminin substrata: when primary mouse skeletal myoblasts are plated onto laminin, more myosin and desmin-positive myoblasts (myo+ cells) develop than on plates coated with fibronectin or collagen. In clonal cultures virtually all cells differentiate into postmitotic, fusion-capable myo + myoblasts on laminin after 3 days. In contrast, on fibronectin, the majority of the cells becomes myosin- and desmin-negative, partially due to proliferation of undifferentiated myoblast precursor cells, partially due to dedifferentiation or modulation of myoblasts into fibroblast-like myo- cells. Loss of the myogenic phenotype on fibronectin was also observed in cloned mouse myoblasts and in cultures of a differentiating mouse satellite cell line, MM14Dy, confirming that the appearance of desmin-negative cells is a result of myoblast modulation and not due simply to overgrowth by muscle fibroblasts. In the light of other effects of laminin on myoblasts, such as the stimulation of migration, differentiation and proliferation, our findings are consistent with the notion that laminin and fibronectin may be counteracting factors in the control of muscle differentiation.     

Crystal structure of a coiled-coil domain from human ROCK I

The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535-700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation.     

Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain

SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1. The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.     

The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival

The survival of cultured chick sympathetic neurons and the outgrowth of neurites were stimulated by the basement membrane protein laminin coated onto polyornithine culture substrates. The survival-potentiating activity was dependent on the presence of nerve growth factor. Both effects of laminin could be completely inhibited by affinity-purified antibodies against laminin fragment 3, the product of a limited proteolysis that corresponds to the heparin-binding globular domain at the end of the long arm of the laminin molecule. Antibodies against other laminin fragments were inactive, including those against previously determined cell-binding domains. A large laminin fragment, E8, was produced by brief elastase digestion and shown to consist of fragment 3 and an adjacent rod-like structure. Although lacking the cell binding domains, fragment E8 potentiated both neuronal survival and neurite outgrowth, and these effects could be blocked by antibodies against fragment 3. Weak survival and neurite potentiating activity was also detected in another fragment corresponding to the short arms of laminin, but as these effects were not inhibited by any of the antibodies tested they probably arose de novo during proteolysis. The heparin-binding domain of laminin is therefore responsible for its effects on neurons.     

The coiled-coil domain of TRAF6 is essential for its auto-ubiquitination

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological processes, including adaptive immunity, innate immunity, and bone metabolism. Importantly, it is essential for activating NF-kappaB signaling pathway in response to interleukin-1 and Toll-like receptor ligands. Previously, we characterized TRAF6 to be a ubiquitin ligase. In combination with the ubiquitin conjugating enzyme complex Ubc13/Uev1A, TRAF6 could catalyze the formation on itself of unique Lys-63 linked polyubiquitin chain that positively regulated NF-kappaB signaling pathway. However, it remains unknown how this auto-ubiquitination process is regulated. In this study, we found that the coiled-coil domain of TRAF6 was essential for its auto-ubiquitination and activating NF-kappaB signaling pathway. This domain served not as the specific target where the polyubiquitin chain was linked, but as a specific bridge to recruit Ubc13/Uev1A.     

Morin exerts anti-metastatic,anti-proliferative and anti-adhesive effect in ovarian cancer cells: an in vitro studies

The influence of morin hydrate on changes of proliferative, metastatic, and adhesive potential of human ovarian cancer cells concerning the influence of decitabine, and decitabine with trichostatin A, and in comparison to untreated cells, were analyzed. The effect of morin hydrate, decitabine, and trichostatin A were examined in A2780 and SKOV-3 ovarian cancer cell lines using MTS assay, clonogenic assay, adhesion to endothelial HMEC-1 cells, transwell migration assay and cell cycle analysis. The expression level of epithelial to mesenchymal transition (EMT) markers was quantified using PCR Array in relation to the level of global methylation determined with Methylated DNA Quantification Kit. We observed statistically significant inhibition of adhesive and migratory potential of both cell lines and the accumulation of G0/G1 phase A2780 cells after treatment with morin hydrate. Our studies confirmed the influence of morin hydrate on down-regulation of genes considered as up-regulated during EMT, and up-regulation of some genes considered as down-regulated during EMT in A2780 and SKOV-3 cells. Phenotypic changes were associated with molecular changes in cells, eg. decrease of the expression level of genes associated with adhesion, and an increase of genes down-regulated during EMT, after morin hydrate treatment in comparison to untreated control cells in both cell lines, were observed.

     

Orientation and oligomerization specificity of the Bcr coiled-coil oligomerization domain

The Bcr oligomerization domain, from the Bcr-Abl oncoprotein, is an attractive therapeutic target for treating leukemias because it is required for cellular transformation. The domain homodimerizes via an antiparallel coiled coil with an adjacent short, helical swap domain. Inspection of the coiled-coil sequence does not reveal obvious determinants of helix-orientation specificity, raising the possibility that the antiparallel orientation preference and/or the dimeric oligomerization state are due to interactions of the swap domains. To better understand how structural specificity is encoded in Bcr, coiled-coil constructs containing either an N- or C-terminal cysteine were synthesized without the swap domain. When cross-linked to adopt exclusively parallel or antiparallel orientations, these showed similar circular dichroism spectra. Both constructs formed coiled-coil dimers, but the antiparallel construct was approximately 16 degrees C more stable than the parallel to thermal denaturation. Equilibrium disulfide-exchange studies confirmed that the isolated coiled-coil homodimer shows a very strong preference for the antiparallel orientation. We conclude that the orientation and oligomerization preferences of Bcr are not caused by the presence of the swap domains, but rather are directly encoded in the coiled-coil sequence. We further explored possible determinants of structural specificity by mutating residues in the d position of the coiled-coil core. Some of the mutations caused a change in orientation specificity, and all of the mutations led to the formation of higher-order oligomers. In the absence of the swap domain, these residues play an important role in disfavoring alternate states and are especially important for encoding dimeric oligomerization specificity.     

The anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of cell surface calreticulin

Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17-35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but not crt(-/-) cells respond to hep I/TSP. Responsiveness can be restored by incubation of cells with exogenous calreticulin or by transfection with calreticulin. Thrombospondin forms complexes with the CRT-N-domain that are enhanced by physiologic levels of calcium and zinc. Consistent with thrombospondin/CRT-N-domain binding, only the CRT-N-domain blocks hep I- and thrombospondin-stimulated focal adhesion disassembly. A series of glutathione S-transferase-N-domain mutants were used to map the sequence within the N-domain that interacts with TSP/hep I. A construct containing aa 1-43 but not a construct of aa 1-31 supported thrombospondin binding and focal adhesion disassembly. A series of overlapping peptides were used to further map the thrombospondin-binding site. Peptides spanning aa 19-36 (RWIESKHKSDFGKFVLSS) blocked hep I-stimulated focal adhesion disassembly, indicating that the TSP/hep I-binding site is located to this sequence in calreticulin. A mutant fusion protein lacking aa 19-36 (glutathione S-transferase-CRTDeltahep I) failed to restore responsiveness to hep I in crt(-/-) cells, bind thrombospondin, or competitively block focal adhesion disassembly, providing evidence for the role of this calreticulin sequence in mediating thrombospondin signaling.