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Sample records for coiled-coil interactions contribute

  1. The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins

    SciTech Connect

    Caillat, Christophe; Fish, Alexander; Pefani, Dafni-Eleftheria; Taraviras, Stavros; Lygerou, Zoi; Perrakis, Anastassis

    2015-10-31

    The GemC1 coiled-coil structure has subtle differences compared with its homologues Geminin and Idas. Co-expression experiments in cells and biophysical stability analysis of the Geminin-family coiled coils suggest that the GemC1 coiled coil alone is unstable. GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin–Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.

  2. IMPROVED COILED-COIL DESIGN ENHANCES INTERACTION WITH BCR-ABL AND INDUCES APOPTOSIS

    PubMed Central

    Dixon, Andrew S.; Miller, Geoffrey D.; Bruno, Benjamin J.; Constance, Jonathan E.; Woessner, David W.; Fidler, Trevor P.; Robertson, James C.; Cheatham, Thomas E.; Lim, Carol S.

    2012-01-01

    The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).1,2 The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.3, 4 We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.5 A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homo-dimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homo-dimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications. PMID:22136227

  3. Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

    SciTech Connect

    Wilbur, Jeremy D.; Hwang, Peter K.; Brodsky, Frances M.; Fletterick, Robert J.

    2010-03-01

    Variable packing interaction related to the conformational flexibility within the huntingtin-interacting protein 1 coiled coil domain. Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington’s disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coil domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.

  4. The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins

    PubMed Central

    Caillat, Christophe; Fish, Alexander; Pefani, Dafni-Eleftheria; Taraviras, Stavros; Lygerou, Zoi; Perrakis, Anastassis

    2015-01-01

    GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin–Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells. PMID:26527144

  5. Data-Driven Prediction and Design of bZIP Coiled-Coil Interactions

    PubMed Central

    Potapov, Vladimir; Kaplan, Jenifer B.; Keating, Amy E.

    2015-01-01

    Selective dimerization of the basic-region leucine-zipper (bZIP) transcription factors presents a vivid example of how a high degree of interaction specificity can be achieved within a family of structurally similar proteins. The coiled-coil motif that mediates homo- or hetero-dimerization of the bZIP proteins has been intensively studied, and a variety of methods have been proposed to predict these interactions from sequence data. In this work, we used a large quantitative set of 4,549 bZIP coiled-coil interactions to develop a predictive model that exploits knowledge of structurally conserved residue-residue interactions in the coiled-coil motif. Our model, which expresses interaction energies as a sum of interpretable residue-pair and triplet terms, achieves a correlation with experimental binding free energies of R = 0.68 and significantly out-performs other scoring functions. To use our model in protein design applications, we devised a strategy in which synthetic peptides are built by assembling 7-residue native-protein heptad modules into new combinations. An integer linear program was used to find the optimal combination of heptads to bind selectively to a target human bZIP coiled coil, but not to target paralogs. Using this approach, we designed peptides to interact with the bZIP domains from human JUN, XBP1, ATF4 and ATF5. Testing more than 132 candidate protein complexes using a fluorescence resonance energy transfer assay confirmed the formation of tight and selective heterodimers between the designed peptides and their targets. This approach can be used to make inhibitors of native proteins, or to develop novel peptides for applications in synthetic biology or nanotechnology. PMID:25695764

  6. A coiled-coil interaction mediates cauliflower mosaic virus cell-to-cell movement

    PubMed Central

    Stavolone, Livia; Villani, Maria Elena; Leclerc, Denis; Hohn, Thomas

    2005-01-01

    The function of the virion-associated protein (VAP) of cauliflower mosaic virus (CaMV) has long been only poorly understood. VAP is associated with the virion but is dispensable for virus morphogenesis and replication. It mediates virus transmission by aphids through simultaneous interaction with both the aphid transmission factor and the virion. However, although insect transmission is not fundamental to CaMV survival, VAP is indispensable for spreading the virus infection within the host plant. We used a GST pull-down technique to demonstrate that VAP interacts with the viral movement protein through coiled-coil domains and surface plasmon resonance to measure the interaction kinetics. We mapped the movement protein coiled-coil to the C terminus of the protein and proved that it self-assembles as a trimer. Immunogold labeling/electron microscopy revealed that the VAP and viral movement protein colocalize on CaMV particles within plasmodesmata. These results highlight the multifunctional potential of the VAP protein conferred by its efficient coiled-coil interaction system and show a plant virus possessing a surface-exposed protein (VAP) mediating viral entry into host cells. PMID:15837934

  7. A coiled-coil interaction mediates cauliflower mosaic virus cell-to-cell movement

    NASA Astrophysics Data System (ADS)

    Stavolone, Livia; Villani, Maria Elena; Leclerc, Denis; Hohn, Thomas

    2005-04-01

    The function of the virion-associated protein (VAP) of cauliflower mosaic virus (CaMV) has long been only poorly understood. VAP is associated with the virion but is dispensable for virus morphogenesis and replication. It mediates virus transmission by aphids through simultaneous interaction with both the aphid transmission factor and the virion. However, although insect transmission is not fundamental to CaMV survival, VAP is indispensable for spreading the virus infection within the host plant. We used a GST pull-down technique to demonstrate that VAP interacts with the viral movement protein through coiled-coil domains and surface plasmon resonance to measure the interaction kinetics. We mapped the movement protein coiled-coil to the C terminus of the protein and proved that it self-assembles as a trimer. Immunogold labeling/electron microscopy revealed that the VAP and viral movement protein colocalize on CaMV particles within plasmodesmata. These results highlight the multifunctional potential of the VAP protein conferred by its efficient coiled-coil interaction system and show a plant virus possessing a surface-exposed protein (VAP) mediating viral entry into host cells. movement protein | virion-associated protein | Biacore

  8. Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

    NASA Astrophysics Data System (ADS)

    Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

    2016-04-01

    Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes

  9. Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

    NASA Astrophysics Data System (ADS)

    Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

    2016-04-01

    Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes

  10. The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle

    PubMed Central

    Qadota, Hiroshi; Mayans, Olga; Matsunaga, Yohei; McMurry, Jonathan L.; Wilson, Kristy J.; Kwon, Grace E.; Stanford, Rachel; Deehan, Kevin; Tinley, Tina L.; Ngwa, Verra M.; Benian, Guy M.

    2016-01-01

    UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89’s SH3 domain and residues 294–376 of paramyosin and has a KD of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89’s SH3 is α-helical and lacks prolines. Homology modeling of UNC-89’s SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a “skip residue,” which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity. PMID:27009202

  11. Interactions of HIV-1 Inhibitory Peptide T20 with the gp41 N-HR Coiled Coil*S⃞

    PubMed Central

    Champagne, Kelly; Shishido, Akira; Root, Michael J.

    2009-01-01

    Cellular entry of human immunodeficiency virus type 1 (HIV-1) involves fusion of viral and cellular membranes and is mediated by structural transitions in viral glycoprotein gp41. The antiviral C-peptide T20 targets the gp41 N-terminal heptad repeat region (N-HR), blocking gp41 conformational changes essential for the entry process. To probe the T20 structure-activity relationship, we engineered a molecular mimic of the entire gp41 N-HR coiled coil using the 5-Helix design strategy. T20 bound this artificial protein (denoted 5H-ex) with nanomolar affinity (KD = 30 nm), close to its IC50 concentration (∼3 nm) but much weaker than the affinity of a related inhibitory C-peptide C37 (KD = 0.0007 nm). T20/C37 competitive binding assays confirmed that T20 interacts with the hydrophobic groove on the surface of the N-HR coiled coil outside of a deep pocket region crucial for C37 binding. We used 5H-ex to investigate how the T20 N and C termini contributed to the inhibitor binding activity. Mutating three aromatic residues at the T20 C terminus (WNWF → ANAA) had no effect on affinity, suggesting that these amino acids do not participate in T20 binding to the gp41 N-HR. The results support recent evidence pointing to a different role for these residues in T20 inhibition (Peisajovich, S. G., Gallo, S. A., Blumenthal, R., and Shai, Y. (2003) J. Biol. Chem. 278, 21012–21017; Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612–9620). By contrast, mutations near the T20 N terminus substantially influenced inhibitor binding strength. When Ile was substituted for Thr in the second T20 position, a 40-fold increase in binding affinity was measured (KD = 0.75 nm). The effect of this affinity enhancement on T20 inhibitory potency varied among different viral strains. The original T20 and the higher affinity T20 variant had similar potency against wild type HIV-1. However, the higher affinity T20

  12. Tropomyosin is an interaction partner of the Drosophila coiled coil protein yuri gagarin.

    PubMed

    Texada, Michael J; Simonette, Rebecca A; Deery, William J; Beckingham, Kathleen M

    2011-02-15

    The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin "cones" that mediate spermatid individualization. We used the tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuri(F64), failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions. PMID:21126519

  13. TROPOMYOSIN IS AN INTERACTION PARTNER OF THE DROSOPHILA COILED COIL PROTEIN YURI GAGARIN

    PubMed Central

    Texada, Michael J.; Simonette, Rebecca A.; Deery, William J.; Beckingham, Kathleen M.

    2011-01-01

    The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin “cones” that mediate spermatid individualization. We used tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuriF64, failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions. PMID:21126519

  14. Crystal Structure of Cytomegalovirus IE1 Protein Reveals Targeting of TRIM Family Member PML via Coiled-Coil Interactions

    PubMed Central

    Sevvana, Madhumati; Otto, Victoria; Schilling, Eva-Maria; Stump, Joachim D.; Müller, Regina; Reuter, Nina; Sticht, Heinrich; Muller, Yves A.; Stamminger, Thomas

    2014-01-01

    PML nuclear bodies (PML-NBs) are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV) expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1CORE) flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less susceptible to

  15. Peptidyl Materials Formed Through Click Chemistry Enhanced Coiled-Coil Interactions

    NASA Astrophysics Data System (ADS)

    Koehler, Kenneth

    2014-03-01

    Biologically derived materials offer a level of sophistication synthetically fabricated materials have only attempted to mimic. This level of complexity may be found in materials such as peptides. Implementing new theory and modeling, peptides with the propensity to form coiled-coil (CC) bundles were designed and synthesized. Through the use of this de novo approach, modeling allowed prediction of the feasibility to include non-natural amino acids conducive to click chemistry into the peptide. Amino acids showcasing thiol or alkyne functionalities were considered owing to the ability of these moieties to participate in the thiol-ene and copper click reactions respectively. Once synthesized, the peptides decorated with these clickable motifs were placed in solution and allowed to self-assemble into CC's. CD spectroscopy and DLS experiments confirmed the formation and assembly of CC's. Click reactions were then incited to link the CC assemblies together and form a network with predictable dimensionality and pore size between CC bundles. To incite network formation, click reactions between CC side chain residues and suitably functionalized crosslinkers were implemented. The linking of coiled-coils and material formation were assessed using DLS and TEM.

  16. SAS-6 coiled-coil structure and interaction with SAS-5 suggest a regulatory mechanism in C. elegans centriole assembly

    PubMed Central

    Qiao, Renping; Cabral, Gabriela; Lettman, Molly M; Dammermann, Alexander; Dong, Gang

    2012-01-01

    The centriole is a conserved microtubule-based organelle essential for both centrosome formation and cilium biogenesis. Five conserved proteins for centriole duplication have been identified. Two of them, SAS-5 and SAS-6, physically interact with each other and are codependent for their targeting to procentrioles. However, it remains unclear how these two proteins interact at the molecular level. Here, we demonstrate that the short SAS-5 C-terminal domain (residues 390–404) specifically binds to a narrow central region (residues 275–288) of the SAS-6 coiled coil. This was supported by the crystal structure of the SAS-6 coiled-coil domain (CCD), which, together with mutagenesis studies, indicated that the association is mediated by synergistic hydrophobic and electrostatic interactions. The crystal structure also shows a periodic charge pattern along the SAS-6 CCD, which gives rise to an anti-parallel tetramer. Overall, our findings establish the molecular basis of the specific interaction between SAS-5 and SAS-6, and suggest that both proteins individually adopt an oligomeric conformation that is disrupted upon the formation of the hetero-complex to facilitate the correct assembly of the nine-fold symmetric centriole. PMID:23064147

  17. Molecular basis of coiled-coil formation.

    PubMed

    Steinmetz, Michel O; Jelesarov, Ilian; Matousek, William M; Honnappa, Srinivas; Jahnke, Wolfgang; Missimer, John H; Frank, Sabine; Alexandrescu, Andrei T; Kammerer, Richard A

    2007-04-24

    Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed "trigger sequence," that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach, we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen-bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation. PMID:17438295

  18. The Conserved RIC-3 Coiled-Coil Domain Mediates Receptor-specific Interactions with Nicotinic Acetylcholine Receptors

    PubMed Central

    Biala, Yoav; Liewald, Jana F.; Ben-Ami, Hagit Cohen; Gottschalk, Alexander

    2009-01-01

    RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function. To examine the role of CC-I, we studied effects of its deletion on Caenorhabditis elegans nAChRs in vivo. Presence of CC-I promoted maturation of particular nAChRs expressed in body-wall muscle, whereas it was not required for other nAChR subtypes expressed in neurons or pharyngeal muscles. This effect is receptor-specific, because it could be reproduced after heterologous expression. Consistently, coimmunoprecipitation analysis showed that CC-I enhances the interaction of RIC-3 with a nAChR that requires CC-I in vivo; thus CC-I appears to enhance affinity of RIC-3 to specific nAChRs. However, we found that this function of CC-I is redundant with functions of sequences downstream to CC-I, potentially a second coiled-coil. Alternative splicing in both vertebrates and invertebrates generates RIC-3 transcripts that lack the entire C-terminus, or only CC-I. Thus, our results suggest that RIC-3 alternative splicing enables subtype specific regulation of nAChR maturation. PMID:19116311

  19. CCBuilder: an interactive web-based tool for building, designing and assessing coiled-coil protein assemblies

    PubMed Central

    Wood, Christopher W.; Bruning, Marc; Ibarra, Amaurys Á.; Bartlett, Gail J.; Thomson, Andrew R.; Sessions, Richard B.; Brady, R Leo; Woolfson, Derek N.

    2014-01-01

    Motivation: The ability to accurately model protein structures at the atomistic level underpins efforts to understand protein folding, to engineer natural proteins predictably and to design proteins de novo. Homology-based methods are well established and produce impressive results. However, these are limited to structures presented by and resolved for natural proteins. Addressing this problem more widely and deriving truly ab initio models requires mathematical descriptions for protein folds; the means to decorate these with natural, engineered or de novo sequences; and methods to score the resulting models. Results: We present CCBuilder, a web-based application that tackles the problem for a defined but large class of protein structure, the α-helical coiled coils. CCBuilder generates coiled-coil backbones, builds side chains onto these frameworks and provides a range of metrics to measure the quality of the models. Its straightforward graphical user interface provides broad functionality that allows users to build and assess models, in which helix geometry, coiled-coil architecture and topology and protein sequence can be varied rapidly. We demonstrate the utility of CCBuilder by assembling models for 653 coiled-coil structures from the PDB, which cover >96% of the known coiled-coil types, and by generating models for rarer and de novo coiled-coil structures. Availability and implementation: CCBuilder is freely available, without registration, at http://coiledcoils.chm.bris.ac.uk/app/cc_builder/ Contact: D.N.Woolfson@bristol.ac.uk or Chris.Wood@bristol.ac.uk PMID:25064570

  20. A Coiled-coil Clamp Controls Both Conformation and Clustering of Stromal Interaction Molecule 1 (STIM1)*

    PubMed Central

    Fahrner, Marc; Muik, Martin; Schindl, Rainer; Butorac, Carmen; Stathopulos, Peter; Zheng, Le; Jardin, Isaac; Ikura, Mitsuhiko; Romanin, Christoph

    2014-01-01

    Store-operated Ca2+ entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Ca2+ entry occurs through the plasma membrane resident Ca2+ channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca2+ store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) interactions in controlling STIM1 quiescent as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai1 channels. PMID:25342749

  1. An intramolecular interaction between the FHA domain and a coiled coil negatively regulates the kinesin motor KIF1A

    PubMed Central

    Lee, Jae-Ran; Shin, Hyewon; Choi, Jeonghoon; Ko, Jaewon; Kim, Seho; Lee, Hyun Woo; Kim, Karam; Rho, Seong-Hwan; Lee, Jun Hyuck; Song, Hye-Eun; Eom, Soo Hyun; Kim, Eunjoon

    2004-01-01

    Motor proteins not actively involved in transporting cargoes should remain inactive at sites of cargo loading to save energy and remain available for loading. KIF1A/Unc104 is a monomeric kinesin known to dimerize into a processive motor at high protein concentrations. However, the molecular mechanisms underlying monomer stabilization and monomer-to-dimer transition are not well understood. Here, we report an intramolecular interaction in KIF1A between the forkhead-associated (FHA) domain and a coiled-coil domain (CC2) immediately following the FHA domain. Disrupting this interaction by point mutations in the FHA or CC2 domains leads to a dramatic accumulation of KIF1A in the periphery of living cultured neurons and an enhancement of the microtubule (MT) binding and self-multimerization of KIF1A. In addition, point mutations causing rigidity in the predicted flexible hinge disrupt the intramolecular FHA–CC2 interaction and increase MT binding and peripheral accumulation of KIF1A. These results suggest that the intramolecular FHA–CC2 interaction negatively regulates KIF1A activity by inhibiting MT binding and dimerization of KIF1A, and point to a novel role of the FHA domain in the regulation of kinesin motors. PMID:15014437

  2. Study on the interaction between methyl jasmonate and the coiled-coil domain of rice blast resistance protein Pi36 by spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Liu, Xin Q.; Zhang, Dan; Zhang, Xiang M.; Wang, Chun T.; Liu, Xue Q.; Tan, Yan P.; Wu, Yun H.

    2012-03-01

    Interaction between the coiled-coil domain of rice blast resistance protein Pi36 and methyl-jasmonate (MeJA) was studied by fluorescence and UV-vis spectroscopic techniques. The quenching mechanism of fluorescence of MeJA by this domain was discussed to be a static quenching procedure. Fluorescence quenching was explored to measure the number of binding sites n and apparent binding constants K. The thermodynamics parameters ΔH, ΔG, ΔS were also calculated. The results indicate the binding reaction was not entropy-driven but enthalpy-driven, and hydrophobic binding played major role in the interaction. The binding sites of MeJA with the coiled-coil structural domain of rice blast resistance protein Pi36 were found to approach the microenvironment of both Tyr and Trp by the synchronous fluorescence spectrometry. The distance r between donor (the coiled-coil domain of rice blast resistance protein Pi36) and acceptor (MeJA) was obtained according to Förster theory of non-radioactive energy transfer.

  3. Disruption of Bcr-Abl Coiled Coil Oligomerization by Design*

    PubMed Central

    Dixon, Andrew S.; Pendley, Scott S.; Bruno, Benjamin J.; Woessner, David W.; Shimpi, Adrian A.; Cheatham, Thomas E.; Lim, Carol S.

    2011-01-01

    Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention. PMID:21659527

  4. SUMO modification of TBK1 at the adaptor-binding C-terminal coiled-coil domain contributes to its antiviral activity.

    PubMed

    Saul, Vera V; Niedenthal, Rainer; Pich, Andreas; Weber, Friedemann; Schmitz, M Lienhard

    2015-01-01

    The non-canonical IKK kinase TBK1 serves as an important signal transmitter of the antiviral interferon response, but is also involved in the regulation of further processes such as autophagy. The activity of TBK1 is regulated by posttranslational modifications comprising phosphorylation and ubiquitination. This study identifies SUMOylation as a novel posttranslational TBK1 modification. TBK1 kinase activity is required to allow the attachment of SUMO1 or SUMO2/3 proteins. Since TBK1 does not bind to the E2 enzyme Ubc9, this modification most likely proceeds via trans-SUMOylation. Mass spectrometry allowed identifying K694 as the SUMO acceptor site, a residue located in the C-terminal coiled-coil domain which is exclusively responsible for the association with the adaptor proteins NAP1, Sintbad and TANK. SUMO modification at K694 contributes to the antiviral function of TBK1 and accordingly the viral protein Gam1 antagonizes this posttranslational modification. PMID:25409927

  5. The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis

    PubMed Central

    2014-01-01

    Background During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. Results Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation. Conclusions As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation. PMID

  6. α/β coiled coils

    PubMed Central

    Hartmann, Marcus D; Mendler, Claudia T; Bassler, Jens; Karamichali, Ioanna; Ridderbusch, Oswin; Lupas, Andrei N; Hernandez Alvarez, Birte

    2016-01-01

    Coiled coils are the best-understood protein fold, as their backbone structure can uniquely be described by parametric equations. This level of understanding has allowed their manipulation in unprecedented detail. They do not seem a likely source of surprises, yet we describe here the unexpected formation of a new type of fiber by the simple insertion of two or six residues into the underlying heptad repeat of a parallel, trimeric coiled coil. These insertions strain the supercoil to the breaking point, causing the local formation of short β-strands, which move the path of the chain by 120° around the trimer axis. The result is an α/β coiled coil, which retains only one backbone hydrogen bond per repeat unit from the parent coiled coil. Our results show that a substantially novel backbone structure is possible within the allowed regions of the Ramachandran space with only minor mutations to a known fold. DOI: http://dx.doi.org/10.7554/eLife.11861.001 PMID:26771248

  7. Coiled-coil networking shapes cell molecular machinery

    PubMed Central

    Wang, Yongqiang; Zhang, Xinlei; Zhang, Hong; Lu, Yi; Huang, Haolong; Dong, Xiaoxi; Chen, Jinan; Dong, Jiuhong; Yang, Xiao; Hang, Haiying; Jiang, Taijiao

    2012-01-01

    The highly abundant α-helical coiled-coil motif not only mediates crucial protein–protein interactions in the cell but is also an attractive scaffold in synthetic biology and material science and a potential target for disease intervention. Therefore a systematic understanding of the coiled-coil interactions (CCIs) at the organismal level would help unravel the full spectrum of the biological function of this interaction motif and facilitate its application in therapeutics. We report the first identified genome-wide CCI network in Saccharomyces cerevisiae, which consists of 3495 pair-wise interactions among 598 predicted coiled-coil regions. Computational analysis revealed that the CCI network is specifically and functionally organized and extensively involved in the organization of cell machinery. We further show that CCIs play a critical role in the assembly of the kinetochore, and disruption of the CCI network leads to defects in kinetochore assembly and cell division. The CCI network identified in this study is a valuable resource for systematic characterization of coiled coils in the shaping and regulation of a host of cellular machineries and provides a basis for the utilization of coiled coils as domain-based probes for network perturbation and pharmacological applications. PMID:22875988

  8. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N' and Regulates Light-Dependent Cell Death.

    PubMed

    Hamel, Louis-Philippe; Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei; Moffett, Peter

    2016-05-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N', which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N' results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N' is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. PMID:26951433

  9. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N′ and Regulates Light-Dependent Cell Death1[OPEN

    PubMed Central

    Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei

    2016-01-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N′, which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N′ results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N′ is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. PMID:26951433

  10. Transforming acidic coiled-coil 3 and Aurora-A interact in human thyrocytes and their expression is deregulated in thyroid cancer tissues

    PubMed Central

    Ulisse, Salvatore; Baldini, Enke; Toller, Matteo; Delcros, Jean-Guy; Guého, Aurélie; Curcio, Francesco; De Antoni, Enrico; Giacomelli, Laura; Ambesi-Impiombato, Francesco S; Bocchini, Sarah; D'Armiento, Massimino; Arlot-Bonnemains, Yannick

    2007-01-01

    Aurora-A kinase has recently been shown to be deregulated in thyroid cancer cells and tissues. Among the Aurora-A substrates identified, transforming acidic coiled-coil (TACC3), a member of the TACC family, plays an important role in cell cycle progression and alterations of its expression occur in different cancer tissues. In this study, we demonstrated the expression of the TACC3 gene in normal human thyroid cells (HTU5), and its modulation at both mRNA and protein levels during cell cycle. Its expression was found, with respect to HTU5 cells, unchanged in cells derived from a benign thyroid follicular tumor (HTU42), and significantly reduced in cell lines derived from follicular (FTC-133), papillary (B-CPAP), and anaplastic thyroid carcinomas (CAL-62 and 8305C). Moreover, in 16 differentiated thyroid cancer tissues, TACC3 mRNA levels were found, with respect to normal matched tissues, reduced by twofold in 56% of cases and increased by twofold in 44% of cases. In the same tissues, a correlation between the expression of the TACC3 and Aurora-A mRNAs was observed. TACC3 and Aurora-A interact in vivo in thyroid cells and both proteins localized onto the mitotic structure of thyroid cells. Finally, TACC3 localization on spindle microtubule was no more observed following the inhibition of Aurora kinase activity by VX-680. We propose that Aurora-A and TACC3 interaction is important to control the mitotic spindle organization required for proper chromosome segregation. PMID:17914111

  11. A Parallel Coiled-Coil Tetramer with Offset Helices

    SciTech Connect

    Liu,J.; Deng, Y.; Zheng, Q.; Cheng, C.; Kallenbach, N.; Lu, M.

    2006-01-01

    Specific helix-helix interactions are fundamental in assembling the native state of proteins and in protein-protein interfaces. Coiled coils afford a unique model system for elucidating principles of molecular recognition between {alpha} helices. The coiled-coil fold is specified by a characteristic seven amino acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Nonpolar side chains spaced three and four residues apart are referred to as the 3-4 hydrophobic repeat. The presence of apolar amino acids at the e or g positions (corresponding to a 3-3-1 hydrophobic repeat) can provide new possibilities for close-packing of {alpha}-helices that includes examples such as the lac repressor tetramerization domain. Here we demonstrate that an unprecedented coiled-coil interface results from replacement of three charged residues at the e positions in the dimeric GCN4 leucine zipper by nonpolar valine side chains. Equilibrium circular dichroism and analytical ultracentrifugation studies indicate that the valine-containing mutant forms a discrete {alpha}-helical tetramer with a significantly higher stability than the parent leucine-zipper molecule. The 1.35 {angstrom} resolution crystal structure of the tetramer reveals a parallel four-stranded coiled coil with a three-residue interhelical offset. The local packing geometry of the three hydrophobic positions in the tetramer conformation is completely different from that seen in classical tetrameric structures yet bears resemblance to that in three-stranded coiled coils. These studies demonstrate that distinct van der Waals interactions beyond the a and d side chains can generate a diverse set of helix-helix interfaces and three-dimensional supercoil structures.

  12. Contributed Review: Absolute spectral radiance calibration of fiber-optic shock-temperature pyrometers using a coiled-coil irradiance standard lamp.

    PubMed

    Fat'yanov, O V; Asimow, P D

    2015-10-01

    We describe an accurate and precise calibration procedure for multichannel optical pyrometers such as the 6-channel, 3-ns temporal resolution instrument used in the Caltech experimental geophysics laboratory. We begin with a review of calibration sources for shock temperatures in the 3000-30,000 K range. High-power, coiled tungsten halogen standards of spectral irradiance appear to be the only practical alternative to NIST-traceable tungsten ribbon lamps, which are no longer available with large enough calibrated area. However, non-uniform radiance complicates the use of such coiled lamps for reliable and reproducible calibration of pyrometers that employ imaging or relay optics. Careful analysis of documented methods of shock pyrometer calibration to coiled irradiance standard lamps shows that only one technique, not directly applicable in our case, is free of major radiometric errors. We provide a detailed description of the modified Caltech pyrometer instrument and a procedure for its absolute spectral radiance calibration, accurate to ±5%. We employ a designated central area of a 0.7× demagnified image of a coiled-coil tungsten halogen lamp filament, cross-calibrated against a NIST-traceable tungsten ribbon lamp. We give the results of the cross-calibration along with descriptions of the optical arrangement, data acquisition, and processing. We describe a procedure to characterize the difference between the static and dynamic response of amplified photodetectors, allowing time-dependent photodiode correction factors for spectral radiance histories from shock experiments. We validate correct operation of the modified Caltech pyrometer with actual shock temperature experiments on single-crystal NaCl and MgO and obtain very good agreement with the literature data for these substances. We conclude with a summary of the most essential requirements for error-free calibration of a fiber-optic shock-temperature pyrometer using a high-power coiled tungsten halogen

  13. Contributed Review: Absolute spectral radiance calibration of fiber-optic shock-temperature pyrometers using a coiled-coil irradiance standard lamp

    SciTech Connect

    Fat’yanov, O. V. Asimow, P. D.

    2015-10-15

    We describe an accurate and precise calibration procedure for multichannel optical pyrometers such as the 6-channel, 3-ns temporal resolution instrument used in the Caltech experimental geophysics laboratory. We begin with a review of calibration sources for shock temperatures in the 3000-30 000 K range. High-power, coiled tungsten halogen standards of spectral irradiance appear to be the only practical alternative to NIST-traceable tungsten ribbon lamps, which are no longer available with large enough calibrated area. However, non-uniform radiance complicates the use of such coiled lamps for reliable and reproducible calibration of pyrometers that employ imaging or relay optics. Careful analysis of documented methods of shock pyrometer calibration to coiled irradiance standard lamps shows that only one technique, not directly applicable in our case, is free of major radiometric errors. We provide a detailed description of the modified Caltech pyrometer instrument and a procedure for its absolute spectral radiance calibration, accurate to ±5%. We employ a designated central area of a 0.7× demagnified image of a coiled-coil tungsten halogen lamp filament, cross-calibrated against a NIST-traceable tungsten ribbon lamp. We give the results of the cross-calibration along with descriptions of the optical arrangement, data acquisition, and processing. We describe a procedure to characterize the difference between the static and dynamic response of amplified photodetectors, allowing time-dependent photodiode correction factors for spectral radiance histories from shock experiments. We validate correct operation of the modified Caltech pyrometer with actual shock temperature experiments on single-crystal NaCl and MgO and obtain very good agreement with the literature data for these substances. We conclude with a summary of the most essential requirements for error-free calibration of a fiber-optic shock-temperature pyrometer using a high-power coiled tungsten halogen

  14. Contributed Review: Absolute spectral radiance calibration of fiber-optic shock-temperature pyrometers using a coiled-coil irradiance standard lamp

    NASA Astrophysics Data System (ADS)

    Fat'yanov, O. V.; Asimow, P. D.

    2015-10-01

    We describe an accurate and precise calibration procedure for multichannel optical pyrometers such as the 6-channel, 3-ns temporal resolution instrument used in the Caltech experimental geophysics laboratory. We begin with a review of calibration sources for shock temperatures in the 3000-30 000 K range. High-power, coiled tungsten halogen standards of spectral irradiance appear to be the only practical alternative to NIST-traceable tungsten ribbon lamps, which are no longer available with large enough calibrated area. However, non-uniform radiance complicates the use of such coiled lamps for reliable and reproducible calibration of pyrometers that employ imaging or relay optics. Careful analysis of documented methods of shock pyrometer calibration to coiled irradiance standard lamps shows that only one technique, not directly applicable in our case, is free of major radiometric errors. We provide a detailed description of the modified Caltech pyrometer instrument and a procedure for its absolute spectral radiance calibration, accurate to ±5%. We employ a designated central area of a 0.7× demagnified image of a coiled-coil tungsten halogen lamp filament, cross-calibrated against a NIST-traceable tungsten ribbon lamp. We give the results of the cross-calibration along with descriptions of the optical arrangement, data acquisition, and processing. We describe a procedure to characterize the difference between the static and dynamic response of amplified photodetectors, allowing time-dependent photodiode correction factors for spectral radiance histories from shock experiments. We validate correct operation of the modified Caltech pyrometer with actual shock temperature experiments on single-crystal NaCl and MgO and obtain very good agreement with the literature data for these substances. We conclude with a summary of the most essential requirements for error-free calibration of a fiber-optic shock-temperature pyrometer using a high-power coiled tungsten halogen

  15. Transport Vesicle Tethering at the Trans Golgi Network: Coiled Coil Proteins in Action

    PubMed Central

    Cheung, Pak-yan P.; Pfeffer, Suzanne R.

    2016-01-01

    The Golgi complex is decorated with so-called Golgin proteins that share a common feature: a large proportion of their amino acid sequences are predicted to form coiled-coil structures. The possible presence of extensive coiled coils implies that these proteins are highly elongated molecules that can extend a significant distance from the Golgi surface. This property would help them to capture or trap inbound transport vesicles and to tether Golgi mini-stacks together. This review will summarize our current understanding of coiled coil tethers that are needed for the receipt of transport vesicles at the trans Golgi network (TGN). How do long tethering proteins actually catch vesicles? Golgi-associated, coiled coil tethers contain numerous binding sites for small GTPases, SNARE proteins, and vesicle coat proteins. How are these interactions coordinated and are any or all of them important for the tethering process? Progress toward understanding these questions and remaining, unresolved mysteries will be discussed. PMID:27014693

  16. Immune responses to coiled coil supramolecular biomaterials

    PubMed Central

    Rudra, Jai S.; Tripathi, Pulak; Hildeman, David A.; Jung, Jangwook P.; Collier, Joel H.

    2010-01-01

    Self-assembly has been increasingly utilized in recent years to create peptide-based biomaterials for 3D cell culture, tissue engineering, and regenerative medicine, but the molecular determinants of these materials' immunogenicity have remained largely unexplored. In this study, a set of molecules that self-assembled through coiled coil oligomerization was designed and synthesized, and immune responses against them were investigated in mice. Experimental groups spanned a range of oligomerization behaviors and included a peptide from the coiled coil region of mouse fibrin that did not form supramolecular structures, an engineered version of this peptide that formed coiled coil bundles, and a peptide-PEG-peptide triblock bioconjugate that formed coiled coil multimers and supramolecular aggregates. In mice, the native peptide and engineered peptide did not produce any detectable antibody response, and none of the materials elicited detectable peptide-specific T cell responses, as evidenced by the absence of IL-2 and interferon-gamma in cultures of peptide-challenged splenocytes or draining lymph node cells. However, specific antibody responses were elevated in mice injected with the multimerizing peptide-PEG-peptide. Minimal changes in secondary structure were observed between the engineered peptide and the triblock peptide-PEG-peptide, making it possible that the triblock's multimerization was responsible for this antibody response. PMID:20708258

  17. A Non-perturbing Probe of Coiled Coil Formation Based on Electron Transfer Mediated Fluorescence Quenching.

    PubMed

    Watson, Matthew D; Peran, Ivan; Raleigh, Daniel P

    2016-07-01

    Coiled coils are abundant in nature, occurring in ∼3% of proteins across sequenced genomes, and are found in proteins ranging from transcription factors to structural proteins. The motif continues to be an important model system for understanding protein-protein interactions and is finding increased use in bioinspired materials and synthetic biology. Knowledge of the thermodynamics of self-assembly, particularly the dissociation constant KD, is essential for the application of designed coiled coils and for understanding the in vivo specificity of natural coiled coils. Standard methods for measuring KD typically rely on concentration dependent circular dichroism (CD). Fluorescence methods are an attractive alternative; however Trp is rarely found in an interior position of a coiled coil, and appending unnatural fluorophores can perturb the system. We demonstrate a simple, non-perturbing method to monitor coiled coil formation using p-cyanophenylalanine (FCN) and selenomethionine (MSe), the Se analogue of Met. FCN fluorescence can be selectively excited and is effectively quenched by electron transfer with MSe. Both FCN and MSe represent minimally perturbing substitutions in coiled coils. MSe quenching of FCN fluorescence is shown to offer a non-perturbing method for following coiled coil formation and for accurately determining dissociation constants. The method is validated using a designed heterodimeric coiled coil. The KD deduced by fluorescence monitored titration is in excellent agreement with the value deduced from concentration dependent CD measurements to within the uncertainty of the measurement. However, the fluorescence approach requires less protein, is less time-consuming, can be applied to lower concentrations and could be applied to high throughput screens. PMID:27258904

  18. Kinking the coiled coil--negatively charged residues at the coiled-coil interface.

    PubMed

    Straussman, Ravid; Ben-Ya'acov, Ami; Woolfson, Derek N; Ravid, Shoshana

    2007-03-01

    The coiled coil is one of the most common protein-structure motifs. It is believed to be adopted by 3-5% of all amino acids in proteins. It comprises two or more alpha-helical chains wrapped around one another. The sequences of most coiled coils are characterized by a seven-residue (heptad) repeat, denoted (abcdefg)(n). Residues at the a and d positions define the helical interface (core) and are usually hydrophobic, though about 20% are polar or charged. We show that parallel coiled-coils have a unique pattern of their negatively charged residues at the core positions: aspartic acid is excluded from these positions while glutamic acid is not. In contrast the antiparallel structures are more permissive in their amino acid usage. We show further, and for the first time, that incorporation of Asp but not Glu into the a positions of a parallel coiled coil creates a flexible hinge and that the maximal hinge angle is being directly related to the number of incorporated mutations. These new computational and experimental observations will be of use in improving protein-structure predictions, and as rules to guide rational design of novel coiled-coil motifs and coiled coil-based materials. PMID:17207815

  19. A Synthetic Coiled-Coil Interactome Provides Heterospecific Modules for Molecular Engineering

    SciTech Connect

    Reinke, Aaron W.; Grant, Robert A.; Keating, Amy E.

    2010-06-21

    The versatile coiled-coil protein motif is widely used to induce and control macromolecular interactions in biology and materials science. Yet the types of interaction patterns that can be constructed using known coiled coils are limited. Here we greatly expand the coiled-coil toolkit by measuring the complete pairwise interactions of 48 synthetic coiled coils and 7 human bZIP coiled coils using peptide microarrays. The resulting 55-member protein 'interactome' includes 27 pairs of interacting peptides that preferentially heteroassociate. The 27 pairs can be used in combinations to assemble sets of 3 to 6 proteins that compose networks of varying topologies. Of special interest are heterospecific peptide pairs that participate in mutually orthogonal interactions. Such pairs provide the opportunity to dimerize two separate molecular systems without undesired crosstalk. Solution and structural characterization of two such sets of orthogonal heterodimers provide details of their interaction geometries. The orthogonal pair, along with the many other network motifs discovered in our screen, provide new capabilities for synthetic biology and other applications.

  20. High-resolution structures of a heterochiral coiled coil

    PubMed Central

    Mortenson, David E.; Steinkruger, Jay D.; Kreitler, Dale F.; Perroni, Dominic V.; Sorenson, Gregory P.; Huang, Lijun; Mittal, Ritesh; Yun, Hyun Gi; Travis, Benjamin R.; Mahanthappa, Mahesh K.; Forest, Katrina T.; Gellman, Samuel H.

    2015-01-01

    Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein–protein interactions. Coiled–coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled–coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern. PMID:26460035

  1. Tropomyosin lysine reactivities and relationship to coiled-coil structure.

    PubMed

    Hitchcock-DeGregori, S E; Lewis, S F; Chou, T M

    1985-06-18

    We have carried out a detailed analysis of tropomyosin structure using lysines as specific probes for the protein surface in regions of the molecule that have not been investigated by other methods. We have measured the relative reactivities of lysines in rabbit skeletal muscle alpha, alpha-tropomyosin with acetic anhydride using a competitive labeling procedure. We have identified 37 of 39 lysines and find that they range 20-fold in reactivity. The observed reactivities are related to the coiled-coil model of the tropomyosin molecule [Crick, F.H.C. (1953) Acta Crystallogr. 6, 689-697; McLachlan, A.D., Stewart, M., & Smillie, L.B. (1975) J. Mol. Biol. 98, 281-291] and other available chemical and physical information about the structure. In most cases, the observed lysine reactivities can be explained by allowable interactions with neighboring amino acid side chains on the same or facing alpha-helix. However, we found no correlation between reactivity and helical position of a given lysine. For example, lysines in the outer helical positions included lysines of low as well as high reactivity, indicating that they vary widely in their accessibility to solvent and that the coiled coil is heterogeneous along its length. Furthermore, the middle of the molecule (residues 126-182) that is susceptible to proteolysis and known to be the least stable region of the protein also contains some of the least and most reactive lysines. We have discussed the implications of our results on our understanding the structures of tropomyosin and other coiled-coil proteins as well as globular proteins containing helical regions. PMID:3927977

  2. Crystal Structure of the Central Coiled-Coil Domain from Human Liprin-[beta]2

    SciTech Connect

    Stafford, Ryan L.; Tang, Ming-Yun; Sawaya, Michael R.; Phillips, Martin L.; Bowie, James U.

    2012-02-07

    Liprins are a conserved family of scaffolding proteins important for the proper regulation and development of neuronal synapses. Humans have four liprin-{alpha}s and two liprin-{beta}s which all contain long coiled-coil domains followed by three tandem SAM domains. Complex interactions between the coiled-coil and SAM domains are thought to create liprin scaffolds, but the structural and biochemical properties of these domains remain largely uncharacterized. In this study we find that the human liprin-{beta}2 coiled-coil forms an extended dimer. Several protease-resistant subdomains within the liprin-{beta}1 and liprin-{beta}2 coiled-coils were also identified. A 2.0 {angstrom} crystal structure of the central, protease-resistant core of the liprin-{beta}2 coiled-coil reveals a parallel helix orientation. These studies represent an initial step toward determining the overall architecture of liprin scaffolds and understanding the molecular basis for their synaptic functions.

  3. Finding the Golgi: Golgin Coiled-Coil Proteins Show the Way.

    PubMed

    Gillingham, Alison K; Munro, Sean

    2016-06-01

    The Golgi apparatus lies at the centre of the secretory pathway. It consists of a series of flattened compartments typically organised into a stack that, in mammals, is connected to additional stacks to form a Golgi ribbon. The Golgi is responsible for the maturation and modification of proteins and lipids, and receives and exports vesicles to and from multiple destinations within the cell. This complex trafficking network requires that only the correct vesicles fuse with the correct destination membrane. Recently, a group of coiled-coil proteins called golgins were shown to not only capture incoming vesicles but to also provide specificity to the tethering step. This raises many interesting questions about how they interact with other components of membrane traffic, some of which may also contribute to specificity. PMID:26972448

  4. Subunit b-Dimer of the Escherichia coli ATP Synthase Can Form Left-Handed Coiled-Coils

    PubMed Central

    Wise, John G.; Vogel, Pia D.

    2008-01-01

    One remaining challenge to our understanding of the ATP synthase concerns the dimeric coiled-coil stator subunit b of bacterial synthases. The subunit b-dimer has been implicated in important protein interactions that appear necessary for energy conservation and that may be instrumental in energy conservation during rotary catalysis by the synthase. Understanding the stator structure and its interactions with the rest of the enzyme is crucial to the understanding of the overall catalytic mechanism. Controversy exists on whether subunit b adopts a classic left-handed or a presumed right-handed dimeric coiled-coil and whether or not staggered pairing between nonhomologous residues in the homodimer is required for intersubunit packing. In this study we generated molecular models of the Escherichia coli subunit b-dimer that were based on the well-established heptad-repeat packing exhibited by left-handed, dimeric coiled-coils by employing simulated annealing protocols with structural restraints collected from known structures. In addition, we attempted to create hypothetical right-handed coiled-coil models and left- and right-handed models with staggered packing in the coiled-coil domains. Our analyses suggest that the available structural and biochemical evidence for subunit b can be accommodated by classic left-handed, dimeric coiled-coil quaternary structures. PMID:18326648

  5. Antiparallel Four-Stranded Coiled Coil Specified by a 3-3-1 Hyrdrophobic Heptad Repeat

    SciTech Connect

    Deng,Y.; Liu, J.; Zheng, Q.; Eliezer, D.; Kallenbach, N.; Lu, M.

    2006-01-01

    Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.

  6. Forced Unfolding of the Coiled-Coils of Fibrinogen by Single-Molecule AFM

    NASA Astrophysics Data System (ADS)

    Brown, Andre; Litvinov, Rustem; Discher, Dennis; Weisel, John

    2007-03-01

    A blood clot needs to have the right degree of stiffness and plasticity for hemostasis, but the origin of these mechanical properties is unknown. Here we report the first measurements using single molecule atomic force microscopy (AFM) to study the forced unfolding of fibrinogen to begin addressing this problem. To generate longer reproducible curves than are possible using monomer, factor XIIIa cross-linked, single chain fibrinogen oligomers were used. When extended under force, these oligomers showed sawtooth shaped force-extension patterns characteristic of unfolding proteins with a peak-to-peak separation of approximately 26 nm, consistent with the independent unfolding of the coiled-coils. These results were then reproduced using a Monte Carlo simulation with parameters in the same range as those previously used for unfolding globular domains. In particular, we found that the refolding time was negligible on experimental time and force scales in contrast to previous work on simpler coiled-coils. We suggest that this difference may be due to fibrinogen's structurally and topologically more complex coiled-coils and that an interaction between the alpha C and central domains may be involved. These results suggest a new functional property of fibrinogen and that the coiled-coil is more than a passive structural element of this molecule.

  7. Essential role of coiled-coils for aggregation and activity of Q/N-rich prions and polyQ proteins

    PubMed Central

    Fiumara, Ferdinando; Fioriti, Luana

    2012-01-01

    SUMMARY The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming β-sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled-coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical cross-linking, we found that Q/N-rich and polyQ peptides form α-helical coiled-coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled-coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases. PMID:21183075

  8. Protein destabilization by electrostatic repulsions in the two-stranded alpha-helical coiled-coil/leucine zipper.

    PubMed Central

    Kohn, W. D.; Kay, C. M.; Hodges, R. S.

    1995-01-01

    The destabilizing effect of electrostatic repulsions on protein stability has been studied by using synthetic two-stranded alpha-helical coiled-coils as a model system. The native coiled-coil consists of two identical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain disulfide bridge. This peptide, designed to contain no intrahelical or interhelical electrostatic interactions, forms a stable coiled-coil structure at 20 degrees C in benign medium (50 mM KCl, 25 mM PO4, pH 7) with a [urea]1/2 value of 6.1 M. Four mutant coiled-coils were designed to contain one or two Glu substitutions for Gln per polypeptide chain. The resulting coiled-coils contained potential i to i' + 5 Glu-Glu interchain repulsions (denoted as peptide E2(15,20)), i to i' + 2 Glu-Glu interchain repulsions (denoted E2(20,22)), or no interchain ionic interactions (denoted E2(13,22) and E1(20)). The stabilities of the coiled-coils were determined by measuring the ellipticities at 222 nm as a function of urea or guanidine hydrochloride concentration at 20 degrees C in the presence and absence of an interchain disulfide bridge. At pH 7, in the presence of urea, the stabilities of E2(13,22) and E2(20,22) were identical suggesting that the potential i to i' + 2 interchain Glu-Glu repulsion in the E2(20,22) coiled-coil does not occur. In contrast, the mutant E2(15,20) is substantially less stable than E2(13,22) or E2(15,20) by 0.9 kcal/mol due to the presence of two i to i' + 5 interchain Glu-Glu repulsions, which destabilize the coiled-coil by 0.45 kcal/mol each. At pH 3 the coiled-coils were found to increase in stability as the number of Glu substitutions were increased. This, combined with reversed-phase HPLC results at pH 7 and pH 2, supports the conclusion that the protonated Glu side chains present at low pH are significantly more hydrophobic than Gln side chains which are in turn more hydrophobic than the ionized

  9. X-Ray Crystal Structure of a TRPM Assembly Domain Reveals An Antiparallel Four-Stranded Coiled-Coil

    SciTech Connect

    Fujiwara, Y.; Minor, D.L.; Jr.

    2009-05-18

    Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold.

  10. X-ray crystal structure of a TRPM assembly domain reveals an antiparallel four-stranded coiled-coil

    PubMed Central

    Fujiwara, Yuichiro; Minor, Daniel L.

    2008-01-01

    Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Previous studies have shown that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of the TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold. PMID:18782578

  11. X-ray crystal structure of a TRPM assembly domain reveals an antiparallel four-stranded coiled-coil.

    PubMed

    Fujiwara, Yuichiro; Minor, Daniel L

    2008-11-21

    Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold. PMID:18782578

  12. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    SciTech Connect

    Singh, Pratibha; Savithri, H.S.

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  13. Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins.

    PubMed

    Waldman, Vincent M; Stanage, Tyler H; Mims, Alexandra; Norden, Ian S; Oakley, Martha G

    2015-06-01

    The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans. PMID:25664627

  14. Coiled coil rich proteins (Ccrp) influence molecular pathogenicity of Helicobacter pylori.

    PubMed

    Schätzle, Sarah; Specht, Mara; Waidner, Barbara

    2015-01-01

    Pathogenicity of the human pathogen Helicobacter pylori relies on its capacity to adapt to a hostile environment and to escape the host response. Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its contribution to virulence. In this study we have explored the influence of coiled coil rich proteins (Ccrp) cytoskeletal elements on pathogenicity factors of H. pylori. Deletion of any of the ccrp resulted in a strongly decreased activity of the main pathogenicity factor urease. We further investigated their role using in vitro co-culture experiments with the human gastric adenocarcinoma cell line AGS modeling H. pylori - host cell interactions. Intriguingly, host cell showed only a weak "scattering/hummingbird" phenotype, in which host cells are transformed from a uniform polygonal shape into a severely elongated state characterized by the formation of needle-like projections, after co-incubation with any ccrp deletion mutant. Furthermore, co-incubation with the ccrp59 mutant resulted in reduced type IV secretion system associated activities, e.g. IL-8 production and CagA translocation/phosphorylation. Thus, in addition to their role in maintaining the helical cell shape of H. pylori Ccrp proteins influence many cellular processes and are thereby crucial for the virulence of this human pathogen. PMID:25822999

  15. Growth factor identity is encoded by discrete coiled coil rotamers in the EGFR juxtamembrane region

    PubMed Central

    Doerner, Amy; Scheck, Rebecca; Schepartz, Alanna

    2015-01-01

    Summary Binding of the growth factor TGF-α to the EGFR extracellular domain is encoded through the formation of a unique anti-parallel coiled coil within the juxtamembrane segment. This new coiled coil is an ‘inside-out’ version of the coiled coil formed in the presence of EGF. A third, intermediary coiled coil interface is formed in the juxtamembrane segment when EGFR is stimulated with betacellulin. The seven growth factors that activate EGFR in mammalian systems (EGF, TGF-α, epigen, epiregulin, betacellulin, heparin-binding EGF, and amphiregulin) fall into distinct categories in which the structure of the coiled coil induced within the juxtamembrane segment correlates with cell state. The observation that coiled coil state tracks with the downstream signaling profiles for each ligand provides evidence for growth factor functional selectivity by EGFR. Encoding growth factor identity in alternative coiled coil rotamers provides a simple and elegant method for communicating chemical information across the plasma membrane. PMID:26091170

  16. Coiled-coil intermediate filament stutter instability and molecular unfolding.

    PubMed

    Arslan, Melis; Qin, Zhao; Buehler, Markus J

    2011-05-01

    Intermediate filaments (IFs) are the key components of cytoskeleton in eukaryotic cells and are critical for cell mechanics. The building block of IFs is a coiled-coil alpha-helical dimer, consisting of several domains that include linkers and other structural discontinuities. One of the discontinuities in the dimer's coiled-coil region is the so-called 'stutter' region. The stutter is a region where a variation of the amino acid sequence pattern from other parts of the alpha-helical domains of the protein is found. It was suggested in earlier works that due to this sequence variation, the perfect coiled-coil arrangement ceases to exist. Here, we show using explicit water molecular dynamics and well-tempered metadynamics that for the coil2 domain of vimentin IFs the stutter is more stable in a non-alpha-helical, unfolded state. This causes a local structural disturbance in the alpha helix, which has a global effect on the nanomechanics of the structure. Our analysis suggests that the stutter features an enhanced tendency to unfolding even under the absence of external forces, implying a much greater structural instability than previously assumed. As a result it features a smaller local bending stiffness than other segments and presents a seed for the initiation of molecular bending and unfolding at large deformation. PMID:21516532

  17. Effect of chain length on the formation and stability of synthetic alpha-helical coiled coils.

    PubMed

    Su, J Y; Hodges, R S; Kay, C M

    1994-12-27

    A series of polypeptides containing 9, 12, 16, 19, 23, 26, 30, 33, and 35 amino acid residues was designed to investigate the effects of peptide chain length on the formation and stability of two-stranded alpha-helical dimers or coiled coils. These peptides were synthesized by the solid-phase method, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and characterized by RP-HPLC, amino acid composition analysis, and mass spectrometry. The amphipathic alpha-helical peptides were designed to dimerize by interchain hydrophobic interactions at positions a and d and interchain salt bridges between lysine and glutamic acid residues at positions e and g of the repeating heptad sequence of Glu-Ile-Glu-Ala-Leu-Lys-Ala (g-a-b-c-d-e-f). The ability of these peptides to form alpha-helical structures in the presence and absence of a helix-inducing reagent (trifluoroethanol) was monitored by circular dichroism spectroscopy. The helicity of the peptides increased with increasing chain length in a cooperative manner. A minimum of three heptads corresponding to six helical turns was required for a peptide to adopt the two-stranded alpha-helical coiled coil conformation in aqueous medium. The increased stability of the peptides as a result of an increase in hydrophobic interactions (chain length) was demonstrated by the shift in the transitions of the guanidine hydrochloride (Gdn.HCl) denaturation and thermal unfolding profiles. The concentrations of denaturant (Gdn.HCl) required to achieve 50% denaturation are 3.2, 4.9, 6.9, and 7.5 M for peptides 23r, 26r, 30r, and 33r, respectively, in aqueous medium. However, the effect of a chain length increase on coiled-coil stability was not additive. The melting temperature, Tm, at which 50% of the helicity is lost, increased by 34 degrees C in changing the peptide chain length from 23 to 26; however, that shift was only 14 degrees C when the chain length was increased from 30 to 33 residues. These results are

  18. Designed coiled coils promote folding of a recombinant bacterial collagen.

    PubMed

    Yoshizumi, Ayumi; Fletcher, Jordan M; Yu, Zhuoxin; Persikov, Anton V; Bartlett, Gail J; Boyle, Aimee L; Vincent, Thomas L; Woolfson, Derek N; Brodsky, Barbara

    2011-05-20

    Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat. PMID:21454493

  19. Transmembrane and coiled-coil domain family 1 is a novel protein of the endoplasmic reticulum.

    PubMed

    Zhang, Chao; Kho, Yik-Shing; Wang, Zhe; Chiang, Yan Ting; Ng, Gary K H; Shaw, Pang-Chui; Wang, Yuzhuo; Qi, Robert Z

    2014-01-01

    The endoplasmic reticulum (ER) is a continuous membrane network in eukaryotic cells comprising the nuclear envelope, the rough ER, and the smooth ER. The ER has multiple critical functions and a characteristic structure. In this study, we identified a new protein of the ER, TMCC1 (transmembrane and coiled-coil domain family 1). The TMCC family consists of at least 3 putative proteins (TMCC1-3) that are conserved from nematode to human. We show that TMCC1 is an ER protein that is expressed in diverse human cell lines. TMCC1 contains 2 adjacent transmembrane domains near the C-terminus, in addition to coiled-coil domains. TMCC1 was targeted to the rough ER through the transmembrane domains, whereas the N-terminal region and C-terminal tail of TMCC1 were found to reside in the cytoplasm. Moreover, the cytosolic region of TMCC1 formed homo- or hetero-dimers or oligomers with other TMCC proteins and interacted with ribosomal proteins. Notably, overexpression of TMCC1 or its transmembrane domains caused defects in ER morphology. Our results suggest roles of TMCC1 in ER organization. PMID:24454821

  20. Selective amine labeling of cell surface proteins guided by coiled-coil assembly.

    PubMed

    Yano, Yoshiaki; Furukawa, Nami; Ono, Satoshi; Takeda, Yuki; Matsuzaki, Katsumi

    2016-11-01

    Covalent labeling of target proteins in living cells is useful for both fluorescence live-cell imaging and the subsequent biochemical analyses of the proteins. Here, we report an efficient method for the amine labeling of membrane proteins on the cell surface, guided by a noncovalent coiled-coil interaction. A carboxyl sulfosuccinimidyl ester introduced at the C-terminus of the coiled-coil probe reacted with target proteins under mild labeling conditions ([probe] = 150 nM, pH 7.4, 25°C) for 20 min. Various fluorescent moieties with different hydrophobicities are available for covalent labeling with high signal/background labeling ratios. Using this method, oligomeric states of glycophorin A (GpA) were compared in mammalian CHO-K1 cells and sodium dodecyl sulfate (SDS) micelles. In the cell membranes, no significant self-association of GpA was detected, whereas SDS-PAGE suggested partial dimerization of the proteins. Membrane cholesterol was found to be an important factor that suppressed the dimerization of GpA. Thus, the covalent functionality enables direct comparison of the oligomeric state of membrane proteins under various conditions. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 484-490, 2016. PMID:26285787

  1. Dimerization of the DYT6 dystonia protein, THAP1, requires residues within the coiled-coil domain.

    PubMed

    Sengel, Cem; Gavarini, Sophie; Sharma, Nutan; Ozelius, Laurie J; Bragg, D Cristopher

    2011-09-01

    Thanatos-associated [THAP] domain-containing apoptosis-associated protein 1 (THAP1) is a DNA-binding protein that has been recently associated with DYT6 dystonia, a hereditary movement disorder involving sustained, involuntary muscle contractions. A large number of dystonia-related mutations have been identified in THAP1 in diverse patient populations worldwide. Previous reports have suggested that THAP1 oligomerizes with itself via a C-terminal coiled-coil domain, raising the possibility that DYT6 mutations in this region might affect this interaction. In this study, we examined the ability of wild-type THAP1 to bind itself and the effects on this interaction of the following disease mutations: C54Y, F81L, ΔF132, T142A, I149T, Q154fs180X, and A166T. The results confirmed that wild-type THAP1 associated with itself and most of the DYT6 mutants tested, except for the Q154fs180X variant, which loses most of the coiled-coil domain because of a frameshift at position 154. However, deletion of C-terminal residues after position 166 produced a truncated variant of THAP1 that was able to bind the wild-type protein. The interaction of THAP1 with itself therefore required residues within a 13-amino acid region (aa 154-166) of the coiled-coil domain. Further inspection of this sequence revealed elements highly consistent with previous descriptions of leucine zippers, which serve as dimerization domains in other transcription factor families. Based on this similarity, a structural model was generated to predict how hydrophobic residues in this region may mediate dimerization. These observations offer additional insight into the role of the coiled-coil domain in THAP1, which may facilitate future analyses of DYT6 mutations in this region. PMID:21752024

  2. Dimerization of the DYT6 dystonia protein, THAP1, requires residues within the coiled-coil domain

    PubMed Central

    Sengel, Cem; Gavarini, Sophie; Sharma, Nutan; Ozelius, Laurie J.; Bragg, D. Cristopher

    2011-01-01

    THAP1 is a DNA binding protein that has been recently associated with DYT6 dystonia, a hereditary movement disorder involving sustained, involuntary muscle contractions. A large number of dystonia-related mutations have been identified in THAP1 in diverse patient populations worldwide. Previous reports have suggested that THAP1 oligomerizes with itself via a C-terminal coiled-coil domain, raising the possibility that DYT6 mutations in this region might affect this interaction. In this study we examined the ability of wild-type THAP1 to bind itself and the effects on this interaction of the following disease mutations: C54Y, F81L, ΔF132, T142A, I149T, Q154fs180X, and A166T. The results confirmed that wild-type THAP1 associated with itself and most of the DYT6 mutants tested, except for the Q154fs180X variant, which loses most of the coiled-coil domain due to a frameshift at position 154. However, deletion of C-terminal residues after position 166 produced a truncated variant of THAP1 that was able to bind the wild-type protein. The interaction of THAP1 with itself therefore required residues within a 13-amino acid region (aa 154–166) of the coiled-coil domain. Further inspection of this sequence revealed elements highly consistent with previous descriptions of leucine zippers, which serve as dimerization domains in other transcription factor families. Based on this similarity, a structural model was generated to predict how hydrophobic residues in this region may mediate dimerization. These observations offer additional insight into the role of the coiled-coil domain in THAP1, which may facilitate future analyses of DYT6 mutations in this region. PMID:21752024

  3. The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation

    PubMed Central

    Lee, Mihwa; Sadowska, Agata; Bekere, Indra; Ho, Diwei; Gully, Benjamin S.; Lu, Yanling; Iyer, K. Swaminathan; Trewhella, Jill; Fox, Archa H.; Bond, Charles S.

    2015-01-01

    SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism. PMID:25765647

  4. Tailoring Supramolecular Peptide-Poly(ethylene glycol) Hydrogels by Coiled Coil Self-Assembly and Self-Sorting.

    PubMed

    Dånmark, Staffan; Aronsson, Christopher; Aili, Daniel

    2016-06-13

    Physical hydrogels are extensively used in a wide range of biomedical applications. However, different applications require hydrogels with different mechanical and structural properties. Tailoring these properties demands exquisite control over the supramolecular interactions involved. Here we show that it is possible to control the mechanical properties of hydrogels using de novo designed coiled coil peptides with different affinities for dimerization. Four different nonorthogonal peptides, designed to fold into four different coiled coil heterodimers with dissociation constants spanning from μM to pM, were conjugated to star-shaped 4-arm poly(ethylene glycol) (PEG). The different PEG-coiled coil conjugates self-assemble as a result of peptide heterodimerization. Different combinations of PEG-peptide conjugates assemble into PEG-peptide networks and hydrogels with distinctly different thermal stabilities, supramolecular, and rheological properties, reflecting the peptide dimer affinities. We also demonstrate that it is possible to rationally modulate the self-assembly process by means of thermodynamic self-sorting by sequential additions of nonpegylated peptides. The specific interactions involved in peptide dimerization thus provides means for programmable and reversible self-assembly of hydrogels with precise control over rheological properties, which can significantly facilitate optimization of their overall performance and adaption to different processing requirements and applications. PMID:27219681

  5. Structural basis for cargo binding and autoinhibition of Bicaudal-D1 by a parallel coiled-coil with homotypic registry

    SciTech Connect

    Terawaki, Shin-ichi; Yoshikane, Asuka; Higuchi, Yoshiki; Wakamatsu, Kaori

    2015-05-01

    Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein mediating the attachment of specific cargo to cytoplasmic dynein. It plays an essential role in minus end-directed intracellular transport along microtubules. The third C-terminal coiled-coil region of BICD1 (BICD1 CC3) has an important role in cargo sorting, including intracellular vesicles associating with the small GTPase Rab6 and the nuclear pore complex Ran binding protein 2 (RanBP2), and inhibiting the association with cytoplasmic dynein by binding to the first N-terminal coiled-coil region (CC1). The crystal structure of BICD1 CC3 revealed a parallel homodimeric coiled-coil with asymmetry and complementary knobs-into-holes interactions, differing from Drosophila BicD CC3. Furthermore, our binding study indicated that BICD1 CC3 possesses a binding surface for two distinct cargos, Rab6 and RanBP2, and that the CC1-binding site overlaps with the Rab6-binding site. These findings suggest a molecular basis for cargo recognition and autoinhibition of BICD proteins during dynein-dependent intracellular retrograde transport. - Highlights: • BICD1 CC3 is a parallel homodimeric coiled-coil with axial asymmetry. • The coiled-coil packing of BICD1 CC3 is adapted to the equivalent heptad position. • BICD1 CC3 has distinct binding sites for two classes of cargo, Rab6 and RanBP2. • The CC1-binding site of BICD1 CC3 overlaps with the Rab6-binding site.

  6. A Switch from Parallel to Antiparallel Strand Orientation in a Coiled-Coil X-Ray Structure via Two Core Hydrophobic Mutations

    PubMed Central

    Malashkevich, Vladimir N.; Higgins, Chelsea D.; Almo, Steven C.; Lai, Jonathan R.

    2016-01-01

    The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative α-helical orientation and oligomer size. Here we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimerization (trimer or tetramer). In the X-ray structure, a third, partially-ordered α-helix is weakly associated with the antiparallel dimer and analytical ultracentrifugation experiments indicate the peptide forms a well-defined tetramer in solution. The HA2-Del-L2seM sequence is closely related to a parent model peptide, HA2-Del, which we previously reported adopts a parallel trimer; HA2-Del-L2seM differs by only hydrophobic leucine to selenomethione mutations and thus this subtle difference is sufficient to switch both relative α-helical topology and number of α-helices participating in the coiled-coil. Comparison of the X-ray structures of HA2-Del-L2seM (reported here) with the HA2-Del parent (reported previously) reveals novel interactions involving the selenomethionine residues that promote antiparallel coiled-coil configuration and preclude parallel trimer formation. These novel atomic insights are instructive for understanding subtle features that can affect coiled-coil topology and provide additional information for design of antiparallel coiled-coils. PMID:25753192

  7. Structure of Leishmania donovani coronin coiled coil domain reveals an antiparallel 4 helix bundle with inherent asymmetry.

    PubMed

    Nayak, Ashok Ranjan; Karade, Sharanbasappa Shrimant; Srivastava, Vijay Kumar; Rana, Ajay Kumar; Gupta, C M; Sahasrabuddhe, Amogh A; Pratap, J Venkatesh

    2016-07-01

    Coiled coils are ubiquitous structural motifs that serve as a platform for protein-protein interactions and play a central role in myriad physiological processes. Though the formation of a coiled coil requires only the presence of suitably spaced hydrophobic residues, sequence specificities have also been associated with specific oligomeric states. RhXXhE is one such sequence motif, associated with parallel trimers, found in coronins and other proteins. Coronin, present in all eukaryotes, is an actin-associated protein involved in regulating actin turnover. Most eukaryotic coronins possess the RhXXhE trimerization motif. However, a unique feature of parasitic kinetoplastid coronin is that the positions of R and E are swapped within their coiled coil domain, but were still expected to form trimers. To understand the role of swapped motif in oligomeric specificity, we determined the X-ray crystal structure of Leishmania donovani coronin coiled coil domain (LdCoroCC) at 2.2Å, which surprisingly, reveals an anti-parallel tetramer assembly. Small angle X-ray scattering studies and chemical crosslinking confirm the tetramer in solution and is consistent with the oligomerization observed in the full length protein. Structural analyses reveal that LdCoroCC possesses an inherent asymmetry, in that one of the helices of the bundle is axially shifted with respect to the other three. The analysis also identifies steric reasons that cause this asymmetry. The bundle adapts an extended a-d-e core packing, the e residue being polar (with an exception) which results in a thermostable bundle with polar and apolar interfaces, unlike the existing a-d-e core antiparallel homotetramers with apolar core. Functional implications of the anti-parallel association in kinetoplastids are discussed. PMID:26940672

  8. N-Terminal Coiled-Coil Structure of ATPase Subunits of 26S Proteasome Is Crucial for Proteasome Function

    PubMed Central

    Inobe, Tomonao; Genmei, Reiko

    2015-01-01

    The proteasome is an essential proteolytic machine in eukaryotic cells, where it removes damaged proteins and regulates many cellular activities by degrading ubiquitinated proteins. Its heterohexameric AAA+ ATPase Rpt subunits play a central role in proteasome activity by the engagement of substrate unfolding and translocation for degradation; however, its detailed mechanism remains poorly understood. In contrast to AAA+ ATPase domains, their N-terminal regions of Rpt subunits substantially differ from each other. Here, to investigate the requirements and roles of the N-terminal regions of six Rpt subunits derived from Saccharomyces cerevisiae, we performed systematic mutational analysis using conditional knockdown yeast strains for each Rpt subunit and bacterial heterologous expression system of the base subcomplex. We showed that the formation of the coiled-coil structure was the most important for the N-terminal region of Rpt subunits. The primary role of coiled-coil structure would be the maintenance of the ring structure with the defined order. However, the coiled-coil region would be also be involved in substrate recognition and an interaction between lid and base subcomplexes. PMID:26208326

  9. L1 retrotransposition requires rapid ORF1p oligomerization, a novel coiled coil-dependent property conserved despite extensive remodeling

    PubMed Central

    Naufer, M. Nabuan; Callahan, Kathryn E.; Cook, Pamela R.; Perez-Gonzalez, Cesar E.; Williams, Mark C.; Furano, Anthony V.

    2016-01-01

    Detailed mechanistic understanding of L1 retrotransposition is sparse, particularly with respect to ORF1p, a coiled coil-mediated homotrimeric nucleic acid chaperone that can form tightly packed oligomers on nucleic acids. Although the coiled coil motif is highly conserved, it is uniquely susceptible to evolutionary change. Here we studied three ORF1 proteins: a modern human one (111p), its resuscitated primate ancestor (555p) and a mosaic modern protein (151p) wherein 9 of the 30 coiled coil substitutions retain their ancestral state. While 111p and 555p equally supported retrotransposition, 151p was inactive. Nonetheless, they were fully active in bulk assays of nucleic acid interactions including chaperone activity. However, single molecule assays showed that 151p trimers form stably bound oligomers on ssDNA at <1/10th the rate of the active proteins, revealing that oligomerization rate is a novel critical parameter of ORF1p activity in retrotransposition conserved for at least the last 25 Myr of primate evolution. PMID:26673717

  10. Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

    PubMed Central

    Hamed, Elham; Keten, Sinan

    2014-01-01

    Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies. PMID:25028889

  11. De Novo Design of Ln(III) Coiled Coils for Imaging Applications

    PubMed Central

    2014-01-01

    A new peptide sequence (MB1) has been designed which, in the presence of a trivalent lanthanide ion, has been programmed to self-assemble to form a three stranded metallo-coiled coil, Ln(III)(MB1)3. The binding site has been incorporated into the hydrophobic core using natural amino acids, restricting water access to the lanthanide. The resulting terbium coiled coil displays luminescent properties consistent with a lack of first coordination sphere water molecules. Despite this the gadolinium coiled coil, the first to be reported, displays promising magnetic resonance contrast capabilities. PMID:24405157

  12. Molecular basis of the STIL coiled coil oligomerization explains its requirement for de-novo formation of centrosomes in mammalian cells

    PubMed Central

    David, Ahuvit; Amartely, Hadar; Rabinowicz, Noa; Shamir, Mai; Friedler, Assaf; Izraeli, Shai

    2016-01-01

    The STIL protein is essential for centriole replication and for the non-templated, de novo centriole biogenesis that is required for mammalian embryogenesis. Here we performed quantitative biophysical and structural analysis of the central short coiled coil domain (CCD) of STIL that is critical for its function. Using biophysical, biochemical and cell biology approaches, we identified the specific residues in the CCD that mediate the oligomerization, centrosomal localization and protein interactions of STIL. We characterized the structural properties of the coiled coil peptide using circular dichroism spectroscopy and size exclusion chromatography. We identified two regions in this domain, containing eight hydrophobic residues, which mediate the coiled coil oligomerization. Mutations in these residues destabilized the coiled coil thermodynamically but in most cases did not affect its secondary structure. Reconstituting mouse embryonic fibroblasts lacking endogenous Stil, we show that STIL oligomerization mediated by these residues is not only important for the centrosomal functions of STIL during the canonical duplication process but also for de-novo formation of centrosomes. PMID:27075531

  13. The Golgin Family of Coiled-Coil Tethering Proteins

    PubMed Central

    Witkos, Tomasz M.; Lowe, Martin

    2016-01-01

    The golgins are a family of predominantly coiled-coil proteins that are localized to the Golgi apparatus. Golgins are present in all eukaryotes, suggesting an evolutionary conserved function. Golgins are anchored to the Golgi membrane by their carboxy terminus and are predicted to adopt an extended conformation that projects into the surrounding cytoplasm. This arrangement is ideal for the capture or tethering of nearby membranes or cytoskeletal elements. Golgin-mediated tethering is thought to be important for vesicular traffic at the Golgi apparatus, the maintenance of Golgi architecture, as well as the positioning of the Golgi apparatus within cells. In addition to acting as tethers, some golgins can also sequester various factors at the Golgi membrane, allowing for the spatiotemporal regulation of downstream cellular functions. Although it is now established that golgins are membrane and cytoskeleton tethers, the mechanisms underlying tethering remain poorly defined. Moreover, the importance of golgin-mediated tethering in a physiological context remains to be fully explored. This review will describe our current understanding of golgin function, highlighting recent progress that has been made, and goes on to discuss outstanding questions and potential avenues for future research with regard to this family of conserved Golgi-associated proteins. PMID:26793708

  14. pH sensitive coiled coils: a strategy for enhanced liposomal drug delivery.

    PubMed

    Reja, Rahi M; Khan, Mohsina; Singh, Sumeet K; Misra, Rajkumar; Shiras, Anjali; Gopi, Hosahudya N

    2016-03-01

    Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic lysosomal compartments in comparison with liposomes without coiled coils. PMID:26876788

  15. Coiled-coil protein composition of 22 proteomes – differences and common themes in subcellular infrastructure and traffic control

    PubMed Central

    Rose, Annkatrin; Schraegle, Shannon J; Stahlberg, Eric A; Meier, Iris

    2005-01-01

    Background Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previously identified all long coiled-coil proteins from the model plant Arabidopsis thaliana and have established a searchable Arabidopsis coiled-coil protein database. Results Here, we have identified all proteins with long coiled-coil domains from 21 additional fully sequenced genomes. Because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. Comparing and grouping all long coiled-coil proteins from 22 genomes, the kingdom-specificity of coiled-coil protein families was determined. At the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms. Conclusion MultiCoil predicts proteins with extended coiled-coil domains (more than 250 amino acids) to be largely absent from bacterial genomes, but present in archaea and eukaryotes. The structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. Motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell. PMID:16288662

  16. Critical evaluation of in silico methods for prediction of coiled-coil domains in proteins.

    PubMed

    Li, Chen; Ching Han Chang, Catherine; Nagel, Jeremy; Porebski, Benjamin T; Hayashida, Morihiro; Akutsu, Tatsuya; Song, Jiangning; Buckle, Ashley M

    2016-03-01

    Coiled-coils refer to a bundle of helices coiled together like strands of a rope. It has been estimated that nearly 3% of protein-encoding regions of genes harbour coiled-coil domains (CCDs). Experimental studies have confirmed that CCDs play a fundamental role in subcellular infrastructure and controlling trafficking of eukaryotic cells. Given the importance of coiled-coils, multiple bioinformatics tools have been developed to facilitate the systematic and high-throughput prediction of CCDs in proteins. In this article, we review and compare 12 sequence-based bioinformatics approaches and tools for coiled-coil prediction. These approaches can be categorized into two classes: coiled-coil detection and coiled-coil oligomeric state prediction. We evaluated and compared these methods in terms of their input/output, algorithm, prediction performance, validation methods and software utility. All the independent testing data sets are available at http://lightning.med.monash.edu/coiledcoil/. In addition, we conducted a case study of nine human polyglutamine (PolyQ) disease-related proteins and predicted CCDs and oligomeric states using various predictors. Prediction results for CCDs were highly variable among different predictors. Only two peptides from two proteins were confirmed to be CCDs by majority voting. Both domains were predicted to form dimeric coiled-coils using oligomeric state prediction. We anticipate that this comprehensive analysis will be an insightful resource for structural biologists with limited prior experience in bioinformatics tools, and for bioinformaticians who are interested in designing novel approaches for coiled-coil and its oligomeric state prediction. PMID:26177815

  17. Crystal Structure of a Super Leucine Zipper an Extended Two-Stranded Super Long Coiled Coil

    SciTech Connect

    J Diao

    2011-12-31

    Coiled coil is a ubiquitous structural motif in proteins, with two to seven alpha helices coiled together like the strands of a rope, and coiled coil folding and assembly is not completely understood. A GCN4 leucine zipper mutant with four mutations of K3A, D7A, Y17W, and H18N has been designed, and the crystal structure has been determined at 1.6 {angstrom} resolution. The peptide monomer shows a helix trunk with short curved N- and C-termini. In the crystal, two monomers cross in 35{sup o} and form an X-shaped dimer, and each X-shaped dimer is welded into the next one through sticky hydrophobic ends, thus forming an extended two-stranded, parallel, super long coiled coil rather than a discrete, two-helix coiled coil of the wild-type GCN4 leucine zipper. Leucine residues appear at every seventh position in the super long coiled coil, suggesting that it is an extended super leucine zipper. Compared to the wild-type leucine zipper, the N-terminus of the mutant has a dramatic conformational change and the C-terminus has one more residue Glu 32 determined. The mutant X-shaped dimer has a large crossing angle of 35{sup o} instead of 18{sup o} in the wild-type dimer. The results show a novel assembly mode and oligomeric state of coiled coil, and demonstrate that mutations may affect folding and assembly of the overall coiled coil. Analysis of the formation mechanism of the super long coiled coil may help understand and design self-assembling protein fibers.

  18. Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel*

    PubMed Central

    Fujiwara, Yuichiro; Takeshita, Kohei; Nakagawa, Atsushi; Okamura, Yasushi

    2013-01-01

    Oxidation is an important biochemical defense mechanism, but it also elicits toxicity; therefore, oxidation must be under strict control. In phagocytotic events in neutrophils, the voltage-gated H+ (Hv) channel is a key regulator of the production of reactive oxygen species against invading bacteria. The cytoplasmic domain of the Hv channel forms a dimeric coiled coil underpinning a dimerized functional unit. Importantly, in the alignment of the coiled-coil core, a conserved cysteine residue forms a potential intersubunit disulfide bond. In this study, we solved the crystal structures of the coiled-coil domain in reduced, oxidized, and mutated (Cys → Ser) states. The crystal structures indicate that a pair of Cys residues forms an intersubunit disulfide bond dependent on the redox conditions. CD spectroscopy revealed that the disulfide bond increases the thermal stability of the coiled-coil protein. We also reveal that two thiol modifier molecules are able to bind to Cys in a redox-dependent manner without disruption of the dimeric coiled-coil assembly. Thus, the biochemical properties of the cytoplasmic coiled-coil domain in the Hv channel depend on the redox condition, which may play a role in redox sensing in the phagosome. PMID:23667254

  19. AAFreqCoil: a new classifier to distinguish parallel dimeric and trimeric coiled coils.

    PubMed

    Wang, Xiaofeng; Zhou, Yuan; Yan, Renxiang

    2015-07-01

    Coiled coils are characteristic rope-like protein structures, constituted by one or more heptad repeats. Native coiled-coil structures play important roles in various biological processes, while the designed ones are widely employed in medicine and industry. To date, two major oligomeric states (i.e. dimeric and trimeric states) of a coiled-coil structure have been observed, plausibly exerting different biological functions. Therefore, exploration of the relationship between heptad repeat sequences and coiled coil structures is highly important. In this paper, we develop a new method named AAFreqCoil to classify parallel dimeric and trimeric coiled coils. Our method demonstrated its competitive performance when benchmarked based on 10-fold cross validation and jackknife cross validation. Meanwhile, the rules that can explicitly explain the prediction results of the test coiled coil can be extracted from the AAFreqCoil model for a better explanation of user predictions. A web server and stand-alone program implementing the AAFreqCoil algorithm are freely available at . PMID:25918905

  20. Natural templates for coiled-coil biomaterials from praying mantis egg cases.

    PubMed

    Walker, Andrew A; Weisman, Sarah; Kameda, Tsunenori; Sutherland, Tara D

    2012-12-10

    Whereas there is growing interest in producing biomaterials containing coiled-coils, relatively few studies have made use of naturally occurring fibrous proteins. In this study, we have characterized fibrous proteins used by mother praying mantises to produce an extensive covering for their eggs called an ootheca and demonstrate the production of artificial ootheca using recombinantly produced proteins. Examination of natural oothecae by infrared spectroscopy and solid-state nuclear magnetic resonance revealed the material to consist of proteins organized predominately as coiled-coils. Two structural proteins, Mantis Fibroin 1 and Mantis Fibroin 2, were identified in ootheca from each of three species. Between species, the primary sequences of both proteins had diverged considerably, but other features were tightly conserved, including low molecular weight, high abundance of Ala, Glu, Lys, and Ser, and a triblock-like architecture with extensive central coiled-coil domain. Mantis fibroin hydrophobic cores had an unusual composition containing high levels of alanine and aromatic residues. Recombinantly produced mantis fibroins folded into coiled-coils in solution and could be fabricated into solid materials with high coiled-coil content. The structural features of mantis fibroins and their straightforward recombinant production make them promising templates for the production of coiled-coil biomimetics materials. PMID:23137042

  1. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    SciTech Connect

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio; Ichinose, Junji; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Nakajima, Jun; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2015-02-13

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  2. Structural domains of vault proteins: a role for the coiled coil domain in vault assembly.

    PubMed

    van Zon, Arend; Mossink, Marieke H; Schoester, Martijn; Scheffer, George L; Scheper, Rik J; Sonneveld, Pieter; Wiemer, Erik A C

    2002-03-01

    Vaults consist of multiple copies of three proteins (MVP, VPARP, and TEP1) and several untranslated RNAs. The function of vaults is unknown but the typical and evolutionary conserved structure indicates a role in intracellular transport. Although all vault components have been identified and characterized, not much is known about vault protein assembly. In this study we identified and analyzed structural domains involved in vault assembly with emphasis on protein-protein interactions. Using a yeast two-hybrid system, we demonstrate within MVP an intramolecular binding site and show that MVP molecules interact with each other via their coiled coil domain. We show that purified MVP is able to bind calcium, most likely at calcium-binding EF-hands. No interactions could be detected between TEP1 and other vault proteins. However, the N-terminal half of MVP binds to a specific domain in the C-terminus of VPARP. Furthermore, VPARP contains amino acid stretches mediating intramolecular binding. PMID:11855821

  3. Crystal Structure of a Coiled-Coil Domain from Human ROCK I

    PubMed Central

    Tu, Daqi; Li, Yiqun; Song, Hyun Kyu; Toms, Angela V.; Gould, Christopher J.; Ficarro, Scott B.; Marto, Jarrod A.; Goode, Bruce L.; Eck, Michael J.

    2011-01-01

    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. PMID:21445309

  4. Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif

    PubMed Central

    Hirtreiter, Angela; Damsma, Gerke E.; Cheung, Alan C. M.; Klose, Daniel; Grohmann, Dina; Vojnic, Erika; Martin, Andrew C. R.; Cramer, Patrick; Werner, Finn

    2010-01-01

    Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA–RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5—the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5. PMID:20197319

  5. Accessing Three-Dimensional Crystals with Incorporated Guests through Metal-Directed Coiled-Coil Peptide Assembly.

    PubMed

    Nepal, Manish; Sheedlo, Michael J; Das, Chittaranjan; Chmielewski, Jean

    2016-08-31

    Obtaining three-dimensional (3D) protein and peptide crystals on demand requires a precisely orchestrated hierarchical assembly of biopolymer building blocks. In this work, we disclose a metal-ion-mediated strategy to assemble trimeric coiled-coil peptides in a head-to-tail fashion into linear strands with interstrand interactions. This design led to hexagonal 3D peptide crystal formation within 30 min in the presence of divalent metal ions. The crystal morphology could be controlled by varying the metal ion/peptide ratio, resulting in hexagonal discs to rods. Diffraction studies elucidated the head-to-tail arrangement of the coiled-coil linear strands and their hexagonal, antiparallel packing within the crystal. Unsatisfied ligands at the hexagonal ends of the crystals were harnessed as a powerful means to direct His-tagged fluorophores to distinct locations within the crystals. Overall, the designed hierarchical assembly provides a facile means to obtain 3D peptide crystals and incorporate His-tag-based cargoes and may have potential use in drug delivery and sensor design. PMID:27500907

  6. X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides.

    PubMed

    Spencer, Ryan K; Hochbaum, Allon I

    2016-06-14

    The self-assembly of peptides and proteins into higher-ordered structures is encoded in the amino acid sequence of each peptide or protein. Understanding the relationship among the amino acid sequence, the assembly dynamics, and the structure of well-defined peptide oligomers expands the synthetic toolbox for these structures. Here, we present the X-ray crystallographic structure and solution behavior of de novo peptides that form antiparallel coiled-coil hexamers (ACC-Hex) by an interaction motif neither found in nature nor predicted by existing peptide design software. The 1.70 Å X-ray crystallographic structure of peptide 1a shows six α-helices associating in an antiparallel arrangement around a central axis comprising hydrophobic and aromatic residues. Size-exclusion chromatography studies suggest that peptides 1 form stable oligomers in solution, and circular dichroism experiments show that peptides 1 are stable to relatively high temperatures. Small-angle X-ray scattering studies of the solution behavior of peptide 1a indicate an equilibrium of dimers, hexamers, and larger aggregates in solution. The structures presented here represent a new motif of biomolecular self-assembly not previously observed for de novo peptides and suggest supramolecular design principles for material scaffolds based on coiled-coil motifs containing aromatic residues. PMID:27192036

  7. An engineered coiled-coil polypeptide assembled onto quantum dots for targeted cell imaging

    NASA Astrophysics Data System (ADS)

    Yao, Ming-Hao; Yang, Jie; Song, Ji-Tao; Zhang, Lin; Fang, Bi-Yun; Zhao, Dong-Hui; Xia, Rui-Xue; Jin, Rui-Mei; Zhao, Yuan-Di; Liu, Bo

    2015-12-01

    Quantum dot (QD)-polypeptide probes have been developed through the specific metal-affinity interaction between polypeptides appended with N-terminal polyhistidine sequences and CdSe/ZnS core-shell QDs. The size and charge of a QD-polypeptide can be tuned by using different coiled-coil polypeptides. Compared to glutathione-capped QDs (QD-GSH), QD-polypeptide probes showed an approximately two- to three-fold luminescence increase, and the luminescence increase was not obviously related to the charge of the polypeptide. QD-polypeptide probes with different charge have a great effect on nonspecific cellular uptake. QD-polypeptide probes with negative charge exhibited lower nonspecific cellular uptake in comparison to the QD-GSH, while positively charged QD-polypeptide probes presented higher cellular uptake than the QD-GSH. A targeted QD-ARGD probe can obviously increase targeted cellular uptake in α v β 3 overexpressing HeLa cells compared to QD-A. In addition, QD-polypeptide probes showed lower in vitro cytotoxicity compared to the original QDs. These results demonstrate that these QD-polypeptide probes with high specific cellular uptake, high fluorescence intensity and low background noise are expected to have great potential applications in targeted cell imaging.

  8. pH sensitive coiled coils: a strategy for enhanced liposomal drug delivery

    NASA Astrophysics Data System (ADS)

    Reja, Rahi M.; Khan, Mohsina; Singh, Sumeet K.; Misra, Rajkumar; Shiras, Anjali; Gopi, Hosahudya N.

    2016-02-01

    Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic lysosomal compartments in comparison with liposomes without coiled coils.Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic

  9. A coiled-coil domain acts as a molecular ruler in LPS chain length regulation

    PubMed Central

    Tuukkanen, Anne; Danciu, Iulia; Svergun, Dmitri I.; Hussain, Rohanah; Liu, Huanting; Whitfield, Chris; Naismith, James H.

    2014-01-01

    Long-chain bacterial polysaccharides play important roles in pathogenicity. In Escherichia coli O9a, a model for ABC transporter dependent polysaccharide assembly, a large extracellular carbohydrate with a narrow distribution of size is polymerized from monosaccharides by a complex of two proteins, WbdA (polymerase) and WbdD (terminating protein). Such careful control of polymerization is recurring theme in biology. Combining crystallography and small angle X-ray scattering, we show that the C-terminal domain of WbdD contains an extended coiled-coil that physically separates WbdA from the catalytic domain of WbdD. The effects of insertions and deletions within the coiled-coil region were analyzed in vivo, revealing that polymer size is controlled by varying the length of the coiled-coil domain. Thus, the coiled-coil domain of WbdD functions as a molecular ruler that, along with WbdA:WbdD stoichiometry, controls the chain length of a model bacterial polysaccharide. PMID:25504321

  10. Self-sorting heterodimeric coiled coil peptides with defined and tuneable self-assembly properties

    PubMed Central

    Aronsson, Christopher; Dånmark, Staffan; Zhou, Feng; Öberg, Per; Enander, Karin; Su, Haibin; Aili, Daniel

    2015-01-01

    Coiled coils with defined assembly properties and dissociation constants are highly attractive components in synthetic biology and for fabrication of peptide-based hybrid nanomaterials and nanostructures. Complex assemblies based on multiple different peptides typically require orthogonal peptides obtained by negative design. Negative design does not necessarily exclude formation of undesired species and may eventually compromise the stability of the desired coiled coils. This work describe a set of four promiscuous 28-residue de novo designed peptides that heterodimerize and fold into parallel coiled coils. The peptides are non-orthogonal and can form four different heterodimers albeit with large differences in affinities. The peptides display dissociation constants for dimerization spanning from the micromolar to the picomolar range. The significant differences in affinities for dimerization make the peptides prone to thermodynamic social self-sorting as shown by thermal unfolding and fluorescence experiments, and confirmed by simulations. The peptides self-sort with high fidelity to form the two coiled coils with the highest and lowest affinities for heterodimerization. The possibility to exploit self-sorting of mutually complementary peptides could hence be a viable approach to guide the assembly of higher order architectures and a powerful strategy for fabrication of dynamic and tuneable nanostructured materials. PMID:26370878

  11. The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer

    PubMed Central

    Sanchez, Jacint G.; Okreglicka, Katarzyna; Chandrasekaran, Viswanathan; Welker, Jordan M.; Sundquist, Wesley I.; Pornillos, Owen

    2014-01-01

    Tripartite motif (TRIM) proteins make up a large family of coiled-coil-containing RING E3 ligases that function in many cellular processes, particularly innate antiviral response pathways. Both dimerization and higher-order assembly are important elements of TRIM protein function, but the atomic details of TRIM tertiary and quaternary structure have not been fully understood. Here, we present crystallographic and biochemical analyses of the TRIM coiled-coil and show that TRIM proteins dimerize by forming interdigitating antiparallel helical hairpins that position the N-terminal catalytic RING domains at opposite ends of the dimer and the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, symmetric pattern of flanking heptad and central hendecad repeats that appear to be conserved across the entire TRIM family. Our studies reveal how the coiled-coil organizes TRIM25 to polyubiquitylate the RIG-I/viral RNA recognition complex and how dimers of the TRIM5α protein are arranged within hexagonal arrays that recognize the HIV-1 capsid lattice and restrict retroviral replication. PMID:24550273

  12. Self-Assembling Peptide-Polymer Hydrogels Designed From the Coiled Coil Region of Fibrin

    PubMed Central

    Jing, Peng; Rudra, Jai S.; Herr, Andrew B.; Collier, Joel H.

    2010-01-01

    Biomaterials constructed from self-assembling peptides, peptide derivatives, and peptide-polymer conjugates are receiving increasing attention as defined matrices for tissue engineering, controlled therapeutic release, and in vitro cell expansion, but many are constructed from peptide structures not typically found in the human extracellular matrix. Here we report a self-assembling biomaterial constructed from a designed peptide inspired by the coiled coil domain of human fibrin, the major protein constituent of blood clots and the provisional scaffold of wound healing. Targeted substitutions were made in the residues forming the interface between coiled coil strands for a 37-amino acid peptide from human fibrinogen to stabilize the coiled coil peptide bundle, while the solvent-exposed residues were left unchanged to provide a surface similar to that of the native protein. This peptide, which self-assembled into coiled coil dimers and tetramers, was then used to produce triblock peptide-PEG-peptide bioconjugates that self-assembled into viscoelastic hydrogel biomaterials. PMID:18712921

  13. Coiled-Coil Irregularities and Instabilities in Group A Streptococcus M1 Are Required for Virulence

    SciTech Connect

    McNamara, Case; Zinkernagel, Annelies S.; Macheboeuf, Pauline; Cunningham, Madeleine W.; Nizet, Victor; Ghosh, Partho

    2008-07-21

    Antigenically variable M proteins are major virulence factors and immunogens of the human pathogen group A Streptococcus (GAS). Here, we report the -3 angstrom resolution structure of a GAS M1 fragment containing the regions responsible for eliciting type-specific, protective immunity and for binding fibrinogen, which promotes M1 proinflammatory and antiphagocytic functions. The structure revealed substantial irregularities and instabilities throughout the coiled coil of the M1 fragment. Similar structural irregularities occur in myosin and tropomyosin, explaining the patterns of cross-reactivity seen in autoimmune sequelae of GAS infection. Sequence idealization of a large segment of the M1 coiled coil enhanced stability but diminished fibrinogen binding, proinflammatory effects, and antibody cross-reactivity, whereas it left protective immunogenicity undiminished. Idealized M proteins appear to have promise as vaccine immunogens.

  14. Short peptide tag for covalent protein labeling based on coiled coils.

    PubMed

    Wang, Jianpeng; Yu, Yongsheng; Xia, Jiang

    2014-01-15

    To label proteins covalently, one faces a trade-off between labeling a protein specifically and using a small tag. Often one must compromise one parameter for the other or use additional components, such as an enzyme, to satisfy both requirements. Here, we report a new reaction that covalently labels proteins by using engineered coiled-coil peptides. Harnessing the concept of "proximity-induced reactivity", the 21-amino-acid three-heptad peptides CCE/CCK were modified with a nucleophilic cysteine and an α-chloroacetyl group at selected positions. When pairs of coiled coils associated, an irreversible covalent bond spontaneously formed between the peptides. The specificity of the cross-linking reaction was characterized, the probes were improved by making them bivalent, and the system was used to label a protein in vitro and receptors on the surface of mammalian cells. PMID:24341800

  15. Application of Coiled Coil Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft Model.

    PubMed

    Yang, Jian; Shimada, Yasuhito; Olsthoorn, René C L; Snaar-Jagalska, B Ewa; Spaink, Herman P; Kros, Alexander

    2016-08-23

    The complementary coiled coil forming peptides E4 [(EIAALEK)4] and K4 [(KIAALKE)4] are known to trigger liposomal membrane fusion when tethered to lipid vesicles in the form of lipopeptides. In this study, we examined whether these coiled coil forming peptides can be used for drug delivery applications. First, we prepared E4 peptide modified liposomes containing the far-red fluorescent dye TO-PRO-3 iodide (E4-Lipo-TP3) and confirmed that E4-liposomes could deliver TP3 into HeLa cells expressing K4 peptide on the membrane (HeLa-K) under cell culture conditions in a selective manner. Next, we prepared doxorubicin-containing E4-liposomes (E4-Lipo-DOX) and confirmed that E4-liposomes could also deliver DOX into HeLa-K cells. Moreover, E4-Lipo-DOX showed enhanced cytotoxicity toward HeLa-K cells compared to free doxorubicin. To prove the suitability of E4/K4 coiled coil formation for in vivo drug delivery, we injected E4-Lipo-TP3 or E4-Lipo-DOX into zebrafish xenografts of HeLa-K. As a result, E4-liposomes delivered TP3 to the implanted HeLa-K cells, and E4-Lipo-DOX could suppress cancer proliferation in the xenograft when compared to nontargeted conditions (i.e., zebrafish xenograft with free DOX injection). These data demonstrate that coiled coil formation enables drug selectivity and efficacy in vivo. It is envisaged that these findings are a step forward toward biorthogonal targeting systems as a tool for clinical drug delivery. PMID:27504667

  16. Dynamics of the coiled-coil unfolding transition of myosin rod probed by dissipation force spectrum.

    PubMed

    Taniguchi, Yukinori; Khatri, Bhavin S; Brockwell, David J; Paci, Emanuele; Kawakami, Masaru

    2010-07-01

    The motor protein myosin II plays a crucial role in muscle contraction. The mechanical properties of its coiled-coil region, the myosin rod, are important for effective force transduction during muscle function. Previous studies have investigated the static elastic response of the myosin rod. However, analogous to the study of macroscopic complex fluids, how myosin will respond to physiological time-dependent loads can only be understood from its viscoelastic response. Here, we apply atomic force microscopy using a magnetically driven oscillating cantilever to measure the dissipative properties of single myosin rods that provide unique dynamical information about the coiled-coil structure as a function of force. We find that the friction constant of the single myosin rod has a highly nontrivial variation with force; in particular, the single-molecule friction constant is reduced dramatically and increases again as it passes through the coiled-uncoiled transition. This is a direct indication of a large free-energy barrier to uncoiling, which may be related to a fine-tuned dynamic mechanosignaling response to large and unexpected physiological loads. Further, from the critical force at which the minimum in friction occurs we determine the asymmetry of the bistable landscape that controls uncoiling of the coiled coil. This work highlights the sensitivity of the dissipative signal in force unfolding to dynamic molecular structure that is hidden to the elastic signal. PMID:20655854

  17. Leveraging intrinsic chain anisotropy to align coil-coil block copolymers with magnetic fields

    NASA Astrophysics Data System (ADS)

    Rokhlenko, Yekaterina; Zhang, Kai; Gopinadhan, Manesh; Larson, Steve; Majewski, Pawel; Yager, Kevin; Gopalan, Padma; O'Hern, Corey; Osuji, Chinedum

    Magnetic field alignment of block copolymers (BCPs) has typically relied on the presence of liquid crystalline or crystalline assemblies to provide sufficient magnetic anisotropy to drive alignment. Recent experiments however show that alignment is also possible in simple coil-coil BCPs. In particular, alignment of lamellae was observed in poly(styrene-b-4-vinylpyridine) (PS-P4VP) on cooling across the order-disorder transition at field strengths as low as 1 T, with alignment improving markedly with increasing field strength and decreasing cooling rate. Here we discuss the intrinsic chain anisotropy which drives the observed alignment, and its display as a net microdomain anisotropy due to chain tethering at the block interface. We use in-situ X-ray scattering to study the phase behavior and temperature-, time-, and field- dependent dynamics of magnetic alignment in coil-coil BCPs, highlighting the important roles of chain anisotropy and grain size in alignment. For the right combination of field strength and grain size, we can leverage intrinsic chain anisotropy to magnetically direct self-assembly in other coil-coil systems, including cylinder-forming poly(styrene-b-dimethylsiloxane). Field alignment of PS-P4VP with PEO and other blends provides a route to form functional materials such as nanoporous films and ion conducting polymers.

  18. Cloning and expression analysis of mouse Cclp1, a new gene encoding a coiled-coil-like protein.

    PubMed

    Noben-Trauth, K; Naggert, J K; Nishina, P M

    1997-05-30

    Here we describe the nucleotide sequence and expression pattern of a novel gene termed Coiled-coil-like protein 1 (Cclp1). A 2646bp open reading frame encodes a 882 amino acid protein with a predicted coiled-coil domain at the amino terminus. Cclp1 is expressed in a variety of adult tissues and during different stages of embryogenesis. The broad expression pattern suggests a general cellular function of CCLP1. PMID:9199242

  19. Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein.

    PubMed

    Boudko, Sergei P; Londer, Yuri Y; Letarov, Andrei V; Sernova, Natalia V; Engel, Juergen; Mesyanzhinov, Vadim V

    2002-02-01

    Fibritin is a segmented coiled-coil homotrimer of the 486-residue product of phage T4 gene wac. This protein attaches to a phage particle by the N-terminal region and forms fibrous whiskers of 530 A, which perform a chaperone function during virus assembly. The short C-terminal region has a beta-annulus-like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N-termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements. The analysis of DSC curves indicates that full-length fibritin exhibits three thermal-heat-absorption peaks centred at 321 K (Delta H=1390 kJ x mol trimer(-1)), at 336 K (Delta H=7600 kJ x mol trimer(-1)), and at 345 K (Delta H=515 kJ x mol trimer(-1)). These transitions were assigned to the N-terminal, segmented coiled-coil, and C-terminal functional domains, respectively. The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy Delta Hres=21 kJ x mol residue(-1). The ratio of Delta HVH/Delta Hcal for the coiled-coil part of the 120-, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively. This gives an indication of the decrease of the 'all-or-none' character of the transition with increasing protein size. The deletion of the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region. According to CD data, full-length fibritin and all the mutants truncated from the N-termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C-terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C-terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon. PMID

  20. STIM1/Orai1 coiled-coil interplay in the regulation of store-operated calcium entry

    PubMed Central

    Stathopulos, Peter B.; Schindl, Rainer; Fahrner, Marc; Zheng, Le; Gasmi-Seabrook, Geneviève M.; Muik, Martin; Romanin, Christoph; Ikura, Mitsuhiko

    2013-01-01

    Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE). The cytosolic region of STIM1 contains a long putative coiled-coil (CC)1 segment and shorter CC2 and CC3 domains. Here we present solution nuclear magnetic resonance structures of a trypsin-resistant CC1–CC2 fragment in the apo and Orai1-bound states. Each CC1–CC2 subunit forms a U-shaped structure that homodimerizes through antiparallel interactions between equivalent α-helices. The CC2:CC2′ helix pair clamps two identical acidic Orai1 C-terminal helices at opposite ends of a hydrophobic/basic STIM–Orai association pocket. STIM1 mutants disrupting CC1:CC1′ interactions attenuate, while variants promoting CC1 stability spontaneously activate Orai1 currents. CC2 mutations cause remarkable variability in Orai1 activation because of a dual function in binding Orai1 and autoinhibiting STIM1 oligomerization via interactions with CC3. We conclude that SOCE is activated through dynamic interplay between STIM1 and Orai1 helices. PMID:24351972

  1. Type I macrophage scavenger receptor contains α-helical and collagen-like coiled coils

    NASA Astrophysics Data System (ADS)

    Kodama, Tatsuhiko; Freeman, Mason; Rohrer, Lucia; Zabrecky, James; Matsudaira, Paul; Krieger, Monty

    1990-02-01

    The macrophage scavenger receptor is a trimeric membrane glycoprotein with unusual ligand-binding properties which has been implicated in the development of atherosclerosis. The trimeric structure of the bovine type I scavenger receptor, deduced by complementary DNA cloning, contains three extracellular C-terminal cysteine-rich domains connected to the transmembrane domain by a long fibrous stalk. This stalk structure, composed of an a-helical coiled coil and a collagen-like triple helix, has not previously been observed in an integral membrane protein.

  2. Structural characterization of the C-terminal coiled-coil domains of wild-type and kidney disease-associated mutants of apolipoprotein L1.

    PubMed

    Sharma, Alok K; Friedman, David J; Pollak, Martin R; Alper, Seth L

    2016-05-01

    Trypanosomes that cause sleeping sickness endocytose apolipoprotein L1 (APOL1)-containing trypanolytic factors from human serum, leading to trypanolytic death through generation of APOL1-associated lytic pores in trypanosomal membranes. The trypanosome Trypanosoma brucei rhodesiense counteracts trypanolysis by expressing the surface protein serum response-associated (SRA), which can bind APOL1 common variant G0 to block its trypanolytic activity. However, two missense variants in the C terminal predicted coiled-coil (CC) domains of human APOL1 G1 (S342G/I384M) and G2 (ΔN388Y389) decrease or abrogate APOL1 binding to T. brucei rhodesiense SRA, thus preserving APOL1 trypanolytic activity. These evolutionarily selected APOL1 missense variants, found at a high frequency in some populations of African descent, also confer elevated risk of kidney disease. Understanding the SRA-APOL1 interaction and the role of APOL1 G1 and G2 variants in kidney disease demands structural characterization of the APOL1 CC domain. Using CD, heteronuclear NMR, and molecular dynamics (MD) simulation on structural homology models, we report here unique and dynamic solution conformations of nephropathy variants G1 and G2 as compared with the common variant G0. Conformational plasticity in G1 and G2 CC domains led to interhelical α1-α2 approximation coupled with secondary structural changes and delimited motional properties absent in the G0 CC domain. The G1 substitutions conferred local structural changes principally along helix α1, whereas the G2 deletion altered the structure of both helix α2 and helix α1. These dynamic features of APOL1 CC variants likely reflect their intrinsic structural properties, and should help interpret future APOL1 structural studies and define the contribution of APOL1 risk variants to kidney disease. PMID:26945671

  3. Protein-based hydrogels self-assembled from genetically engineered triblock polypeptides containing coiled-coil domains

    NASA Astrophysics Data System (ADS)

    Xu, Chunyu

    Protein-based biomaterials have great potential in biomedical applications due to their similar composition with biological organisms. Environment-sensitive hydrogels based on proteins can undergo sol-gel transition due to the conformational change of the proteins in response to external stimuli. The physical properties of these hydrogels can be tailored by modification of the protein structures. Two major hypotheses were made in this dissertation. One was that coiled-coil folding motifs could be a good candidate for physical crosslinking in protein-based hydrogels, and the other was that the conformational change of coiled-coils in response to external stimuli could mediate the sol-gel transition of the protein-based hydrogels. The first part established synthesis strategies of the coiled-coil containing proteins using a genetic engineering technique. An important observation was made that the fusion sequence on the proteins could influence the thermal stability of the proteins. In the second part of the research, the self-assembly of hydrogels from a series of triblock polypeptides containing coiled-coils was evaluated. It was found that the hydrogels had a porous interconnected network microstructure. The hydrogels responded to temperature and pH, which correlated to the temperature- and pH-triggered structural transition of the coiled-coil domains. In addition, the formation of hydrogels was reversible in the present or absence of guanidine hydrochloride (GdnHCl). The last part of the research attempted to explore the relationship between the structure of the protein polymers and the physical property of the hydrogels, and to investigate the parameters influencing the hydrogel formation and physical properties. Triblock and diblock polypeptides were designed to contain different lengths of coiled-coil domains. Tyrosine residues were incorporated at selected solvent-exposed positions in order to increase the hydrophobicity of the coiled-coil domains. The

  4. The coiled-coil domain of the Nop56/58 core protein is dispensable for sRNP assembly but is critical for archaeal box C/D sRNP-guided nucleotide methylation

    PubMed Central

    Zhang, Xinxin; Champion, Erica A.; Tran, Elizabeth J.; Brown, Bernard A.; Baserga, Susan J.; Maxwell, E. Stuart

    2006-01-01

    Archaeal box C/D sRNAs guide the methylation of specific nucleotides in archaeal ribosomal and tRNAs. Three Methanocaldococcus jannaschii sRNP core proteins (ribosomal protein L7, Nop56/58, and fibrillarin) bind the box C/D sRNAs to assemble the sRNP complex, and these core proteins are essential for nucleotide methylation. A distinguishing feature of the Nop56/58 core protein is the coiled-coil domain, established by α-helices 4 and 5, that facilitates Nop56/58 self-dimerization in vitro. The function of this coiled-coil domain has been assessed for box C/D sRNP assembly, sRNP structure, and sRNP-guided nucleotide methylation by mutating or deleting this protein domain. Protein pull-down experiments demonstrated that Nop56/58 self-dimerization and Nop56/58 dimerization with the core protein fibrillarin are mutually exclusive protein:protein interactions. Disruption of Nop56/58 homodimerization by alteration of specific amino acids or deletion of the entire coiled-coil domain had no obvious effect upon core protein binding and sRNP assembly. Site-directed mutation of the Nop56/58 homodimerization domain also had no apparent effect upon either box C/D RNP- or C′/D′ RNP-guided nucleotide modification. However, deletion of this domain disrupted guided methylation from both RNP complexes. Nuclease probing of the sRNP assembled with Nop56/58 proteins mutated in the coiled-coil domain indicated that while functional complexes were assembled, box C/D and C′/D′ RNPs were altered in structure. Collectively, these experiments revealed that the self-dimerization of the Nop56/58 coiled-coil domain is not required for assembly of a functional sRNP, but the coiled-coil domain is important for the establishment of wild-type box C/D and C′/D′ RNP structure essential for nucleotide methylation. PMID:16601205

  5. Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association

    PubMed Central

    Jiang, Yan; Ling, Junjie; Hettiarachchi, Chamari; Tellgren-Roth, Christian; Wei, Ning; Deng, Xing Wang

    2015-01-01

    CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) functions as an E3 ubiquitin ligase and mediates a variety of developmental processes in Arabidopsis by targeting a number of key regulators for ubiquitination and degradation. Here, we identify a novel COP1 interacting protein, COP1 SUPPRESSOR 2 (CSU2). Loss of function mutations in CSU2 suppress the constitutive photomorphogenic phenotype of cop1-6 in darkness. CSU2 directly interacts with COP1 via their coiled-coil domains and is recruited by COP1 into nuclear speckles in living plant cells. Furthermore, CSU2 inhibits COP1 E3 ubiquitin ligase activity in vitro, and represses COP1 mediated turnover of HY5 in cell-free extracts. We propose that in csu2 cop1-6 mutants, the lack of CSU2’s repression of COP1 allows the low level of COP1 to exhibit higher activity that is sufficient to prevent accumulation of HY5 in the dark, thus restoring the etiolated phenotype. In addition, CSU2 is required for primary root development under normal light growth condition. PMID:26714275

  6. Structure of a Designed, Right-Handed Coiled-Coil Tetramer Containing All Biological Amino Acids

    SciTech Connect

    Sales, M.; Plecs, J.J.; Holton, J.M.; Alber, T.

    2009-06-04

    The previous design of an unprecedented family of two-, three-, and four-helical, right-handed coiled coils utilized nonbiological amino acids to efficiently pack spaces in the oligomer cores. Here we show that a stable, right-handed parallel tetrameric coiled coil, called RH4B, can be designed entirely using biological amino acids. The X-ray crystal structure of RH4B was determined to 1.1 {angstrom} resolution using a designed metal binding site to coordinate a single Yb{sup 2+} ion per 33-amino acid polypeptide chain. The resulting experimental phases were particularly accurate, and the experimental electron density map provided an especially clear, unbiased view of the molecule. The RH4B structure closely matched the design, with equivalent core rotamers and an overall root-mean-square deviation for the N-terminal repeat of the tetramer of 0.24 {angstrom}. The clarity and resolution of the electron density map, however, revealed alternate rotamers and structural differences between the three sequence repeats in the molecule. These results suggest that the RH4B structure populates an unanticipated variety of structures.

  7. The Orientations of Large Aspect-Ratio Coiled-Coil Proteins Attached to Gold Nanostructures.

    PubMed

    Chang, Jae-Byum; Kim, Yong Ho; Thompson, Evan; No, Young Hyun; Kim, Nam Hyeong; Arrieta, Jose; Manfrinato, Vitor R; Keating, Amy E; Berggren, Karl K

    2016-03-01

    Methods for patterning biomolecules on a substrate at the single molecule level have been studied as a route to sensors with single-molecular sensitivity or as a way to probe biological phenomena at the single-molecule level. However, the arrangement and orientation of single biomolecules on substrates has been less investigated. Here, the arrangement and orientation of two rod-like coiled-coil proteins, cortexillin and tropomyosin, around patterned gold nanostructures is examined. The high aspect ratio of the coiled coils makes it possible to study their orientations and to pursue a strategy of protein orientation via two-point attachment. The proteins are anchored to the surfaces using thiol groups, and the number of cysteine residues in tropomyosin is varied to test how this variation affects the structure and arrangement of the surface-attached proteins. Molecular dynamics studies are used to interpret the observed positional distributions. Based on initial studies of protein attachment to gold post structures, two 31-nm-long tropomyosin molecules are aligned between the two sidewalls of a trench with a width of 68 nm. Because the approach presented in this study uses one of twenty natural amino acids, this method provides a convenient way to pattern biomolecules on substrates using standard chemistry. PMID:26799936

  8. Improvement of probe peptides for coiled-coil labeling by introducing phosphoserines.

    PubMed

    Ono, Satoshi; Yano, Yoshiaki; Matsuzaki, Katsumi

    2012-01-01

    We have developed a method of rapidly labeling membrane proteins in living cells using a high-affinity heterodimeric coiled-coil construct containing an E3 tag (EIAALEK)(3) genetically fused to the target protein and a K4 probe (KIAALKE)(4) labeled with a fluorophore such as tetramethylrhodamine (TMR) at its N-terminus (TMR-K4). However, coiled-coil labeling cannot be applied to highly negatively charged cell lines such as HEK293, because of the nonspecific adsorption of the positively charged K4 probes to cell membranes. To reduce the net positive charge, we synthesized new probes that include phosphoserine residues (pSer) between the K4 sequence and TMR fluorophore (TMR-(pSer)(n)-K4, [n = 1-3]). The affinity of the pSer-introduced probes was comparable to that of the TMR-K4 probe. However, the TMR-(pSer)(2)-K4 and TMR-(pSer)(3)-K4 probes tended to aggregate during labeling. In contrast, TMR-pSer-K4, which was as soluble as TMR-K4, achieved higher signal/background ratios (30-100) for four host cell lines (HEK293, HeLa, SH-SY5Y, and PC12) than did TMR-K4 (~10 for HEK293 cells), demonstrating that the improved probe can be used for various types of cells. PMID:22782565

  9. Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles.

    PubMed

    Indelicato, Giuliana; Wahome, Newton; Ringler, Philippe; Müller, Shirley A; Nieh, Mu-Ping; Burkhard, Peter; Twarock, Reidun

    2016-02-01

    Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications. PMID:26840729

  10. An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors

    PubMed Central

    Shekhawat, Sujan S.; Porter, Jason R.; Sriprasad, Akshay; Ghosh, Indraneel

    2009-01-01

    Proteases are widely studied as they are integral players in cell cycle control and apoptosis. We report a new approach for the design of a family of genetically encoded turn-on protease biosensors. In our design, an auto-inhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of split-protein reporters. Utilizing this new auto-inhibition design paradigm, we present the rational construction and optimization of three generations of protease biosensors, with the final design providing a 1000 fold increase in bioluminescent signal upon addition of the TEV protease. We demonstrate the generality of the approach utilizing two different split-protein reporters, firefly luciferase and beta-lactamase, while also testing our design in the context of a therapeutically relevant protease, caspase-3. Finally, we present a dual-protease sensor geometry that allows for the use of these turn-on sensors as potential AND logic gates. Thus these studies potentially provide a new method for the design and implementation of genetically encoded turn-on protease sensors while also providing a general auto-inhibited coiled-coil strategy for controlling the activity of fragmented proteins. PMID:19803505

  11. Directed surface attachment of nanomaterials via coiled-coil-driven self-assembly

    NASA Astrophysics Data System (ADS)

    White, Simon J.; Johnson, Steven; Szymonik, Michal; Wardingley, Richard A.; Pye, Douglas; Davies, A. Giles; Wälti, Christoph; Stockley, Peter G.

    2012-12-01

    Numerous nanoscale devices and materials have been fabricated in recent years using a variety of biological scaffolds. However, the interfacing of these devices and materials into existing circuits and ordered arrays has proved problematic. Here, we describe a simple solution to this problem using self-assembly of the peptide coiled-coil heterodimer ACID:BASE to immobilize M13 bacteriophage particles to specific locations on a patterned gold surface. Surface plasmon resonance demonstrated that free ACID peptides will assemble onto a surface derivatized with BASE. We then displayed the ACID peptide on the pIX coat protein of M13 and showed that these phage particles permit formation of the coiled-coil resulting in specific surface attachment. The ACID:immobilized BASE affinities appear to be similar for free peptide and phage-displayed ACID. Finally, we fabricated two gold electrodes, separated by a 200 nm gap, coated one of them with BASE and showed that this allows localization of the M13:ACID onto the functionalized electrode.

  12. Directed surface attachment of nanomaterials via coiled-coil-driven self-assembly.

    PubMed

    White, Simon J; Johnson, Steven; Szymonik, Michal; Wardingley, Richard A; Pye, Douglas; Davies, A Giles; Wälti, Christoph; Stockley, Peter G

    2012-12-14

    Numerous nanoscale devices and materials have been fabricated in recent years using a variety of biological scaffolds. However, the interfacing of these devices and materials into existing circuits and ordered arrays has proved problematic. Here, we describe a simple solution to this problem using self-assembly of the peptide coiled-coil heterodimer ACID:BASE to immobilize M13 bacteriophage particles to specific locations on a patterned gold surface. Surface plasmon resonance demonstrated that free ACID peptides will assemble onto a surface derivatized with BASE. We then displayed the ACID peptide on the pIX coat protein of M13 and showed that these phage particles permit formation of the coiled-coil resulting in specific surface attachment. The ACID:immobilized BASE affinities appear to be similar for free peptide and phage-displayed ACID. Finally, we fabricated two gold electrodes, separated by a 200 nm gap, coated one of them with BASE and showed that this allows localization of the M13:ACID onto the functionalized electrode. PMID:23154792

  13. Evidence of α-helical coiled coils and β-sheets in hornet silk.

    PubMed

    Kameda, Tsunenori; Nemoto, Takashi; Ogawa, Tetsuya; Tosaka, Masatoshi; Kurata, Hiroki; Schaper, Andreas K

    2014-03-01

    α-Helical coiled coil and β-sheet complexes are essential structural building elements of silk proteins produced by different species of the Hymenoptera. Beside X-ray scattering at wide and small angles we applied cryo-electron diffraction and microscopy to demonstrate the presence and the details of such structures in silk of the giant hornet Vespa mandarinia japonica. Our studies on the assembly of the fibrous silk proteins and their internal organization in relation to the primary chain structure suggest a 172 Å pitch supercoil consisting of four intertwined alanine-rich α-helical strands. The axial periodicity may adopt even multiples of the pitch value. Coiled coil motifs form the largest portion of the hornet silk structure and are aligned nearly parallel to the cocoon fiber axis in the same way as the membrane-like parts of the cocoon are molecularly orientated in the spinning direction. Supercoils were found to be associated with β-crystals, predominantly localized in the l-serine-rich chain sequences terminating each of the four predominant silk proteins. Such β-sheet blocks are considered resulting from transformation of random coil molecular sequences due to the action of elongational forces during the spinning process. PMID:24345346

  14. Immunogenicity of coiled-coil based drug-free macromolecular therapeutics

    PubMed Central

    Kverka, Miloslav; Hartley, Jonathan M.; Chu, Te-Wei; Yang, Jiyuan; Heidchen, Regina; Kopeček, Jindřich

    2014-01-01

    A two-component CD20 (non-internalizing) receptor crosslinking system based on the biorecognition of complementary coiled-coil forming peptides was evaluated. Exposure of B cells to Fab’-peptide1 conjugate decorates the cell surface with peptide1; further exposure of the decorated cells to P-(peptide2)x (P is the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone) results in the formation of coiled-coil heterodimers at the cell surface with concomitant induction of apoptosis. The aim of this study was to determine the potential immunogenicity of this therapeutic system that does not contain low molecular weight drugs. Enantiomeric peptides (L- and D-CCE and L- and D-CCK), HPMA copolymer-peptide conjugates, and Fab’ fragment-peptide conjugates were synthesized and the immunological properties of peptide conjugates evaluated in vitro on RAW264.7 macrophages and in vivo on immunocompetent BALB/c mice. HPMA copolymer did not induce immune response in vitro and in vivo. Administration of P-peptide conjugates with strong adjuvant resulted in antibody response directed to the peptide. Fab’ was responsible for macrophage activation of Fab’-peptide conjugates and a major factor in the antibody induction following i.v. administration of Fab’ conjugates. There was no substantial difference in the ability of conjugates of D-peptides and conjugates of L-peptides to induce Ab response. PMID:24767787

  15. ELKS controls the pool of readily releasable vesicles at excitatory synapses through its N-terminal coiled-coil domains

    PubMed Central

    Held, Richard G; Liu, Changliang; Kaeser, Pascal S

    2016-01-01

    In a presynaptic nerve terminal, synaptic strength is determined by the pool of readily releasable vesicles (RRP) and the probability of release (P) of each RRP vesicle. These parameters are controlled at the active zone and vary across synapses, but how such synapse specific control is achieved is not understood. ELKS proteins are enriched at vertebrate active zones and enhance P at inhibitory hippocampal synapses, but ELKS functions at excitatory synapses are not known. Studying conditional knockout mice for ELKS, we find that ELKS enhances the RRP at excitatory synapses without affecting P. Surprisingly, ELKS C-terminal sequences, which interact with RIM, are dispensable for RRP enhancement. Instead, the N-terminal ELKS coiled-coil domains that bind to Liprin-α and Bassoon are necessary to control RRP. Thus, ELKS removal has differential, synapse-specific effects on RRP and P, and our findings establish important roles for ELKS N-terminal domains in synaptic vesicle priming. DOI: http://dx.doi.org/10.7554/eLife.14862.001 PMID:27253063

  16. Identification of a region in the coiled-coil domain of Smc3 that is essential for cohesin activity.

    PubMed

    Orgil, Ola; Mor, Hadar; Matityahu, Avi; Onn, Itay

    2016-07-27

    The cohesin complex plays an important role in sister chromatin cohesion. Cohesin's core is composed of two structural maintenance of chromosome (SMC) proteins, called Smc1 and Smc3. SMC proteins are built from a globular hinge domain, a rod-shaped domain composed of long anti-parallel coiled-coil (CC), and a second globular adenosine triphosphatase domain called the head. The functions of both head and hinge domains have been studied extensively, yet the function of the CC region remains elusive. We identified a mutation in the CC of smc3 (L217P) that disrupts the function of the protein. Cells carrying the smc3-L217P allele have a strong cohesion defect and complexes containing smc3-L217P are not loaded onto the chromosomes. However, the mutation does not affect inter-protein interactions in either the core complex or with the Scc2 loader. We show by molecular dynamics and biochemistry that wild-type Smc3 can adopt distinct conformations, and that adenosine triphosphate (ATP) induces the conformational change. The L217P mutation restricts the ability of the mutated protein to switch between the conformations. We suggest that the function of the CC is to transfer ATP binding/hydrolysis signals between the head and the hinge domains. The results provide a new insight into the mechanism of cohesin activity. PMID:27307603

  17. Coiled-Coils at the Edge of Configurational Heterogeneity. Structural Analyses of Parallel and Antiparallel Homotetrameric Coiled-Coils Reveal Configurational Sensitivity to a Single Solvent-Exposed Amino Acid Substitution.†§

    PubMed Central

    Yadav, Maneesh K.; Leman, Luke J.; Price, Daniel J.; Brooks, Charles L.; Stout, C. David; Ghadiri, M. Reza

    2007-01-01

    A detailed understanding of the mechanisms by which particular amino acid sequences can give rise to more than one folded structure, such as for proteins that undergo large conformational changes or misfolding, is a long-standing objective of protein chemistry. Here we describe the crystal structures of a single coiled-coil peptide in distinct parallel and antiparallel tetrameric configurations and further describe the parallel or antiparallel crystal structures of several related peptide sequences; the antiparallel tetrameric assemblies represents the first crystal structures of GCN4-derived peptides exhibiting such a configuration. Intriguingly, substitution of a single solvent-exposed residue enabled the parallel coiled-coil tetramer GCN4-pLI to populate the antiparallel configuration, suggesting that the two configurations are close enough in energy for subtle sequence changes to have important structural consequences. We present a structural analysis of the small changes to helix register and side chain conformations that accommodate the two configurations, and have supplemented these results using solution studies and a molecular dynamics energetic analysis using a replica exchange methodology. Considering the previous examples of structural nonspecificity in coiled-coil peptides, the findings reported here not only emphasize the predisposition of the coiled-coil motif to adopt multiple configurations, but also call attention to the associated risk that observed crytstal structures may not represent the only (or even the major) species present in solution. PMID:16584182

  18. Gene Delivery from Supercharged Coiled-coil Protein and Cationic Lipid Hybrid Complex

    PubMed Central

    More, Haresh T.; Frezzo, Joseph A.; Dai, Jisen; Yamano, Seiichi; Montclare, Jin K.

    2014-01-01

    A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP•DNA(8:1)•FG lipoproteoplex complex demonstrated enhanced transfection of β-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat•FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP•DNA(8:1)•FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery. PMID:24875765

  19. Designed Coiled-Coil Peptides Inhibit the Type Three Secretion System of Enteropathogenic Escherichia coli

    PubMed Central

    Larzábal, Mariano; Mercado, Elsa C.; Vilte, Daniel A.; Salazar-González, Hector; Cataldi, Angel; Navarro-Garcia, Fernando

    2010-01-01

    Background Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry. Methods/Principal Findings We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled–coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments. Conclusions Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis. PMID:20140230

  20. pH-Dependent Assembly and Segregation of the Coiled-Coil Segments of Yeast Putative Cargo Receptors Emp46p and Emp47p

    PubMed Central

    Noda, Masanori; Kajino, Megumi; Kim, Akemi; Kurimoto, Eiji; Sato, Ken; Nakano, Akihiko; Kobayashi, Yuji; Yagi, Hirokazu; Uchiyama, Susumu; Kato, Koichi

    2015-01-01

    Emp46p and Emp47p are yeast putative cargo receptors that recycle between the endoplasmic reticulum and the Golgi apparatus. These receptors can form complexes in a pH-dependent manner, but their molecular mechanisms remain unclear. Here, we successfully reproduced their interactions in vitro solely with their coiled-coil segments, which form stable heterotetramers in the neutral condition but segregate at lower pH. Mutational data identified a key glutamate residue of Emp46p that serves as the pH-sensing switch of their oligomer formation. Our findings elucidate the mechanisms of the dynamic cargo receptor interactions in the secretory pathway and the design framework of the environment-responsive molecular assembly and disassembly systems. PMID:26447473

  1. Truncated and Helix-Constrained Peptides with High Affinity and Specificity for the cFos Coiled-Coil of AP-1

    PubMed Central

    Rao, Tara; Ruiz-Gómez, Gloria; Hill, Timothy A.; Hoang, Huy N.; Fairlie, David P.; Mason, Jody M.

    2013-01-01

    Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity. Here we combine structural stability and target specificity of proteins, with low cost and rapid synthesis of small molecules, towards meeting the significant challenge of binding coiled coil proteins in transcriptional regulation. By iteratively truncating a Jun-based peptide from 37 to 22 residues, strategically incorporating i→i+4 helix-inducing constraints, and positioning unnatural amino acids, we have produced short, water-stable, α-helical peptides that bind cFos. A three-dimensional NMR-derived structure for one peptide (24) confirmed a highly stable α-helix which was resistant to proteolytic degradation in serum. These short structured peptides are entropically pre-organized for binding with high affinity and specificity to cFos, a key component of the oncogenic transcriptional regulator Activator Protein-1 (AP-1). They competitively antagonized the cJun–cFos coiled-coil interaction. Truncating a Jun-based peptide from 37 to 22 residues decreased the binding enthalpy for cJun by ∼9 kcal/mol, but this was compensated by increased conformational entropy (TΔS ≤7.5 kcal/mol). This study demonstrates that rational design of short peptides constrained by α-helical cyclic pentapeptide modules is able to retain parental high helicity, as well as high affinity and specificity for cFos. These are important steps towards small antagonists of the cJun-cFos interaction that mediates gene transcription in cancer and inflammatory diseases. PMID:23544065

  2. Structural Basis of HIV-1 Neutralization by Affinity Matured Fabs Directed against the Internal Trimeric Coiled-Coil of gp41

    SciTech Connect

    Gustchina, Elena; Li, Mi; Louis, John M.; Anderson, D.Eric; Lloyd, John; Frisch, Christian; Bewley, Carole A.; Gustchina, Alla; Wlodawer, Alexander; Clore, G.Marius

    2010-12-03

    The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naive human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.

  3. Missense mutation in DISC1 C-terminal coiled-coil has GSK3β signaling and sex-dependent behavioral effects in mice

    PubMed Central

    Dachtler, James; Elliott, Christina; Rodgers, R. John; Baillie, George S.; Clapcote, Steven J.

    2016-01-01

    Disrupted-in-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and affective disorders. The full-length DISC1 protein consists of an N-terminal ‘head’ domain and a C-terminal tail domain that contains several predicted coiled-coils, structural motifs involved in protein-protein interactions. To probe the in vivo effects of missense mutation of DISC1’s C-terminal tail, we tested mice carrying mutation D453G within a predicted α-helical coiled-coil region. We report that, relative to wild-type littermates, female DISC1D453G mice exhibited novelty-induced hyperlocomotion, an anxiogenic profile in the elevated plus-maze and open field tests, and reduced social exploration of unfamiliar mice. Male DISC1D453G mice displayed a deficit in passive avoidance, while neither males nor females exhibited any impairment in startle reactivity or prepulse inhibition. Whole brain homogenates showed normal levels of DISC1 protein, but decreased binding of DISC1 to GSK3β, decreased phospho-inhibition of GSK3β at serine 9, and decreased levels of β-catenin in DISC1D453G mice of either sex. Interrupted GSK3β signaling may thus be part of the mechanism underlying the behavioral phenotype associated with D453G, in common with the previously described N-terminal domain mutations Q31L and L100P in mice, and the schizophrenia risk-conferring variant R264Q in humans. PMID:26728762

  4. Missense mutation in DISC1 C-terminal coiled-coil has GSK3β signaling and sex-dependent behavioral effects in mice.

    PubMed

    Dachtler, James; Elliott, Christina; Rodgers, R John; Baillie, George S; Clapcote, Steven J

    2016-01-01

    Disrupted-in-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and affective disorders. The full-length DISC1 protein consists of an N-terminal 'head' domain and a C-terminal tail domain that contains several predicted coiled-coils, structural motifs involved in protein-protein interactions. To probe the in vivo effects of missense mutation of DISC1's C-terminal tail, we tested mice carrying mutation D453G within a predicted α-helical coiled-coil region. We report that, relative to wild-type littermates, female DISC1(D453G) mice exhibited novelty-induced hyperlocomotion, an anxiogenic profile in the elevated plus-maze and open field tests, and reduced social exploration of unfamiliar mice. Male DISC1(D453G) mice displayed a deficit in passive avoidance, while neither males nor females exhibited any impairment in startle reactivity or prepulse inhibition. Whole brain homogenates showed normal levels of DISC1 protein, but decreased binding of DISC1 to GSK3β, decreased phospho-inhibition of GSK3β at serine 9, and decreased levels of β-catenin in DISC1(D453G) mice of either sex. Interrupted GSK3β signaling may thus be part of the mechanism underlying the behavioral phenotype associated with D453G, in common with the previously described N-terminal domain mutations Q31L and L100P in mice, and the schizophrenia risk-conferring variant R264Q in humans. PMID:26728762

  5. Rapid Identification of Malaria Vaccine Candidates Based on α-Helical Coiled Coil Protein Motif

    PubMed Central

    Villard, Viviane; Agak, George W.; Frank, Géraldine; Jafarshad, Ali; Servis, Catherine; Nébié, Issa; Sirima, Sodiomon B.; Felger, Ingrid; Arevalo-Herrera, Myriam; Herrera, Socrates; Heitz, Frederic; Bäcker, Volker; Druilhe, Pierre; Kajava, Andrey V.; Corradin, Giampietro

    2007-01-01

    To identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally “native” epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens. PMID:17653272

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

    PubMed

    Kelsey, Jessica S; Fastman, Nathan M; Noratel, Elizabeth F; Blumberg, Daphne D

    2012-09-01

    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. PMID:22809629

  7. The Coiled-Coil Domain of EHD2 Mediates Inhibition of LeEix2 Endocytosis and Signaling

    PubMed Central

    Bar, Maya; Sharfman, Miya; Schuster, Silvia; Avni, Adi

    2009-01-01

    Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis. PMID:19936242

  8. The coiled-coil domain of EHD2 mediates inhibition of LeEix2 endocytosis and signaling.

    PubMed

    Bar, Maya; Sharfman, Miya; Schuster, Silvia; Avni, Adi

    2009-01-01

    Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis. PMID:19936242

  9. Mechanical Transition from α-Helical Coiled-Coils to β-Sheets in Fibrin(ogen)

    PubMed Central

    Zhmurov, Artem; Kononova, Olga; Litvinov, Rustem I.; Dima, Ruxandra I.; Barsegov, Valeri; Weisel, John W.

    2012-01-01

    We characterized the α-to-β transition in α-helical coiled-coil connectors of human fibrin(ogen) molecule using biomolecular simulations of their forced elongation, and theoretical modeling. The force (F) - extension (X) profiles show three distinct regimes: (1) the elastic regime, in which the coiled-coils act as entropic springs (F < 100–125 pN; X < 7–8 nm); (2) the constant-force plastic regime, characterized by a force-plateau (F≈150 pN; X≈10–35 nm); and (3) the non-linear regime (F >175–200 pN; X > 40–50 nm). In the plastic regime, the three-stranded α-helices undergo a non-cooperative phase transition to form parallel three-stranded β-sheets. The critical extension of α-helices is 0.25 nm, and the energy difference between the α-helices and β-sheets is 4.9 kcal/mol per helical pitch. The soft α-to-β phase transition in coiled-coils might be a universal mechanism underlying mechanical properties of filamentous α-helical proteins. PMID:22953986

  10. A thermodynamic model for the helix-coil transition coupled to dimerization of short coiled-coil peptides.

    PubMed Central

    Qian, H

    1994-01-01

    A simple thermodynamic formalism is presented to model the conformational transition between a random-coil monomeric peptide and a coiled-coil helical dimer. The coiled-coil helical dimer is the structure of a class of proteins also called leucine zipper, which has been studied intensively in recent years. Our model, which is appropriate particularly for short peptides, is an alternative to the theory developed by Skolnick and Holtzer. Using the present formalism, we discuss the multi-equilibriatory nature of this transition and provide an explanation for the apparent two-state behavior of coiled-coil formation when the helix-coil transition is coupled to dimerization. It is found that such coupling between multi-equilibria and a true two-state transition can simplify the data analysis, but care must be taken in using the overall association constant to determine helix propensities (w) of single residues. Successful use of the two-state model does not imply that the helix-coil transition is all-or-none. The all-or-none assumption can provide good numerical estimates when w is around unity (0.35 < or = w < or = 1.35), but when w is small (w < 0.01), similar estimations can lead to large errors. The theory of the helix-coil transition in denaturation experiments is also discussed. PMID:7919005

  11. Balance between Coiled-Coil Stability and Dynamics Regulates Activity of BvgS Sensor Kinase in Bordetella

    PubMed Central

    Lesne, E.; Krammer, E.-M.; Dupre, E.; Locht, C.; Lensink, M. F.

    2016-01-01

    ABSTRACT The two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS) domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil’s dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases. PMID:26933056

  12. High-resolution spot-scan electron microscopy of microcrystals of an alpha-helical coiled-coil protein.

    PubMed

    Bullough, P A; Tulloch, P A

    1990-09-01

    We describe the electron microscopy of a crystalline assembly of an alpha-helical coiled-coil protein extracted from the ootheca of the praying mantis. Electron diffraction patterns of unstained crystals show crystal lattice sampling of the coiled-coil molecular transform to a resolution beyond 1.5 A. Using a "spot-scan" method of electron imaging, micrographs of unstained crystals have been obtained that visibly diffract laser light from crystal spacings as small as 4.3 A. A projection map was calculated to 4 A using electron diffraction amplitudes and phases from computer-processed images. The projection map clearly shows modulations in density arising from the 5.1 A alpha-helical repeat, the first time this type of modulation has been revealed by electron microscopy. The crystals have p2 plane group symmetry with a = 92.4 A, b = 150.7 A, y = 92.4 degrees. Examination of tilted specimens shows that c is approximately 18 A, indicating that the unit cell is only one molecule thick. A preliminary interpretation shows tightly packed molecules some 400 A long lying with their long axes in the plane of the projection. The molecules have a coiled-coil configuration for most of their length. The possible modes of packing of the molecules in three dimensions are discussed. PMID:2398496

  13. Structural Correlation of the Neck Coil with the Coiled-coil (CC1)-Forkhead-associated (FHA) Tandem for Active Kinesin-3 KIF13A.

    PubMed

    Ren, Jinqi; Huo, Lin; Wang, Wenjuan; Zhang, Yong; Li, Wei; Lou, Jizhong; Xu, Tao; Feng, Wei

    2016-02-12

    Processive kinesin motors often contain a coiled-coil neck that controls the directionality and processivity. However, the neck coil (NC) of kinesin-3 is too short to form a stable coiled-coil dimer. Here, we found that the coiled-coil (CC1)-forkhead-associated (FHA) tandem (that is connected to NC by Pro-390) of kinesin-3 KIF13A assembles as an extended dimer. With the removal of Pro-390, the NC-CC1 tandem of KIF13A unexpectedly forms a continuous coiled-coil dimer that can be well aligned into the CC1-FHA dimer. The reverse introduction of Pro-390 breaks the NC-CC1 coiled-coil dimer but provides the intrinsic flexibility to couple NC with the CC1-FHA tandem. Mutations of either NC, CC1, or the FHA domain all significantly impaired the motor activity. Thus, the three elements within the NC-CC1-FHA tandem of KIF13A are structurally interrelated to form a stable dimer for activating the motor. This work also provides the first direct structural evidence to support the formation of a coiled-coil neck by the short characteristic neck domain of kinesin-3. PMID:26680000

  14. Linear free-energy analysis of mercury(II) and cadmium(II) binding to three-stranded coiled coils.

    PubMed

    Ghosh, Debdip; Lee, Kyung-Hoon; Demeler, Borries; Pecoraro, Vincent L

    2005-08-01

    Investigators have studied how proteins enforce nonstandard geometries on metal centers to assess the question of how protein structures can define the coordination geometry and binding affinity of an active-site metal cofactor. We have shown that cysteine-substituted versions of the TRI peptide series [AcG-(LKALEEK)(4)G-NH(2)] bind Hg(II) and Cd(II) in geometries that are different from what is normally found with thiol ligands in aqueous solution. A fundamental question has been whether this structural perturbation is due to protein influence or a change in the metal geometry preference. To address this question, we have completed linear free-energy analyses that correlate the association of three-stranded coiled coils in the absence of a metal with the binding affinity of the peptides to the heavy metals, Hg(II) and Cd(II). In this paper, six new members of this family have been synthesized, replacing core leucine residues with smaller and less hydrophobic residues, consequently leading to varying degrees of self-association affinities. At the same time, studies with some smaller and longer sequenced peptides have also been examined. All of these peptides are seen to sequester Hg(II) and Cd(II) in an uncommon trigonal environment. For both metals, the binding is strong with micromolar dissociation constants. For binding of Hg(II) to the peptides, the dissociation constants range from 2.4 x 10(-)(5) M for Baby L12C to 2.5 x 10(-)(9) M for Grand L9C for binding of the third thiolate to a linear Hg(II)(pep)(2) species. The binding of Hg(II) to the peptide Grand L9C is similar in energetics for metal binding in the metalloregulatory protein, mercury responsive (merR), displaying approximately 50% trigonal Hg(II) formation at nanomolar metal concentrations. Approximately, 11 kcal/mol of the Hg(II)(Grand L9C)(3)(-) stability is due to peptide interactions, whereas only 1-4 kcal/mol stabilization results from Hg(II)(RS)(2) binding the third thiolate ligand. This

  15. Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction

    PubMed Central

    Pelassa, Ilaria; Corà, Davide; Cesano, Federico; Monje, Francisco J.; Montarolo, Pier Giorgio; Fiumara, Ferdinando

    2014-01-01

    The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins owing to genetic mutations induces protein aggregation and toxicity, causing at least 18 human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone β-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ and polyQA repeats have a phylogenetically graded association in human and non-human proteomes and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form—alone or with polyQ and polyQA—CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2—a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion—and found that the stability of its polyQ/polyA CC controls its aggregation, localization and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases. PMID:24497578

  16. Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction.

    PubMed

    Pelassa, Ilaria; Corà, Davide; Cesano, Federico; Monje, Francisco J; Montarolo, Pier Giorgio; Fiumara, Ferdinando

    2014-07-01

    The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins owing to genetic mutations induces protein aggregation and toxicity, causing at least 18 human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone β-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ and polyQA repeats have a phylogenetically graded association in human and non-human proteomes and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form--alone or with polyQ and polyQA--CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2--a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion--and found that the stability of its polyQ/polyA CC controls its aggregation, localization and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases. PMID:24497578

  17. Novel Anti-Nicotine Vaccine Using a Trimeric Coiled-Coil Hapten Carrier

    PubMed Central

    Miller, Keith D.; Roque, Richard; Clegg, Christopher H.

    2014-01-01

    Tobacco addiction represents one of the largest public health problems in the world and is the leading cause of cancer and heart disease, resulting in millions of deaths a year. Vaccines for smoking cessation have shown considerable promise in preclinical models, although functional antibody responses induced in humans are only modestly effective in preventing nicotine entry into the brain. The challenge in generating serum antibodies with a large nicotine binding capacity is made difficult by the fact that this drug is non-immunogenic and must be conjugated as a hapten to a protein carrier. To circumvent the limitations of traditional carriers like keyhole limpet hemocyanin (KLH), we have synthesized a short trimeric coiled-coil peptide (TCC) that creates a series of B and T cell epitopes with uniform stoichiometry and high density. Here we compared the relative activities of a TCC-nic vaccine and two control KLH-nic vaccines using Alum as an adjuvant or GLA-SE, which contains a synthetic TLR4 agonist formulated in a stable oil-in-water emulsion. The results showed that the TCC's high hapten density correlated with a better immune response in mice as measured by anti-nicotine Ab titer, affinity, and specificity, and was responsible for a reduction in anti-carrier immunogenicity. The Ab responses achieved with this synthetic vaccine resulted in a nicotine binding capacity in serum that could prevent >90% of a nicotine dose equivalent to three smoked cigarettes (0.05 mg/kg) from reaching the brain. PMID:25494044

  18. Design and characterization of the anion-sensitive coiled-coil peptide.

    PubMed Central

    Hoshino, M.; Yumoto, N.; Yoshikawa, S.; Goto, Y.

    1997-01-01

    As a model for analyzing the role of charge repulsion in proteins and its shielding by the solvent, we designed a peptide of 27 amino acid residues that formed a homodimeric coiled-coil. The interface between the coils consisted of hydrophobic Leu and Val residues, and 10 Lys residues per monomer were incorporated into the positions exposed to solvent. During the preparation of a disulfide-linked dimer in which the two peptides were linked in parallel by the two disulfide bonds located at the N and C terminals, a cyclic monomer with an intramolecular disulfide bond was also obtained. On the basis of CD and 1H-NMR, the conformational stabilities of these isomers and several reference peptides were examined. Whereas all these peptides were unfolded in the absence of salt at pH 4.7 and 20 degrees C, the addition of NaClO4 cooperatively stabilized the alpha-helical conformation. The crosslinking of the peptides by disulfide bonds significantly decreased the midpoint salt concentration of the transition. The 1H-NMR spectra in the presence of NaClO4 suggested that, whereas the disulfide-bonded dimer assumed a native-like conformation, the cyclic monomer assumed a molten globule-like conformation with disordered side chains. However, the cyclic monomer exhibited cooperative transitions against temperature and Gdn-HCl that were only slightly less cooperative than those of the disulfide-bonded parallel dimer. These results indicate that the charge repulsion critically destabilizes the native-like state as well as the molten globule-like state, and that the solvent-dependent charge repulsion may be useful for controlling the conformation of designed peptides. PMID:9232640

  19. A Single Missense Mutation in a Coiled-Coil Domain of Escherichia coli Ribosomal Protein S2 Confers a Thermosensitive Phenotype That Can Be Suppressed by Ribosomal Protein S1

    PubMed Central

    Aseev, Leonid V.; Chugunov, Anton O.; Efremov, Roman G.

    2013-01-01

    Ribosomal protein S2 is an essential component of translation machinery, and its viable mutated variants conferring distinct phenotypes serve as a valuable tool in studying the role of S2 in translation regulation. One of a few available rpsB mutants, rpsB1, shows thermosensitivity and ensures enhanced expression of leaderless mRNAs. In this study, we identified the nature of the rpsB1 mutation. Sequencing of the rpsB1 allele revealed a G-to-A transition in the part of the rpsB gene which encodes a coiled-coil domain of S2. The resulting E132K substitution resides in a highly conserved site, TKKE, a so-called N-terminal capping box, at the beginning of the second alpha helix. The protruding coiled-coil domain of S2 is known to provide binding with 16S rRNA in the head of the 30S subunit and, in addition, to interact with a key mRNA binding protein, S1. Molecular dynamics simulations revealed a detrimental impact of the E132K mutation on the coiled-coil structure and thereby on the interactions between S2 and 16S rRNA, providing a clue for the thermosensitivity of the rpsB1 mutant. Using a strain producing a leaderless lacZ transcript from the chromosomal lac promoter, we demonstrated that not only the rpsB1 mutation generating S2/S1-deficient ribosomes but also the rpsA::IS10 mutation leading to partial deficiency in S1 alone increased translation efficiency of the leaderless mRNA by about 10-fold. Moderate overexpression of S1 relieved all these effects and, moreover, suppressed the thermosensitive phenotype of rpsB1, indicating the role of S1 as an extragenic suppressor of the E132K mutation. PMID:23104805

  20. Crystal Structure of the Signaling Helix Coiled-coil Domain of the b1 Subunit of the Soluble guanylyl Cyclase

    SciTech Connect

    Ma, X.; Beuve, A; van den Akker, F

    2010-01-01

    The soluble guanylyl cyclase (sGC) is a heterodimeric enzyme that, upon activation by nitric oxide, stimulates the production of the second messenger cGMP. Each sGC subunit harbor four domains three of which are used for heterodimerization: H-NOXA/H-NOBA domain, coiled-coil domain (CC), and catalytic guanylyl cyclase domain. The CC domain has previously been postulated to be part of a larger CC family termed the signaling helix (S-helix) family. Homodimers of sGC have also been observed but are not functionally active yet are likely transient awaiting their intended heterodimeric partner. To investigate the structure of the CC S-helix region, we crystallized and determined the structure of the CC domain of the sGC{beta}1 subunit comprising residues 348-409. The crystal structure was refined to 2.15 {angstrom} resolution. The CC structure of sGC{beta}1 revealed a tetrameric arrangement comprised of a dimer of CC dimers. Each monomer is comprised of a long a-helix, a turn near residue P399, and a short second a-helix. The CC structure also offers insights as to how sGC homodimers are not as stable as (functionally) active heterodimers via a possible role for inter-helix salt-bridge formation. The structure also yielded insights into the residues involved in dimerization. In addition, the CC region is also known to harbor a number of congenital and man-made mutations in both membrane and soluble guanylyl cyclases and those function-affecting mutations have been mapped onto the CC structure. This mutant analysis indicated an importance for not only certain dimerization residue positions, but also an important role for other faces of the CC dimer which might perhaps interact with adjacent domains. Our results also extend beyond guanylyl cyclases as the CC structure is, to our knowledge, the first S-helix structure and serves as a model for all S-helix containing family members.

  1. Adenovirus E4-ORF3-dependent relocalization of TIF1{alpha} and TIF1{gamma} relies on access to the Coiled-Coil motif

    SciTech Connect

    Vink, Elizabeth I.; Yondola, Mark A.; Wu, Kai; Hearing, Patrick

    2012-01-20

    The adenovirus E4-ORF3 protein promotes viral replication by relocalizing cellular proteins into nuclear track structures, interfering with potential anti-viral activities. E4-ORF3 targets transcriptional intermediary factor 1 alpha (TIF1{alpha}), but not homologous TIF1{beta}. Here, we introduce TIF1{gamma} as a novel E4-ORF3-interacting partner. E4-ORF3 relocalizes endogenous TIF1{gamma} in virus-infected cells in vivo and binds to TIF1{gamma} in vitro. We used the homologous nature, yet differing binding capabilities, of these proteins to study how E4-ORF3 targets proteins for track localization. We mapped the ability of E4-ORF3 to interact with specific TIF1 subdomains, demonstrating that E4-ORF3 interacts with the Coiled-Coil domains of TIF1{alpha}, TIF1{beta}, and TIF1{gamma}, and that the C-terminal half of TIF1{beta} interferes with this interaction. The results of E4-ORF3-directed TIF1 protein relocalization assays performed in vivo were verified using coimmunoprecipitation assays in vitro. These results suggest that E4-ORF3 targets proteins for relocalization through a loosely homologous sequence dependent on accessibility.

  2. A Coiled-Coil Enabled Split-Luciferase Three-Hybrid System: Applied Toward Profiling Inhibitors of Protein Kinases

    PubMed Central

    Jester, Benjamin W.; Cox, Kurt J.; Gaj, Alicia; Shomin, Carolyn D.; Porter, Jason R.; Ghosh, Indraneel

    2010-01-01

    The 518 protein kinases encoded in the human genome are exquisitely regulated and their aberrant function(s) are often associated with human disease. Thus, in order to advance therapeutics and to probe signal transduction cascades there is considerable interest in the development of inhibitors that can selectively target protein kinases. However, identifying specific compounds against such a large array of protein kinases is difficult to routinely achieve utilizing traditional activity assays, where purified protein kinases are necessary. Toward a simple, rapid, and practical method for identifying specific inhibitors, we describe the development and application of a split-protein methodology utilizing a coiled-coil assisted three-hybrid system. In this approach, a protein kinase of interest is attached to the C-terminal fragment of split-firefly luciferase and the coiled-coil Fos, which is specific for the coiled-coil Jun, is attached to the N-terminal fragment. Upon addition of Jun conjugated to a pan-kinase inhibitor such as staurosporine, a three-hybrid complex is established with concomitant reassembly of the split-luciferase enzyme. An inhibitor can be potentially identified by the commensurate loss in split-luciferase activity by displacement of the modified staurosporine. We demonstrate that this new three-hybrid approach is potentially general by testing protein kinases from the different kinase families. To interrogate whether this method allows for screening inhibitors, we tested six different protein kinases against a library of 80 known protein kinase inhibitors. Finally, we demonstrate that this three-hybrid system can potentially provide a rapid method for structure/function analysis as well as aid in the identification of allosteric inhibitors. PMID:20669947

  3. Second coiled-coil domain of KCNQ channel controls current expression and subfamily specific heteromultimerization by salt bridge networks.

    PubMed

    Nakajo, Koichi; Kubo, Yoshihiro

    2008-06-15

    KCNQ channels carry the slowly activating, voltage-dependent M-current in excitable cells such as neurons. Although the KCNQ2 homomultimer can form a functional voltage-gated K(+) channel, heteromultimerization with KCNQ3 produces a > 10-fold increase in current amplitude. All KCNQ channels contain double coiled-coil domains (TCC1 and TCC2, or A-domain Head and Tail), of which TCC2 (A-domain Tail) is thought to be important for subunit recognition, channel assembly and surface expression. The mechanism by which TCC2 recognizes and associates with its partner is not fully understood, however. Our aim in the present study was to elucidate the recognition mechanism by examining the phenotypes of TCC2-deletion mutants, TCC2-swapped chimeras and point mutants. Electrophysiological analysis using Xenopus oocytes under two-electrode voltage clamp revealed that homotetrameric KCNQ3 TCC2 is a negative regulator of current expression in the absence of KCNQ2 TCC2. Recent structural analysis of KCNQ4 TCC2 revealed the presence of intercoil salt bridge networks. We therefore swapped the sign of the charged residues reportedly involved in the salt bridge formation and functionally confirmed that the intercoil salt bridge network is responsible for the subunit recognition between KCNQ2 and KCNQ3. Finally, we constructed TCC2-swapped KCNQ2/KCNQ3 mutants with KCNQ1 TCC2 or GCN4-pLI, a coiled-coil domain from an unrelated protein, and found that TCC2 is substitutable and even GCN4-pLI can work as a substitute for TCC2. Our present data provide some new insights into the role played by TCC2 during current expression, and also provide functional evidence of the importance of the intercoil salt bridge network for subunit recognition and coiled-coil formation, as is suggested by recent crystallographic data. PMID:18440995

  4. Missense mutation in immunodeficient patients shows the multifunctional roles of coiled-coil domain 3 (CC3) in STIM1 activation

    PubMed Central

    Maus, Mate; Jairaman, Amit; Stathopulos, Peter B.; Muik, Martin; Fahrner, Marc; Weidinger, Carl; Benson, Melina; Fuchs, Sebastian; Ehl, Stephan; Romanin, Christoph; Ikura, Mitsuhiko; Prakriya, Murali; Feske, Stefan

    2015-01-01

    Store-operated Ca2+ entry (SOCE) is a universal Ca2+ influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca2+ stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca2+ channel ORAI1 and the ER Ca2+ sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER–PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1–ORAI1 interactions. Thus, despite its constitutive localization at ER–PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1–ORAI1 binding required for ORAI1 activation. PMID:25918394

  5. Missense mutation in immunodeficient patients shows the multifunctional roles of coiled-coil domain 3 (CC3) in STIM1 activation.

    PubMed

    Maus, Mate; Jairaman, Amit; Stathopulos, Peter B; Muik, Martin; Fahrner, Marc; Weidinger, Carl; Benson, Melina; Fuchs, Sebastian; Ehl, Stephan; Romanin, Christoph; Ikura, Mitsuhiko; Prakriya, Murali; Feske, Stefan

    2015-05-12

    Store-operated Ca(2+) entry (SOCE) is a universal Ca(2+) influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca(2+) stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca(2+) channel ORAI1 and the ER Ca(2+) sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER-PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1-ORAI1 interactions. Thus, despite its constitutive localization at ER-PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1-ORAI1 binding required for ORAI1 activation. PMID:25918394

  6. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics

    NASA Astrophysics Data System (ADS)

    Woo, Sun Young; Lee, Hwankyu

    2016-03-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect.

  7. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics.

    PubMed

    Woo, Sun Young; Lee, Hwankyu

    2016-01-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect. PMID:26926570

  8. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics

    PubMed Central

    Woo, Sun Young; Lee, Hwankyu

    2016-01-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect. PMID:26926570

  9. Crystallographic characterization of the C-terminal coiled-coil region of mouse Bicaudal-D1 (BICD1)

    PubMed Central

    Terawaki, Shin-ichi; Ootsuka, Hiroki; Higuchi, Yoshiki; Wakamatsu, Kaori

    2014-01-01

    Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein which is evolutionarily conserved from Drosophila to mammals and facilitates the attachment of specific cargo factors to the dynein motor complex. The C-terminal coiled-coil region (CC3) of BICD1 plays an important role in sorting cargo, linking proteins such as the small GTPase Rab6 and the nuclear pore complex component Ran-binding protein 2 (RanBP2) to the dynein motor complex. This report describes the crystallization and X-ray data collection of the BICD1 CC3 region, as well as the preparation of the complex of BICD1 CC3 with a constitutively active mutant of Rab6. The crystals of the BICD1 CC3 region belonged to space group C2, with unit-cell parameters a = 59.0, b = 36.8, c = 104.3 Å, α = γ = 90, β = 99.8°. The X-ray diffraction data set was collected to 1.50 Å resolution. PMID:25084392

  10. A coiled-coil motif in non-structural protein 3 (NS3) of bluetongue virus forms an oligomer.

    PubMed

    Chacko, Nirmal; Mohanty, Nihar Nalini; Biswas, Sanchay Kumar; Chand, Karam; Yogisharadhya, Revanaiah; Pandey, Awadh Bihari; Mondal, Bimalendu; Shivachandra, Sathish Bhadravati

    2015-10-01

    Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus. PMID:26318174

  11. Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus.

    PubMed

    Wanke, Dierk; Hohenstatt, Mareike L; Dynowski, Marek; Bloss, Ulrich; Hecker, Andreas; Elgass, Kirstin; Hummel, Sabine; Hahn, Achim; Caesar, Katharina; Schleifenbaum, Frank; Harter, Klaus; Berendzen, Kenneth W

    2011-01-01

    GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms. PMID:21347358

  12. Alanine Zipper-Like Coiled-Coil Domains Are Necessary for Homotypic Dimerization of Plant GAGA-Factors in the Nucleus and Nucleolus

    PubMed Central

    Bloss, Ulrich; Hecker, Andreas; Elgass, Kirstin; Hummel, Sabine; Hahn, Achim; Caesar, Katharina; Schleifenbaum, Frank; Harter, Klaus; Berendzen, Kenneth W.

    2011-01-01

    GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms. PMID:21347358

  13. Intermediate filament mechanics in vitro and in the cell: From coiled coils to filaments, fibers and networks

    PubMed Central

    Köster, Sarah; Weitz, David; Goldman, Robert D.; Aebi, Ueli; Herrmann, Harald

    2015-01-01

    Summary Intermediate filament proteins form filaments, fibers and networks both in the cytoplasm and the nucleus of metazoan cells. Their general structural building plan accommodates highly varying amino acid sequences to yield extended dimeric α-helical coiled coils of highly conserved design. These “rod” particles are the basic building blocks of intrinsically flexible, filamentous structures that are able to resist high mechanical stresses, i.e. bending and stretching to a considerable degree, both in vitro and in the cell. Biophysical and computer modeling studies are beginning to unfold detailed structural and mechanical insights into these major supramolecular assemblies of cell architecture, not only in the “test tube” but also in the cellular and tissue context. PMID:25621895

  14. Multimodality Imaging of Coiled-Coil Mediated Self-Assembly in a “Drug Free” Therapeutic System

    PubMed Central

    Zhang, Rui; Yang, Jiyuan; Chu, Te-Wei; Hartley, Jonathan M.; Kopeček, Jindřich

    2015-01-01

    We used two complementary coiled-coil peptides CCE/CCK to develop a “drug free” therapeutic system, which can specifically kill cancer cells without a drug. CCE was attached to the Fab’ fragment of anti-CD20 1F5 antibody (Fab’-CCE), and CCK was conjugated in multiple grafts to poly[N-(2-hydroxypropyl)methacrylamide] (P-(CCK)x). Two conjugates are consecutively administered: First, Fab’-CCE coats peptide CCE at CD20 antigen of lymphoma cell surface; second, CCE/CCK biorecognition between Fab’-CCE and P-(CCK)x leads to coiled-coil formation, CD20 crosslinking, membrane reorganization, and ultimately cell apoptosis. To prove that two conjugates can assemble at cell surface, multiple fluorescence imaging studies were performed, including 2-channel FMT, 3D confocal microscopy, and 4-color FACS. Confocal microscopy showed co-localization of two fluorescently labeled conjugates on non-Hodgkin's lymphoma (NHL) Raji cell surface, indicating “two-step” targeting specificity. The fluorescent images also revealed that these two conjugates could disrupt normal membrane lipid distribution and form lipid raft clusters, leading to cancer cell apoptosis. This “two-step” biorecognition capacity was further demonstrated in a NHL xenograft model, using fluorescent images at whole-body, tissue and cell levels. We also found that delaying injection of P-(CCK)x could significantly enhance targeting efficacy. This high-specificity therapeutics provide a safe option to treat NHL and other B cell malignancies. PMID:25612325

  15. The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

    SciTech Connect

    Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V.

    2012-03-26

    Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.

  16. Insights on the structure and stability of Licanantase: a trimeric acid-stable coiled-coil lipoprotein from Acidithiobacillus thiooxidans

    PubMed Central

    Abarca, Fernando; Gutierrez-Maldonado, Sebastian E.; Parada, Pilar; Martinez, Patricio; Maass, Alejandro

    2014-01-01

    Licanantase (Lic) is the major component of the secretome of Acidithiobacillus thiooxidans when grown in elemental sulphur. When used as an additive, Lic improves copper recovery from bioleaching processes. However, this recovery enhancement is not fully understood. In this context, our aim is to predict the 3D structure of Lic, to shed light on its structure-function relationships. Bioinformatics analyses on the amino acid sequence of Lic showed a great similarity with Lpp, an Escherichia coli Lipoprotein that can form stable trimers in solution. Lic and Lpp share the secretion motif, intracellular processing and alpha helix structure, as well as the distribution of hydrophobic residues in heptads forming a hydrophobic core, typical of coiled-coil structures. Cross-linking experiments showed the presence of Lic trimers, supporting our predictions. Taking the in vitro and in silico evidence as a whole, we propose that the most probable structure for Lic is a trimeric coiled-coil. According to this prediction, a suitable model for Lic was produced using the de novo algorithm “Rosetta Fold-and-Dock”. To assess the structural stability of our model, Molecular Dynamics (MD) and Replica Exchange MD simulations were performed using the structure of Lpp and a 14-alanine Lpp mutant as controls, at both acidic and neutral pH. Our results suggest that Lic was the most stable structure among the studied proteins in both pH conditions. This increased stability can be explained by a higher number of both intermonomer hydrophobic contacts and hydrogen bonds, key elements for the stability of Lic’s secondary and tertiary structure. PMID:25165619

  17. Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

    PubMed Central

    Tegoni, Matteo; Yu, Fangting; Bersellini, Manuela; Penner-Hahn, James E.; Pecoraro, Vincent L.

    2012-01-01

    One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)3+/2+, where Cu(I/II) is embeded in α-helical coiled coils, as a model for the CuT2 center of copper nitrite reductase. In Cu(I/II)(TRIL23H)3+/2+, Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)3 active site is characterized in both oxidation states, revealing similarities to the CuT2 site in the natural enzyme. The species Cu(II)(TRIL23H)32+ in aqueous solution can be reduced to Cu(I)(TRIL23H)3+ using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)32+ acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed. PMID:23236170

  18. Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils.

    PubMed

    Tegoni, Matteo; Yu, Fangting; Bersellini, Manuela; Penner-Hahn, James E; Pecoraro, Vincent L

    2012-12-26

    One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)(3)(+/2+), where Cu(I/II) is embeded in α-helical coiled coils, as a model for the Cu(T2) center of copper nitrite reductase. In Cu(I/II)(TRIL23H)(3)(+/2+), Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)(3) active site is characterized in both oxidation states, revealing similarities to the Cu(T2) site in the natural enzyme. The species Cu(II)(TRIL23H)(3)(2+) in aqueous solution can be reduced to Cu(I)(TRIL23H)(3)(+) using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)(3)(2+) acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed. PMID:23236170

  19. Decorin induces rapid secretion of thrombospondin-1 in basal breast carcinoma cells via inhibition of Ras homolog gene family, member A/Rho-associated coiled-coil containing protein kinase 1.

    PubMed

    Neill, Thomas; Jones, Holly R; Crane-Smith, Zoe; Owens, Rick T; Schaefer, Liliana; Iozzo, Renato V

    2013-05-01

    Pathological neovascularization relies on an imbalance between potent proangiogenic agents and equally effective antiangiogenic cues. Collectively, these factors contribute to an angiogenic niche within the tumor microenvironment. Oncogenic events and hypoxia contribute to augmented levels of angiokines, and thereby activate the so-called angiogenic switch to promote aggressive tumorigenic and metastatic growth. Soluble decorin functions as a paracrine pan-inhibitor of receptor tyrosine kinases, such as Met and epidermal growth factor receptor, and thus is capable of suppressing angiogenesis under normoxia. This leads to noncanonical repression of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor A (VEGFA), and concurrent induction of thrombospondin-1. The substantial induction of endogenous tumor cell-derived thrombospondin-1, a potent antiangiogenic effector, led us to the discovery of an unexpected secretory phenotype occurring very rapidly (within 5 min) after decorin treatment of the triple-negative basal breast carcinoma cell line MDA-MB-231. Surprisingly, the effect was not mediated by Met receptor antagonism, as initially hypothesized, but required epidermal growth factor receptor signaling to achieve swift and robust thrombospondin-1 release. Furthermore, this effect was ultimately dependent on the prompt degradation of Ras homolog gene family member A, via the 26S proteasome, leading to direct inactivation of Rho-associated coiled-coil containing protein kinase 1. The latter led to derepression of thrombospondin-1 secretion. Collectively, these data provide a novel mechanistic role for Rho-associated coiled-coil containing protein kinase 1, in addition to providing the first conclusive evidence of decorin exclusively targeting a receptor tyrosine kinase to achieve a specific effect. The overall effects of soluble decorin on the tumor microenvironment would cause an immediately-early as well as a sustained antiangiogenic response

  20. Analysis of the coding sequence and expression of the coiled-coil α-helical rod protein 1 gene in normal and neoplastic epithelial cervical cells

    PubMed Central

    PACHOLSKA-BOGALSKA, JOANNA; MYGA-NOWAK, MAGDALENA; CIEPŁUCH, KATARZYNA; JÓZEFIAK, AGATA; KWAŒNIEWSKA, ANNA; GOźDZICKA-JÓZEFIAK, ANNA

    2012-01-01

    The role of the CCHCR1 (coiled-coil α-helical rod protein 1) protein in the cell is poorly understood. It is thought to be engaged in processes such as proliferation and differentiation of epithelial cells, tissue-specific gene transcription and steroidogenesis. It is supposed to participate in keratinocyte transformation. It has also been found that this protein interacts with the E2 protein of human papilloma virus type 16 (HPV16). The oncogenic HPV forms, such as HPV16, are known to be necessary but not sufficient agents in the development of cervical carcinoma. In the present study, the CCHCR1 gene coding sequence and its expression was analyzed in normal, precancerous and cervical cancer cells. Changes in the non-coding region were found in 20.3% of the examined probes from women with cervical cancer or precancerous lesions and in 16.67% of the control probes. Most of the detected changes were single nucleotide polymorphisms (SNPs). Changes in the coding region were found in 22.8% of the probes with cervical cancer and in 16.67% of the control probes and all of them were SNPs. The level of CCHCR1 transcripts was determined using the real-time PCR method and the highest gene expression was detected in the H-SIL group and slightly decreased in the cervical carcinoma cells, compared with the control probes. It suggests that CCHCR1 could have a role in the process of cervical epithelial cell transformation, but this suggestion must be confirmed experimentally. PMID:22218424

  1. The coiled-coil domain containing protein CCDC151 is required for the function of IFT-dependent motile cilia in animals.

    PubMed

    Jerber, Julie; Baas, Dominique; Soulavie, Fabien; Chhin, Brigitte; Cortier, Elisabeth; Vesque, Christine; Thomas, Joëlle; Durand, Bénédicte

    2014-02-01

    Cilia are evolutionarily conserved organelles endowed with essential physiological and developmental functions. In humans, disruption of cilia motility or signaling leads to complex pleiotropic genetic disorders called ciliopathies. Cilia motility requires the assembly of multi-subunit motile components such as dynein arms, but mechanisms underlying their assembly pathway and transport into the axoneme are still largely unknown. We identified a previously uncharacterized coiled-coil domain containing protein CCDC151, which is evolutionarily conserved in motile ciliated species and shares ancient features with the outer dynein arm-docking complex 2 of Chlamydomonas. In Drosophila, we show that CG14127/CCDC151 is associated with motile intraflagellar transport (IFT)-dependent cilia and required for geotaxis behavior of adult flies. In zebrafish, Ccdc151 is expressed in tissues with motile cilia, and morpholino-induced depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts. We demonstrate that Ccdc151 is required for proper motile function of cilia in the Kupffer's vesicle and in the pronephros by controlling dynein arm assembly, showing that Ccdc151 is a novel player in the control of IFT-dependent dynein arm assembly in animals. However, we observed that CCDC151 is also implicated in other cellular functions in vertebrates. In zebrafish, ccdc151 is involved in proper orientation of cell divisions in the pronephros and genetically interacts with prickle1 in this process. Furthermore, knockdown experiments in mammalian cells demonstrate that CCDC151 is implicated in the regulation of primary cilium length. Hence, CCDC151 is required for motile cilia function in animals but has acquired additional non-motile functions in vertebrates. PMID:24067530

  2. A systematic study of fundamentals in α-helical coiled coil mimicry by alternating sequences of β- and γ-amino acids.

    PubMed

    Rezaei Araghi, Raheleh; Baldauf, Carsten; Gerling, Ulla I M; Cadicamo, Cosimo Damiano; Koksch, Beate

    2011-08-01

    Aimed at understanding the crucially important structural features for the integrity of α-helical mimicry by βγ-sequences, an α-amino acid sequence in a native peptide was substituted by differently arranged βγ-sequences. The self- and hetero-assembly of a series of αβγ-chimeric sequences based on a 33-residue GCN4-derived peptide was investigated by means of molecular dynamics, circular dichroism, and a disulfide exchange assay. Despite the native-like behavior of βγ alternating sequences such as retention of α-helix dipole and the formation of 13-membered α-helix turns, the αβγ-chimeras with different βγ substitution patterns do not equally mimic the structural behavior of the native parent peptide in solution. The preservation of the key residue contacts such as van der Waals interactions and intrahelical H-bonding, which can be met only by particular substitution patterns, thermodynamically favor the adoption of coiled coil folding motif. In this study, we show how successfully the destabilizing structural consequences of α → βγ modification can be harnessed by reducing the solvent-exposed hydrophobic surface area and placing of suitably long and bulky helix-forming side chains at the hydrophobic core. The pairing of αβγ-chimeric sequences with the native wild-type are thermodynamically allowed in the case of ideal arrangement of β- and γ-residues. This indicates a similarity in local side chain packing of β- and γ-amino acids at the helical interface of αβγ-chimeras and the native α-peptide. Consequently, the backbone extended residues are able to participate in classical "knob-into-hole" packing with native α-peptide. PMID:21638022

  3. TACC2 (transforming acidic coiled-coil protein 2) in breast carcinoma as a potent prognostic predictor associated with cell proliferation.

    PubMed

    Onodera, Yoshiaki; Takagi, Kiyoshi; Miki, Yasuhiro; Takayama, Ken-Ichi; Shibahara, Yukiko; Watanabe, Mika; Ishida, Takanori; Inoue, Satoshi; Sasano, Hironobu; Suzuki, Takashi

    2016-08-01

    Transforming acidic coiled-coil protein 2 (TACC2) belongs to TACC family proteins and involved in a variety of cellular processes through interactions with some molecules involved in centrosomes/microtubules dynamics. Mounting evidence suggests that TACCs is implicated in the progression of some human malignancies, but significance of TACC2 protein in breast carcinoma is still unknown. Therefore, in this study, we examined the clinical significance of TACC2 in breast carcinoma and biological functions by immunohistochemistry and in vitro experiments. Immunohistochemistry for TACC2 was performed in 154 cases of invasive ductal carcinoma. MCF-7 and MDA-MB-453 breast carcinoma cell lines were transfected with small interfering RNA (siRNA) for TACC2, and subsequently, cell proliferation, 5-Bromo-2'-deoxyuridine (BrdU), and invasion assays were performed. TACC2 immunoreactivity was detected in 78 out of 154 (51%) breast carcinoma tissues, and it was significantly associated with Ki-67 LI. The immunohistochemical TACC2 status was significantly associated with increased incidence of recurrence and breast cancer-specific death of the patients, and multivariate analyses demonstrated TACC2 status as an independent prognostic factor for both disease-free and breast cancer-specific survival. Subsequent in vitro experiments showed that TACC2 significantly increased the proliferation activity of MCF-7 and MDA-MB-453. These results suggest that TACC2 plays an important role in the cell proliferation of breast carcinoma and therefore immunohistochemical TACC2 status is a candidate of worse prognostic factor in breast cancer cases. PMID:27333920

  4. Biogenesis of the Secretory Granule: Chromogranin a Coiled-Coil Structure Results in Unusual Physical Properties And Suggests a Mechanism for Granule Core Condensation

    SciTech Connect

    Mosley, C.A.; Taupenot, L.; Biswas, N.; Taulane, J.P.; Olson, N.H.; Vaingankar, S.M.; Wen, G.; Schork, N.J.; Ziegler, M.G.; Mahata, S.K.; O'Connor, D.T.

    2009-06-03

    The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein 'packing' in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a binding

  5. Insights into the coiled-coil organization of the Hendra virus phosphoprotein from combined biochemical and SAXS studies.

    PubMed

    Beltrandi, Matilde; Blocquel, David; Erales, Jenny; Barbier, Pascale; Cavalli, Andrea; Longhi, Sonia

    2015-03-01

    Nipah and Hendra viruses are recently emerged paramyxoviruses belonging to the Henipavirus genus. The Henipavirus phosphoprotein (P) consists of a large intrinsically disordered domain and a C-terminal domain (PCT) containing alternating disordered and ordered regions. Among these latter is the P multimerization domain (PMD). Using biochemical, analytical ultracentrifugation and small-angle X-ray scattering (SAXS) studies, we show that Hendra virus (HeV) PMD forms an elongated coiled-coil homotrimer in solution, in agreement with our previous findings on Nipah virus (NiV) PMD. However, the orientation of the N-terminal region differs from that observed in solution for NiV PMD, consistent with the ability of this region to adopt different conformations. SAXS studies provided evidence for a trimeric organization also in the case of PCT, thus extending and strengthening our findings on PMD. The present results are discussed in light of conflicting reports in the literature pointing to a tetrameric organization of paramyxoviral P proteins. PMID:25637789

  6. Synthesis, morphology, and sensory applications of multifunctional rod-coil-coil triblock copolymers and their electrospun nanofibers.

    PubMed

    Chiu, Yu-Cheng; Chen, Yougen; Kuo, Chi-Ching; Tung, Shih-Huang; Kakuchi, Toyoji; Chen, Wen-Chang

    2012-07-25

    We report the synthesis, morphology, and applications of conjugated rod-coil-coil triblock copolymers, polyfluorene-block-poly(N-isopropylacrylamide)-block-poly(N-methylolacrylamide) (PF-b-PNIPAAm-b-PNMA), prepared by atom transfer radical polymerization first and followed by click coupling reaction. The blocks of PF, PNIPAAm, and PNMA were designed for fluorescent probing, hydrophilic thermo-responsive and chemically cross-linking, respectively. In the following, the electrospun (ES) nanofibers of PF-b-PNIPAAm-b-PNMA were prepared in pure water using a single-capillary spinneret. The SAXS and TEM results suggested the lamellar structure of the PF-b-PNIPAAm-b-PNMA along the fiber axis. These obtained nanofibers showed outstanding wettability and dimension stability in the aqueous solution, and resulted in a reversible on/off transition on photoluminescence as the temperatures varied. Furthermore, the high surface/volume ratio of the ES nanofibers efficiently enhanced the temperature-sensitivity and responsive speed compared to those of the drop-cast film. The results indicated that the ES nanofibers of the conjugated rod-coil block copolymers would have potential applications for multifunctional sensory devices. PMID:22712723

  7. Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

    PubMed

    Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

    2015-10-21

    Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments. PMID:26367072

  8. Magnetic Field Alignment of PS-P4VP: a Non-Liquid Crystalline Coil-Coil Block Copolymer

    NASA Astrophysics Data System (ADS)

    Rokhlenko, Yekaterina; Zhang, Kai; Larson, Steven; Gopalan, Padma; O'Hern, Corey; Osuji, Chinedum

    2015-03-01

    Magnetic fields provide the ability to control alignment of self-assembled soft materials such as block copolymers. Most prior work in this area has relied on the presence of ordered assemblies of anisotropic liquid crystalline species to ensure sufficient magnetic anisotropy to drive alignment. Recent experiments with poly(styrene-b-4-vinylpyridine), a non-liquid crystalline BCP, however, show field-induced alignment of a lamellar microstructure during cooling across the order-disorder transition. Using in situ x-ray scattering, we examine the roles of field strength and cooling rate on the alignment response of this low MW coil-coil BCP. Alignment is first observed at field strengths as low as 1 Tesla and improves markedly with both increasing field strength and slower cooling. We present a geometric argument to illustrate the origin of a finite, non-trivial magnetic susceptibility anisotropy for highly stretched surface-tethered polymer chains and corroborate this using coarse-grained molecular dynamics simulations. We rationalize the magnetic field response of the system in terms of the mobility afforded by the absence of entanglements, the intrinsic anisotropy resulting from the stretched polymer chains and sterically constrained conjugated rings, and the large grain size in these low molecular weight materials.

  9. Biochemical and structural studies of the oligomerization domain of the Nipah virus phosphoprotein: evidence for an elongated coiled-coil homotrimer.

    PubMed

    Blocquel, David; Beltrandi, Matilde; Erales, Jenny; Barbier, Pascale; Longhi, Sonia

    2013-11-01

    Nipah virus (NiV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The NiV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that is the substrate used by the polymerase for transcription and replication. The polymerase is recruited onto the nucleocapsid via its cofactor, the phosphoprotein (P). The NiV P protein has a modular organization, with alternating disordered and ordered domains. Among these latter, is the P multimerization domain (PMD) that was predicted to adopt a coiled-coil conformation. Using both biochemical and biophysical approaches, we show that NiV PMD forms a highly stable and elongated coiled-coil trimer, a finding in striking contrast with respect to the PMDs of Paramyxoviridae members investigated so far that were all found to tetramerize. The present results therefore represent the first report of a paramyxoviral P protein forming trimers. PMID:24074578

  10. The C-terminal region of the transcriptional regulator THAP11 forms a parallel coiled-coil domain involved in protein dimerization.

    PubMed

    Cukier, Cyprian D; Maveyraud, Laurent; Saurel, Olivier; Guillet, Valérie; Milon, Alain; Gervais, Virginie

    2016-06-01

    Thanatos associated protein 11 (THAP11) is a cell cycle and cell growth regulator differentially expressed in cancer cells. THAP11 belongs to a distinct family of transcription factors recognizing specific DNA sequences via an atypical zinc finger motif and regulating diverse cellular processes. Outside the extensively characterized DNA-binding domain, THAP proteins vary in size and predicted domains, for which structural data are still lacking. We report here the crystal structure of the C-terminal region of human THAP11 protein, providing the first 3D structure of a coiled-coil motif from a THAP family member. We further investigate the stability, dynamics and oligomeric properties of the determined structure combining molecular dynamics simulations and biophysical experiments. Our results show that the C-ter region of THAP11 forms a left-handed parallel homo-dimeric coiled-coil structure possessing several unusual features. PMID:26975212

  11. Heteronuclear NMR assignments and secondary structure of the coiled coil trimerization domain from cartilage matrix protein in oxidized and reduced forms.

    PubMed Central

    Wiltscheck, R.; Kammerer, R. A.; Dames, S. A.; Schulthess, T.; Blommers, M. J.; Engel, J.; Alexandrescu, A. T.

    1997-01-01

    The C-terminal oligomerization domain of chicken cartilage matrix protein is a trimeric coiled coil comprised of three identical 43-residue chains. NMR spectra of the protein show equivalent magnetic environments for each monomer, indicating a parallel coiled coil structure with complete threefold symmetry. Sequence-specific assignments for 1H-, 15N-, and 13C-NMR resonances have been obtained from 2D 1H NOESY and TOCSY spectra, and from 3D HNCA, 15N NOESY-HSQC, and HCCH-TOCSY spectra. A stretch of alpha-helix encompassing five heptad repeats (35 residues) has been identified from intra-chain HN-HN and HN-H alpha NOE connectivities. 3JHNH alpha coupling constants, and chemical shift indices. The alpha-helix begins immediately downstream of inter-chain disulfide bonds between residues Cys 5 and Cys 7, and extends to near the C-terminus of the molecule. The threefold symmetry of the molecule is maintained when the inter-chain disulfide bonds that flank the N-terminus of the coiled coil are reduced. Residues Ile 21 through Glu 36 show conserved chemical shifts and NOE connectivities, as well as strong protection from solvent exchange in the oxidized and reduced forms of the protein. By contrast, residues Ile 10 through Val 17 show pronounced chemical shift differences between the oxidized and reduced protein. Strong chemical exchange NOEs between HN resonances and water indicate solvent exchange on time scales faster than 10 s, and suggests a dynamic fraying of the N-terminus of the coiled coil upon reduction of the disulfide bonds. Possible roles for the disulfide crosslinks of the oligomerization domain in the function of cartilage matrix protein are proposed. PMID:9260286

  12. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    SciTech Connect

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-07-20

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.

  13. Solid-supported polymer bilayers formed by coil-coil block copolymers.

    PubMed

    Yang, Yan-Ling; Tsao, Heng-Kwong; Sheng, Yu-Jane

    2016-08-14

    The formation and physical properties of solid-supported polymer bilayers (SPBs) on an adhesive substrate have been explored by dissipative particle dynamics simulations. A SPB is developed by the adsorption of vesicles formed by diblock copolymers in a selective solvent. The adsorbed vesicle can remain intact or become ruptured into a SPB, depending on the interaction between solvophobic blocks and solvent and the interaction between solvophilic blocks and the substrate. The morphological phase diagram of adsorbed vesicles is acquired. The influence of polymer adhesion strength and solvophobicity on the geometrical and mechanical properties of a SPB is systematically studied as well. It is found that vesicular disruption is easily triggered for strong adhesion strength. Moreover, for strong adhesion strength and weak solvophobicity, the fluctuation of membrane height is impeded while the area of fluctuation is enhanced. PMID:27418114

  14. Defining the minimum size of a hydrophobic cluster in two-stranded α-helical coiled-coils: Effects on protein stability

    PubMed Central

    Lu, Stephen M.; Hodges, Robert S.

    2004-01-01

    The α-helical coiled-coil motif is characterized by a heptad repeat pattern (abcdefg)n in which residues a and d form the hydrophobic core. Long coiled-coils (e.g., tropomyosin, 284 residues per polypeptide chain) typically do not have a continuous hydrophobic core of stabilizing residues, but rather one that consists of alternating clusters of stabilizing and destabilizing residues. We have arbitrarily defined a cluster as a minimum of three consecutive stabilizing or destabilizing residues in the hydrophobic core. We report here on a series of two-stranded, disulfide-bridged parallel α-helical coiled-coils that contain a central cassette of three consecutive hydrophobic core positions (d, a, and d) with a destabilizing cluster of three consecutive Ala residues in the hydrophobic core on each side of the cassette. The effect of adding one to three stabilizing hydrophobes in these positions (Leu or Ile; denoted as •) was investigated. Alanine residues (denoted as ○) are used to represent destabilizing residues. The peptide with three Ala residues in the d a d cassette positions (○○○) was among the least stable coiled-coil (Tm = 39.3°C and Urea1/2 = 1.9 M). Surprisingly, the addition of one stabilizing hydrophobe (Leu) to the cassette or two stabilizing hydrophobes (Leu), still interspersed by an Ala in the cassette (•○•), also did not lead to any gain in stability. However, peptides with two adjacent hydrophobes in the cassette (••○)(○••) did show a gain in stability of 0.9 kcal/mole over the peptide with two interspersed hydrophobes (•○•). Because the latter three peptides have the same inherent hydrophobicity, the juxtaposition of stabilizing hydrophobes leads to a synergistic effect, and thus a clustering effect. The addition of a third stabilizing hydrophobe to the cassette (•••) resulted in a further synergistic gain in stability of 1.7 kcal/mole (Tm = 54.1°C and Urea1/2 = 3.3M). Therefore, the role of hydrophobicity

  15. Identification of BECN1 and ATG14 Coiled-Coil Interface Residues That Are Important for Starvation-Induced Autophagy.

    PubMed

    Mei, Yang; Su, Minfei; Sanishvili, Ruslan; Chakravarthy, Srinivas; Colbert, Christopher L; Sinha, Sangita C

    2016-08-01

    Autophagy, an essential eukaryotic homeostasis pathway, allows the sequestration of unwanted, damaged, or harmful cytoplasmic components in vesicles called autophagosomes, permitting subsequent lysosomal degradation and nutrient recycling. Autophagosome nucleation is mediated by class III phosphatidylinositol-3-kinase complexes that include two key autophagy proteins, BECN1/Beclin 1 and ATG14/BARKOR, which form parallel heterodimers via their coiled-coil domains (CCDs). Here we present the 1.46 Å X-ray crystal structure of the antiparallel, human BECN1 CCD homodimer, which represents BECN1 oligomerization outside the autophagosome nucleation complex. We use circular dichroism and small-angle X-ray scattering (SAXS) to show that the ATG14 CCD is significantly disordered but becomes more helical in the BECN1:ATG14 heterodimer, although it is less well-folded than the BECN1 CCD homodimer. SAXS also indicates that the BECN1:ATG14 heterodimer is more curved than other BECN1-containing CCD dimers, which has important implications for the structure of the autophagosome nucleation complex. A model of the BECN1:ATG14 CCD heterodimer that agrees well with the SAXS data shows that BECN1 residues at the homodimer interface are also responsible for heterodimerization, allowing us to identify ATG14 interface residues. Finally, we verify the role of BECN1 and ATG14 interface residues in binding by assessing the impact of point mutations of these residues on co-immunoprecipitation of the partner and demonstrate that these mutations abrogate starvation-induced upregulation of autophagy but do not impact basal autophagy. Thus, this research provides insights into structures of the BECN1 CCD homodimer and the BECN1:ATG14 CCD heterodimer and identifies interface residues that are important for BECN1:ATG14 heterodimerization and for autophagy. PMID:27383850

  16. Stability and specificity of heterodimer formation for the coiled-coil neck regions of the motor proteins Kif3A and Kif3B: the role of unstructured oppositely charged regions

    PubMed Central

    Chana, M.S.; Tripet, B.P.; Mant, C.T.; Hodges, R.

    2005-01-01

    We investigated the folding, stability, and specificity of dimerization of the neck regions of the kinesin-like proteins Kif3A (residues 356–416) and Kif3B (residues 351–411). We showed that the complementary charged regions found in the hinge regions (which directly follow the neck regions) of these proteins do not adopt any secondary structure in solution. We then explored the ability of the complementary charged regions to specify heterodimer formation for the neck region coiled-coils found in Kif3A and Kif3B. Redox experiments demonstrated that oppositely charged regions specified the formation of a heterodimeric coiled-coil. Denaturation studies with urea demonstrated that the negatively charged region of Kif3A dramatically destabilized its neck coiled-coil (urea1/2 value of 3.9 m compared with 6.7 m for the coiled-coil alone). By comparison, the placement of a positively charged region C-terminal to the neck coiled-coil of Kif3B had little effect on stability (urea1/2 value of 8.2 m compared with 8.8 m for the coiled-coil alone). The pairing of complementary charged regions leads to specific heterodimer formation where the stability of the heterodimeric neck coiled-coil with charged regions had similar stability (urea1/2 value of 7.8 m) to the most stable homodimer (Kif3B) with charged regions (urea1/2 value of 8.0 m) and dramatically more stable than the Kif3A homodimer with charged regions (urea1/2, value of 3.9 m). The heterodimeric coiled-coil with charged extensions has essentially the same stability as the heterodimeric coiled-coil on its own (urea1/2 values of 7.8 and 8.1 m, respectively) suggesting that specificity of heterodimerization is driven by non-specific attraction of the oppositely unstructured charged regions without affecting stability of the heterodimeric coiled-coil. PMID:15705165

  17. Structural Comparisons of Apo- and Metalated Three-Stranded Coiled Coils Clarify Metal Binding Determinants in Thiolate Containing Designed Peptides

    SciTech Connect

    Chakraborty, Saumen; Touw, Debra S.; Peacock, Anna F.A.; Stuckey, Jeanne; Pecoraro, Vincent L.

    2010-11-05

    Over the past two decades, designed metallopeptides have held the promise for understanding a variety of fundamental questions in metallobiochemistry; however, these dreams have not yet been realized because of a lack of structural data to elaborate the protein scaffolds before metal complexation and the resultant metalated structures which ultimately exist. This is because there are few reports of structural characterization of such systems either in their metalated or nonmetalated forms and no examples where an apo structure and the corresponding metalated peptide assembly have both been defined by X-ray crystallography. Herein we present X-ray structures of two de novo designed parallel three-stranded coiled coils (designed using the heptad repeat (a {yields} g)) CSL9C (CS = Coil Ser) and CSL19C in their nonmetalated forms, determined to 1.36 and 2.15 {angstrom} resolutions, respectively. Leucines from either position 9 (a site) or 19 (d site) are replaced by cysteine to generate the constructs CSL9C and CSL19C, respectively, yielding thiol-rich pockets at the hydrophobic interior of these peptides, suitable to bind heavy metals such as As(III), Hg(II), Cd(II), and Pb(II). We use these structures to understand the inherent structural differences between a and d sites to clarify the basis of the observed differential spectroscopic behavior of metal binding in these types of peptides. Cys side chains of (CSL9C){sub 3} show alternate conformations and are partially preorganized for metal binding, whereas cysteines in (CSL19C){sub 3} are present as a single conformer. Zn(II) ions, which do not coordinate or influence Cys residues at the designed metal sites but are essential for forming X-ray quality crystals, are bound to His and Glu residues at the crystal packing interfaces of both structures. These 'apo' structures are used to clarify the changes in metal site organization between metalated As(CSL9C){sub 3} and to speculate on the differential basis of Hg

  18. Metal-binding properties and structural characterization of a self-assembled coiled coil: formation of a polynuclear Cd-thiolate cluster.

    PubMed

    Zaytsev, Daniil V; Morozov, Vasily A; Fan, Jiufeng; Zhu, Xianchun; Mukherjee, Madhumita; Ni, Shuisong; Kennedy, Michael A; Ogawa, Michael Y

    2013-02-01

    This paper describes the design, characterization, and metal-binding properties of a 32-residue polypeptide called AQ-C16C19. The sequence of this peptide is composed of four repeats of the seven residue sequence Ile-Ala-Ala-Leu-Glu-Gln-Lys but with a Cys-X-X-Cys metal-binding motif substituted at positions 16-19. Size exclusion chromatography with multiangle light scattering detection (SEC-MALS) and circular dichroism (CD) spectroscopy studies showed that the apo peptide exhibits a pH-dependent oligomerization state in which a three-stranded α-helical coiled coil is dominant between pH5.4 and 8.5. The Cd(2+)-binding properties of the AQ-C16C19 peptide were studied by ultraviolet-visible spectroscopy (UV-vis), electrospray ionization mass spectrometry (ESI MS), and (113)Cd NMR techniques. The holoprotein was found to contain a polynuclear cadmium-thiolate center formed within the hydrophobic core of the triple-stranded α-helical coiled-coil structure. The X-ray crystal structure of the Cd-loaded peptide, resolved at 1.85Å resolution, revealed an adamantane-like configuration of the polynuclear metal center consisting of four cadmium ions, six thiolate sulfur ligands from cysteine residues and four oxygen-donor ligands. Three of these are from glutamic acid residues and one is from an exogenous water molecule. Thus, each cadmium ion is coordinated in a distorted tetrahedral S(3)O geometry. The metal cluster was found to form cooperatively at pH5.4 but in a stepwise fashion at pH>7. The results demonstrate that synthetic coiled-coils can be designed to incorporate multinuclear metal clusters, a proof-of-concept for their potential use in developing synthetic metalloenzymes and multi-electron redox agents. PMID:23160144

  19. Cholera toxin B subunit-five-stranded α-helical coiled-coil fusion protein: "five-to-five" molecular chimera displays robust physicochemical stability.

    PubMed

    Arakawa, Takeshi; Harakuni, Tetsuya

    2014-09-01

    To create a physicochemically stable cholera toxin (CT) B subunit (CTB), it was fused to the five-stranded α-helical coiled-coil domain of cartilage oligomeric matrix protein (COMP). The chimeric fusion protein (CTB-COMP) was expressed in Pichia pastoris, predominantly as a pentamer, and retained its affinity for the monosialoganglioside GM1, a natural receptor of CT. The fusion protein displayed thermostability, tolerating the boiling temperature of water for 10min, whereas unfused CTB readily dissociated to its monomers and lost its affinity for GM1. The fusion protein also displayed resistance to strong acid at pHs as low as 0.1, and to the protein denaturant sodium dodecyl sulfate at concentrations up to 10%. Intranasal administration of the fusion protein to mice induced anti-B subunit serum IgG, even after the protein was boiled, whereas unfused CTB showed no thermostable mucosal immunogenicity. This study demonstrates that CTB fused to a pentameric α-helical coiled coil has a novel physicochemical phenotype, which may provide important insight into the molecular design of enterotoxin-B-subunit-based vaccines and vaccine delivery molecules. PMID:25045819

  20. Methods for Solving Highly Symmetric De Novo Designed Metalloproteins: Crystallographic Examination of a Novel Three-Stranded Coiled-Coil Structure Containing d-Amino Acids.

    PubMed

    Ruckthong, L; Stuckey, J A; Pecoraro, V L

    2016-01-01

    The core objective of de novo metalloprotein design is to define metal-protein relationships that control the structure and function of metal centers by using simplified proteins. An essential requirement to achieve this goal is to obtain high resolution structural data using either NMR or crystallographic studies in order to evaluate successful design. X-ray crystal structures have proven that a four heptad repeat scaffold contained in the three-stranded coiled coil (3SCC), called CoilSer (CS), provides an excellent motif for modeling a three Cys binding environment capable of chelating metals into geometries that resemble heavy metal sites in metalloregulatory systems. However, new generations of more complicated designs that feature, for example, a d-amino acid or multiple metal ligand sites in the helical sequence require a more stable construct. In doing so, an extra heptad was introduced into the original CS sequence, yielding a GRAND-CoilSer (GRAND-CS) to retain the 3SCC folding. An apo-(GRAND-CSL12DLL16C)3 crystal structure, designed for Cd(II)S3 complexation, proved to be a well-folded parallel 3SCC. Because this structure is novel, protocols for crystallization, structural determination, and refinements of the apo-(GRAND-CSL12DLL16C)3 are described. This report should be generally useful for future crystallographic studies of related coiled-coil designs. PMID:27586331

  1. An Evolutionarily Conserved Family of Virion Tail Needles Related to Bacteriophage P22 gp26: Correlation between Structural Stability and Length of the -Helical Trimeric Coiled Coil

    SciTech Connect

    Bhardwaj, A.; Walker-Kopp, N; Casjens, S; Cingolani, G

    2009-01-01

    Bacteriophages of the Podoviridae family use short noncontractile tails to inject their genetic material into Gram-negative bacteria. In phage P22, the tail contains a thin needle, encoded by the phage gene 26, which is essential both for stabilization and for ejection of the packaged viral genome. Bioinformatic analysis of the N-terminal domain of gp26 (residues 1-60) led us to identify a family of genes encoding putative homologues of the tail needle gp26. To validate this idea experimentally and to explore their diversity, we cloned the gp26-like gene from phages HK620, Sf6 and HS1, and characterized these gene products in solution. All gp26-like factors contain an elongated {alpha}-helical coiled-coil core consisting of repeating, adjacent trimerization heptads and form trimeric fibers with length ranging between about 240 to 300 {angstrom}. gp26 tail needles display a high level of structural stability in solution, with Tm (temperature of melting) between 85 and 95 C. To determine how the structural stability of these phage fibers correlates with the length of the {alpha}-helical core, we investigated the effect of insertions and deletions in the helical core. In the P22 tail needle, we identified an 85-residue-long helical domain, termed MiCRU (minimal coiled-coil repeat unit), that can be inserted in-frame inside the gp26 helical core, preserving the straight morphology of the fiber. Likewise, we were able to remove three quarters of the helical core of the HS1 tail needle, minimally decreasing the stability of the fiber. We conclude that in the gp26 family of tail needles, structural stability increases nonlinearly with the length of the {alpha}-helical core. Thus, the overall stability of these bacteriophage fibers is not solely dependent on the number of trimerization repeats in the {alpha}-helical core.

  2. Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subunit vaccine against egg-drop syndrome.

    PubMed

    Harakuni, Tetsuya; Andoh, Kiyohiko; Sakamoto, Ryu-Ichi; Tamaki, Yukihiro; Miyata, Takeshi; Uefuji, Hirotaka; Yamazaki, Ken-Ichi; Arakawa, Takeshi

    2016-06-01

    Egg-drop syndrome (EDS) virus is an avian adenovirus that causes a sudden drop in egg production and in the quality of the eggs when it infects chickens, leading to substantial economic losses in the poultry industry. Inactivated EDS vaccines produced in embryonated duck eggs or cell culture systems are available for the prophylaxis of EDS. However, recombinant subunit vaccines that are efficacious and inexpensive are a desirable alternative. In this study, we engineered chimeric fusion proteins in which the trimeric fiber knob domain lacking the triple β-spiral motif in the fiber shaft region was genetically fused to trimeric coiled coils, such as those of the engineered form of the GCN4 leucine zipper peptide or chicken cartilage matrix protein (CMP). The fusion proteins were expressed predominantly as soluble trimeric proteins in Escherichia coli at levels of 15-80mg/L of bacterial culture. The single immunization of chickens with the purified fusion proteins, at a dose equivalent to 10μg of the knob moiety, elicited serum antibodies with high hemagglutination inhibition (HI) activities, similar to those induced by an inactivated EDS vaccine. A dose-response analysis indicated that a single immunization with as little as 1μg of the knob moiety of the CMP-knob fusion protein was as effective as the inactivated vaccine in inducing antibodies with HI activity. The immunization of laying hens had no apparent adverse effects on egg production and effectively prevented clinical symptoms of EDS when the chickens were challenged with pathogenic EDS virus. This study demonstrates that the knob domain lacking the shaft sequence but fused to a trimeric coiled coil is a promising candidate subunit vaccine for the prophylaxis of EDS in chickens. PMID:27105561

  3. Coiled-coil domain of PML is essential for the aberrant dynamics of PML-RAR{alpha}, resulting in sequestration and decreased mobility of SMRT

    SciTech Connect

    Huang Ying; Qiu Jihui; Chen Guoqiang; Dong Shuo

    2008-01-11

    Promyelocytic leukemia-retinoic acid receptor {alpha} (PML-RAR{alpha}) is the most frequent RAR{alpha} fusion protein in acute promyelocytic leukemia (APL). Our previous study has demonstrated that, compared with RAR{alpha}, PML-RAR{alpha} had reduced intranuclear mobility accompanied with mislocalization. To understand the molecular basis for the altered dynamics of PML-RAR{alpha} fusion protein, we performed FRAP analysis at a single cell level. Results indicated that three known sumoylation site mutated PML-RAR{alpha} had same intracellular localization and reduced mobility as wild-type counterpart. The coiled-coil domain of PML is responsible for the aberrant dynamics of PML-RAR{alpha}. In addition, we revealed that co-repressor SMRT co-localized with PML-RAR{alpha}, resulting in the immobilization of SMRT while ATRA treatment eliminated their association and reversed the immobile effect of SMRT. Furthermore, co-activator CBP, co-localized with PML-RAR{alpha} in an ATRA-independent way, was demonstrated as a high dynamic intranuclear molecule. These results would shed new insights for the molecular mechanisms of PML-RAR{alpha}-associated leukemogenesis.

  4. Control of Smc Coiled Coil Architecture by the ATPase Heads Facilitates Targeting to Chromosomal ParB/parS and Release onto Flanking DNA.

    PubMed

    Minnen, Anita; Bürmann, Frank; Wilhelm, Larissa; Anchimiuk, Anna; Diebold-Durand, Marie-Laure; Gruber, Stephan

    2016-03-01

    Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome-presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc. PMID:26904953

  5. Control of Smc Coiled Coil Architecture by the ATPase Heads Facilitates Targeting to Chromosomal ParB/parS and Release onto Flanking DNA

    PubMed Central

    Minnen, Anita; Bürmann, Frank; Wilhelm, Larissa; Anchimiuk, Anna; Diebold-Durand, Marie-Laure; Gruber, Stephan

    2016-01-01

    Summary Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome—presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc. PMID:26904953

  6. The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus

    PubMed Central

    Kracklauer, Martin P.; Wiora, Heather M.; Deery, William J.; Chen, Xin; Bolival, Benjamin; Romanowicz, Dwight; Simonette, Rebecca A.; Fuller, Margaret T.; Fischer, Janice A.; Beckingham, Kathleen M.

    2010-01-01

    Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface. PMID:20647369

  7. The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus.

    PubMed

    Kracklauer, Martin P; Wiora, Heather M; Deery, William J; Chen, Xin; Bolival, Benjamin; Romanowicz, Dwight; Simonette, Rebecca A; Fuller, Margaret T; Fischer, Janice A; Beckingham, Kathleen M

    2010-08-15

    Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface. PMID:20647369

  8. Forced expression of desmin and desmin mutants in cultured cells: impact of myopathic missense mutations in the central coiled-coil domain on network formation.

    PubMed

    Bär, Harald; Kostareva, Anna; Sjöberg, Gunnar; Sejersen, Thomas; Katus, Hugo A; Herrmann, Harald

    2006-05-15

    We recently demonstrated that inherited disease-causing mutations clustered in the alpha-helical coiled-coil "rod" domain of the muscle-specific intermediate filament (IF) protein desmin display a wide range of inhibitory effects on regular in vitro assembly. In these studies, we showed that individual mutations exhibited phenotypes that were not, with respect to the severity of interference, predictable by our current knowledge of the structural design of IF proteins. Moreover, the behavior of some mutated proteins in a standard tissue culture cell expression system was found to be even more complex. Here, we systematically investigate the behavior of these disease mutants in four different cell types: three not containing desmin or the related IF protein vimentin and the standard fibroblast line 3T3, which has an extensive vimentin system. The ability of the mutants to form filaments in the vimentin-free cells varies considerably, and only the mutants forming IFs in vitro generate extended filamentous networks. Furthermore, these latter mutants integrate into the 3T3 vimentin network but all the others do not. Instead, they cause the endogenous network of 3T3 vimentin to reorganize into perinuclear bundles. In addition, most of these assembly-deficient mutant desmins completely segregate from the vimentin system. Instead, the small round to fibrillar particles formed distribute independently throughout the cytoplasm as well as between the collapsed vimentin filament arrays in the perinuclear area. PMID:16519886

  9. Antibody elicited against the gp41 N-heptad repeat (NHR) coiled-coil can neutralize HIV-1 with modest potency but non-neutralizing antibodies also bind to NHR mimetics

    SciTech Connect

    Nelson, Josh D.; Kinkead, Heather; Brunel, Florence M.; Leaman, Dan; Jensen, Richard; Louis, John M.; Maruyama, Toshiaki; Bewley, Carole A.; Bowdish, Katherine; Clore, G. Marius; Dawson, Philip E.; Frederickson, Shana; Mage, Rose G.; Richman, Douglas D.; Burton, Dennis R.; Zwick, Michael B.

    2008-07-20

    Following CD4 receptor binding to the HIV-1 envelope spike (Env), the conserved N-heptad repeat (NHR) region of gp41 forms a coiled-coil that is a precursor to the fusion reaction. Although it has been a target of drug and vaccine design, there are few monoclonal antibody (mAb) tools with which to probe the antigenicity and immunogenicity specifically of the NHR coiled-coil. Here, we have rescued HIV-1-neutralizing anti-NHR mAbs from immune phage display libraries that were prepared (i) from b9 rabbits immunized with a previously described mimetic of the NHR coiled-coil, N35{sub CCG}-N13, and (ii) from an HIV-1 infected individual. We describe a rabbit single-chain Fv fragment (scFv), 8K8, and a human Fab, DN9, which specifically recognize NHR coiled-coils that are unoccupied by peptide corresponding to the C-heptad repeat or CHR region of gp41 (e.g. C34). The epitopes of 8K8 and DN9 were found to partially overlap with that of a previously described anti-NHR mAb, IgG D5; however, 8K8 and DN9 were much more specific than D5 for unoccupied NHR trimers. The mAbs, including a whole IgG 8K8 molecule, neutralized primary HIV-1 of clades B and C in a pseudotyped virus assay with comparable, albeit relatively modest potency. Finally, a human Fab T3 and a rabbit serum (both non-neutralizing) were able to block binding of D5 and 8K8 to a gp41 NHR mimetic, respectively, but not the neutralizing activity of these mAbs. We conclude from these results that NHR coiled-coil analogs of HIV-1 gp41 elicit many Abs during natural infection and through immunization, but that due to limited accessibility to the corresponding region on fusogenic gp41 few can neutralize. Caution is therefore required in targeting the NHR for vaccine design. Nevertheless, the mAb panel may be useful as tools for elucidating access restrictions to the NHR of gp41 and in designing potential improvements to mimetics of receptor-activated Env.

  10. The DNA rearrangement that generates the TRK-T3 oncogene involves a novel gene on chromosome 3 whose product has a potential coiled-coil domain.

    PubMed Central

    Greco, A; Mariani, C; Miranda, C; Lupas, A; Pagliardini, S; Pomati, M; Pierotti, M A

    1995-01-01

    Oncogenic rearrangements of the NTRK1 gene (also designated TRKA), encoding one of the receptors for the nerve growth factor, are frequently detected in thyroid carcinomas. Such rearrangements fuse the NTRK1 tyrosine kinase domain to 5'-end sequences belonging to different genes. In previously reported studies we have demonstrated that NTRK1 oncogenic activation involves two genes, TPM3 and TPR, both localized similarly to the receptor tyrosine kinase, on the q arm of chromosome 1. Here we report the characterization of a novel NTRK1-derived thyroid oncogene, named TRK-T3. A cDNA clone, capable of transforming activity, was isolated from a transformant cell line. Sequence analysis revealed that TRK-T3 contains 1,412 nucleotides of NTRK1 preceded by 598 nucleotides belonging to a novel gene that we have named TFG (TRK-fused gene). The TRK-T3 amino acid sequence displays, within the TFG region, a coiled-coil motif that could endow the oncoprotein with the capability to form complexes. The TRK-T3 oncogene encodes a 68-kDa cytoplasmic protein reacting with NTRK1-specific antibodies. By sedimentation gradient experiments the TRK-T3 oncoprotein was shown to form, in vivo, multimeric complexes, most likely trimers or tetramers. The TFG gene is ubiquitously expressed and is located on chromosome 3. The breakpoint producing the TRK-T3 oncogene occurs within exons of both the TFG gene and the NTRK1 gene and produces a chimeric exon that undergoes alternative splicing. Molecular analysis of the NTRK1 rearranged fragments indicated that the chromosomal rearrangement is reciprocal and balanced and involves loss of a few nucleotides of germ line sequences. PMID:7565764

  11. Functional and Mechanistic Analyses of Biomimetic Aminoacyl Transfer Reactions in de novo Designed Coiled Coil Peptides via Rational Active Site Engineering

    PubMed Central

    Leman, Luke J.; Weinberger, Dana A.; Huang, Zheng-Zheng; Wilcoxen, Keith M.; Ghadiri, M. Reza

    2008-01-01

    Ribosomes and nonribosomal peptide synthetases (NRPSs) carry out instructed peptide synthesis through a series of directed intermodular aminoacyl transfer reactions. We recently reported the design of coiled-coil assemblies that could functionally mimic the elementary aminoacyl loading and intermodular aminoacyl transfer steps of NRPSs. These peptides were designed initially to accelerate aminoacyl transfer mainly through catalysis by approximation by closely juxtaposing four active site moieties, two each from adjacent noncovalently-associated helical modules. In our designs peptide self-assembly positions a cysteine residue that is used to covalently capture substrates from solution via transthiolesterification (substrate loading step to generate the aminoacyl donor site) adjacent to an aminoacyl acceptor site provided by a covalently tethered amino acid or modeled by the ε-amine of an active site lysine. However, through systematic functional analyses of 48 rationally designed peptide sequences, we have now determined that the substrate loading and intermodular aminoacyl transfer steps can be significantly influenced (up to ~103-fold) by engineering changes in the active site microenvironment through amino acid substitutions and variations in the inter-residue distances and geometry. Mechanistic studies based on 15N-NMR and kinetic analysis further indicate that certain active site constellations furnish an unexpectedly large pKa depression (1.5 pH units) of the aminoacyl-acceptor moiety, helping to explain the observed high rates of aminoacyl transfer in those constructs. Taken together, our studies demonstrate the feasibility of engineering efficient de novo peptide sequences possessing active sites and functions reminiscent of those in natural enzymes. PMID:17302417

  12. Contribution of hydrophobic interactions to protein stability.

    PubMed

    Pace, C Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R; Shirley, Bret A; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J Martin; Grimsley, Gerald R

    2011-05-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6±0.3 kcal/mol per -CH(2)- group), than to the stability of a large protein, VlsE (1.6±0.3 kcal/mol per -CH(2)- group). (2) Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kilocalories per mole) Phe18 (3.9), Met13 (3.1), Phe7 (2.9), Phe11 (2.7), and Leu21 (2.7). (3) Based on the Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a -CH(2)- group on folding contributes, on average, 1.1±0.5 kcal/mol to protein stability. (4) The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔG(tr) values from water to cyclohexane. (5) For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60±4% and hydrogen bonds contribute 40±4% to protein stability. (6) Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominantly by hydrophobic interactions. PMID:21377472

  13. Titins in C.elegans with unusual features: coiled-coil domains, novel regulation of kinase activity and two new possible elastic regions.

    PubMed

    Flaherty, Denise B; Gernert, Kim M; Shmeleva, Nataliya; Tang, Xuexin; Mercer, Kristina B; Borodovsky, Mark; Benian, Guy M

    2002-10-25

    We report that there are previously unrecognized proteins in Caenorhabditis elegans that are similar to the giant muscle proteins called titins, and these are encoded by a single approximately 90kb gene. The gene structure was predicted by GeneMark.hmm and then experimentally verified. The Ce titin gene encodes polypeptides of 2.2MDa, 1.2MDa and 301kDa. The 2.2MDa isoform resembles twitchin and UNC-89 in that it contains multiple Ig (56) and FnIII (11) domains, and a single protein kinase domain. In addition, however, the 2.2MDa isoform contains four classes of short, 14-51 residue, repeat motifs arranged mostly in many tandem copies. One of these tandem repeat regions is similar to the PEVK regions of vertebrate and fly titins. As the PEVK region is one of the main elastic elements of the titins and is also composed of short tandem repeats, this suggests that the repeat motifs in the Ce titins may have a similar elastic function. An interesting aspect of the two largest Ce titin isoforms, is that in contrast to other members of the twitchin/titin family, there are multiple regions which are likely to form coiled-coil structure. In transgenic animals, the first approximately 100 residues of the largest isoforms targets to dense bodies, the worm analogs of Z-discs. Anti-Ce titin antibodies show localization to muscle I-bands beginning at the L2-L3 larval stages and this pattern continues into adult muscle. Ce titins may not have a role in early myofibril assembly: (1) Ce titins are too short to span half a sarcomere, and the onset of their expression is well after the initial assembly of thick filaments. (2) Ce titins are not localized to I-bands in embryonic or L1 larval muscle. The Ce titin protein kinase domain is most similar to the kinase domains of the twitchins and projectin. The Ce titin kinase has protein kinase activity in vitro, and this activity is regulated by a novel mechanism. PMID:12381307

  14. Electrostatic Contributions to the Stability of the GCN4 Leucine Zipper Structure

    PubMed Central

    Matousek, William M.; Ciani, Barbara; Fitch, Carolyn A.; Bertrand García-Moreno, E.; Kammerer, Richard A.; Alexandrescu, Andrei T.

    2007-01-01

    Summary Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pKa values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pKa values for all groups that ionize between pH 1 and 13 in the 33-residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pKa values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pKa differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pKa values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pKa predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil. PMID:17920624

  15. Contribution of Hydrophobic Interactions to Protein Stability

    PubMed Central

    Pace, C. Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R.; Shirley, Bret A.; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J. Martin; Grimsley, Gerald R.

    2011-01-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin head piece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compare our results with previous studies and reach the following conclusions. 1. Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mole per –CH2– group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per –CH2– group). 2. Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kcal/mol): Phe 18 (3.9), Met 13 (3.1), Phe 7 (2.9), Phe 11 (2.7), and Leu 21 (2.7). 3. Based on Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a –CH2– group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. 4. The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔGtr values from water to cyclohexane. 5. For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds 40 ± 4% to protein stability. 6. Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominately by hydrophobic interactions. PMID:21377472

  16. Effect of coiled-coil peptides on the function of the type III secretion system-dependent activity of enterohemorragic Escherichia coli O157:H7 and Citrobacter rodentium.

    PubMed

    Larzábal, Mariano; Zotta, Elsa; Ibarra, Cristina; Rabinovitz, Bettina C; Vilte, Daniel A; Mercado, Elsa C; Cataldi, Ángel

    2013-01-01

    Many animal and human pathogenic Gram-negative bacteria such as Salmonella, Yersinia, enterohemorrhagic Escherichia coli (EHEC), and enteropathogenic Escherichia coli (EPEC) possess a type III secretion system (TTSS) that is used to deliver virulence proteins directly into the host cell. Recent evidence has suggested that CoilA and CoilB, two synthetic peptides corresponding to coiled-coil domains of the translocator protein EspA, are effective in inhibiting the action of TTSS from EPEC. In the current study, the action of these coiled-coil peptides on the TTSS of EHEC O157:H7 and Citrobacter rodentium was examined. CoilA and CoilB showed to be effective in reducing the red blood cell lysis mediated by EHEC O157:H7 and the in vitro secretion of translocator proteins EspB and EspD by EHEC O157:H7 and EspD by C. rodentium. Treatment of mice with CoilA and CoilB peptides prevented colon damage when the animals were inoculated with C. rodentium. Colon samples of the non-treated group showed areas with loss of superficial epithelium, damaged cells, and endoluminal mononuclear inflammatory infiltrate, consistent with histological lesions induced by C. rodentium, whereas mice treated with the synthetic peptides displayed normal surface epithelium showing a similar structure as the uninfected control group. These encouraging results prompt us to test coiled-coil peptides as treatment or vaccines in other models of bacterial infections in future work. PMID:23312797

  17. Crystal Structure of C-Terminal Coiled-Coil Domain of SYCP1 Reveals Non-Canonical Anti-Parallel Dimeric Structure of Transverse Filament at the Synaptonemal Complex

    PubMed Central

    Jeong, Jae-Hee; Kim, Yeon-Gil; Park, Hyun Ho

    2016-01-01

    The synaptonemal complex protein 1 (SYCP1) is the main structural element of transverse filaments (TFs) of the synaptonemal complex (SC), which is a meiosis-specific complex structure formed at the synapse of homologue chromosomes to hold them together. The N-terminal domain of SYCP1 is known to be located within the central elements (CEs), whereas the C-terminal domain is located toward lateral elements (LEs). SYCP1 is a well-known meiosis marker that is also known to be a prognostic marker in the early stage of several cancers including breast, gliomas, and ovarian cancers. The structure of SC, especially the TF structure formed mainly by SYCP1, remains unclear without any structural information. To elucidate a molecular basis of SC formation and function, we first solved the crystal structure of C-terminal coiled-coil domain of SYCP1. The coiled-coil domain of SYCP1 forms asymmetric, anti-parallel dimers in solution. PMID:27548613

  18. Crystal Structure of C-Terminal Coiled-Coil Domain of SYCP1 Reveals Non-Canonical Anti-Parallel Dimeric Structure of Transverse Filament at the Synaptonemal Complex.

    PubMed

    Seo, Eun Kyung; Choi, Jae Young; Jeong, Jae-Hee; Kim, Yeon-Gil; Park, Hyun Ho

    2016-01-01

    The synaptonemal complex protein 1 (SYCP1) is the main structural element of transverse filaments (TFs) of the synaptonemal complex (SC), which is a meiosis-specific complex structure formed at the synapse of homologue chromosomes to hold them together. The N-terminal domain of SYCP1 is known to be located within the central elements (CEs), whereas the C-terminal domain is located toward lateral elements (LEs). SYCP1 is a well-known meiosis marker that is also known to be a prognostic marker in the early stage of several cancers including breast, gliomas, and ovarian cancers. The structure of SC, especially the TF structure formed mainly by SYCP1, remains unclear without any structural information. To elucidate a molecular basis of SC formation and function, we first solved the crystal structure of C-terminal coiled-coil domain of SYCP1. The coiled-coil domain of SYCP1 forms asymmetric, anti-parallel dimers in solution. PMID:27548613

  19. Quantitative Analyses of Cryptochrome-mBMAL1 Interactions

    PubMed Central

    Czarna, Anna; Breitkreuz, Helena; Mahrenholz, Carsten C.; Arens, Julia; Strauss, Holger M.; Wolf, Eva

    2011-01-01

    The mammalian cryptochromes mCRY1 and mCRY2 act as transcriptional repressors within the 24-h transcription-translational feedback loop of the circadian clock. The C-terminal tail and a preceding predicted coiled coil (CC) of the mCRYs as well as the C-terminal region of the transcription factor mBMAL1 are involved in transcriptional feedback repression. Here we show by fluorescence polarization and isothermal titration calorimetry that purified mCRY1/2CCtail proteins form stable heterodimeric complexes with two C-terminal mBMAL1 fragments. The longer mBMAL1 fragment (BMAL490) includes Lys-537, which is rhythmically acetylated by mCLOCK in vivo. mCRY1 (but not mCRY2) has a lower affinity to BMAL490 than to the shorter mBMAL1 fragment (BMAL577) and a K537Q mutant version of BMAL490. Using peptide scan analysis we identify two mBMAL1 binding epitopes within the coiled coil and tail regions of mCRY1/2 and document the importance of positively charged mCRY1 residues for mBMAL1 binding. A synthetic mCRY coiled coil peptide binds equally well to the short and to the long (wild-type and K537Q mutant) mBMAL1 fragments. In contrast, a peptide including the mCRY1 tail epitope shows a lower affinity to BMAL490 compared with BMAL577 and BMAL490(K537Q). We propose that Lys-537mBMAL1 acetylation enhances mCRY1 binding by affecting electrostatic interactions predominantly with the mCRY1 tail. Our data reveal different molecular interactions of the mCRY1/2 tails with mBMAL1, which may contribute to the non-redundant clock functions of mCRY1 and mCRY2. Moreover, our study suggests the design of peptidic inhibitors targeting the interaction of the mCRY1 tail with mBMAL1. PMID:21521686

  20. 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.

    PubMed

    Schéele, Susanne; Sasaki, Takako; Arnal-Estapé, Anna; Durbeej, Madeleine; Ekblom, Peter

    2006-07-01

    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. PMID:16631359

  1. Full-length Gαq-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain

    SciTech Connect

    Lyon, Angeline M.; Dutta, Somnath; Boguth, Cassandra A.; Skiniotis, Georgios; Tesmer, John J.G.

    2014-08-21

    Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.

  2. Full-length Gα(q)-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain.

    PubMed

    Lyon, Angeline M; Dutta, Somnath; Boguth, Cassandra A; Skiniotis, Georgios; Tesmer, John J G

    2013-03-01

    Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis. PMID:23377541

  3. F-actin asymmetry and the endoplasmic reticulum-associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo.

    PubMed

    Berends, Christian W H; Muñoz, Javier; Portegijs, Vincent; Schmidt, Ruben; Grigoriev, Ilya; Boxem, Mike; Akhmanova, Anna; Heck, Albert J R; van den Heuvel, Sander

    2013-07-01

    The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα-GPR-LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα-GPR-LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division. PMID:23699393

  4. F-actin asymmetry and the endoplasmic reticulum–associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo

    PubMed Central

    Berends, Christian W. H.; Muñoz, Javier; Portegijs, Vincent; Schmidt, Ruben; Grigoriev, Ilya; Boxem, Mike; Akhmanova, Anna; Heck, Albert J. R.; van den Heuvel, Sander

    2013-01-01

    The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα–GPR–LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα–GPR–LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division. PMID:23699393

  5. Modes of interaction among yeast Nej1, Lif1 and Dnl4 proteins and comparison to human XLF, XRCC4 and Lig4.

    PubMed

    Deshpande, Rajashree A; Wilson, Thomas E

    2007-10-01

    The nonhomologous end joining (NHEJ) pathway of double-strand break repair depends on DNA ligase IV and its interacting partner protein XRCC4 (Lif1 in yeast). A third yeast protein, Nej1, interacts with Lif1 and supports NHEJ, similar to the distantly related mammalian Nej1 orthologue XLF (also known as Cernunnos). XRCC4/Lif1 and XLF/Nej1 are themselves related and likely fold into similar coiled-coil structures, which suggests many possible modes of interaction between these proteins. Using yeast two-hybrid and co-precipitation methods we examined these interactions and the protein domains required to support them. Results suggest that stable coiled-coil homodimers are a predominant form of XLF/Nej1, just as for XRCC4/Lif1, but that similar heterodimers are not. XLF-XRCC4 and Nej1-Lif1 interactions were instead mediated independently of the coiled coil, and by different regions of XLF and Nej1. Specifically, the globular head of XRCC4/Lif1 interacted with N- and C-terminal domains of XLF and Nej1, respectively. Direct interactions between XLF/Nej1 and DNA ligase IV were also observed, but again appeared qualitatively different than the stable coiled-coil-mediated interaction between XRCC4/Lif1 and DNA ligase IV. The implications of these findings for DNA ligase IV function are considered in light of the evolutionary pattern in the XLF/XRCC4 and XLF/Nej1 family. PMID:17567543

  6. The Conserved Glycine-Rich Segment Linking the N-Terminal Fusion Peptide to the Coiled Coil of Human T-Cell Leukemia Virus Type 1 Transmembrane Glycoprotein gp21 Is a Determinant of Membrane Fusion Function

    PubMed Central

    Wilson, Kirilee A.; Bär, Séverine; Maerz, Anne L.; Alizon, Marc; Poumbourios, Pantelis

    2005-01-01

    Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an ∼90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment. PMID:15767455

  7. The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function.

    PubMed

    Wilson, Kirilee A; Bär, Séverine; Maerz, Anne L; Alizon, Marc; Poumbourios, Pantelis

    2005-04-01

    Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an approximately 90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment. PMID:15767455

  8. The WTX Tumor Suppressor Interacts with the Transcriptional Corepressor TRIM28*

    PubMed Central

    Kim, Woo Jae; Wittner, Ben S.; Amzallag, Arnaud; Brannigan, Brian W.; Ting, David T.; Ramaswamy, Sridhar; Maheswaran, Shyamala; Haber, Daniel A.

    2015-01-01

    WTX encodes a tumor suppressor implicated in the pediatric kidney cancer Wilms tumor and in mesenchymal differentiation with potentially distinct functions in the cytoplasm, at the plasma membrane, and in the nucleus. Although modulating components of the WNT signaling pathway is a proposed function for cytoplasmic and membrane-bound WTX, its nuclear properties are not well understood. Here we report that the transcriptional corepressor TRIM28 is the major binding partner for nuclear WTX. WTX interacted with the coiled coil domain of TRIM28 required for its binding to Krüppel-associated box domains of transcription factors and for its chromatin recruitment through its own coiled coil and proline-rich domains. Knockdown of endogenous WTX reduced the recruitment of TRIM28 to a chromatinized reporter sequence and its ability to repress a target transcript. In mouse embryonic stem cells where TRIM28 plays a major role in repressing endogenous retroviruses and long interspersed elements, knockdown of either TRIM28 or WTX combined with single molecule RNA sequencing revealed a highly significant shared set of differentially regulated transcripts, including derepression of non-coding repetitive sequences and their neighboring protein encoding genes (p < 1e−20). In mesenchymal precursor cells, depletion of WTX and TRIM28 resulted in analogous β-catenin-independent defects in adipogenic and osteogenic differentiation, and knockdown of WTX reduced TRIM28 binding to Pparγ promoter. Together, the physical and functional interaction between WTX and TRIM28 suggests that the nuclear fraction of WTX plays a role in epigenetic silencing, an effect that may contribute to its function as a regulator of cellular differentiation and tumorigenesis. PMID:25882849

  9. Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1.

    PubMed

    Povelones, Michael; Upton, Leanna M; Sala, Katarzyna A; Christophides, George K

    2011-04-01

    Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition. PMID:21533217

  10. An aromatic amino acid in the coiled-coil 1 domain plays a crucial role in the auto-inhibitory mechanism of STIM1.

    PubMed

    Yu, Junwei; Zhang, Haining; Zhang, Mingshu; Deng, Yongqiang; Wang, Huiyu; Lu, Jingze; Xu, Tao; Xu, Pingyong

    2013-09-15

    STIM1 (stromal interaction molecule 1) is one of the key elements that mediate store-operated Ca²⁺ entry via CRAC (Ca²⁺- release-activated Ca²⁺) channels in immune and non-excitable cells. Under physiological conditions, the intramolecular auto-inhibitions in STIM1 C- and STIM1 N-termini play essential roles in keeping STIM1 in an inactive state. However, the auto-inhibitory mechanism of the STIM1 C-terminus is still unclear. In the present study, we first predicted a short inhibitory domain (residues 310-317) in human STIM1 that might determine the different localizations of human STIM1 from Caenorhabditis elegans STIM1 in resting cells. Next, we confirmed the prediction and further identified an aromatic amino acid residue, Tyr³¹⁶, that played a crucial role in maintaining STIM1 in a closed conformation in quiescent cells. Full-length STIM1-Y316A formed constitutive clusters near the plasma membrane and activated the CRAC channel in the resting state when co-expressed with Orai1. The introduction of a Y316A mutation caused the higher-order oligomerization of the in vitro purified STIM1 fragment containing both the auto-inhibitory domain and CAD(CRAC-activating domain).We propose that the Tyr³¹⁶ residue may be involved in the auto-inhibitory mechanism of the STIM1 C-terminus in the quiescent state. This inhibition could be achieved either by interacting with the CAD using hydrogen and/or hydrophobic bonds, or by an intermolecular interaction using repulsive forces, which maintained a dimeric STIM1. PMID:23795811

  11. Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen.

    PubMed

    Veena, Mariswamy; Melvin, Prasad; Prabhu, Sreedhara Ashok; Shailasree, Sekhar; Shetty, Hunthrike Shekar; Kini, Kukkundoor Ramachandra

    2016-03-01

    Downy mildew caused by Sclerospora graminicola is a devastating disease of pearl millet. Based on candidate gene approach, a set of 22 resistance gene analogues were identified. The clone RGPM 301 (AY117410) containing a partial sequence shared 83% similarity to rice R-proteins. A full-length R-gene RGA RGPM 301 of 3552 bp with 2979 bp open reading frame encoding 992 amino acids was isolated by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach. It had a molecular mass of 113.96 kDa and isoelectric point (pI) of 8.71. The sequence alignment and phylogenetic analysis grouped it to a non-TIR NBS LRR group. The quantitative real-time PCR (qRT-PCR) analysis revealed higher accumulation of the transcripts following inoculation with S. graminicola in the resistant cultivar (IP18296) compared to susceptible cultivar (7042S). Further, significant induction in the transcript levels were observed when treated with abiotic elicitor β-aminobutyric acid (BABA) and biotic elicitor Pseudomonas fluorescens. Exogenous application of phytohormones jasmonic acid or salicylic acid also up-regulated the expression levels of RGA RGPM 301. The treatment of cultivar IP18296 with mitogen-activated protein kinase (MPK) inhibitors (PD98059 and U0126) suppressed the levels of RGA RGPM 301. A 3.5 kb RGA RGPM 301 which is a non-TIR NBS-LRR protein was isolated from pearl millet and its up-regulation during downy mildew interaction was demonstrated by qRT-PCR. These studies indicate a role for this RGA in pearl millet downy mildew interaction. PMID:26842722

  12. Solving coiled-coil protein structures

    DOE PAGESBeta

    Dauter, Zbigniew

    2015-02-26

    With the availability of more than 100,000 entries stored in the Protein Data Bank (PDB) that can be used as search models, molecular replacement (MR) is currently the most popular method of solving crystal structures of macromolecules. Significant methodological efforts have been directed in recent years towards making this approach more powerful and practical. This resulted in the creation of several computer programs, highly automated and user friendly, that are able to successfully solve many structures even by researchers who, although interested in structures of biomolecules, are not very experienced in crystallography.

  13. Vibrational solvatochromism. III. Rigorous treatment of the dispersion interaction contribution.

    PubMed

    Błasiak, Bartosz; Cho, Minhaeng

    2015-10-28

    A rigorous first principles theory of vibrational solvatochromism including the intermolecular dispersion interaction, which is based on the effective fragment potential method, is developed. The present theory is an extended version of our previous vibrational solvatochromism model that took into account the Coulomb, exchange-repulsion, and induction interactions. We show that the frequency shifts of the amide I mode of N-methylacetamide in H2O and CDCl3, when combined with molecular dynamics simulations, can be quantitatively reproduced by the theory, which indicates that the dispersion interaction contribution to the vibrational frequency shift is not always negligibly small. Nonetheless, the reason that the purely Coulombic interaction model for vibrational solvatochromism works well for describing amide I mode frequency shifts in polar solvents is because the electrostatic contribution is strong and highly sensitive to the relative orientation of surrounding solvent molecules, which is in stark contrast with polarization, dispersion, and exchange-repulsion contributions. It is believed that the theory presented and discussed here will be of great use in quantitatively describing vibrational solvatochromism and electrochromism of infrared probes in not just polar solvent environments but also in biopolymers such as proteins. PMID:26520502

  14. Vibrational solvatochromism. III. Rigorous treatment of the dispersion interaction contribution

    NASA Astrophysics Data System (ADS)

    Błasiak, Bartosz; Cho, Minhaeng

    2015-10-01

    A rigorous first principles theory of vibrational solvatochromism including the intermolecular dispersion interaction, which is based on the effective fragment potential method, is developed. The present theory is an extended version of our previous vibrational solvatochromism model that took into account the Coulomb, exchange-repulsion, and induction interactions. We show that the frequency shifts of the amide I mode of N-methylacetamide in H2O and CDCl3, when combined with molecular dynamics simulations, can be quantitatively reproduced by the theory, which indicates that the dispersion interaction contribution to the vibrational frequency shift is not always negligibly small. Nonetheless, the reason that the purely Coulombic interaction model for vibrational solvatochromism works well for describing amide I mode frequency shifts in polar solvents is because the electrostatic contribution is strong and highly sensitive to the relative orientation of surrounding solvent molecules, which is in stark contrast with polarization, dispersion, and exchange-repulsion contributions. It is believed that the theory presented and discussed here will be of great use in quantitatively describing vibrational solvatochromism and electrochromism of infrared probes in not just polar solvent environments but also in biopolymers such as proteins.

  15. Magnetic interactions in strongly correlated systems: Spin and orbital contributions

    SciTech Connect

    Secchi, A.; Lichtenstein, A.I.; Katsnelson, M.I.

    2015-09-15

    We present a technique to map an electronic model with local interactions (a generalized multi-orbital Hubbard model) onto an effective model of interacting classical spins, by requiring that the thermodynamic potentials associated to spin rotations in the two systems are equivalent up to second order in the rotation angles, when the electronic system is in a symmetry-broken phase. This allows to determine the parameters of relativistic and non-relativistic magnetic interactions in the effective spin model in terms of equilibrium Green’s functions of the electronic model. The Hamiltonian of the electronic system includes, in addition to the non-relativistic part, relativistic single-particle terms such as the Zeeman coupling to an external magnetic field, spin–orbit coupling, and arbitrary magnetic anisotropies; the orbital degrees of freedom of the electrons are explicitly taken into account. We determine the complete relativistic exchange tensors, accounting for anisotropic exchange, Dzyaloshinskii–Moriya interactions, as well as additional non-diagonal symmetric terms (which may include dipole–dipole interaction). The expressions of all these magnetic interactions are determined in a unified framework, including previously disregarded features such as the vertices of two-particle Green’s functions and non-local self-energies. We do not assume any smallness in spin–orbit coupling, so our treatment is in this sense exact. Finally, we show how to distinguish and address separately the spin, orbital and spin–orbital contributions to magnetism, providing expressions that can be computed within a tight-binding Dynamical Mean Field Theory.

  16. A Bub1–Mad1 interaction targets the Mad1–Mad2 complex to unattached kinetochores to initiate the spindle checkpoint

    PubMed Central

    Moyle, Mark W.; Kim, Taekyung; Hattersley, Neil; Espeut, Julien; Cheerambathur, Dhanya K.; Oegema, Karen

    2014-01-01

    Recruitment of Mad1–Mad2 complexes to unattached kinetochores is a central event in spindle checkpoint signaling. Despite its importance, the mechanism that recruits Mad1–Mad2 to kinetochores is unclear. In this paper, we show that MAD-1 interacts with BUB-1 in Caenorhabditis elegans. Mutagenesis identified specific residues in a segment of the MAD-1 coiled coil that mediate the BUB-1 interaction. In addition to unattached kinetochores, MAD-1 localized between separating meiotic chromosomes and to the nuclear periphery. Mutations in the MAD-1 coiled coil that selectively disrupt interaction with BUB-1 eliminated MAD-1 localization to unattached kinetochores and between meiotic chromosomes, both of which require BUB-1, and abrogated checkpoint signaling. The identified MAD-1 coiled-coil segment interacted with a C-terminal region of BUB-1 that contains its kinase domain, and mutations in this region prevented MAD-1 kinetochore targeting independently of kinase activity. These results delineate an interaction between BUB-1 and MAD-1 that targets MAD-1–MAD-2 complexes to kinetochores and is essential for spindle checkpoint signaling. PMID:24567362

  17. Interaction of the Spo20 Membrane-Sensor Motif with Phosphatidic Acid and Other Anionic Lipids, and Influence of the Membrane Environment

    PubMed Central

    Horchani, Habib; de Saint-Jean, Maud; Barelli, Hélène; Antonny, Bruno

    2014-01-01

    The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context. PMID:25426975

  18. Person-Environment Interactions Contributing to Nursing Home Resident Falls

    PubMed Central

    Hill, Elizabeth E.; Nguyen, Tam H.; Shaha, Maya; Wenzel, Jennifer A.; DeForge, Bruce R.; Spellbring, Ann Marie

    2011-01-01

    Although approximately 50% of nursing home residents fall annually, the surrounding circumstances remain inadequately understood. This study explored nursing staff perspectives of person, environment, and interactive circumstances surrounding nursing home falls. Focus groups were conducted at two nursing homes in the mid-Atlantic region with the highest and lowest fall rates among corporate facilities. Two focus groups were conducted per facility: one with licensed nurses and one with geriatric nursing assistants. Thematic and content analysis revealed three themes and 11 categories. Three categories under the Person theme were Change in Residents’ Health Status, Decline in Residents’ Abilities, and Residents’ Behaviors and Personality Characteristics. There were five Nursing Home Environment categories: Design Safety, Limited Space, Obstacles, Equipment Misuse and Malfunction, and Staff and Organization of Care. Three Interactions Leading to Falls categories were identified: Reasons for Falls, Time of Falls, and High-Risk Activities. Findings highlight interactions between person and environment factors as significant contributors to resident falls. PMID:20077985

  19. The Interactive Account of ventral occipitotemporal contributions to reading

    PubMed Central

    Price, Cathy J.; Devlin, Joseph T.

    2011-01-01

    The ventral occipitotemporal cortex (vOT) is involved in the perception of visually presented objects and written words. The Interactive Account of vOT function is based on the premise that perception involves the synthesis of bottom-up sensory input with top-down predictions that are generated automatically from prior experience. We propose that vOT integrates visuospatial features abstracted from sensory inputs with higher level associations such as speech sounds, actions and meanings. In this context, specialization for orthography emerges from regional interactions without assuming that vOT is selectively tuned to orthographic features. We discuss how the Interactive Account explains left vOT responses during normal reading and developmental dyslexia; and how it accounts for the behavioural consequences of left vOT damage. PMID:21549634

  20. Gene-Environment Interactions in Stress Response Contribute Additively to a Genotype-Environment Interaction

    PubMed Central

    Matsui, Takeshi; Ehrenreich, Ian M.

    2016-01-01

    How combinations of gene-environment interactions collectively give rise to genotype-environment interactions is not fully understood. To shed light on this problem, we genetically dissected an environment-specific poor growth phenotype in a cross of two budding yeast strains. This phenotype is detectable when certain segregants are grown on ethanol at 37°C (‘E37’), a condition that differs from the standard culturing environment in both its carbon source (ethanol as opposed to glucose) and temperature (37°C as opposed to 30°C). Using recurrent backcrossing with phenotypic selection, we identified 16 contributing loci. To examine how these loci interact with each other and the environment, we focused on a subset of four loci that together can lead to poor growth in E37. We measured the growth of all 16 haploid combinations of alleles at these loci in all four possible combinations of carbon source (ethanol or glucose) and temperature (30 or 37°C) in a nearly isogenic population. This revealed that the four loci act in an almost entirely additive manner in E37. However, we also found that these loci have weaker effects when only carbon source or temperature is altered, suggesting that their effect magnitudes depend on the severity of environmental perturbation. Consistent with such a possibility, cloning of three causal genes identified factors that have unrelated functions in stress response. Thus, our results indicate that polymorphisms in stress response can show effects that are intensified by environmental stress, thereby resulting in major genotype-environment interactions when multiple of these variants co-occur. PMID:27437938

  1. A testis-specific and testis developmentally regulated tumor protein D52 (TPD52)-like protein TPD52L3/hD55 interacts with TPD52 family proteins

    SciTech Connect

    Cao Qinhong; Chen Jie; Zhu Li; Liu Yun; Zhou Zuomin; Sha Jiahao; Wang Shui; Li Jianmin . E-mail: jianminli@njmu.edu.cn

    2006-06-09

    Tumor protein D52-like proteins (TPD52) are small coiled-coil motif bearing proteins that were first identified in breast cancer. TPD52 and related proteins have been implicated in cell proliferation, apoptosis, and vesicle trafficking. To date, three human TPD52 members had been identified, named hD52 (TPD52), hD53 (TPD52L1), and hD54 (TPD52L2). The most important characteristic of the protein family is a highly conserved coiled-coil motif that is required for homo- and heteromeric interaction with other TPD52-like proteins. Herein, we identified a novel TPD52-like sequence (TPD52L3, or hD55) in human testis using cDNA microarray. Sequence analysis of the deduced protein suggests that hD55 contains a coiled-coil motif and is highly conserved compared with other TPD52-like sequences. Yeast two-hybrid and GST pull-down assays revealed that hD55 interacts with hD52, hD53, hD54, and itself. cDNA microarray detection found that hD55 was expressed at 5.6-fold higher levels in adult testis than in fetal testis. Additionally, the expression profile shows that hD55 is testis-specific, indicating a potential role for hD55 in testis development and spermatogenesis.

  2. Electrostatic forces contribute to interactions between trp repressor dimers.

    PubMed Central

    Martin, K S; Royer, C A; Howard, K P; Carey, J; Liu, Y C; Matthews, K; Heyduk, E; Lee, J C

    1994-01-01

    The trp repressor of Escherichia coli (TR), although generally considered to be dimeric, has been shown by fluorescence anisotropy of extrinsically labeled protein to undergo oligomerization in solution at protein concentrations in the micromolar range (Fernando, T., and C. A. Royer 1992. Biochemistry. 31:3429-3441). Providing evidence that oligomerization is an intrinsic property of TR, the present studies using chemical cross-linking, analytical ultracentrifugation, and molecular sieve chromatography demonstrate that unmodified TR dimers form higher order aggregates. Tetramers and higher order species were observed in chemical cross-linking experiments at concentrations between 1 and 40 microM. Results from analytical ultracentrifugation and gel filtration chromatography were consistent with average molecular weight values between tetramer and dimer, although no plateaus in the association were evident over the concentration ranges studied, indicating that higher order species are populated. Analytical ultracentrifugation data in presence of corepressor imply that corepressor binding destabilizes the higher order aggregates, an observation that is consistent with the earlier fluorescence work. Through the investigation of the salt and pH dependence of oligomerization, the present studies have revealed an electrostatic component to the interactions between TR dimers. Images FIGURE 1 PMID:8038388

  3. Genetic interactions contribute less than additive effects to quantitative trait variation in yeast

    PubMed Central

    Bloom, Joshua S.; Kotenko, Iulia; Sadhu, Meru J.; Treusch, Sebastian; Albert, Frank W.; Kruglyak, Leonid

    2015-01-01

    Genetic mapping studies of quantitative traits typically focus on detecting loci that contribute additively to trait variation. Genetic interactions are often proposed as a contributing factor to trait variation, but the relative contribution of interactions to trait variation is a subject of debate. Here we use a very large cross between two yeast strains to accurately estimate the fraction of phenotypic variance due to pairwise QTL–QTL interactions for 20 quantitative traits. We find that this fraction is 9% on average, substantially less than the contribution of additive QTL (43%). Statistically significant QTL–QTL pairs typically have small individual effect sizes, but collectively explain 40% of the pairwise interaction variance. We show that pairwise interaction variance is largely explained by pairs of loci at least one of which has a significant additive effect. These results refine our understanding of the genetic architecture of quantitative traits and help guide future mapping studies. PMID:26537231

  4. Shaping Learner Contributions in an EFL Classroom: Implications for L2 Classroom Interactional Competence

    ERIC Educational Resources Information Center

    Can Daskin, Nilüfer

    2015-01-01

    This study investigated the interactional patterns for shaping learner contributions in an EFL classroom with reference to Walsh's classroom interactional competence (CIC). In doing so, an EFL class at an English preparatory school in a Turkish state university was both videotaped and audiotaped in the course of six classroom hours. Conversation…

  5. An N-terminal glycine-rich sequence contributes to retrovirus trimer of hairpins stability

    SciTech Connect

    Wilson, Kirilee A.; Maerz, Anne L.; Baer, Severine; Drummer, Heidi E.; Poumbourios, Pantelis . E-mail: apoumbourios@burnet.edu.au

    2007-08-10

    Retroviral transmembrane proteins (TMs) contain a glycine-rich segment linking the N-terminal fusion peptide and coiled coil core. Previously, we reported that the glycine-rich segment (Met-326-Ser-337) of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, is a determinant of membrane fusion function [K.A. Wilson, S. Baer, A.L. Maerz, M. Alizon, P. Poumbourios, The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function, J. Virol. 79 (2005) 4533-4539]. Here we show that the reduced fusion activity of an I334A mutant correlated with a decrease in stability of the gp21 trimer of hairpins conformation, in the context of a maltose-binding protein-gp21 chimera. The stabilizing influence of Ile-334 required the C-terminal membrane-proximal sequence Trp-431-Ser-436. Proline substitution of four of five Gly residues altered gp21 trimer of hairpins stability. Our data indicate that flexibility within and hydrophobic interactions mediated by this region are determinants of gp21 stability and membrane fusion function.

  6. An N-terminal glycine-rich sequence contributes to retrovirus trimer of hairpins stability.

    PubMed

    Wilson, Kirilee A; Maerz, Anne L; Bär, Séverine; Drummer, Heidi E; Poumbourios, Pantelis

    2007-08-10

    Retroviral transmembrane proteins (TMs) contain a glycine-rich segment linking the N-terminal fusion peptide and coiled coil core. Previously, we reported that the glycine-rich segment (Met-326-Ser-337) of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, is a determinant of membrane fusion function [K.A. Wilson, S. Bär, A.L. Maerz, M. Alizon, P. Poumbourios, The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function, J. Virol. 79 (2005) 4533-4539]. Here we show that the reduced fusion activity of an I334A mutant correlated with a decrease in stability of the gp21 trimer of hairpins conformation, in the context of a maltose-binding protein-gp21 chimera. The stabilizing influence of Ile-334 required the C-terminal membrane-proximal sequence Trp-431-Ser-436. Proline substitution of four of five Gly residues altered gp21 trimer of hairpins stability. Our data indicate that flexibility within and hydrophobic interactions mediated by this region are determinants of gp21 stability and membrane fusion function. PMID:17577584

  7. Professional Interaction, Relevant Practical Experience, and Intellectual Contributions at Nondoctoral AACSB-Accredited Accounting Programs

    ERIC Educational Resources Information Center

    Arlinghaus, Barry P.

    2008-01-01

    In this article, the author discusses a survey of faculty members at nondoctoral AACSB-accredited accounting programs in the United States. The purpose of the survey was to determine the environment for professional interaction and relevant experience in light of institutional demands for intellectual contributions. The findings show that the…

  8. Early Markers of Language and Attention: Mutual Contributions and the Impact of Parent-Infant Interactions

    ERIC Educational Resources Information Center

    Gartstein, Maria A.; Crawford, Jennifer; Robertson, Christopher D.

    2008-01-01

    This study was conducted to explore the contribution of attentional skills to early language, and the influence of early language markers on the development of attention, simultaneously examining the impact of parent-child interaction factors (reciprocity/synchrony and sensitivity/responsivity), including their potential moderator effects. All…

  9. Intersegment interactions and helix-coil transition within the generalized model of polypeptide chains approach

    NASA Astrophysics Data System (ADS)

    Badasyan, A. V.; Hayrapetyan, G. N.; Tonoyan, Sh. A.; Mamasakhlisov, Y. Sh.; Benight, A. S.; Morozov, V. F.

    2009-09-01

    The generalized model of polypeptide chains is extended to describe the helix-coil transition in a system comprised of two chains interacting side-by-side. The Hamiltonian of the model takes into account four possible types of interactions between repeated units of the two chains, i.e., helix-helix, helix-coil, coil-helix, and coil-coil. Analysis reveals when the energy Ihh+Icc of (h-h, c-c) interactions overwhelms the energy Ihc+Ich of mixed (h-c, c-h) interactions, the correlation length rises substantially, resulting in narrowing of the transition interval. In the opposite case, when Ihh+Iccinteractions from the same theoretical perspective.

  10. Assessing Energetic Contributions to Binding from a Disordered Region in a Protein-Protein Interaction

    SciTech Connect

    S Cho; C Swaminathan; D Bonsor; M Kerzic; R Guan; J Yang; C Kieke; P Anderson; D Kranz; et al.

    2011-12-31

    Many functional proteins are at least partially disordered prior to binding. Although the structural transitions upon binding of disordered protein regions can influence the affinity and specificity of protein complexes, their precise energetic contributions to binding are unknown. Here, we use a model protein-protein interaction system in which a locally disordered region has been modified by directed evolution to quantitatively assess the thermodynamic and structural contributions to binding of disorder-to-order transitions. Through X-ray structure determination of the protein binding partners before and after complex formation and isothermal titration calorimetry of the interactions, we observe a correlation between protein ordering and binding affinity for complexes along this affinity maturation pathway. Additionally, we show that discrepancies between observed and calculated heat capacities based on buried surface area changes in the protein complexes can be explained largely by heat capacity changes that would result solely from folding the locally disordered region. Previously developed algorithms for predicting binding energies of protein-protein interactions, however, are unable to correctly model the energetic contributions of the structural transitions in our model system. While this highlights the shortcomings of current computational methods in modeling conformational flexibility, it suggests that the experimental methods used here could provide training sets of molecular interactions for improving these algorithms and further rationalizing molecular recognition in protein-protein interactions.

  11. Disorder and structure in the Rab11 binding domain of Rab11 family interacting protein 2.

    PubMed

    Wei, Jie; Liu, Yuqi; Bose, Kakoli; Henry, Gillian D; Baleja, James D

    2009-01-27

    Rab11 plays a central role in plasma membrane recycling which returns cellular receptors for reuse at the cell surface. A recently identified family of Rab11 interacting proteins (FIP) includes FIP2. The C-terminal region of FIP2 is essential for colocalization with Rab11 on early endosomes and for enabling formation of higher-order oligomers. Rab11 binding and oligomerization of FIP2 are separable. Here we have determined the three-dimensional structure of the 40-residue coiled-coil oligomerization domain of FIP2 in the absence of Rab11 using NMR methods. The N-terminal half showed strong NOE cross-peaks and well-dispersed NMR resonances, whereas the C-terminal half had fewer NOE cross-peaks and less chemical shift dispersion. The 10 C-terminal residues were mostly disordered. The final structures of the dimer had favorable Ramachandran angles and a root-mean-square deviation of 0.59 +/- 0.13 A over superimposed backbone residues. The structure allows a comparison to a structure of FIP2 in complex with Rab11 that was determined crystallographically. In complex with Rab11, the C-terminal residues are not disordered but have a helical structure that predicts residual dipolar coupling constants that are incompatible with those measured on the unbound FIP2. In both structures, a histidine residue is found at the normally hydrophobic position of the heptad repeat of the coiled coil, and here we show its ionization destabilizes the coiled-coil structure. Together, these data allow us to build a model in which the binding of FIP family proteins to Rab11 can be described in terms of conformational changes and that suggests new modes of regulation. PMID:19119858

  12. A bivariate mann-whitney approach for unraveling genetic variants and interactions contributing to comorbidity.

    PubMed

    Wen, Yalu; Schaid, Daniel J; Lu, Qing

    2013-04-01

    Although comorbidity among complex diseases (e.g., drug dependence syndromes) is well documented, genetic variants contributing to the comorbidity are still largely unknown. The discovery of genetic variants and their interactions contributing to comorbidity will likely shed light on underlying pathophysiological and etiological processes, and promote effective treatments for comorbid conditions. For this reason, studies to discover genetic variants that foster the development of comorbidity represent high-priority research projects, as manifested in the behavioral genetics studies now underway. The yield from these studies can be enhanced by adopting novel statistical approaches, with the capacity of considering multiple genetic variants and possible interactions. For this purpose, we propose a bivariate Mann-Whitney (BMW) approach to unravel genetic variants and interactions contributing to comorbidity, as well as those unique to each comorbid condition. Through simulations, we found BMW outperformed two commonly adopted approaches in a variety of underlying disease and comorbidity models. We further applied BMW to datasets from the Study of Addiction: Genetics and Environment, investigating the contribution of 184 known nicotine dependence (ND) and alcohol dependence (AD) single nucleotide polymorphisms (SNPs) to the comorbidity of ND and AD. The analysis revealed a candidate SNP from CHRNA5, rs16969968, associated with both ND and AD, and replicated the findings in an independent dataset with a P-value of 1.06 × 10(-03) . PMID:23334941

  13. Seeds of strategic and interactional psychotherapies: seminal contributions of Milton H. Erickson.

    PubMed

    Zeig, J K; Geary, B B

    1990-10-01

    The life and work of Milton H. Erickson exerts a considerable influence upon the development of strategic and interactional psychotherapies. In this paper we trace the historical course of Erickson's impact in these areas from his early associations with Gregory Bateson and Margaret Mead through his contributions to the ideologies of Jay Haley and practitioners at the Mental Research Institute. We have identified seven philosophical and methodological realms which represent the incorporation of Ericksonian principles into strategic and interactional family therapy models. PMID:2270835

  14. XTACC3-XMAP215 association reveals an asymmetric interaction promoting microtubule elongation.

    PubMed

    Mortuza, Gulnahar B; Cavazza, Tommaso; Garcia-Mayoral, Maria Flor; Hermida, Dario; Peset, Isabel; Pedrero, Juan G; Merino, Nekane; Blanco, Francisco J; Lyngsø, Jeppe; Bruix, Marta; Pedersen, Jan Skov; Vernos, Isabelle; Montoya, Guillermo

    2014-01-01

    chTOG is a conserved microtubule polymerase that catalyses the addition of tubulin dimers to promote microtubule growth. chTOG interacts with TACC3, a member of the transforming acidic coiled-coil (TACC) family. Here we analyse their association using the Xenopus homologues, XTACC3 (TACC3) and XMAP215 (chTOG), dissecting the mechanism by which their interaction promotes microtubule elongation during spindle assembly. Using SAXS, we show that the TACC domain (TD) is an elongated structure that mediates the interaction with the C terminus of XMAP215. Our data suggest that one TD and two XMAP215 molecules associate to form a four-helix coiled-coil complex. A hybrid methods approach was used to define the precise regions of the TACC heptad repeat and the XMAP215 C terminus required for assembly and functioning of the complex. We show that XTACC3 can induce the recruitment of larger amounts of XMAP215 by increasing its local concentration, thereby promoting efficient microtubule elongation during mitosis. PMID:25262927

  15. Genetic Interactions Involving Five or More Genes Contribute to a Complex Trait in Yeast

    PubMed Central

    Taylor, Matthew B.; Ehrenreich, Ian M.

    2014-01-01

    Recent research suggests that genetic interactions involving more than two loci may influence a number of complex traits. How these ‘higher-order’ interactions arise at the genetic and molecular levels remains an open question. To provide insights into this problem, we dissected a colony morphology phenotype that segregates in a yeast cross and results from synthetic higher-order interactions. Using backcrossing and selective sequencing of progeny, we found five loci that collectively produce the trait. We fine-mapped these loci to 22 genes in total and identified a single gene at each locus that caused loss of the phenotype when deleted. Complementation tests or allele replacements provided support for functional variation in these genes, and revealed that pre-existing genetic variants and a spontaneous mutation interact to cause the trait. The causal genes have diverse functions in endocytosis (END3), oxidative stress response (TRR1), RAS-cAMP signalling (IRA2), and transcriptional regulation of multicellular growth (FLO8 and MSS11), and for the most part have not previously been shown to exhibit functional relationships. Further efforts uncovered two additional loci that together can complement the non-causal allele of END3, suggesting that multiple genotypes in the cross can specify the same phenotype. Our work sheds light on the complex genetic and molecular architecture of higher-order interactions, and raises questions about the broader contribution of such interactions to heritable trait variation. PMID:24784154

  16. β-arrestin-1 contributes to brown fat function and directly interacts with PPARα and PPARγ

    PubMed Central

    Wang, Congcong; Zeng, Xianglu; Zhou, Zhaocai; Zhao, Jian; Pei, Gang

    2016-01-01

    The peroxisome proliferator-activated receptor (PPAR) family plays central roles in brown adipose tissue (BAT) adipogenesis and contributes to body temperature maintenance. The transcriptional activity of PPAR family has been shown to be tightly controlled by cellular signal networks. β-arrestins function as major secondary messengers of G protein-coupled receptors (GPCR) signaling by functional interactions with diverse proteins. Here, we report that β-arrestin-1 knock-out mice show enhanced cold tolerance. We found that β-arrestin-1 directly interacts with PPARα and PPARγ through a LXXXLXXXL motif, while D371 in PPARα and L311/N312/D380 in PPARγ are required for their interactions with β-arrestin-1. Further mechanistic studies showed that β-arrestin-1 promotes PPARα- but represses PPARγ-mediated transcriptional activities, providing potential regulatory pathway for BAT function. PMID:27301785

  17. β-arrestin-1 contributes to brown fat function and directly interacts with PPARα and PPARγ.

    PubMed

    Wang, Congcong; Zeng, Xianglu; Zhou, Zhaocai; Zhao, Jian; Pei, Gang

    2016-01-01

    The peroxisome proliferator-activated receptor (PPAR) family plays central roles in brown adipose tissue (BAT) adipogenesis and contributes to body temperature maintenance. The transcriptional activity of PPAR family has been shown to be tightly controlled by cellular signal networks. β-arrestins function as major secondary messengers of G protein-coupled receptors (GPCR) signaling by functional interactions with diverse proteins. Here, we report that β-arrestin-1 knock-out mice show enhanced cold tolerance. We found that β-arrestin-1 directly interacts with PPARα and PPARγ through a LXXXLXXXL motif, while D371 in PPARα and L311/N312/D380 in PPARγ are required for their interactions with β-arrestin-1. Further mechanistic studies showed that β-arrestin-1 promotes PPARα- but represses PPARγ-mediated transcriptional activities, providing potential regulatory pathway for BAT function. PMID:27301785

  18. CFL3D Contribution to the AIAA Supersonic Shock Boundary Layer Interaction Workshop

    NASA Technical Reports Server (NTRS)

    Rumsey, Christopher L.

    2010-01-01

    This paper documents the CFL3D contribution to the AIAA Supersonic Shock Boundary Layer Interaction Workshop, held in Orlando, Florida in January 2010. CFL3D is a Reynolds-averaged Navier-Stokes code. Four shock boundary layer interaction cases are computed using a one-equation turbulence model widely used for other aerodynamic problems of interest. Two of the cases have experimental data available at the workshop, and two of the cases do not. The effect of grid, flux scheme, and thin-layer approximation are investigated. Comparisons are made to the available experimental data. All four cases exhibit strong three-dimensional behavior in and near the interaction regions, resulting from influences of the tunnel side-walls.

  19. Conformational Contribution to the Heat Capacity of Interacting System of Carbohydrate Polymer - Water.

    NASA Astrophysics Data System (ADS)

    Pyda, Marek; Wunderlich, Bernhard

    2001-03-01

    Based on the measured heat capacities of amorphous, dry starch and starch with low concentration of water above the partial glass transition of starch, the calculated Cp has been estimated from its vibrational, external, and conformational contributions. The conformational part is evaluated from a fit of the experimental Cp of starch and starch-water, decreased by the vibrational and the external Cp to a one-dimensional Ising-type model for two discrete states, and stiffness, cooperativity, and degeneracy parameters. These differences above the glass transition are interpreted as contributions of different conformational heat capacities from interacting chains of carbohydrate with water. The vibrational contribution was calculated as the heat capacity contributions from group and skeletal vibrations. The external contribution was computed based on thermal expansivity and compressibility as a function of temperature from experimental data of the partial liquid state of both dry starch and starch-water. The calculated and experimental heat capacities of starch-water and dry starch are compared over the whole range of temperatures measurements from 8 to 490 K. NSF, Polymers Program, DMR-9703692, and the Div. of Mat. Sci., BES, DOE at ORNL, managed UT-Batelle, LLC, for the U.S. Department of Energy, under contract number DOE- AC05-00OR22725.

  20. PMLRAR homodimers: distinct DNA binding properties and heteromeric interactions with RXR.

    PubMed Central

    Perez, A; Kastner, P; Sethi, S; Lutz, Y; Reibel, C; Chambon, P

    1993-01-01

    Fusion proteins (named PMLRAR) between PML and the retinoic acid receptor alpha (RAR alpha) are generated as a result of the t(15;17) chromosomal translocation found in acute promyelocytic leukemia (APL). We show here that PMLRAR proteins exist in solution as stable homodimers whose formation is mediated by a presumptive coiled coil in the PML moiety. In contrast to RAR alpha, which requires heterodimerization with RXR for efficient DNA binding, PMLRAR homodimers can bind to target sequences in the absence of RXR, and the binding pattern of PMLRAR homodimeric complexes to directly repeated motif (DR) response elements with 1-5 bp spacers is different from that of RAR/RXR heterodimeric complexes. We show that the presence of RXR induces the formation of PMLRAR/RXR heteromeric complexes which bind to DNA via one RAR DNA binding domain (DBD) and one RXR DBD, like 'classical' RAR/RXR heterodimers. PMLRAR interaction with RXR occurs in solution and in transfected cultured Cos cells, and PMLRAR is able to sequester RXR efficiently in the cytoplasm, suggesting that dominant 'inactivation' of RXR may be a possible mechanism of action for PMLRAR. Accordingly, we show that PMLRAR can both prevent the binding of the vitamin D3 receptor (VDR) to a target sequence in vitro and inhibit vitamin D3-dependent activation of a VDR-responsive reporter gene in transfected cells. These results suggest that both the distinct DNA binding properties of PMLRAR homodimers and the sequestration of RXR by PMLRARs may contribute to the molecular mechanisms which underlie the pathogenesis of APL. We also report that RXR alpha transcripts are down-regulated by RA-treatment in promyelocytic cells. Images PMID:8393784

  1. Conditioned place preference for social interaction in rats: contribution of sensory components

    PubMed Central

    Kummer, Kai; Klement, Sabine; Eggart, Vincent; Mayr, Michael J.; Saria, Alois; Zernig, Gerald

    2011-01-01

    A main challenge in the therapy of drug dependent individuals is to help them reactivate interest in non-drug-associated activities. We previously developed a rat experimental model based on the conditioned place preference (CPP) paradigm in which only four 15-min episodes of social interaction with a gender- and weight-matched male Sprague Dawley rat (1) reversed CPP from cocaine to social interaction despite continuing cocaine training and (2) prevented the reinstatement of cocaine CPP. In the present study, we investigated which of the sensory modalities of the composite stimulus “social interaction” contributes most to the rats' preference for it. If touch was limited by steel bars spaced at a distance of 2 cm and running across the whole length of a partitioning, CPP was still acquired, albeit to a lesser degree. If both rats were placed on the same side of a partitioning, rats did not develop CPP for social interaction. Thus, decreasing the available area for social interaction from 750 to 375 cm2 prevented the acquisition of CPP to social interaction despite the fact that animals could touch each other more intensely than through the bars of the partitioning. When touch was fully restricted by a glass screen dividing the conditioning chambers, and the only sensory modalities left were visual and olfactory cues, place preference shifted to place aversion. Overall, our findings indicate that the major rewarding sensory component of the composite stimulus “social interaction” is touch (taction). PMID:22232578

  2. ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein–protein interactions with HPRT1

    PubMed Central

    Vasiliou, Vasilis; Sandoval, Monica; Backos, Donald S.; Jackson, Brian C.; Chen, Ying; Reigan, Philip; Lanaspa, Miguel A.; Johnson, Richard J.; Koppaka, Vindhya; Thompson, David C.

    2013-01-01

    Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers. PMID:23348497

  3. Social inequalities in health: measuring the contribution of housing deprivation and social interactions for Spain

    PubMed Central

    2012-01-01

    Introduction Social factors have been proved to be main determinants of individuals’ health. Recent studies have also analyzed the contribution of some of those factors, such as education and job status, to socioeconomic inequalities in health. The aim of this paper is to provide new evidence about the factors driving socioeconomic inequalities in health for the Spanish population by including housing deprivation and social interactions as health determinants. Methods Cross-sectional study based on the Spanish sample of European Statistics on Income and Living Conditions (EU-SILC) for 2006. The concentration index measuring income-related inequality in health is decomposed into the contribution of each determinant. Several models are estimated to test the influence of different regressors for three proxies of ill-health. Results Health inequality favouring the better-off is observed in the distribution of self-assessed health, presence of chronic diseases and presence of limiting conditions. Inequality is mainly explained, besides age, by social factors such as labour status and financial deprivation. Housing deprivation contributes to pro-rich inequality in a percentage ranging from 7.17% to 13.85%, and social interactions from 6.16% to 10.19%. The contribution of some groups of determinants significantly differs depending on the ill-health variable used. Conclusions Health inequalities can be mostly reduced or shaped by policy, as they are mainly explained by social determinants such as labour status, education and other socioeconomic conditions. The major role played on health inequality by variables taking part in social exclusion points to the need to focus on the most vulnerable groups. JEL Codes H51, I14, I18 PMID:23241384

  4. Bacteria–bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance

    PubMed Central

    Fraune, Sebastian; Anton-Erxleben, Friederike; Augustin, René; Franzenburg, Sören; Knop, Mirjam; Schröder, Katja; Willoweit-Ohl, Doris; Bosch, Thomas CG

    2015-01-01

    Epithelial surfaces of most animals are colonized by diverse microbial communities. Although it is generally agreed that commensal bacteria can serve beneficial functions, the processes involved are poorly understood. Here we report that in the basal metazoan Hydra, ectodermal epithelial cells are covered with a multilayered glycocalyx that provides a habitat for a distinctive microbial community. Removing this epithelial microbiota results in lethal infection by the filamentous fungus Fusarium sp. Restoring the complex microbiota in gnotobiotic polyps prevents pathogen infection. Although mono-associations with distinct members of the microbiota fail to provide full protection, additive and synergistic interactions of commensal bacteria are contributing to full fungal resistance. Our results highlight the importance of resident microbiota diversity as a protective factor against pathogen infections. Besides revealing insights into the in vivo function of commensal microbes in Hydra, our findings indicate that interactions among commensal bacteria are essential to inhibit pathogen infection. PMID:25514534

  5. Contribution of excited states to stellar weak-interaction rates in odd-A nuclei

    NASA Astrophysics Data System (ADS)

    Sarriguren, P.

    2016-05-01

    Weak-interaction rates, including β decay and electron capture, are studied in several odd-A nuclei in the p f -shell region at various densities and temperatures of astrophysical interest. Special attention is paid to the relative contribution to these rates of thermally populated excited states in the decaying nucleus. The nuclear structure involved in the weak processes is studied within a quasiparticle random-phase approximation with residual interactions in both particle-hole and particle-particle channels on top of a deformed Skyrme Hartree-Fock mean field with pairing correlations. In the range of densities and temperatures considered, it is found that the total rates do not differ much from the rates of the ground state fully populated. In any case, the changes are not larger than the uncertainties due to the nuclear-model dependence of the rates.

  6. Bacteria-bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance.

    PubMed

    Fraune, Sebastian; Anton-Erxleben, Friederike; Augustin, René; Franzenburg, Sören; Knop, Mirjam; Schröder, Katja; Willoweit-Ohl, Doris; Bosch, Thomas C G

    2015-07-01

    Epithelial surfaces of most animals are colonized by diverse microbial communities. Although it is generally agreed that commensal bacteria can serve beneficial functions, the processes involved are poorly understood. Here we report that in the basal metazoan Hydra, ectodermal epithelial cells are covered with a multilayered glycocalyx that provides a habitat for a distinctive microbial community. Removing this epithelial microbiota results in lethal infection by the filamentous fungus Fusarium sp. Restoring the complex microbiota in gnotobiotic polyps prevents pathogen infection. Although mono-associations with distinct members of the microbiota fail to provide full protection, additive and synergistic interactions of commensal bacteria are contributing to full fungal resistance. Our results highlight the importance of resident microbiota diversity as a protective factor against pathogen infections. Besides revealing insights into the in vivo function of commensal microbes in Hydra, our findings indicate that interactions among commensal bacteria are essential to inhibit pathogen infection. PMID:25514534

  7. Regulation of COP1 nuclear localization by the COP9 signalosome via direct interaction with CSN1.

    PubMed

    Wang, Xiping; Li, Wenjun; Piqueras, Raquel; Cao, Kaiming; Deng, Xing Wang; Wei, Ning

    2009-05-01

    COP1 and COP9 signalosome (CSN) are key regulators of plant light responses and development. Deficiency in either COP1 or CSN causes a constitutive photomorphogenic phenotype. Through coordinated actions of nuclear- and cytoplasmic-localization signals, COP1 can respond to light signals by differentially partitions between nuclear and cytoplasmic compartments. Previous genetic analysis in Arabidopsis indicated that the nuclear localization of COP1 requires CSN, an eight-subunit heteromeric complex. However the mechanism underlying the functional relationship between COP1 and CSN is unknown. We report here that COP1 weakly associates with CSN in vivo. Furthermore, we report on the direct interaction involving the coiled-coil domain of COP1 and the N-terminal domain of the CSN1 subunit. In onion epidermal cells, expression of CSN1 can stimulate nuclear localization of GUS-COP1, and the N-terminal domain of CSN1 is necessary and sufficient for this function. Moreover, CSN1-induced COP1 nuclear localization requires the nuclear-localization sequences of COP1, as well as its coiled-coil domain, which contains both the cytoplasmic localization sequences and the CSN1 interacting domain. We also provide genetic evidence that the CSN1 N-terminal domain is specifically required for COP1 nuclear localization in Arabidopsis hypocotyl cells. This study advances our understanding of COP1 localization, and the molecular interactions between COP1 and CSN. PMID:19175768

  8. Analysis of the Contribution of Individual Substituents in 4,6-Aminoglycoside-Ribosome Interaction

    PubMed Central

    Hobbie, Sven N.; Pfister, Peter; Brüll, Christian; Westhof, Eric; Böttger, Erik C.

    2005-01-01

    The 4,6-disubstituted 2-deoxystreptamines interfere with protein biosynthesis by specifically targeting the ribosomal A site. These drugs show subtle variations in the chemical groups of rings I, II, and III. In the present study we used site-directed mutagenesis to generate mutant strains of Mycobacterium smegmatis mc2155 SMR5 ΔrrnB with mutations in its single rRNA allele, rrnA. This genetic procedure gives rise to strains carrying homogeneous populations of mutant ribosomes and was used to study the contribution of individual chemical substituents to the binding of 4,6-disubstituted aminoglycosides. X-ray crystal structures of geneticin and tobramycin complexed to oligonucleotides containing the minimal bacterial ribosomal A site were used for interpretation of MICs determined for a panel of 4,6-aminoglycosides, including tobramycin, kanamycin A, kanamycin B, amikacin, gentamicin, and geneticin. Surprisingly, the considerable differences present within ring III did not seem to alter the interaction of the drug with the ribosome, as determined by site-directed mutagenesis of the A site. In contrast, subtle variations in ring I significantly influenced binding: (i) a 4′-hydroxyl moiety participates in the proper drug target interaction; and (ii) a 2′-amino group contributes an additional positive charge to ring I, making the drug less susceptible to any kind of sequence alteration within the decoding site, most notably, to conformational changes induced by transversion of U1495 to 1495A. The 4-amino-2-hydroxyl-1-oxobutyl extension at position 1 of ring II of amikacin provides an additional anchor and renders amikacin less dependent on the structural conformation of nucleotide U1406 compared to the dependencies of other kanamycins. Overall, the set of interactions forming the complex between drug substituents and nucleotides of the A site constitutes a network in which the interactions can partly compensate for each other when they are disrupted. PMID:16304180

  9. Isolating the non-polar contributions to the intermolecular potential for water-alkane interactions

    NASA Astrophysics Data System (ADS)

    Ballal, Deepti; Venkataraman, Pradeep; Fouad, Wael A.; Cox, Kenneth R.; Chapman, Walter G.

    2014-08-01

    Intermolecular potential models for water and alkanes describe pure component properties fairly well, but fail to reproduce properties of water-alkane mixtures. Understanding interactions between water and non-polar molecules like alkanes is important not only for the hydrocarbon industry but has implications to biological processes as well. Although non-polar solutes in water have been widely studied, much less work has focused on water in non-polar solvents. In this study we calculate the solubility of water in different alkanes (methane to dodecane) at ambient conditions where the water content in alkanes is very low so that the non-polar water-alkane interactions determine solubility. Only the alkane-rich phase is simulated since the fugacity of water in the water rich phase is calculated from an accurate equation of state. Using the SPC/E model for water and TraPPE model for alkanes along with Lorentz-Berthelot mixing rules for the cross parameters produces a water solubility that is an order of magnitude lower than the experimental value. It is found that an effective water Lennard-Jones energy ɛW/k = 220 K is required to match the experimental water solubility in TraPPE alkanes. This number is much higher than used in most simulation water models (SPC/E—ɛW/k = 78.2 K). It is surprising that the interaction energy obtained here is also higher than the water-alkane interaction energy predicted by studies on solubility of alkanes in water. The reason for this high water-alkane interaction energy is not completely understood. Some factors that might contribute to the large interaction energy, such as polarizability of alkanes, octupole moment of methane, and clustering of water at low concentrations in alkanes, are examined. It is found that, though important, these factors do not completely explain the anomalously strong attraction between alkanes and water observed experimentally.

  10. Genetic variations and miRNA-target interactions contribute to natural phenotypic variations in Populus.

    PubMed

    Chen, Jinhui; Xie, Jianbo; Chen, Beibei; Quan, Mingyang; Li, Ying; Li, Bailian; Zhang, Deqiang

    2016-10-01

    Variation in regulatory factors, including microRNAs (miRNAs), contributes to variation in quantitative and complex traits. However, in plants, variants in miRNAs and their target genes that contribute to natural phenotypic variation, and the underlying regulatory networks, remain poorly characterized. We investigated the associations and interactions of single-nucleotide polymorphisms (SNPs) in miRNAs and their target genes with phenotypes in 435 individuals from a natural population of Populus. We used RNA-seq to identify 217 miRNAs differentially expressed in a tension wood system, and identified 1196 candidate target genes; degradome sequencing confirmed 60 of the target sites. In addition, 72 miRNA-target pairs showed significant co-expression. Gene ontology (GO) term analysis showed that most of the genes in the co-regulated pairs participate in biological regulation. Genome resequencing found 5383 common SNPs (frequency ≥ 0.05) in 139 miRNAs and 31 037 SNPs in 819 target genes. Single-SNP association analyses identified 232 significant associations between wood traits (P ≤ 0.05) and SNPs in 102 miRNAs and 1387 associations with 478 target genes. Among these, 102 miRNA-target pairs associated with the same traits. Multi-SNP associations found 102 epistatic pairs associated with traits. Furthermore, a reconstructed regulatory network contained 12 significantly co-expressed pairs, including eight miRNAs and nine targets associated with traits. Lastly, both expression and genetic association showed that miR156i, miR156j, miR396a and miR6445b were involved in the formation of tension wood. This study shows that variants in miRNAs and target genes contribute to natural phenotypic variation and annotated roles and interactions of miRNAs and their target genes by genetic association analysis. PMID:27265357