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Sample records for acid n-terminal domain

  1. The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain.

    PubMed

    Ampah-Korsah, Henry; Anderberg, Hanna I; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel. PMID:27379142

  2. The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain.

    PubMed

    Ampah-Korsah, Henry; Anderberg, Hanna I; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.

  3. The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain

    PubMed Central

    Ampah-Korsah, Henry; Anderberg, Hanna I.; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel. PMID:27379142

  4. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    PubMed Central

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-01-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct. PMID:27681031

  5. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    NASA Astrophysics Data System (ADS)

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-09-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct.

  6. γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains*

    PubMed Central

    Brown, Laura E.; Nicholson, Martin W.; Arama, Jessica E.; Thomson, Alex M.

    2016-01-01

    The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABAA receptors (GABAARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABAARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type of α/β subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, β2, and γ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs and α1/β2/γ2-expressing HEK293 cells, the α1, β2, or γ2 ECD each caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABAARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific. PMID:27129275

  7. NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

    PubMed Central

    Parnham, Stuart; Gaines, William A.; Duggan, Brendan M.; Marcotte, William R.

    2011-01-01

    The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all 1H, 13C, and 15N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes. PMID:21152998

  8. pH-sensitive self-associations of the N-terminal domain of NBCe1-A suggest a compact conformation under acidic intracellular conditions.

    PubMed

    Gill, Harindarpal S

    2012-10-01

    NBCe1-A is an integral membrane protein that cotransports Na+ and HCO3 - ions across the basolateral membrane of the proximal tubule. It is essential for maintaining a homeostatic balance of cellular and blood pH. In X-ray diffraction studies, we reported that the cytoplasmic, N-terminal domain of NBCe1-A (NtNBCe1-A) is a dimer. Here, biophysical measurements show that the dimer is in a concentration-dependent dynamic equilibrium among three additional states in solution that are characterized by its hydrodynamic properties, molar masses, emission spectra, binding properties, and stabilities as a function of pH. Under physiological conditions, dimers are in equilibrium with monomers that are pronounced at low concentration and clusters of molecular masses up to 3-5 times that of a dimer that are pronounced at high concentration. The equilibrium can be influenced so that individual dimers predominate in a taut conformation by lowering the pH. Conversely, dimers begin to relax and disassociate into an increasing population of monomers by elevating the pH. A mechanistic diagram for the inter-conversion of these states is given. The self-associations are further supported by surface plasmon resonance (SPR-Biacore) techniques that illustrate NtNBCe1-A molecules transiently bind with one another. Bicarbonate and bicarbonate-analog bisulfite appear to enhance dimerization and induce a small amount of tetramers. A model is proposed, where the Nt responds to pH or bicarbonate fluctuations inside the cell and plays a role in self-association of entire NBCe1-A molecules in the membrane. PMID:22316307

  9. pH-sensitive Self-associations of the N-terminal Domain of NBCe1-A Suggest a Compact Conformation under Acidic Intracellular Conditions

    PubMed Central

    Gill, Harindarpal S

    2012-01-01

    NBCe1-A is an integral membrane protein that cotransports Na+ and HCO3- ions across the basolateral membrane of the proximal tubule. It is essential for maintaining a homeostatic balance of cellular and blood pH. In X-ray diffraction studies, we reported that the cytoplasmic, N-terminal domain of NBCe1-A (NtNBCe1-A) is a dimer. Here, biophysical measurements show that the dimer is in a concentration-dependent dynamic equilibrium among three additional states in solution that are characterized by its hydrodynamic properties, molar masses, emission spectra, binding properties, and stabilities as a function of pH. Under physiological conditions, dimers are in equilibrium with monomers that are pronounced at low concentration and clusters of molecular masses up to 3-5 times that of a dimer that are pronounced at high concentration. The equilibrium can be influenced so that individual dimers predominate in a taut conformation by lowering the pH. Conversely, dimers begin to relax and disassociate into an increasing population of monomers by elevating the pH. A mechanistic diagram for the inter-conversion of these states is given. The self-associations are further supported by surface plasmon resonance (SPR-Biacore) techniques that illustrate NtNBCe1-A molecules transiently bind with one another. Bicarbonate and bicarbonate-analog bisulfite appear to enhance dimerization and induce a small amount of tetramers. A model is proposed, where the Nt responds to pH or bicarbonate fluctuations inside the cell and plays a role in self-association of entire NBCe1-A molecules in the membrane. PMID:22316307

  10. The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain.

    PubMed

    Pedersen, L B; Birkelund, S; Holm, A; Ostergaard, S; Christiansen, G

    1996-02-01

    The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may, in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect the DNA-binding properties of Hc1. PMID:8576073

  11. A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor alpha is essential for transcriptional activation and interaction with the general transcription factor TFIIB.

    PubMed Central

    Hadzic, E; Desai-Yajnik, V; Helmer, E; Guo, S; Wu, S; Koudinova, N; Casanova, J; Raaka, B M; Samuels, H H

    1995-01-01

    The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand

  12. Luminescent and substrate binding activities of firefly luciferase N-terminal domain.

    PubMed

    Zako, Tamotsu; Ayabe, Keiichi; Aburatani, Takahide; Kamiya, Noriho; Kitayama, Atsushi; Ueda, Hiroshi; Nagamune, Teruyuki

    2003-07-30

    Firefly luciferase catalyzes highly efficient emission of light from the substrates luciferin, Mg-ATP, and oxygen. A number of amino acid residues are identified to be important for the luminescent activity, and almost all the key residues are thought to be located in the N-terminal domain (1-437), except one in the C-terminal domain, Lys529, which is thought to be critical for efficient substrate orientation. Here we show that the purified N-terminal domain still binds to the substrates luciferin and ATP with reduced affinity, and retains luminescent activity of up to 0.03% of the wild-type enzyme (WT), indicating that all the essential residues for the activity are located in the N-terminal domain. Also found is low luminescence enhancement by coenzyme A (CoA), which implies a lower product inhibition than in the WT enzyme. These findings have interesting implications for the light emission reaction mechanism of the enzyme, such as reaction intermediates, product inhibition, and the role of the C-terminal domain.

  13. Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains

    PubMed Central

    Jacewicz, Agata

    2015-01-01

    ABSTRACT Mycobacterium smegmatis encodes several DNA repair polymerases that are adept at incorporating ribonucleotides, which raises questions about how ribonucleotides in DNA are sensed and removed. RNase H enzymes, of which M. smegmatis encodes four, are strong candidates for a surveillance role. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of M. smegmatis RnhC, a bifunctional RNase H and acid phosphatase. We report that (i) the RnhC nuclease is stringently specific for RNA:DNA hybrid duplexes; (ii) RnhC does not selectively recognize and cleave DNA-RNA or RNA-DNA junctions in duplex nucleic acid; (iii) RnhC cannot incise an embedded monoribonucleotide or diribonucleotide in duplex DNA; (iv) RnhC can incise tracts of 4 or more ribonucleotides embedded in duplex DNA, leaving two or more residual ribonucleotides at the cleaved 3′-OH end and at least one or two ribonucleotides on the 5′-PO4 end; (v) the RNase H activity is inherent in an autonomous 140-amino-acid (aa) N-terminal domain of RnhC; and (vi) the C-terminal 211-aa domain of RnhC is an autonomous acid phosphatase. The cleavage specificity of RnhC is clearly distinct from that of Escherichia coli RNase H2, which selectively incises at an RNA-DNA junction. Thus, we classify RnhC as a type I RNase H. The properties of RnhC are consistent with a role in Okazaki fragment RNA primer removal or in surveillance of oligoribonucleotide tracts embedded in DNA but not in excision repair of single misincorporated ribonucleotides. IMPORTANCE RNase H enzymes help cleanse the genome of ribonucleotides that are present either as ribotracts (e.g., RNA primers) or as single ribonucleotides embedded in duplex DNA. Mycobacterium smegmatis encodes four RNase H proteins, including RnhC, which is characterized in this study. The nucleic acid substrate and cleavage site specificities of RnhC are consistent with a role in initiating the removal of ribotracts but not in single

  14. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of delta6-desaturated fatty acids in transgenic tobacco.

    PubMed

    Sayanova, O; Smith, M A; Lapinskas, P; Stobart, A K; Dobson, G; Christie, W W; Shewry, P R; Napier, J A

    1997-04-15

    gamma-Linolenic acid (GLA; C18:3 delta(6,9,12)) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a delta6-fatty acid desaturase using linoleic acid (C18:2 delta(9,12)) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the delta6-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 delta(6,9,12,15)) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage delta6-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b5.

  15. Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.

    PubMed Central

    Sanford, J C; Pan, Y; Wessling-Resnick, M

    1995-01-01

    Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue. Images PMID:7749197

  16. N-terminal domain of complexin independently activates calcium-triggered fusion

    PubMed Central

    Lai, Ying; Choi, Ucheor B.; Zhang, Yunxiang; Zhao, Minglei; Pfuetzner, Richard A.; Wang, Austin L.; Brunger, Axel T.

    2016-01-01

    Complexin activates Ca2+-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca2+-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca2+-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca2+-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca2+-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca2+-triggered release. PMID:27444020

  17. N-terminal domain of complexin independently activates calcium-triggered fusion.

    PubMed

    Lai, Ying; Choi, Ucheor B; Zhang, Yunxiang; Zhao, Minglei; Pfuetzner, Richard A; Wang, Austin L; Diao, Jiajie; Brunger, Axel T

    2016-08-01

    Complexin activates Ca(2+)-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca(2+)-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca(2+)-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca(2+)-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca(2+)-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca(2+)-triggered release. PMID:27444020

  18. N-terminal domain of complexin independently activates calcium-triggered fusion.

    PubMed

    Lai, Ying; Choi, Ucheor B; Zhang, Yunxiang; Zhao, Minglei; Pfuetzner, Richard A; Wang, Austin L; Diao, Jiajie; Brunger, Axel T

    2016-08-01

    Complexin activates Ca(2+)-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca(2+)-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca(2+)-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca(2+)-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca(2+)-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca(2+)-triggered release.

  19. The Pitx2c N-terminal domain is a critical interaction domain required for asymmetric morphogenesis

    PubMed Central

    Simard, Annie; Di Giorgio, Luciano; Amen, Melanie; Westwood, Ashley; Amendt, Brad A.; Ryan, Aimee K.

    2010-01-01

    The paired-like homeodomain transcription factor Pitx2c has an essential role in patterning the left-right axis. However, neither its transcriptional targets nor the molecular mechanisms through which it exerts its patterning function are known. Here we provide evidence that the N-terminal domain of Pitx2c is important for this activity. Overexpression of the Pitx2c N-terminus in ovo randomizes the direction of heart looping, the first morphological asymmetry conserved in vertebrate embryos. In addition, the Pitx2c N-terminal domain blocks the ability of Pitx2c to synergize with Nkx2.5 to transactivate the procollagen lysyl hydroxylase (Plod-1) promoter in transient transfection assays. A five amino acid region containing leucine-41 is required for both of these effects. Our data suggest that the Pitx2c N-terminal domain competes with endogenous Pitx2c for binding to a protein interaction partner that is required for the activation of genes that direct asymmetric morphogenesis along the left-right axis. PMID:19681163

  20. Structure of the EMMPRIN N-terminal domain 1: Dimerization via [beta]-strand swapping

    SciTech Connect

    Luo, Jinquan; Teplyakov, Alexey; Obmolova, Galina; Malia, Thomas; Wu, Sheng-Jiun; Beil, Eric; Baker, Audrey; Swencki-Underwood, Bethany; Zhao, Yonghong; Sprenkle, Justin; Dixon, Ken; Sweet, Raymond; Gilliland, Gary L.

    2010-09-27

    Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as Hab18G, CD147, Basigin, M6, and neurothelin, is a membrane glycoprotein expressed on the surface of various cell types and many cancer cells. EMMPRIN stimulates adjacent fibroblasts and tumor cells to produce matrix metalloproteinases and plays an important role in tumor invasion and metastasis, angiogenesis, spermatogensis and fertilization, cell-cell adhesion and communication, and other biological processes (reviewed in Ref. 1 and references therein). It was demonstrated that the EMMPRIN extracellular domain (ECD), which structurally belongs to the IgG superfamily, can form homo-oligomers in a cis dependent manner and the N-terminal domain 1 (residues 22-101) was necessary and sufficient to mediate this interaction. The crystal structure of the ECD of recombinant human EMMPRIN (Hab18G/CD147) expressed in E. coli was reported at 2.8 {angstrom} resolution (Yu et al. 2008). The construct consists of residues 22-205 of the mature protein and has both an N-terminal IgC2 domain (ND1, residues 22-101) and a C-terminal IgC2 domain (ND2, residues 107-205). The two domains are joined by a five amino acid residue linker that constitutes a flexible hinge between the two domains. The crystal form has four copies of the molecule in the asymmetric unit, each of which has a different inter-domain angle that varies from 121{sup o} to 144{sup o}. The two domains each have a conserved disulfide bridge and both are comprised of two {beta}-sheets formed by strands EBA and GFCC, and DEBA and AGFCC for ND1 and ND2, respectively. Based on the crystal packing in this structure, the authors proposed that lateral packing between the two IgG domains of EMMPRIN ECD represents a potential mechanism for cell adhesion. Here we report the 2.0-{angstrom} crystal structure of the N-terminal domain of EMMPRIN ECD (ND1) expressed in mammalian cells. The overall structure of the domain is very similar to that in the full length

  1. The charged region of Hsp90 modulates the function of the N-terminal domain

    PubMed Central

    Scheibel, Thomas; Siegmund, Heiko Ingo; Jaenicke, Rainer; Ganz, Peter; Lilie, Hauke; Buchner, Johannes

    1999-01-01

    Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90. PMID:9990018

  2. Expression and characterization of the intact N-terminal domain of streptokinase.

    PubMed Central

    Azuaga, A. I.; Woodruff, N. D.; Conejero-Lara, F.; Cox, V. F.; Smith, R. A.; Dobson, C. M.

    1999-01-01

    Proteolytic studies have enabled two of the three putative domains of the fibrinolytic protein streptokinase to be isolated and characterized (Conejero-Lara F et al., 1996, Protein Sci 5:2583-2591). The N-terminal domain, however, could not be isolated in these experiments because of its susceptibility to proteolytic cleavage. To complete the biophysical characterization of the domain structure of streptokinase we have overexpressed, purified, and characterized the N-terminal region of the protein, residues 1-146. The results show this is cooperatively folded with secondary structure content and overall stability closely similar to those of the equivalent region in the intact protein. PMID:10048340

  3. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA

    PubMed Central

    Dwivedi, Gajendradhar R.; Srikanth, Kolluru D.; Anand, Praveen; Naikoo, Javed; Srilatha, N. S.; Rao, Desirazu N.

    2015-01-01

    DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA) and double stranded DNA (dsDNA). Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed. PMID:26135134

  4. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA.

    PubMed

    Dwivedi, Gajendradhar R; Srikanth, Kolluru D; Anand, Praveen; Naikoo, Javed; Srilatha, N S; Rao, Desirazu N

    2015-01-01

    DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA) and double stranded DNA (dsDNA). Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed. PMID:26135134

  5. Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae.

    PubMed

    Edwin, Aaron; Persson, Cecilia; Mayzel, Maxim; Wai, Sun Nyunt; Öhman, Anders; Karlsson, B Göran; Sauer-Eriksson, A Elisabeth

    2015-12-01

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N-terminal domain (residues 23-103) that contains two short α-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.

  6. Crystal structure of the Sec18p N-terminal domain

    PubMed Central

    Babor, S. Mariana; Fass, Deborah

    1999-01-01

    Yeast Sec18p and its mammalian orthologue N-ethylmaleimide-sensitive fusion protein (NSF) are hexameric ATPases with a central role in vesicle trafficking. Aided by soluble adapter factors (SNAPs), Sec18p/NSF induces ATP-dependent disassembly of a complex of integral membrane proteins from the vesicle and target membranes (SNAP receptors). During the ATP hydrolysis cycle, the Sec18p/NSF homohexamer undergoes a large-scale conformational change involving repositioning of the most N terminal of the three domains of each protomer, a domain that is required for SNAP-mediated interaction with SNAP receptors. Whether an internal conformational change in the N-terminal domains accompanies their reorientation with respect to the rest of the hexamer remains to be addressed. We have determined the structure of the N-terminal domain from Sec18p by x-ray crystallography. The Sec18p N-terminal domain consists of two β-sheet-rich subdomains connected by a short linker. A conserved basic cleft opposite the linker may constitute a SNAP-binding site. Despite structural variability in the linker region and in an adjacent loop, all three independent molecules in the crystal asymmetric unit have the identical subdomain interface, supporting the notion that this interface is a preferred packing arrangement. However, the linker flexibility allows for the possibility that other subdomain orientations may be sampled. PMID:10611286

  7. The N-terminal domain determines the affinity and specificity of H1 binding to chromatin

    SciTech Connect

    Oeberg, Christine; Belikov, Sergey

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer wt Human histone H1.4 and hH1.4 devoid of N-terminal domain, {Delta}N-hH1.4, were compared. Black-Right-Pointing-Pointer Both histones bind to chromatin, however, {Delta}N-hH1.4 displays lower binding affinity. Black-Right-Pointing-Pointer Interaction of {Delta}N-hH1.4 with chromatin includes a significant unspecific component. Black-Right-Pointing-Pointer N-terminal domain is a determinant of specificity of histone H1 binding to chromatin. -- Abstract: Linker histone H1, one of the most abundant nuclear proteins in multicellular eukaryotes, is a key component of the chromatin structure mainly due to its role in the formation and maintenance of the 30 nm chromatin fiber. It has a three-domain structure; a central globular domain flanked by a short N-terminal domain and a long, highly basic C-terminal domain. Previous studies have shown that the binding abilities of H1 are at large determined by the properties of the C-terminal domain; much less attention has been paid to role of the N-terminal domain. We have previously shown that H1 can be reconstituted via cytoplasmic mRNA injection in Xenopus oocytes, cells that lack somatic H1. The heterologously expressed H1 proteins are incorporated into in vivo assembled chromatin at specific sites and the binding event is monitored as an increase in nucleosomal repeat length (NRL). Using this setup we have here compared the binding properties of wt-H1.4 and hH1.4 devoid of its N-terminal domain ({Delta}N-hH1.4). The {Delta}N-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wild type hH1.4. Our data also indicates that {Delta}N-hH1.4 is more prone to unspecific chromatin binding than the wild type. We conclude that the N-terminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions.

  8. The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

    PubMed

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-05-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats). PMID:25792750

  9. The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

    PubMed

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-05-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats).

  10. Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase

    PubMed Central

    Qiu, Shihong; Ogino, Minako; Luo, Ming

    2015-01-01

    ABSTRACT Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae, Paramyxoviridae, and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. IMPORTANCE Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the

  11. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

    PubMed

    Kereïche, Sami; Kováčik, Lubomír; Bednár, Jan; Pevala, Vladimír; Kunová, Nina; Ondrovičová, Gabriela; Bauer, Jacob; Ambro, Ľuboš; Bellová, Jana; Kutejová, Eva; Raška, Ivan

    2016-01-01

    Lon is an essential, multitasking AAA(+) protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon's N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning. PMID:27632940

  12. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease

    PubMed Central

    Kereïche, Sami; Kováčik, Lubomír; Bednár, Jan; Pevala, Vladimír; Kunová, Nina; Ondrovičová, Gabriela; Bauer, Jacob; Ambro, Ľuboš; Bellová, Jana; Kutejová, Eva; Raška, Ivan

    2016-01-01

    Lon is an essential, multitasking AAA+ protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon’s N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning. PMID:27632940

  13. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

    PubMed

    Kereïche, Sami; Kováčik, Lubomír; Bednár, Jan; Pevala, Vladimír; Kunová, Nina; Ondrovičová, Gabriela; Bauer, Jacob; Ambro, Ľuboš; Bellová, Jana; Kutejová, Eva; Raška, Ivan

    2016-01-01

    Lon is an essential, multitasking AAA(+) protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon's N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning.

  14. Structure of the N-terminal domain of human thioredoxin-interacting protein.

    PubMed

    Polekhina, Galina; Ascher, David Benjamin; Kok, Shie Foong; Beckham, Simone; Wilce, Matthew; Waltham, Mark

    2013-03-01

    Thioredoxin-interacting protein (TXNIP) is one of the six known α-arrestins and has recently received considerable attention owing to its involvement in redox signalling and metabolism. Various stress stimuli such as high glucose, heat shock, UV, H2O2 and mechanical stress among others robustly induce the expression of TXNIP, resulting in the sequestration and inactivation of thioredoxin, which in turn leads to cellular oxidative stress. While TXNIP is the only α-arrestin known to bind thioredoxin, TXNIP and two other α-arrestins, Arrdc4 and Arrdc3, have been implicated in metabolism. Furthermore, owing to its roles in the pathologies of diabetes and cardiovascular disease, TXNIP is considered to be a promising drug target. Based on their amino-acid sequences, TXNIP and the other α-arrestins are remotely related to β-arrestins. Here, the crystal structure of the N-terminal domain of TXNIP is reported. It provides the first structural information on any of the α-arrestins and reveals that although TXNIP adopts a β-arrestin fold as predicted, it is structurally more similar to Vps26 proteins than to β-arrestins, while sharing below 15% pairwise sequence identity with either.

  15. N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

    PubMed Central

    Taggart, David J.; Dayeh, Daniel M.; Fredrickson, Saul W.; Suo, Zucai

    2014-01-01

    The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase

  16. The functional integrity of the serpin domain of C1-inhibitor depends on the unique N-terminal domain, as revealed by a pathological mutant.

    PubMed

    Bos, Ineke G A; Lubbers, Yvonne T P; Roem, Dorina; Abrahams, Jan Pieter; Hack, C Erik; Eldering, Eric

    2003-08-01

    C1-inhibitor (C1-Inh) is a serine protease inhibitor (serpin) with a unique, non-conserved N-terminal domain of unknown function. Genetic deficiency of C1-Inh causes hereditary angioedema. A novel type of mutation (Delta 3) in exon 3 of the C1-Inh gene, resulting in deletion of Asp62-Thr116 in this unique domain, was encountered in a hereditary angioedema pedigree. Because the domain is supposedly not essential for inhibitory activity, the unexpected loss-of-function of this deletion mutant was further investigated. The Delta 3 mutant and three additional mutants starting at Pro76, Gly98, and Ser115, lacking increasing parts of the N-terminal domain, were produced recombinantly. C1-Inh76 and C1-Inh98 retained normal conformation and interaction kinetics with target proteases. In contrast, C1-Inh115 and Delta 3, which both lack the connection between the serpin and the non-serpin domain via two disulfide bridges, were completely non-functional because of a complex-like and multimeric conformation, as demonstrated by several criteria. The Delta 3 mutant also circulated in multimeric form in plasma from affected family members. The C1-Inh mutant reported here is unique in that deletion of an entire amino acid stretch from a domain not shared by other serpins leads to a loss-of-function. The deletion in the unique N-terminal domain results in a "multimerization phenotype" of C1-Inh, because of diminished stability of the central beta-sheet. This phenotype, as well as the location of the disulfide bridges between the serpin and the non-serpin domain of C1-Inh, suggests that the function of the N-terminal region may be similar to one of the effects of heparin in antithrombin III, maintenance of the metastable serpin conformation.

  17. Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of gamma-Gliadin.

    PubMed

    Benitez-Cardoza, Claudia G; Rogniaux, Hélène; Popineau, Yves; Guéguen, Jacques

    2006-04-01

    The gene encoding the repetitive domain located in the N-terminal half of gamma-Gliadin from wheat endosperm has been subcloned into a thioredoxin expression system (pET102/D-Topo). It was over-expressed as fusion protein with thioredoxin in Escherichia coli. Thioredoxin was removed by enterokinase cleavage or by acid cleavage at the respective engineered recognition sites. The soluble N-terminal half of gamma-Gliadin was purified by affinity and reverse-phase chromatography. While, the enterokinase cleavage leaded to only one species detectable by mass spectroscopy, the acid cleavage resulted in a three different length polypeptides, due to the presence of the same number of acid cleavage sites. The secondary structure of the purified protein domain was analysed by circular dichroism, showing an spectral shape common to a Poly(Pro) II conformation. The spectrum is dominated by a large negative peak centred around 201 nm and a broad shoulder centred around 225 nm. Also, the temperature denaturation process was studied. The differences observed in the spectra show two main tendencies, the increment of the shoulder intensity, and the drop of the intensity of the peak around 201. When the sample was cooled down, the change on intensity of the shoulder around 225 was completely reversible and that around the 201 nm peak reached a reversibility of 90%. Such structure and thermal behaviour are characteristic of the repetitive domains of the wheat prolamins.

  18. Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.

    PubMed

    Lamb, Brian M; Mercer, Andrew C; Barbas, Carlos F

    2013-11-01

    Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5'-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5' T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5' T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes.

  19. N-terminal domain of turkey pancreatic lipase is active on long chain triacylglycerols and stabilized by colipase.

    PubMed

    Bou Ali, Madiha; Karray, Aida; Gargouri, Youssef; Ben Ali, Yassine

    2013-01-01

    The gene encoding the TPL N-terminal domain (N-TPL), fused with a His6-tag, was cloned and expressed in Pichia pastoris, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The recombinant protein was successfully expressed and secreted with an expression level of 5 mg/l of culture medium after 2 days of culture. The N-TPL was purified through a one-step Ni-NTA affinity column with a purification factor of approximately 23-fold. The purified N-TPL, with a molecular mass of 35 kDa, had a specific activity of 70 U/mg on tributyrin. Surprisingly, this domain was able to hydrolyse long chain TG with a specific activity of 11 U/mg using olive oil as substrate. This result was confirmed by TLC analysis showing that the N-TPL was able to hydrolyse insoluble substrates as olive oil. N-TPL was unstable at temperatures over 37°C and lost 70% of its activity at acid pH, after 5 min of incubation. The N-TPL exhibited non linear kinetics, indicating its rapid denaturation at the tributyrin-water interface. Colipase increased the N-TPL stability at the lipid-water interface, so the TPL N-terminal domain probably formed functional interactions with colipase despite the absence of the C-terminal domain.

  20. The pro-enzyme C-terminal processing domain of Pholiota nameko tyrosinase is responsible for folding of the N-terminal catalytic domain.

    PubMed

    Moe, Lai Lai; Maekawa, Saya; Kawamura-Konishi, Yasuko

    2015-07-01

    Pholiota nameko (Pholiota microspore) tyrosinase is expressed as a latent 67-kDa pro-tyrosinase, comprising a 42-kDa N-terminal catalytic domain with a binuclear copper centre and a 25-kDa C-terminal domain and is activated by proteolytic digestion of the C-terminal domain. To investigate the role of the C-terminal processing domain of pro-tyrosinase, we constructed a recombinant tyrosinase lacking the C-terminal domain and four recombinant pro-tyrosinase mutants (F515G, H539N, L540G and Y543G) carrying substituted amino acid residues on the C-terminal domain. The recombinant tyrosinase lacking the C-terminal domain had no catalytic activity; whereas the mutant L540G was copper depleted, the other mutants had copper contents similar to that of the wild-type pro-tyrosinase. Proteolytic digestion activated the mutants H539N and Y543G following release of the C-terminal domain, and the resulting tyrosinases had higher K m values for t-butyl catechol than the wild-type pro-tyrosinase. The mutants F515G and L540G were degraded by proteolytic digestion and yielded smaller proteins with no activity. These data suggest that the C-terminal processing domain of P. nameko pro-tyrosinase is essential for correct folding of the N-terminal catalytic domain and acts as an intramolecular chaperone during assembly of the active-site conformation.

  1. Miro's N-terminal GTPase domain is required for transport of mitochondria into axons and dendrites.

    PubMed

    Babic, Milos; Russo, Gary J; Wellington, Andrea J; Sangston, Ryan M; Gonzalez, Migdalia; Zinsmaier, Konrad E

    2015-04-01

    Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiroT460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiroT460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, we show that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, our analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state.

  2. Proline-directed phosphorylation of the dopamine transporter N-terminal domain

    PubMed Central

    Gorentla, Balachandra K.; Moritz, Amy E.; Foster, James D.; Vaughan, Roxanne A.

    2009-01-01

    Phosphorylation of the dopamine transporter (DAT) on N-terminal serines and unidentified threonines occurs concomitantly with PKC- and substrate-induced alterations in transporter activity, subcellular distribution, and dopamine efflux, but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. As one approach to investigating these issues we recombinantly expressed the N-terminal tail of rat DAT (NDAT) and examined its phosphorylation and dephosphorylation properties in vitro. We found that NDAT could be phosphorylated to significant levels by PKCα, PKA, PKG, and CaMKII, which catalyzed serine phosphorylation, and ERK1, JNK, and p38, which catalyzed threonine phosphorylation. We identified Thr53, present in a membrane proximal proline-directed kinase motif as the NDAT site phosphorylated in vitro by ERK1, JNK and p38, and confirmed by peptide mapping and mutagenesis that Thr53 is phosphorylated in vivo. Dephosphorylation studies showed that protein phosphatase 1 catalyzed near-complete in vitro dephosphorylation of PKCα-phosphorylated NDAT, similar to its in vivo and in vitro effects on native DAT. These findings demonstrate the ability of multiple enzymes to directly recognize the DAT N-terminal domain and for kinases to act at multiple distinct sites. The strong correspondence between NDAT and rDAT phosphorylation characteristics suggests the potential for the enzymes that are active on NDAT in vitro to act on DAT in vivo and indicates the usefulness of NDAT for guiding future DAT phosphorylation analyses. PMID:19146407

  3. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

    PubMed

    Obergfell, Kyle P; Seifert, H Steven

    2016-05-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants

  4. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

    PubMed

    Obergfell, Kyle P; Seifert, H Steven

    2016-05-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants

  5. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation

    PubMed Central

    2016-01-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic

  6. Direct interaction of the N-terminal domain of ribosomal protein S1 with protein S2 in Escherichia coli.

    PubMed

    Byrgazov, Konstantin; Manoharadas, Salim; Kaberdina, Anna C; Vesper, Oliver; Moll, Isabella

    2012-01-01

    Despite of the high resolution structure available for the E. coli ribosome, hitherto the structure and localization of the essential ribosomal protein S1 on the 30 S subunit still remains to be elucidated. It was previously reported that protein S1 binds to the ribosome via protein-protein interaction at the two N-terminal domains. Moreover, protein S2 was shown to be required for binding of protein S1 to the ribosome. Here, we present evidence that the N-terminal domain of S1 (amino acids 1-106; S1(106)) is necessary and sufficient for the interaction with protein S2 as well as for ribosome binding. We show that over production of protein S1(106) affects E. coli growth by displacing native protein S1 from its binding pocket on the ribosome. In addition, our data reveal that the coiled-coil domain of protein S2 (S2α(2)) is sufficient to allow protein S1 to bind to the ribosome. Taken together, these data uncover the crucial elements required for the S1/S2 interaction, which is pivotal for translation initiation on canonical mRNAs in gram-negative bacteria. The results are discussed in terms of a model wherein the S1/S2 interaction surface could represent a possible target to modulate the selectivity of the translational machinery and thereby alter the translational program under distinct conditions.

  7. N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization.

    PubMed

    Cadwallader, K A; Paterson, H; Macdonald, S G; Hancock, J F

    1994-07-01

    Plasma membrane targeting of Ras requires CAAX motif modifications together with a second signal from an adjacent polybasic domain or nearby cysteine palmitoylation sites. N-terminal myristoylation is known to restore membrane binding to H-ras C186S (C-186 is changed to S), a mutant protein in which all CAAX processing is abolished. We show here that myristoylated H-ras C186S is a substrate for palmitoyltransferase, despite the absence of C-terminal farnesylation, and that palmitoylation is absolutely required for plasma membrane targeting of myristoylated H-ras. Similarly, the polybasic domain is required for specific plasma membrane targeting of myristoylated K-ras. In contrast, the combination of myristoylation plus farnesylation results in the mislocalization of Ras to numerous intracellular membranes. Ras that is only myristoylated does not bind with a high affinity to any membrane. The specific targeting of Ras to the plasma membrane is therefore critically dependent on signals that are contained in the hypervariable domain but can be supported by N-terminal myristoylation or C-terminal prenylation. Interestingly, oncogenic Ras G12V that is localized correctly to the plasma membrane leads to mitogen-activated protein kinase activation irrespective of the combination of targeting signals used for localization, whereas Ras G12V that is mislocalized to the cytosol or to other membranes activates mitogen-activated protein kinase only if the Ras protein is farnesylated.

  8. Size does matter: 18 amino acids at the N-terminal tip of an amino acid transporter in Leishmania determine substrate specificity

    PubMed Central

    Schlisselberg, Doreen; Mazarib, Eldar; Inbar, Ehud; Rentsch, Doris; Myler, Peter J.; Zilberstein, Dan

    2015-01-01

    Long N-terminal tails of amino acid transporters are known to act as sensors of the internal pool of amino acids and as positive regulators of substrate flux rate. In this study we establish that N-termini of amino acid transporters can also determine substrate specificity. We show that due to alternative trans splicing, the human pathogen Leishmania naturally expresses two variants of the proline/alanine transporter, one 18 amino acid shorter than the other. We demonstrate that the longer variant (LdAAP24) translocates both proline and alanine, whereas the shorter variant (∆18LdAAP24) translocates just proline. Remarkably, co-expressing the hydrophilic N-terminal peptide of the long variant with ∆18LdAAP24 was found to recover alanine transport. This restoration of alanine transport could be mediated by a truncated N-terminal tail, though truncations exceeding half of the tail length were no longer functional. Taken together, the data indicate that the first 18 amino acids of the negatively charged N-terminal LdAAP24 tail are required for alanine transport and may facilitate the electrostatic interactions of the entire negatively charged N-terminal tail with the positively charged internal loops in the transmembrane domain, as this mechanism has been shown to underlie regulation of substrate flux rate for other transporters. PMID:26549185

  9. Chloride transporter KCC2-dependent neuroprotection depends on the N-terminal protein domain.

    PubMed

    Winkelmann, A; Semtner, M; Meier, J C

    2015-01-01

    Neurodegeneration is a serious issue of neurodegenerative diseases including epilepsy. Downregulation of the chloride transporter KCC2 in the epileptic tissue may not only affect regulation of the polarity of GABAergic synaptic transmission but also neuronal survival. Here, we addressed the mechanisms of KCC2-dependent neuroprotection by assessing truncated and mutated KCC2 variants in different neurotoxicity models. The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased chloride transport activity, but they also identify the KCC2 N-terminal domain (NTD) as the relevant minimal KCC2 protein domain that is sufficient for neuroprotection. As ectopic expression of the KCC2-NTD works independently of full-length KCC2-dependent regulation of Cl(-) transport or structural KCC2 C-terminus-dependent regulation of synaptogenesis, our study may pave the way for a selective neuroprotective therapeutic strategy that will be applicable to a wide range of neurodegenerative diseases. PMID:26043076

  10. PACSIN 1 forms tetramers via its N-terminal F-BAR domain.

    PubMed

    Halbach, Arndt; Mörgelin, Matthias; Baumgarten, Maria; Milbrandt, Mark; Paulsson, Mats; Plomann, Markus

    2007-02-01

    The ability of protein kinase C and casein kinase 2 substrate in neurons (PACSIN)/syndapin proteins to self-polymerize is crucial for the simultaneous interactions with more than one Src homology 3 domain-binding partner or with lipid membranes. The assembly of this network has profound effects on the neural Wiskott-Aldrich syndrome protein-mediated attachment of the actin polymerization machinery to vesicle membranes as well as on the movement of the corresponding vesicles. Also, the sensing of vesicle membranes and/or the induction of membrane curvature are more easily facilitated in the presence of larger PACSIN complexes. The N-terminal Fes-CIP homology and Bin-Amphiphysin-Rvs (F-BAR) domains of several PACSIN-related proteins have been shown to mediate self-interactions, whereas studies using deletion mutants derived from closely related proteins led to the view that oligomerization depends on the formation of a trimeric complex via a coiled-coil region present in these molecules. To address whether the model of trimeric complex formation is applicable to PACSIN 1, the protein was recombinantly expressed and tested in four different assays for homologous interactions. The results showed that PACSIN 1 forms tetramers of about 240 kDa, with the self-interaction having a K(D) of 6.4 x 10(-8) M. Ultrastructural analysis of these oligomers after negative staining showed that laterally arranged PACSIN molecules bind to each other via a large globular domain and form a barrel-like structure. Together, these results demonstrate that the N-terminal F-BAR domain of PACSIN 1 forms the contact site for a tetrameric structure, which is able to simultaneously interact with multiple Src homology 3 binding partners. PMID:17288557

  11. N-terminal domains of native multidomain proteins have the potential to assist de novo folding of their downstream domains in vivo by acting as solubility enhancers

    PubMed Central

    Kim, Chul Woo; Han, Kyoung Sim; Ryu, Ki-Sun; Kim, Byung Hee; Kim, Kyun-Hwan; Choi, Seong Il; Seong, Baik L.

    2007-01-01

    The fusion of soluble partner to the N terminus of aggregation-prone polypeptide has been popularly used to overcome the formation of inclusion bodies in the E. coli cytosol. The chaperone-like functions of the upstream fusion partner in the artificial multidomain proteins could occur in de novo folding of native multidomain proteins. Here, we show that the N-terminal domains of three E. coli multidomain proteins such as lysyl-tRNA synthetase, threonyl-tRNA synthetase, and aconitase are potent solubility enhancers for various C-terminal heterologous proteins. The results suggest that the N-terminal domains could act as solubility enhancers for the folding of their authentic C-terminal domains in vivo. Tandem repeat of N-terminal domain or insertion of aspartic residues at the C terminus of the N-terminal domain also increased the solubility of fusion proteins, suggesting that the solubilizing ability correlates with the size and charge of N-terminal domains. The solubilizing ability of N-terminal domains would contribute to the autonomous folding of multidomain proteins in vivo, and based on these results, we propose a model of how N-terminal domains solubilize their downstream domains. PMID:17384228

  12. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain

    PubMed Central

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-01-01

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23–230) as detected by [1H, 15N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn2+-binding to the octarepeat motif. PMID:27341298

  13. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain.

    PubMed

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-01-01

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif. PMID:27341298

  14. Lipid binding ability of human apolipoprotein E N-terminal domain isoforms: correlation with protein stability?

    PubMed

    Weers, Paul M M; Narayanaswami, Vasanthy; Choy, Nicole; Luty, Robert; Hicks, Les; Kay, Cyril M; Ryan, Robert O

    2003-01-01

    Human apolipoprotein (apo) E exists as one of three major isoforms, E2, E3 or E4. Individuals carrying the epsilon 4 allele have an increased risk of heart disease and premature onset of Alzheimer's disease. To investigate the molecular basis for this phenomenon, the N-terminal domain of apoE3, apoE2 and apoE4 were expressed in bacteria, isolated and employed in lipid binding and stability studies. Far UV circular dichroism spectroscopy in buffer at pH 7 revealed a similar amount of alpha-helix secondary structure for the three isoforms. By contrast, differences were noted in apoE-NT isoform-specific transformation of bilayer vesicles of dimyristoylphosphatidylglycerol (DMPG) into discoidal complexes. ApoE4-NT induced transformation was most rapid, followed by apoE3-NT and apoE2-NT. To determine if differences in the rate of apoE-NT induced DMPG vesicle transformation is due to isoform-specific differences in helix bundle stability, guanidine HCl denaturation studies were conducted. The results revealed that apoE2-NT was the most stable, followed by apoE3-NT and apoE4-NT, establishing an inverse correlation between helix bundle stability and DMPG vesicle transformation rate at pH 7. When the zwitterionic dimyristoylphosphatidylcholine (DMPC) was employed as the model lipid surface, interaction of apoE-NT isoforms with the lipid substrate was slow. However, upon lowering the pH from 7 to 3, a dramatic increase in the rate of DMPC vesicle transformation rate was observed for each isoform. To evaluate if the increased DMPC vesicle transformation rates observed at low pH is due to pH-dependent alterations in helix bundle stability, guanidine HCl denaturation studies were performed. ApoE2-NT and apoE3-NT displayed increased resistance to denaturation as a function of decreasing pH, while apoE4-NT showed no change in stability. Studies with the fluorescent probe, 8-anilino-1-naphthalene sulfonic acid, indicated an increase in apoE hydrophobic surface exposure upon

  15. Differentiating N-terminal aspartic and isoaspartic acid residues in peptides.

    PubMed

    Sargaeva, Nadezda P; Lin, Cheng; O'Connor, Peter B

    2011-09-01

    Formation of isoaspartic acid (isoAsp) is a common modification of aspartic acid (Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar properties and identical molecular mass. It was recently shown that they can be differentiated using ion-electron or ion-ion interaction fragmentation methods (ExD) because these methods provide diagnostic fragments c + 57 and z(•) - 57 specific to the isoAsp residue. To date, however, the presence of such fragments has not been explored on peptides with an N-terminal isoAsp residue. To address this question, several N-terminal isoAsp-containing peptides were analyzed using ExD methods alone or combined with chromatography. A diagnostic fragment [M + 2H - 74](+•) was observed for the doubly charged precursor ions with N-terminal isoAsp residues. For some peptides, identification of the N-terminal isoAsp residue was challenging because of the low diagnostic ion peak intensity and the presence of interfering peaks. Supplemental activation was used to improve diagnostic ion detection. Further, N-terminal acetylation was offered as a means to overcome the interference problem by shifting the diagnostic fragment peak to [M + 2H - 116](+•).

  16. Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

    SciTech Connect

    Auperin,T.; Bolduc, G.; Baron, M.; Heroux, A.; Filman, D.; Madoff, L.; Hogle, J.

    2005-01-01

    Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} of the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

  17. Crystal structure of the N-terminal domain of human SIRT7 reveals a three-helical domain architecture.

    PubMed

    Priyanka, Anu; Solanki, Vipul; Parkesh, Raman; Thakur, Krishan Gopal

    2016-10-01

    Human SIRT7 is an NAD(+) dependent deacetylase, which belongs to sirtuin family of proteins. SIRT7, like other sirtuins has conserved catalytic domain and is flanked by N- and C-terminal domains reported to play vital functional roles. Here, we report the crystal structure of the N-terminal domain of human SIRT7 (SIRT7(NTD) ) at 2.3 Å resolution as MBP-SIRT7(NTD) fusion protein. SIRT7(NTD) adopts three-helical domain architecture and comparative structural analyses suggest similarities to some DNA binding motifs and transcription regulators. We also report here the importance of N- and C-terminal domains in soluble expression of SIRT7. Proteins 2016; 84:1558-1563. © 2016 Wiley Periodicals, Inc.

  18. Crystal structure of the N-terminal domain of human SIRT7 reveals a three-helical domain architecture.

    PubMed

    Priyanka, Anu; Solanki, Vipul; Parkesh, Raman; Thakur, Krishan Gopal

    2016-10-01

    Human SIRT7 is an NAD(+) dependent deacetylase, which belongs to sirtuin family of proteins. SIRT7, like other sirtuins has conserved catalytic domain and is flanked by N- and C-terminal domains reported to play vital functional roles. Here, we report the crystal structure of the N-terminal domain of human SIRT7 (SIRT7(NTD) ) at 2.3 Å resolution as MBP-SIRT7(NTD) fusion protein. SIRT7(NTD) adopts three-helical domain architecture and comparative structural analyses suggest similarities to some DNA binding motifs and transcription regulators. We also report here the importance of N- and C-terminal domains in soluble expression of SIRT7. Proteins 2016; 84:1558-1563. © 2016 Wiley Periodicals, Inc. PMID:27287224

  19. Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain

    SciTech Connect

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G.

    2012-03-16

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its 20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

  20. Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

    PubMed Central

    Krieger, James; Bahar, Ivet; Greger, Ingo H.

    2015-01-01

    Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment. PMID:26255587

  1. Retroviral retargeting by envelopes expressing an N-terminal binding domain.

    PubMed Central

    Cosset, F L; Morling, F J; Takeuchi, Y; Weiss, R A; Collins, M K; Russell, S J

    1995-01-01

    We have engineered ecotropic Moloney murine leukemia virus-derived envelopes targeted to cell surface molecules expressed on human cells by the N-terminal insertion of polypeptides able to bind either Ram-1 phosphate transporter (the first 208 amino acids of amphotropic murine leukemia virus surface protein) or epidermal growth factor receptor (EGFR) (the 53 amino acids of EGF). Both envelopes were correctly processed and incorporated into viral particles. Virions carrying these envelopes could specifically bind the new cell surface receptors. Virions targeted to Ram-1 could infect human cells, although the efficiency was reduced compared with that of virions carrying wild-type amphotropic murine leukemia virus envelopes. The infectivity of virions targeted to EGFR was blocked at a postbinding step, and our results suggest that EGFR-bound virions were rapidly trafficked to lysosomes. These data suggest that retroviruses require specific properties of cell surface molecules to allow the release of viral cores into the correct cell compartment. PMID:7666532

  2. Structural polymorphism in the N-terminal oligomerization domain of NPM1

    PubMed Central

    Mitrea, Diana M.; Grace, Christy R.; Buljan, Marija; Yun, Mi-Kyung; Pytel, Nicholas J.; Satumba, John; Nourse, Amanda; Park, Cheon-Gil; Madan Babu, M.; White, Stephen W.; Kriwacki, Richard W.

    2014-01-01

    Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer–pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions. PMID:24616519

  3. Structural polymorphism in the N-terminal oligomerization domain of NPM1.

    PubMed

    Mitrea, Diana M; Grace, Christy R; Buljan, Marija; Yun, Mi-Kyung; Pytel, Nicholas J; Satumba, John; Nourse, Amanda; Park, Cheon-Gil; Madan Babu, M; White, Stephen W; Kriwacki, Richard W

    2014-03-25

    Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer-pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.

  4. Characterization of regions within the N-terminal 6-kilodalton domain of phytochrome A that modulate its biological activity.

    PubMed Central

    Jordan, E T; Marita, J M; Clough, R C; Vierstra, R D

    1997-01-01

    Phytochrome A (phyA) is a red/far-red (FR) light photoreceptor responsible for initiating numerous light-mediated plant growth and developmental responses, especially in FR light-enriched environments. We previously showed that the first 70 amino acids of the polypeptide contain at least two regions with potentially opposite functions (E.T. Jordan, J.R. Cherry, J.M. Walker, R.D. Vierstra [1996] Plant J 9: 243-257). One region is required for activity and correct apoprotein/chromophore interactions, whereas the second appears to regulate phytochrome activity. We have further resolved these functional regions by analysis of N-terminal deletion and alanine-scanning mutants of oat (Avena sativa) phyA in transgenic tobacco (Nicotiana tabacum). The results indicate that the region involved in chromophore/apoprotein interactions contains two separate segments (residues 25-33 and 50-62) also required for biological activity. The region that regulates phyA activity requires only five adjacent serines (Sers) (residues 8-12). Removal or alteration of these Sers generates a photoreceptor that increases the sensitivity of transgenic seedlings to red and FR light more than intact phyA. Taken together, these data identify three distinct regions in the N-terminal domain necessary for photoreceptor activity, and further define the Ser-rich region as an important site for phyA regulation. PMID:9342873

  5. Regulation of Telomere Length Requires a Conserved N-Terminal Domain of Rif2 in Saccharomyces cerevisiae.

    PubMed

    Kaizer, Hannah; Connelly, Carla J; Bettridge, Kelsey; Viggiani, Christopher; Greider, Carol W

    2015-10-01

    The regulation of telomere length equilibrium is essential for cell growth and survival since critically short telomeres signal DNA damage and cell cycle arrest. While the broad principles of length regulation are well established, the molecular mechanism of how these steps occur is not fully understood. We mutagenized the RIF2 gene in Saccharomyces cerevisiae to understand how this protein blocks excess telomere elongation. We identified an N-terminal domain in Rif2 that is essential for length regulation, which we have termed BAT domain for Blocks Addition of Telomeres. Tethering this BAT domain to Rap1 blocked telomere elongation not only in rif2Δ mutants but also in rif1Δ and rap1C-terminal deletion mutants. Mutation of a single amino acid in the BAT domain, phenylalanine at position 8 to alanine, recapitulated the rif2Δ mutant phenotype. Substitution of F8 with tryptophan mimicked the wild-type phenylalanine, suggesting the aromatic amino acid represents a protein interaction site that is essential for telomere length regulation.

  6. N-terminal Domains Elicit Formation of Functional Pmel17 Amyloid Fibrils*

    PubMed Central

    Watt, Brenda; van Niel, Guillaume; Fowler, Douglas M.; Hurbain, Ilse; Luk, Kelvin C.; Stayrook, Steven E.; Lemmon, Mark A.; Raposo, Graça; Shorter, James; Kelly, Jeffery W.; Marks, Michael S.

    2009-01-01

    Pmel17 is a transmembrane protein that mediates the early steps in the formation of melanosomes, the subcellular organelles of melanocytes in which melanin pigments are synthesized and stored. In melanosome precursor organelles, proteolytic fragments of Pmel17 form insoluble, amyloid-like fibrils upon which melanins are deposited during melanosome maturation. The mechanism(s) by which Pmel17 becomes competent to form amyloid are not fully understood. To better understand how amyloid formation is regulated, we have defined the domains within Pmel17 that promote fibril formation in vitro. Using purified recombinant fragments of Pmel17, we show that two regions, an N-terminal domain of unknown structure and a downstream domain with homology to a polycystic kidney disease-1 repeat, efficiently form amyloid in vitro. Analyses of fibrils formed in melanocytes confirm that the polycystic kidney disease-1 domain forms at least part of the physiological amyloid core. Interestingly, this same domain is also required for the intracellular trafficking of Pmel17 to multivesicular compartments within which fibrils begin to form. Although a domain of imperfect repeats (RPT) is required for fibril formation in vivo and is a component of fibrils in melanosomes, RPT is not necessary for fibril formation in vitro and in isolation is unable to adopt an amyloid fold in a physiologically relevant time frame. These data define the structural core of Pmel17 amyloid, imply that the RPT domain plays a regulatory role in timing amyloid conversion, and suggest that fibril formation might be physically linked with multivesicular body sorting. PMID:19840945

  7. Crystal structure of the N-terminal domain of Nup358/RanBP2

    PubMed Central

    Kassube, Susanne A.; Stuwe, Tobias; Lin, Daniel H.; Antonuk, C. Danielle; Napetschnig, Johanna; Blobel, Günter; Hoelz, André

    2014-01-01

    Key steps in mRNA export are the nuclear assembly of messenger ribonucleoprotein particles (mRNPs), the translocation of mRNPs through the nuclear pore complex (NPC), and the mRNP remodeling events at the cytoplasmic side of the NPC. Nup358/RanBP2 is a constituent of the cytoplasmic filaments of the NPC specific to higher eukaryotes and provides a multitude of binding sites for the nucleocytoplasmic transport machinery. Here, we present the crystal structure of the Nup358 N-terminal domain (NTD) at 0.95-Å resolution. The structure reveals an α-helical domain that harbors three central tetratricopeptide repeats (TPR), flanked on each side by an additional solvating amphipathic α helix. Overall, the NTD adopts an unusual extended conformation that lacks the characteristic peptide-binding groove observed in canonical TPR domains. Strikingly, the vast majority of the NTD surface exhibits an evolutionarily conserved, positive electrostatic potential, and we demonstrate that the NTD possesses the capability to bind single-stranded RNA in solution. Together, these data suggest that the NTD contributes to mRNP remodeling events at the cytoplasmic face of the NPC. PMID:23353830

  8. Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio.

    PubMed

    Liebschner, Dorothee; Brzezinski, Krzysztof; Dauter, Miroslawa; Dauter, Zbigniew; Nowak, Marta; Kur, Józef; Olszewski, Marcin

    2012-12-01

    PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains.

  9. Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio

    PubMed Central

    Liebschner, Dorothee; Brzezinski, Krzysztof; Dauter, Miroslawa; Dauter, Zbigniew; Nowak, Marta; Kur, Józef; Olszewski, Marcin

    2012-01-01

    PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains. PMID:23151633

  10. The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA

    PubMed Central

    Poulet, Anaïs; Pisano, Sabrina; Faivre-Moskalenko, Cendrine; Pei, Bei; Tauran, Yannick; Haftek-Terreau, Zofia; Brunet, Frédéric; Le Bihan, Yann-Vaï; Ledu, Marie-Hélène; Montel, Fabien; Hugo, Nicolas; Amiard, Simon; Argoul, Françoise; Chaboud, Annie; Giraud-Panis, Marie-Josèphe

    2012-01-01

    TRF1 and TRF2 are key proteins in human telomeres, which, despite their similarities, have different behaviors upon DNA binding. Previous work has shown that unlike TRF1, TRF2 condenses telomeric, thus creating consequential negative torsion on the adjacent DNA, a property that is thought to lead to the stimulation of single-strand invasion and was proposed to favor telomeric DNA looping. In this report, we show that these activities, originating from the central TRFH domain of TRF2, are also displayed by the TRFH domain of TRF1 but are repressed in the full-length protein by the presence of an acidic domain at the N-terminus. Strikingly, a similar repression is observed on TRF2 through the binding of a TERRA-like RNA molecule to the N-terminus of TRF2. Phylogenetic and biochemical studies suggest that the N-terminal domains of TRF proteins originate from a gradual extension of the coding sequences of a duplicated ancestral gene with a consequential progressive alteration of the biochemical properties of these proteins. Overall, these data suggest that the N-termini of TRF1 and TRF2 have evolved to finely regulate their ability to condense DNA. PMID:22139926

  11. A non-catalytic N-terminal domain negatively influences the nucleotide exchange activity of translation elongation factor 1Bα.

    PubMed

    Trosiuk, Tetiana V; Shalak, Vyacheslav F; Szczepanowski, Roman H; Negrutskii, Boris S; El'skaya, Anna V

    2016-02-01

    Eukaryotic translation elongation factor 1Bα (eEF1Bα) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Bα functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The C-terminal domain of eEF1Bα is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bγ (eEF1Bγ) to form a tight complex. However, eEF1Bγ has been shown to enhance the catalytic activity of eEF1Bα attributed to the C-terminal domain of eEF1Bα. This suggests that the N-terminal domain of eEF1Bα may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Bα and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Bα, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Bα. A similar effect on the catalytic activity of eEF1Bα was observed after its interaction with eEF1Bγ. We suggest that the non-catalytic N-terminal domain of eEF1Bα may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bγ and eEF1Bα N-terminal domains abolishes this inhibitory effect. PMID:26587907

  12. NMR structure of the N-terminal domain of the replication initiator protein DnaA

    SciTech Connect

    Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

    2007-08-07

    DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

  13. Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

    PubMed Central

    Gary, R; Bretscher, A

    1995-01-01

    Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking. Images PMID:7579708

  14. Zinc folds the N-terminal domain of HIV-1 integrase, promotes multimerization, and enhances catalytic activity

    PubMed Central

    Zheng, Ronglan; Jenkins, Timothy M.; Craigie, Robert

    1996-01-01

    The N-terminal domain of HIV-1 integrase contains a pair of His and Cys residues (the HHCC motif) that are conserved among retroviral integrases. Although His and Cys residues are often involved in binding zinc, the HHCC motif does not correspond to any recognized class of zinc binding domain. We have investigated the binding of zinc to HIV-1 integrase protein and find that it binds zinc with a stoichiometry of one zinc per integrase monomer. Analysis of zinc binding to deletion derivatives of integrase locates the binding site to the N-terminal domain. Integrase with a mutation in the HHCC motif does not bind zinc, consistent with coordination of zinc by these residues. The isolated N-terminal domain is disordered in the absence of zinc but, in the presence of zinc, it adopts a secondary structure with a high alpha helical content. Integrase bound by zinc tetramerizes more readily than the apoenzyme and is also more active than the apoenzyme in in vitro integration assays. We conclude that binding of zinc to the HHCC motif stabilizes the folded state of the N-terminal domain of integrase and bound zinc is required for optimal enzymatic activity. PMID:8942990

  15. The N-terminal fingers of chicken GATA-2 and GATA-3 are independent sequence-specific DNA binding domains.

    PubMed

    Pedone, P V; Omichinski, J G; Nony, P; Trainor, C; Gronenborn, A M; Clore, G M; Felsenfeld, G

    1997-05-15

    The GATA family of vertebrate DNA binding regulatory proteins are expressed in diverse tissues and at different times of development. However, the DNA binding regions of these proteins possess considerable homology and recognize a rather similar range of DNA sequence motifs. DNA binding is mediated through two domains, each containing a zinc finger. Previous results have led to the conclusion that although in some cases the N-terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C-terminal finger is both necessary and sufficient for binding. Here we show that although this is true for the N-terminal finger of GATA-1, those of GATA-2 and GATA-3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N-terminal finger. The absence of one of these near the GATA-1 N-terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. Our results suggest that the DNA binding properties of the N-terminal finger may help distinguish GATA-2 and GATA-3 from GATA-1 and the other GATA family members in their selective regulatory roles in vivo. PMID:9184231

  16. Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio

    SciTech Connect

    Liebschner, Dorothee; Brzezinski, Krzysztof; Dauter, Miroslawa; Dauter, Zbigniew; Nowak, Marta; Kur, Józef; Olszewski, Marcin

    2012-12-01

    The N-terminal domain of the PriB protein from the thermophilic bacterium T. tengcongensis (TtePriB) was expressed and its crystal structure has been solved at the atomic resolution of 1.09 Å by direct methods. PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains.

  17. Functional characterization of a special thermophilic multifunctional amylase OPMA-N and its N-terminal domain.

    PubMed

    Li, Fan; Zhu, Xuejun; Li, Yanfei; Cao, Hao; Zhang, Yingjiu

    2011-04-01

    A gene encoding a special thermophilic multifunctional amylase OPMA-N was cloned from Bacillus sp. ZW2531-1. OPMA-N has an additional 124-residue N-terminal domain compared with typical amylases and forms a relatively independent domain with a β-pleated sheet and random coil structure. Here we reported an unusual substrate and product specificities of OPMA-N and the impact of the additional N-terminal domain (1-124 aa) on the function and properties of OPMA-N. Both OPMA-N (12.82 U/mg) and its N-terminal domain-truncated ΔOPMA-N (12.55 U/mg) only degraded starch to produce oligosaccharides including maltose, maltotriose, isomaltotriose, and isomaltotetraose, but not to produce glucose. Therefore, the N-terminal domain did not determine its substrate and product specificities that were probably regulated by its C-terminal β-pleated sheet structure. However, the N-terminal domain of OPMA-N seemed to modulate its catalytic feature, leading to the production of more isomaltotriose and less maltose, and it seemed to contribute to OPMA-N's thermostability since OPMA-N showed higher activity than ΔOPMA-N in a temperature range from 40 to 80°C and the half-life (t(1/2)) was 5 h for OPMA-N and 2 h for ΔOPMA-N at 60°C. Both OPMA-N and ΔOPMA-N were Ca(2+)-independent, but their activities could be influenced by Cu(2+), Ni(2+), Zn(2+), EDTA, SDS (1 mM), or Triton-X100 (1%). Kinetic analysis and starch-adsorption assay indicated that the N-terminal domain of OPMA-N could increase the OPMA-N-starch binding and subsequently increase the catalytic efficiency of OPMA-N for starch. In particular, the N-terminal domain of OPMA-N did not determine its oligomerization, because both OPMA-N and ΔOPMA-N could exist in the forms of monomer, homodimer, and homooligomer at the same time.

  18. Solution structure of Atg8 reveals conformational polymorphism of the N-terminal domain

    SciTech Connect

    Schwarten, Melanie; Stoldt, Matthias; Mohrlueder, Jeannine; Willbold, Dieter

    2010-05-07

    During autophagy a crescent shaped like membrane is formed, which engulfs the material that is to be degraded. This membrane grows further until its edges fuse to form the double membrane covered autophagosome. Atg8 is a protein, which is required for this initial step of autophagy. Therefore, a multistage conjugation process of newly synthesized Atg8 to phosphatidylethanolamine is of critical importance. Here we present the high resolution structure of unprocessed Atg8 determined by nuclear magnetic resonance spectroscopy. Its C-terminal subdomain shows a well-defined ubiquitin-like fold with slightly elevated mobility in the pico- to nanosecond timescale as determined by heteronuclear NOE data. In comparison to unprocessed Atg8, cleaved Atg8{sup G116} shows a decreased mobility behaviour. The N-terminal domain adopts different conformations within the micro- to millisecond timescale. The possible biological relevance of the differences in dynamic behaviours between both subdomains as well as between the cleaved and uncleaved forms is discussed.

  19. Impact of the N-Terminal Domain of STAT3 in STAT3-Dependent Transcriptional Activity

    PubMed Central

    Hu, Tiancen; Yeh, Jennifer E.; Pinello, Luca; Jacob, Jaison; Chakravarthy, Srinivas; Yuan, Guo-Cheng

    2015-01-01

    The transcription factor STAT3 is constitutively active in many cancers, where it mediates important biological effects, including cell proliferation, differentiation, survival, and angiogenesis. The N-terminal domain (NTD) of STAT3 performs multiple functions, such as cooperative DNA binding, nuclear translocation, and protein-protein interactions. However, it is unclear which subsets of STAT3 target genes depend on the NTD for transcriptional regulation. To identify such genes, we compared gene expression in STAT3-null mouse embryonic fibroblasts (MEFs) stably expressing wild-type STAT3 or STAT3 from which NTD was deleted. NTD deletion reduced the cytokine-induced expression of specific STAT3 target genes by decreasing STAT3 binding to their regulatory regions. To better understand the potential mechanisms of this effect, we determined the crystal structure of the STAT3 NTD and identified a dimer interface responsible for cooperative DNA binding in vitro. We also observed an Ni2+-mediated oligomer with an as yet unknown biological function. Mutations on both dimer and Ni2+-mediated interfaces affected the cytokine induction of STAT3 target genes. These studies shed light on the role of the NTD in transcriptional regulation by STAT3 and provide a structural template with which to design STAT3 NTD inhibitors with potential therapeutic value. PMID:26169829

  20. Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter.

    PubMed

    Lee, Youngjin; Min, Choon Kee; Kim, Tae Gyun; Song, Hong Ki; Lim, Yunki; Kim, Dongwook; Shin, Kahee; Kang, Moonkyung; Kang, Jung Youn; Youn, Hyung-Seop; Lee, Jung-Gyu; An, Jun Yop; Park, Kyoung Ryoung; Lim, Jia Jia; Kim, Ji Hun; Kim, Ji Hye; Park, Zee Yong; Kim, Yeon-Soo; Wang, Jimin; Kim, Do Han; Eom, Soo Hyun

    2015-10-01

    The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca(2+) uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.

  1. Highly heterologous region in the N-terminal extracellular domain of reptilian follitropin receptors.

    PubMed

    Akazome, Y; Ogasawara, O; Park, M K; Mori, T

    1996-12-01

    The primary structure of the N-terminal extracellular region of the follitropin receptor (FSH-R), which is thought to be responsible for hormone binding specificity, was determined in three reptilian species (tortoise, gecko, and lizard). Remarkably low sequence homologies were detected in the C-terminal part of the extracellular domain. This region was estimated to be a part of exon 10, which is the last exon of the FSH-R gene. In this region, not only were low homologies detected among the three reptilian species, but also specific deletions and/or insertions were found. In particular, large deletions were detected in squamate (gecko and lizard) FSH-Rs. Phylogenetic analysis indicated that these large deletions occurred recently, i.e., after the Triassic period. In another region characterized, sequence homologies were high, with tortoise-rat homology 78.4%, gecko-rat 64.7%, and lizard-rat 69.1%. In this highly conserved region, however, some reptile-specific alterations were detected, such as the loss of a cysteine residue in putative exon 7 and the existence of potential N-linked glycosylation sites in putative exon 9. PMID:8954771

  2. Highly heterologous region in the N-terminal extracellular domain of reptilian follitropin receptors.

    PubMed

    Akazome, Y; Ogasawara, O; Park, M K; Mori, T

    1996-12-01

    The primary structure of the N-terminal extracellular region of the follitropin receptor (FSH-R), which is thought to be responsible for hormone binding specificity, was determined in three reptilian species (tortoise, gecko, and lizard). Remarkably low sequence homologies were detected in the C-terminal part of the extracellular domain. This region was estimated to be a part of exon 10, which is the last exon of the FSH-R gene. In this region, not only were low homologies detected among the three reptilian species, but also specific deletions and/or insertions were found. In particular, large deletions were detected in squamate (gecko and lizard) FSH-Rs. Phylogenetic analysis indicated that these large deletions occurred recently, i.e., after the Triassic period. In another region characterized, sequence homologies were high, with tortoise-rat homology 78.4%, gecko-rat 64.7%, and lizard-rat 69.1%. In this highly conserved region, however, some reptile-specific alterations were detected, such as the loss of a cysteine residue in putative exon 7 and the existence of potential N-linked glycosylation sites in putative exon 9.

  3. Crystal Structure of the N-Terminal Domain of the Human Protooncogene Nup214/CAN

    SciTech Connect

    Napetschnig,J.; Blobel, G.; Hoelz, A.

    2007-01-01

    The mammalian nuclear pore complex (NPC) is an {approx}120-MDa proteinaceous assembly consisting of {approx}30 proteins and is the sole gate in the nuclear envelope. The human protooncogene Nup214 was first identified as a target for chromosomal translocation involved in leukemogenesis. Nup214 is located on the cytoplasmic face of the NPC and is implicated in anchoring the cytoplasmic filaments of the NPC and recruiting the RNA helicase Ddx19. Here, we present the crystal structure of the human Nup214 N-terminal domain at 1.65-{angstrom} resolution. The structure reveals a seven-bladed {beta}-propeller followed by a 30-residue C-terminal extended peptide segment, which folds back onto the {beta}-propeller and binds to its bottom face. The {beta}-propeller repeats lack any recognizable sequence motif and are distinguished by extensive insertions between the canonical {beta}-strands. We propose a mechanism by which the C-terminal peptide extension is involved in NPC assembly.

  4. Crystal structure of the N-terminal domain of the human protooncogene Nup214/CAN

    PubMed Central

    Napetschnig, Johanna; Blobel, Günter; Hoelz, André

    2007-01-01

    The mammalian nuclear pore complex (NPC) is an ≈120-MDa proteinaceous assembly consisting of ≈30 proteins and is the sole gate in the nuclear envelope. The human protooncogene Nup214 was first identified as a target for chromosomal translocation involved in leukemogenesis. Nup214 is located on the cytoplasmic face of the NPC and is implicated in anchoring the cytoplasmic filaments of the NPC and recruiting the RNA helicase Ddx19. Here, we present the crystal structure of the human Nup214 N-terminal domain at 1.65-Å resolution. The structure reveals a seven-bladed β-propeller followed by a 30-residue C-terminal extended peptide segment, which folds back onto the β-propeller and binds to its bottom face. The β-propeller repeats lack any recognizable sequence motif and are distinguished by extensive insertions between the canonical β-strands. We propose a mechanism by which the C-terminal peptide extension is involved in NPC assembly. PMID:17264208

  5. Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain

    PubMed Central

    Moparthi, Lavanya; Survery, Sabeen; Kreir, Mohamed; Simonsen, Charlotte; Kjellbom, Per; Högestätt, Edward D.; Johanson, Urban; Zygmunt, Peter M.

    2014-01-01

    We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1–688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ9-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1–688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca2+, or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease). PMID:25389312

  6. ClpB N-terminal domain plays a regulatory role in protein disaggregation

    PubMed Central

    Rosenzweig, Rina; Farber, Patrick; Velyvis, Algirdas; Rennella, Enrico; Latham, Michael P.; Kay, Lewis E.

    2015-01-01

    ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB–substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD–substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD–substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD–substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation. PMID:26621746

  7. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    PubMed

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS.

  8. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization

    SciTech Connect

    Shiheido, Hirokazu Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND3{sub 56–58}, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. - Highlights: • BEND3 localizes to the nucleus. • The N-terminal 60 amino acids region of BEND3 contains NLS. • Amino acids located between 56 and 58 of BEND3 (KRK) are part of NLS. • KRK motif is highly conserved among BEND3 homologs.

  9. Structural Diversity of the Active N-Terminal Kinase Domain of p90 Ribosomal S6 Kinase 2

    SciTech Connect

    Malakhova, Margarita; Kurinov, Igor; Liu, Kangdong; Zheng, Duo; D'Angelo, Igor; Shim, Jung-Hyun; Steinman, Valerie; Bode, Ann M.; Dong, Zigang

    2010-10-08

    The p90 ribosomal protein kinase 2 (RSK2) is a highly expressed Ser/Thr kinase activated by growth factors and is involved in cancer cell proliferation and tumor promoter-induced cell transformation. RSK2 possesses two non-identical kinase domains, and the structure of its N-terminal domain (NTD), which is responsible for phosphorylation of a variety of substrates, is unknown. The crystal structure of the NTD RSK2 was determined at 1.8 {angstrom} resolution in complex with AMP-PNP. The N-terminal kinase domain adopted a unique active conformation showing a significant structural diversity of the kinase domain compared to other kinases. The NTD RSK2 possesses a three-stranded {beta}B-sheet inserted in the N-terminal lobe, resulting in displacement of the {alpha}C-helix and disruption of the Lys-Glu interaction, classifying the kinase conformation as inactive. The purified protein was phosphorylated at Ser227 in the T-activation loop and exhibited in vitro kinase activity. A key characteristic is the appearance of a new contact between Lys216 ({beta}B-sheet) and the {beta}-phosphate of AMP-PNP. Mutation of this lysine to alanine impaired both NTDs in vitro and full length RSK2 ex vivo activity, emphasizing the importance of this interaction. Even though the N-terminal lobe undergoes structural re-arrangement, it possesses an intact hydrophobic groove formed between the {alpha}C-helix, the {beta}4-strand, and the {beta}B-sheet junction, which is occupied by the N-terminal tail. The presence of a unique {beta}B-sheet insert in the N-lobe suggests a different type of activation mechanism for RSK2.

  10. Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

    PubMed

    Tamura, Tomokazu; Ruggli, Nicolas; Nagashima, Naofumi; Okamatsu, Masatoshi; Igarashi, Manabu; Mine, Junki; Hofmann, Martin A; Liniger, Matthias; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-09-01

    Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs. PMID:26018962

  11. NMR solution structure of the N-terminal domain of hERG and its interaction with the S4-S5 linker

    SciTech Connect

    Li, Qingxin; Gayen, Shovanlal; Chen, Angela Shuyi; Huang, Qiwei; Raida, Manfred; Kang, CongBao

    2010-12-03

    Research highlights: {yields} The N-terminal domain (NTD, eag domain) containing 135 residues of hERG was expressed and purified from E. coli cells. {yields} Solution structure of NTD was determined with NMR spectroscopy. {yields} The alpha-helical region (residues 13-23) was demonstrated to possess the characteristics of an amphipathic helix. {yields} NMR titration confirmed the interaction between NTD and the peptide from the S4-S5 linker. -- Abstract: The human Ether-a-go-go Related Gene (hERG) potassium channel mediates the rapid delayed rectifier current (IKr) in the cardiac action potential. Mutations in the 135 amino acid residue N-terminal domain (NTD) cause channel dysfunction or mis-translocation. To study the structure of NTD, it was overexpressed and purified from Escherichia coli cells using affinity purification and gel filtration chromatography. The purified protein behaved as a monomer under purification conditions. Far- and near-UV, circular dichroism (CD) and solution nuclear magnetic resonance (NMR) studies showed that the purified protein was well-folded. The solution structure of NTD was obtained and the N-terminal residues 13-23 forming an amphipathic helix which may be important for the protein-protein or protein-membrane interactions. NMR titration experiment also demonstrated that residues from 88 to 94 in NTD are important for the molecular interaction with the peptide derived from the S4-S5 linker.

  12. N-terminal amino acid sequences and some characteristics of fibrinolytic/hemorrhagic metalloproteinases purified from Bothrops jararaca venom.

    PubMed

    Maruyama, Masugi; Sugiki, Masahiko; Anai, Keita; Yoshida, Etsuo

    2002-08-01

    We determined the N-terminal amino acid sequences of the fibrinolytic/hemorrhagic metalloproteinases (jararafibrases I, III and IV) purified from Bothrops jararaca venom. The N-terminal amino acid sequences of jararafibrase I and its degradation products were identical to those of jararhagin, another hemorrhagic metalloproteinase purified from the same snake venom. Together with enzymatic and immunological properties, we concluded that those two enzymes are identical. The N-terminal amino acid sequence of jararafibrase III was quite similar to C-type lectin isolated from Crotalus atrox, and the protein had a hemagglutinating activity on intact rat red blood cells. PMID:12165326

  13. Solution structure and backbone dynamics of the N-terminal region of the calcium regulatory domain from soybean calcium-dependent protein kinase alpha.

    PubMed

    Weljie, Aalim M; Gagné, Stéphane M; Vogel, Hans J

    2004-12-01

    Ca(2+)-dependent protein kinases (CDPKs) are vital Ca(2+)-signaling proteins in plants and protists which have both a kinase domain and a self-contained calcium regulatory calmodulin-like domain (CLD). Despite being very similar to CaM (>40% identity) and sharing the same fold, recent biochemical and structural evidence suggests that the behavior of CLD is distinct from its namesake, calmodulin. In this study, NMR spectroscopy is employed to examine the structure and backbone dynamics of a 168 amino acid Ca(2+)-saturated construct of the CLD (NtH-CLD) in which almost the entire C-terminal domain is exchange broadened and not visible in the NMR spectra. Structural characterization of the N-terminal domain indicates that the first Ca(2+)-binding loop is significantly more open than in a recently reported structure of the CLD complexed with a putative intramolecular binding region (JD) in the CDPK. Backbone dynamics suggest that parts of the third helix exhibit unusually high mobility, and significant exchange, consistent with previous findings that this helix interacts with the C-terminal domain. Dynamics data also show that the "tether" region, consisting of the first 11 amino acids of CLD, is highly mobile and these residues exhibit distinctive beta-type secondary structure, which may help to position the JD and CLD. Finally, the unusual global dynamic behavior of the protein is rationalized on the basis of possible interdomain rearrangements and the highly variable environments of the C- and N-terminal domains.

  14. The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

    PubMed Central

    Richardson, Brian C; Halaby, Steve L; Gustafson, Margaret A; Fromme, J Christopher

    2016-01-01

    The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth. DOI: http://dx.doi.org/10.7554/eLife.12411.001 PMID:26765562

  15. NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5.

    PubMed Central

    Bycroft, M; Grünert, S; Murzin, A G; Proctor, M; St Johnston, D

    1995-01-01

    The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR. Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet. This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way. Images PMID:7628456

  16. Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites

    PubMed Central

    Aregger, Michael; Cowling, Victoria H.

    2013-01-01

    Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5′ phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation. PMID:23863084

  17. Effect of N-terminal glutamic acid and glutamine on fragmentation of peptide ions.

    PubMed

    Godugu, Bhaskar; Neta, Pedatsur; Simón-Manso, Yamil; Stein, Stephen E

    2010-07-01

    A prominent dissociation path for electrospray generated tryptic peptide ions is the dissociation of the peptide bond linking the second and third residues from the amino-terminus. The formation of the resulting b(2) and y(n-2) fragments has been rationalized by specific facile mechanisms. An examination of spectral libraries shows that this path predominates in diprotonated peptides composed of 12 or fewer residues, with the notable exception of peptides containing glutamine or glutamic acid at the N-terminus. To elucidate the mechanism by which these amino acids affect peptide fragmentation, we synthesized peptides of varying size and composition and examined their MS/MS spectra as a function of collision voltage in a triple quadrupole mass spectrometer. Loss of water from N-terminal glutamic acid and glutamine is observed at a lower voltage than any other fragmentation, leading to cyclization of the terminal residue. This cyclization results in the conversion of the terminal amine group to an imide, which has a lower proton affinity. As a result, the second proton is not localized at the N-terminus but is readily transferred to other sites, leading to fragmentation near the center of the peptide. Further confirmation was obtained by examining peptides with N-terminal pyroglutamic acid and N-acetyl peptides. Peptides with N-terminal proline maintain the trend of forming b(2) and y(n-2) because their ring contains an imine rather than imide and has sufficient proton affinity to retain the proton at the N-terminus.

  18. Passive immunization targeting the N-terminal projection domain of tau decreases tau pathology and improves cognition in a transgenic mouse model of Alzheimer disease and tauopathies.

    PubMed

    Dai, Chun-ling; Chen, Xia; Kazim, Syed Faraz; Liu, Fei; Gong, Cheng-Xin; Grundke-Iqbal, Inge; Iqbal, Khalid

    2015-04-01

    Intraneuronal accumulation of abnormally hyperphosphorylated tau in the brain is a histopathological hallmark of Alzheimer's disease and a family of related neurodegenerative disorders collectively called tauopathies. At present there is no effective treatment available for these progressive neurodegenerative diseases which are clinically characterized by dementia in mid to old-age. Here we report the treatment of 14-17-months-old 3xTg-AD mice with tau antibodies 43D (tau 6-18) and 77E9 (tau 184-195) to the N-terminal projection domain of tau or mouse IgG as a control by intraperitoneal injection once a week for 4 weeks, and the effects of the passive immunization on reduction of hyperphosphorylated tau, Aβ accumulation and cognitive performance in these animals. We found that treatment with tau antibodies 43D and 77E9 reduced total tau level, decreased tau hyperphosphorylated at Ser199, Ser202/Thr205 (AT8), Thr205, Ser262/356 (12E8), and Ser396/404 (PHF-1) sites, and a trend to reduce Aβ pathology. Most importantly, targeting N-terminal tau especially by 43D (tau 6-18) improved reference memory in the Morris water maze task in 3xTg-AD mice. We did not observe any abnormality in general physical characteristics of the treated animals with either of the two antibodies during the course of this study. Taken together, our studies demonstrate for the first time (1) that passive immunization targeting normal tau can effectively clear the hyperphosphorylated protein and possibly reduce Aβ pathology from the brain and (2) that targeting N-terminal projection domain of tau containing amino acid 6-18 is especially beneficial. Thus, targeting selective epitopes of N-terminal domain of tau may present a novel effective therapeutic opportunity for Alzheimer disease and other tauopathies.

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

    PubMed

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

    2016-06-02

    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.

  20. Crystallized N-terminal domain of influenza virus matrix protein M1 and method of determining and using same

    NASA Technical Reports Server (NTRS)

    Luo, Ming (Inventor); Sha, Bingdong (Inventor)

    2000-01-01

    The matrix protein, M1, of influenza virus strain A/PR/8/34 has been purified from virions and crystallized. The crystals consist of a stable fragment (18 Kd) of the M1 protein. X-ray diffraction studies indicated that the crystals have a space group of P3.sub.t 21 or P3.sub.2 21. Vm calculations showed that there are two monomers in an asymmetric unit. A crystallized N-terminal domain of M1, wherein the N-terminal domain of M1 is crystallized such that the three dimensional structure of the crystallized N-terminal domain of M1 can be determined to a resolution of about 2.1 .ANG. or better, and wherein the three dimensional structure of the uncrystallized N-terminal domain of M1 cannot be determined to a resolution of about 2.1 .ANG. or better. A method of purifying M1 and a method of crystallizing M1. A method of using the three-dimensional crystal structure of M1 to screen for antiviral, influenza virus treating or preventing compounds. A method of using the three-dimensional crystal structure of M1 to screen for improved binding to or inhibition of influenza virus M1. The use of the three-dimensional crystal structure of the M1 protein of influenza virus in the manufacture of an inhibitor of influenza virus M1. The use of the three-dimensional crystal structure of the M1 protein of influenza virus in the screening of candidates for inhibition of influenza virus M1.

  1. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization.

    PubMed

    Shiheido, Hirokazu; Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356-58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3.

  2. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization.

    PubMed

    Shiheido, Hirokazu; Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356-58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. PMID:25600804

  3. The N-terminal Domain of the Drosophila Mitochondrial Replicative DNA Helicase Contains an Iron-Sulfur Cluster and Binds DNA*

    PubMed Central

    Stiban, Johnny; Farnum, Gregory A.; Hovde, Stacy L.; Kaguni, Laurie S.

    2014-01-01

    The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys68, Cys71, Cys102, and Cys105) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent Kd of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork. PMID:25023283

  4. The N-terminal domain of the Drosophila mitochondrial replicative DNA helicase contains an iron-sulfur cluster and binds DNA.

    PubMed

    Stiban, Johnny; Farnum, Gregory A; Hovde, Stacy L; Kaguni, Laurie S

    2014-08-29

    The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys(68), Cys(71), Cys(102), and Cys(105)) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent Kd of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.

  5. Localization of the N-terminal domain of cauliflower mosaic virus coat protein precursor.

    PubMed

    Champagne, Julie; Benhamou, Nicole; Leclerc, Denis

    2004-07-01

    Cauliflower mosaic virus (CaMV) open reading frame (ORF) IV encodes a coat protein precursor (pre-CP) harboring an N-terminal extension that is cleaved off by the CaMV-encoded protease. In transfected cells, pre-CP is present in the cytoplasm, while the processed form (p44) of CP is targeted to the nucleus, suggesting that the N-terminal extension might be involved in keeping the pre-CP in the cytoplasm for viral assembly. This study reports for the first time the intracellular localization of the N-terminal extension during CaMV infection in Brassica rapa. Immunogold-labeling electron microscopy using polyclonal antibodies directed to the N-terminal extension of the pre-CP revealed that this region is closely associated with viral particles present in small aggregates, which we called small bodies, adjacent to the main inclusion bodies typical of CaMV infection. Based on these results, we propose a model for viral assembly of CaMV.

  6. The SAS-5 N-terminal domain is a tetramer, with implications for centriole assembly in C. elegans.

    PubMed

    Shimanovskaya, Ekaterina; Qiao, Renping; Lesigang, Johannes; Dong, Gang

    2013-07-01

    The centriole is a conserved microtubule-based organelle essential for both centrosome formation and cilium biogenesis. It has a unique 9-fold symmetry and its assembly is governed by at least five component proteins (SPD-2, ZYG-1, SAS-5, SAS-6 and SAS-4), which are recruited in a hierarchical order. Recently published structural studies of the SAS-6 N-terminal domain have greatly advanced our understanding of the mechanisms of centriole assembly. However, it remains unclear how the weak interaction between the SAS-6 N-terminal head groups could drive the assembly of a closed ring-like structure, and what determines the stacking of multiple rings on top one another in centriole duplication. We recently reported that SAS-5 binds specifically to a very narrow region of the SAS-6 central coiled coil through its C-terminal domain (CTD, residues 391-404). Here, we further demonstrate by both static light scattering and small angle X-ray scattering that the SAS-5 N-terminal domain (NTD, residues 1-260) forms a tetramer. Specifically, we found that the tetramer is formed by SAS-5 residues 82-260, whereas residues 1-81 are intrinsically disordered. Taking these results together, we propose a working model for SAS-5-mediated assembly of the multi-layered central tube structure.

  7. Acetylation of the Entamoeba histone H4 N-terminal domain is influenced by short-chain fatty acids that enter trophozoites in a pH-dependent manner.

    PubMed

    Byers, Jennifer; Eichinger, Daniel

    2008-01-01

    Treatment of higher eukaryotic cells with short-chain fatty acids (SCFA) such as butyrate causes decreased levels of histone deacetylase (HDAC) activity and hyperacetylation of histones, and thereby affects gene expression, cell growth and differentiation. Entamoeba parasites encounter high levels of SCFA in the host colon, and in vitro these compounds allow trophozoite stage parasites to multiply but prevent their differentiation into infectious cysts. The Entamoeba invadens IP-1 histone H4 protein has an unusual number of lysines in its N-terminus, and these become hyperacetylated in trophozoites exposed to the HDAC inhibitors trichostatin A (TSA) or HC-toxin, but not in trophozoites exposed to butyrate. We have now found that several other commonly studied isolates of Entamoeba parasites also have an extended set of histone H4 acetylation sites that become hyperacetylated in response to TSA, but hypoacetylated in response to butyrate, suggesting an unusual sensitivity of this parasite's histone modifying enzymes to SCFA. Butyrate was found to enter trophozoites in a pH-dependent manner consistent with diffusive entry of the un-ionised form of the fatty acid into the amoebae. Transit of the Entamoeba organism through areas of the host intestine with distinct pH and SCFA concentrations would therefore result in very different levels of SCFA within the parasite. Entamoeba appears to have acquired unique alterations of its histone acetylation mechanism that may allow for its growth in the presence of varying amounts of the bacterial fermentation products.

  8. The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src.

    PubMed

    Maffei, Mariano; Arbesú, Miguel; Le Roux, Anabel-Lise; Amata, Irene; Roche, Serge; Pons, Miquel

    2015-05-01

    Regulation of c-Src activity by the intrinsically disordered Unique domain has recently been demonstrated. However, its connection with the classical regulatory mechanisms is still missing. Here we show that the Unique domain is part of a long loop closed by the interaction of the SH4 and SH3 domains. The conformational freedom of the Unique domain is further restricted through direct contacts with SH3 that are allosterically modulated by binding of a poly-proline ligand in the presence and in the absence of lipids. Our results highlight the scaffolding role of the SH3 domain for the c-Src N-terminal intrinsically disordered regions and suggest a connection between the regulatory mechanisms involving the SH3 and Unique domains.

  9. Conformation Changes, N-terminal Involvement, and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain*

    PubMed Central

    Wang, Huanchen; Robinson, Howard; Ke, Hengming

    2010-01-01

    The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98–147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes. PMID:20861010

  10. The N-Terminal Domain of Bcl-xL Reversibly Binds Membranes in a pH-Dependent Manner†

    PubMed Central

    Thuduppathy, Guruvasuthevan R.; Terrones, Oihana; Craig, Jeffrey W.; Basañez, Gorka; Hill, R. Blake

    2006-01-01

    Bcl-xL regulates apoptosis by maintaining the integrity of the mitochondrial outer membrane by adopting both soluble and membrane-associated forms. The membrane-associated conformation does not require a conserved, C-terminal transmembrane domain and appears to be inserted into the bilayer of synthetic membranes as assessed by membrane permeabilization and critical surface pressure measurements. Membrane association is reversible and is regulated by the cooperative binding of approximately two protons to the protein. Two acidic residues, Glu153 and Asp156, that lie in a conserved hairpin of Bcl-xLΔTM appear to be important in this process on the basis of a 16% increase in the level of membrane association of the double mutant E153Q/D156N. Contrary to that for the wild type, membrane permeabilization for the mutant is not correlated with membrane association. Monolayer surface pressure measurements suggest that this effect is primarily due to less membrane penetration. These results suggest that E153 and D156 are important for the Bcl-xLΔTM conformational change and that membrane binding can be distinct from membrane permeabilization. Taken together, these studies support a model in which Bcl-xL activity is controlled by reversible insertion of its N-terminal domain into the mitochondrial outer membrane. Future studies with Bcl-xL mutants such as E153Q/D156N should allow determination of the relative contributions of membrane binding, insertion, and permeabilization to the regulation of apoptosis. PMID:17128992

  11. [Chemical synthesis of lactococcin B and functional evaluation of the N-terminal domain using a truncated synthetic analogue].

    PubMed

    Lasta, S; Fajloun, Z; Mansuelle, P; Sabatier, J M; Boudabous, A; Sampieri, F

    2008-01-01

    The lactococcin B (LnB) is a hydrophobic, positively charged bacteriocin, produced by Lactococcus lactis ssp. cremoris 9B4. It consists of a peptidic chain made up of 47 amino acid residues, and inhibits Lactococcus exclusively. In order to study its biological activity a synthetic lactococcin B (LnBs) was obtained by solid-phase chemical synthesis using a Fmoc strategy. LnBs was shown to be indistinguishable from the natural peptide. In addition, a synthetic (7-47) LnBst analogue was obtained by withdrawal of peptidyl-resin after the 41 cycle of LnBs peptide chain assembly. The synthetic N-terminal truncated (7-47) LnBst analogue was found to be inactive on indicator strains. Our results strongly suggest that the first six N-terminal amino acid residues are involved in the bactericidal activity of LnB.

  12. Dissecting functions of the N-terminal domain and GAS-site recognition in STAT3 nuclear trafficking.

    PubMed

    Martincuks, Antons; Fahrenkamp, Dirk; Haan, Serge; Herrmann, Andreas; Küster, Andrea; Müller-Newen, Gerhard

    2016-08-01

    Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression. Cytokine-induced gene transcription greatly depends on tyrosine phosphorylation of STAT3 on a single tyrosine residue with subsequent nuclear accumulation and specific DNA sequence (GAS) recognition. In this study, we analyzed the roles of the conserved STAT3 N-terminal domain (NTD) and GAS-element binding ability of STAT3 in nucleocytoplasmic trafficking. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. Substitution of five key amino acids within the DNA-binding domain rendered STAT3 unable to bind to GAS-elements while still maintaining the ability for nuclear localization. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding, since both wild-type and NTD deletion mutant of STAT3 could precipitate both full-length and autoinhibitory domain (∆IBB) deletion mutants of importin-α5, as well as ∆IBB-α3 and ∆IBB-α7 isoforms independently of IL-6 stimulation. Despite its inability to translocate to the nucleus upon IL-6 stimulation, the NTD lacking mutant still showed nuclear accumulation in resting cells similar to wild-type upon inhibition of nuclear export by leptomycin B. At the same time, blocking the nuclear export pathway could not rescue cytoplasmic trapping of phosphorylated STAT3 molecules without NTD. Moreover, STAT3 mutant with dysfunctional SH2 domain (R609Q) also localized in the nucleus of unstimulated cells after nuclear export blocking, while upon cytokine treatment the

  13. Association of N-terminal domain polymorphisms of the porcine glucocorticoid receptor with carcass composition and meat quality traits.

    PubMed

    Reyer, Henry; Ponsuksili, Siriluck; Wimmers, Klaus; Murani, Eduard

    2014-02-01

    The glucocorticoid receptor (GR) is a ubiquitously acting transcription factor that is responsible for mediating the physiological response to stress and adaptation to environmental conditions. Genetic variation of a GR gene (NR3C1) may therefore contribute to multiple phenotypic alterations and influence relevant traits of animal production. Here, we examined effects of two non-synonymous mutations of the porcine NR3C1, leading to amino acid exchanges p.Glu13Asp (c.39A>C) and p.Val19Leu (c.55G>C) in the N-terminal domain of the GR, on meat quality and carcass composition. In addition, we explored their influence on transcriptional activity of GR in vitro. A commercial crossbreed Pietrain × (German Large White × German Landrace) herd (n = 545) in which genotypes and relevant traits had been collected was used to perform the association analysis. The single nucleotide polymorphism (SNP) c.55G>C was significantly associated with conductivity and meat color scores. These effects were highly consistent considering the physiological relationship between these traits. Association analysis of SNP c.39A>C also revealed significant effects on closely connected meat quality traits. In addition, SNP c.55G>C showed association with carcass traits, mainly those related to muscle deposition. The molecular mechanism of action of both amino acid substitutions remains obscure because neither showed significant influence on transcriptional activity of GR. Our study emphasizes NR3C1 as an important candidate gene for muscle-related traits in pigs, but further work is necessary to clarify the molecular background of the identified associations.

  14. Computational Modeling of Laminin N-Terminal Domains Using Sparse Distance Constraints from Disulfide Bonds and Chemical Cross-Linking

    PubMed Central

    Kalkhof, Stefan; Haehn, Sebastian; Paulsson, Mats; Smyth, Neil; Meiler, Jens; Sinz, Andrea

    2016-01-01

    Basement membranes are thin extracellular protein layers, which separate endothelial and epithelial cells from the underlying connecting tissue. The main non-collagenous components of basement membranes are laminins, trimeric glycoproteins, which form polymeric networks by interactions of their N-terminal (LN) domains; however, no high-resolution structure of laminin LN domains exists so far. To construct models for laminin β1 and γ1 LN domains 14 potentially suited template structures were determined using fold recognition methods. For each target/template-combination comparative models were created with Rosetta. Final models were selected based on their agreement with experimentally obtained distance constraints from natural cross-links, i.e., disulfide bonds as well as chemical cross-links obtained from reactions with two amine-reactive cross-linkers. We predict that laminin β1 and γ1 LN domains share the galactose-binding domain-like fold. PMID:20939100

  15. The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation

    PubMed Central

    Hennig, Janosch; Zou, Peijian; Nössner, Elfriede; Ling, Paul D.; Sattler, Michael; Kempkes, Bettina

    2015-01-01

    Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics. PMID:26024477

  16. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    PubMed

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  17. N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription

    PubMed Central

    Wu, Hongxia; Zu, Shaopo; Sun, Xue; Liu, Yongxiang; Tian, Jin; Qu, Liandong

    2016-01-01

    Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as “shut-off”. However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression. PMID:27447663

  18. N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription.

    PubMed

    Wu, Hongxia; Zu, Shaopo; Sun, Xue; Liu, Yongxiang; Tian, Jin; Qu, Liandong

    2016-01-01

    Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as "shut-off". However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression. PMID:27447663

  19. Dissecting the Functional Role of the N-Terminal Domain of the Human Small Heat Shock Protein HSPB6

    PubMed Central

    Heirbaut, Michelle; Beelen, Steven; Strelkov, Sergei V.; Weeks, Stephen D.

    2014-01-01

    HSPB6 is a member of the human small heat shock protein (sHSP) family, a conserved group of molecular chaperones that bind partially unfolded proteins and prevent them from aggregating. In vertebrate sHSPs the poorly structured N-terminal domain has been implicated in both chaperone activity and the formation of higher-order oligomers. These two functionally important properties are likely intertwined at the sequence level, complicating attempts to delineate the regions that define them. Differing from the prototypical α-crystallins human HSPB6 has been shown to only form dimers in solution making it more amendable to explore the determinants of chaperoning activity alone. Using a systematic and iterative deletion strategy, we have extensively investigated the role of the N-terminal domain on the chaperone activity of this sHSP. As determined by size-exclusion chromatography and small-angle X-ray scattering, most mutants had a dimeric structure closely resembling that of wild-type HSPB6. The chaperone-like activity was tested using three different substrates, whereby no single truncation, except for complete removal of the N-terminal domain, showed full loss of activity, pointing to the presence of multiple sites for binding unfolding proteins. Intriguingly, we found that the stretch encompassing residues 31 to 35, which is nearly fully conserved across vertebrate sHSPs, acts as a negative regulator of activity, as its deletion greatly enhanced chaperoning capability. Further single point mutational analysis revealed an interplay between the highly conserved residues Q31 and F33 in fine-tuning its function. PMID:25157403

  20. Structure of the N-terminal domain of the protein Expansion: an 'Expansion' to the Smad MH2 fold.

    PubMed

    Beich-Frandsen, Mads; Aragón, Eric; Llimargas, Marta; Benach, Jordi; Riera, Antoni; Pous, Joan; Macias, Maria J

    2015-04-01

    Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.

  1. Hexameric ring structure of the N-terminal domain of Mycobacterium tuberculosis DnaB helicase

    SciTech Connect

    Biswas, Tapan; Tsodikov, Oleg V.

    2009-01-15

    Hexameric DnaB helicase unwinds the DNA double helix during replication of genetic material in bacteria. DnaB is an essential bacterial protein; therefore, it is an important potential target for antibacterial drug discovery. We report a crystal structure of the N-terminal region of DnaB from the pathogen Mycobacterium tuberculosis (MtDnaBn), determined at 2.0 {angstrom} resolution. This structure provides atomic resolution details of formation of the hexameric ring of DnaB by two distinct interfaces. An extensive hydrophobic interface stabilizes a dimer of MtDnaBn by forming a four-helix bundle. The other, less extensive, interface is formed between the dimers, connecting three of them into a hexameric ring. On the basis of crystal packing interactions between MtDnaBn rings, we suggest a model of a helicase-primase complex that explains previously observed effects of DnaB mutations on DNA priming.

  2. Hexameric ring structure of the N-terminal domain of Mycobacterium tuberculosis DnaB helicase.

    PubMed

    Biswas, Tapan; Tsodikov, Oleg V

    2008-06-01

    Hexameric DnaB helicase unwinds the DNA double helix during replication of genetic material in bacteria. DnaB is an essential bacterial protein; therefore, it is an important potential target for antibacterial drug discovery. We report a crystal structure of the N-terminal region of DnaB from the pathogen Mycobacterium tuberculosis (MtDnaBn), determined at 2.0 A resolution. This structure provides atomic resolution details of formation of the hexameric ring of DnaB by two distinct interfaces. An extensive hydrophobic interface stabilizes a dimer of MtDnaBn by forming a four-helix bundle. The other, less extensive, interface is formed between the dimers, connecting three of them into a hexameric ring. On the basis of crystal packing interactions between MtDnaBn rings, we suggest a model of a helicase-primase complex that explains previously observed effects of DnaB mutations on DNA priming.

  3. 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

  4. Identification of a Major Dimorphic Region in the Functionally Critical N-Terminal ID1 Domain of VAR2CSA.

    PubMed

    Doritchamou, Justin; Sabbagh, Audrey; Jespersen, Jakob S; Renard, Emmanuelle; Salanti, Ali; Nielsen, Morten A; Deloron, Philippe; Tuikue Ndam, Nicaise

    2015-01-01

    The VAR2CSA protein of Plasmodium falciparum is transported to and expressed on the infected erythrocyte surface where it plays a key role in placental malaria (PM). It is the current leading candidate for a vaccine to prevent PM. However, the antigenic polymorphism integral to VAR2CSA poses a challenge for vaccine development. Based on detailed analysis of polymorphisms in the sequence of its ligand-binding N-terminal region, currently the main focus for vaccine development, we assessed var2csa from parasite isolates infecting pregnant women. The results reveal for the first time the presence of a major dimorphic region in the functionally critical N-terminal ID1 domain. Parasite isolates expressing VAR2CSA with particular motifs present within this domain are associated with gravidity- and parasite density-related effects. These observations are of particular interest in guiding efforts with respect to optimization of the VAR2CSA-based vaccines currently under development. PMID:26393516

  5. Identification of a Major Dimorphic Region in the Functionally Critical N-Terminal ID1 Domain of VAR2CSA

    PubMed Central

    Doritchamou, Justin; Sabbagh, Audrey; Jespersen, Jakob S.; Renard, Emmanuelle; Salanti, Ali; Nielsen, Morten A.; Deloron, Philippe; Tuikue Ndam, Nicaise

    2015-01-01

    The VAR2CSA protein of Plasmodium falciparum is transported to and expressed on the infected erythrocyte surface where it plays a key role in placental malaria (PM). It is the current leading candidate for a vaccine to prevent PM. However, the antigenic polymorphism integral to VAR2CSA poses a challenge for vaccine development. Based on detailed analysis of polymorphisms in the sequence of its ligand-binding N-terminal region, currently the main focus for vaccine development, we assessed var2csa from parasite isolates infecting pregnant women. The results reveal for the first time the presence of a major dimorphic region in the functionally critical N-terminal ID1 domain. Parasite isolates expressing VAR2CSA with particular motifs present within this domain are associated with gravidity- and parasite density-related effects. These observations are of particular interest in guiding efforts with respect to optimization of the VAR2CSA-based vaccines currently under development. PMID:26393516

  6. Structural basis for substrate selectivity in human maltase-glucoamylase and sucrase-isomaltase N-terminal domains.

    PubMed

    Sim, Lyann; Willemsma, Carly; Mohan, Sankar; Naim, Hassan Y; Pinto, B Mario; Rose, David R

    2010-06-01

    Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are small intestinal enzymes that work concurrently to hydrolyze the mixture of linear alpha-1,4- and branched alpha-1,6-oligosaccharide substrates that typically make up terminal starch digestion products. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display overlapping substrate specificities. The N-terminal catalytic domain of human MGAM (ntMGAM) has a preference for short linear alpha-1,4-oligosaccharides, whereas N-terminal SI (ntSI) has a broader specificity for both alpha-1,4- and alpha-1,6-oligosaccharides. Here we present the crystal structure of the human ntSI, in apo form to 3.2 A and in complex with the inhibitor kotalanol to 2.15 A resolution. Structural comparison with the previously solved structure of ntMGAM reveals key active site differences in ntSI, including a narrow hydrophobic +1 subsite, which may account for its additional substrate specificity for alpha-1,6 substrates.

  7. Characterization of the N-Terminal Catalytic Domain of Lytµ1/6, an Endolysin from Streptomyces aureofaciens Phage µ1/6.

    PubMed

    Farkašovská, Jarmila; Godány, Andrej

    2016-10-01

    Previous characterization of Lytµ1/6, an endolysin from Streptomyces aureofaciens phage µ1/6, suggested that the N-terminal domain is responsible for the catalytic activity of Lytµ1/6. Mutational analyses (deletions and site-directed mutagenesis) demonstrated that lytic activity of Lytµ1/6 relies on the N-terminal part of about 200 amino acid residues. Various C-terminally truncated versions of Lytµ1/6 failed to cause lysis, indicating the necessity of the CBD for full enzyme activity. Functional analysis of the point mutants suggested that the residues K27, H31, E109, H176, and D184 were essential for lytic activity of the µ1/6 endolysin. Further characterization of the purified Lytµ1/6 revealed that this endolysin is an N-acetylmuramoyl-L-alanine amidase which seems to be unrelated to any of the known conserved catalytic domains of phage endolysins or bacterial autolysins.

  8. Structure of the mouse galectin-4 N-terminal carbohydrate-recognition domain reveals the mechanism of oligosaccharide recognition

    SciTech Connect

    Krejciríková, Veronika; Pachl, Petr; Fábry, Milan; Malý, Petr; Rezácová, Pavlína; Brynda, Jirí

    2011-11-18

    Galectin-4, a member of the tandem-repeat subfamily of galectins, participates in cell-membrane interactions and plays an important role in cell adhesion and modulation of immunity and malignity. The oligosaccharide specificity of the mouse galectin-4 carbohydrate-recognition domains (CRDs) has been reported previously. In this work, the structure and binding properties of the N-terminal domain CRD1 were further investigated and the crystal structure of CRD1 in complex with lactose was determined at 2.1 {angstrom} resolution. The lactose-binding affinity was characterized by fluorescence measurements and two lactose-binding sites were identified: a high-affinity site with a K{sub d} value in the micromolar range (K{sub d1} = 600 {+-} 70 {mu}M) and a low-affinity site with K{sub d2} = 28 {+-} 10 mM.

  9. Nuclear magnetic resonance characterization of the N-terminal thioredoxin-like domain of protein disulfide isomerase.

    PubMed Central

    Kemmink, J.; Darby, N. J.; Dijkstra, K.; Scheek, R. M.; Creighton, T. E.

    1995-01-01

    A genetically engineered protein consisting of the 120 residues at the N-terminus of human protein disulfide isomerase (PDI) has been characterized by 1H, 13C, and 15N NMR methods. The sequence of this protein is 35% identical to Escherichia coli thioredoxin, and it has been found also to have similar patterns of secondary structure and beta-sheet topology. The results confirm that PDI is a modular, multidomain protein. The last 20 residues of the N-terminal domain of PDI are some of those that are similar to part of the estrogen receptor, yet they appear to be an intrinsic part of the thioredoxin fold. This observation makes it unlikely that any of the segments of PDI with similarities to the estrogen receptor comprise individual domains. PMID:8580850

  10. N-terminal domain of prion protein directs its oligomeric association.

    PubMed

    Trevitt, Clare R; Hosszu, Laszlo L P; Batchelor, Mark; Panico, Silvia; Terry, Cassandra; Nicoll, Andrew J; Risse, Emmanuel; Taylor, William A; Sandberg, Malin K; Al-Doujaily, Huda; Linehan, Jacqueline M; Saibil, Helen R; Scott, David J; Collinge, John; Waltho, Jonathan P; Clarke, Anthony R

    2014-09-12

    The self-association of prion protein (PrP) is a critical step in the pathology of prion diseases. It is increasingly recognized that small non-fibrillar β-sheet-rich oligomers of PrP may be of crucial importance in the prion disease process. Here, we characterize the structure of a well defined β-sheet-rich oligomer, containing ∼12 PrP molecules, and often enclosing a central cavity, formed using full-length recombinant PrP. The N-terminal region of prion protein (residues 23-90) is required for the formation of this distinct oligomer; a truncated form comprising residues 91-231 forms a broad distribution of aggregated species. No infectivity or toxicity was found using cell and animal model systems. This study demonstrates that examination of the full repertoire of conformers and assembly states that can be accessed by PrP under specific experimental conditions should ideally be done using the full-length protein. PMID:25074940

  11. Identification of Two Binding Domains, One for Peptidoglycan and Another for a Secondary Cell Wall Polymer, on the N-Terminal Part of the S-Layer Protein SbsB from Bacillus stearothermophilus PV72/p2

    PubMed Central

    Sára, Margit; Egelseer, Eva M.; Dekitsch, Christine; Sleytr, Uwe B.

    1998-01-01

    First studies on the structure-function relationship of the S-layer protein from B. stearothermophilus PV72/p2 revealed the coexistence of two binding domains on its N-terminal part, one for peptidoglycan and another for a secondary cell wall polymer (SCWP). The peptidoglycan binding domain is located between amino acids 1 to 138 of the mature S-layer protein comprising a typical S-layer homologous domain. The SCWP binding domain lies between amino acids 240 to 331 and possesses a high serine plus glycine content. PMID:9852032

  12. Solution structure and membrane-binding property of the N-terminal tail domain of human annexin I.

    PubMed

    Yoon, M K; Park, S H; Won, H S; Na, D S; Lee, B J

    2000-11-10

    The conformational preferences of AnxI(N26), a peptide corresponding to residues 2-26 of human annexin I, were investigated using CD and NMR spectroscopy. CD results showed that AnxI(N26) adopts a mainly alpha-helical conformation in membrane-mimetic environments, TFE/water and SDS micelles, while a predominantly random structure with slight helical propensity in aqueous buffer. The helical region of AnxI(N26) showed a nearly identical conformation between in TFE/water and in SDS micelles, except for the orientation of the Trp-12 side-chain, which was quite different between the two. The N-terminal region of the AnxI(N26) helix showed a typical amphipathic nature, which could be stabilized by the neighboring hydrophobic cluster. The helical stability of the peptide in SDS micelles was increased by addition of calcium ions. These results suggest that the N-terminal tail domain of human annexin I interacts with biological membranes in a partially calcium-dependent manner.

  13. The Scavenger Receptor SSc5D Physically Interacts with Bacteria through the SRCR-Containing N-Terminal Domain

    PubMed Central

    Bessa Pereira, Catarina; Bocková, Markéta; Santos, Rita F.; Santos, Ana Mafalda; Martins de Araújo, Mafalda; Oliveira, Liliana; Homola, Jiří; Carmo, Alexandre M.

    2016-01-01

    The scavenger receptor cysteine-rich (SRCR) family comprises a group of membrane-attached or secreted proteins that contain one or more modules/domains structurally similar to the membrane distal domain of type I macrophage scavenger receptor. Although no all-inclusive biological function has been ascribed to the SRCR family, some of these receptors have been shown to recognize pathogen-associated molecular patterns (PAMP) of bacteria, fungi, or other microbes. SSc5D is a recently described soluble SRCR receptor produced by monocytes/macrophages and T lymphocytes, consisting of an N-terminal portion, which contains five SRCR modules, and a large C-terminal mucin-like domain. Toward establishing a global common role for SRCR domains, we interrogated whether the set of five SRCR domains of SSc5D displayed pattern recognition receptor (PRR) properties. For that purpose, we have expressed in a mammalian expression system the N-terminal SRCR-containing moiety of SSc5D (N-SSc5D), thus excluding the mucin-like domain likely by nature to bind microorganisms, and tested the capacity of the SRCR functional groups to physically interact with bacteria. Using conventional protein–bacteria binding assays, we showed that N-SSc5D had a superior capacity to bind to Escherichia coli strains RS218 and IHE3034 compared with that of the extracellular domains of the SRCR proteins CD5 and CD6 (sCD5 and sCD6, respectively), and similar E. coli-binding properties as Spα, a proven PRR of the SRCR family. We have further designed a more sensitive, real-time, and label-free surface plasmon resonance (SPR)-based assay and examined the capacity of N-SSc5D, Spα, sCD5, and sCD6 to bind to different bacteria. We demonstrated that N-SSc5D compares with Spα in the capacity to bind to E. coli and Listeria monocytogenes, and further that it can distinguish between pathogenic E. coli RS218 and IHE3034 strains and the non-pathogenic laboratory E. coli strain BL21(DE3). Our work thus advocates the

  14. Alteration of Substrate Specificity: The Variable N-Terminal Domain of Tobacco Ca2+-Dependent Protein Kinase Is Important for Substrate Recognition[W

    PubMed Central

    Ito, Takeshi; Nakata, Masaru; Fukazawa, Jutarou; Ishida, Sarahmi; Takahashi, Yohsuke

    2010-01-01

    Protein kinases are major signaling molecules that are involved in a variety of cellular processes. However, the molecular mechanisms whereby protein kinases discriminate specific substrates are still largely unknown. Ca2+-dependent protein kinases (CDPKs) play central roles in Ca2+ signaling in plants. Previously, we found that a tobacco (Nicotiana tabacum) CDPK1 negatively regulated the transcription factor REPRESSION OF SHOOT GROWTH (RSG), which is involved in gibberellin feedback regulation. Here, we found that the variable N-terminal domain of CDPK1 is necessary for the recognition of RSG. A mutation (R10A) in the variable N-terminal domain of CDPK1 reduced both RSG binding and RSG phosphorylation while leaving kinase activity intact. Furthermore, the R10A mutation suppressed the in vivo function of CDPK1. The substitution of the variable N-terminal domain of an Arabidopsis thaliana CDPK, At CPK9, with that of Nt CDPK1 conferred RSG kinase activities. This chimeric CDPK behaved according to the identity of the variable N-terminal domain in transgenic plants. Our results open the possibility of engineering the substrate specificity of CDPK by manipulation of the variable N-terminal domain, enabling a rational rewiring of cellular signaling pathways. PMID:20442373

  15. Structural studies of the N-terminal fragments of the WW domain: Insights into co-translational folding of a beta-sheet protein

    PubMed Central

    Hanazono, Yuya; Takeda, Kazuki; Miki, Kunio

    2016-01-01

    Nascent proteins fold co-translationally because the folding speed and folding pathways are limited by the rate of ribosome biosynthesis in the living cell. In addition, though full-length proteins can fold all their residues during the folding process, nascent proteins initially fold only with the N-terminal residues. However, the transient structure and the co-translational folding pathway are not well understood. Here we report the atomic structures of a series of N-terminal fragments of the WW domain with increasing amino acid length. Unexpectedly, the structures indicate that the intermediate-length fragments take helical conformations even though the full-length protein has no helical regions. The circular dichroism spectra and theoretical calculations also support the crystallographic results. This suggests that the short-range interactions are more decisive in the structure formation than the long-range interactions for short nascent proteins. In the course of the peptide extension, the helical structure change to the structure mediated by the long-range interactions at a particular polypeptide length. Our results will provide unique information for elucidating the nature of co-translational folding. PMID:27698466

  16. The N-terminal domain of the repressor of Staphylococcus aureus phage Φ11 possesses an unusual dimerization ability and DNA binding affinity.

    PubMed

    Biswas, Anindya; Mandal, Sukhendu; Sau, Subrata

    2014-01-01

    Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors. PMID:24747758

  17. The N-Terminal Domain of the Repressor of Staphylococcus aureus Phage Φ11 Possesses an Unusual Dimerization Ability and DNA Binding Affinity

    PubMed Central

    Biswas, Anindya; Mandal, Sukhendu; Sau, Subrata

    2014-01-01

    Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors. PMID:24747758

  18. Immunological and protective effects of Bordetella bronchiseptica subunit vaccines based on the recombinant N-terminal domain of dermonecrotic toxin.

    PubMed

    Wang, Chuanwen; Liu, Liping; Zhang, Zhen; Yan, Zhengui; Yu, Cuilian; Shao, Mingxu; Jiang, Xiaodong; Chi, Shanshan; Wei, Kai; Zhu, Ruiliang

    2015-10-01

    Dermonecrotic toxin (DNT) produced by Bordetella bronchiseptica (B. bronchiseptica) can cause clinical turbinate atrophy in swine and induce dermonecrotic lesions in model mice. We know that the N-terminal of DNT molecule contains the receptor-binding domain, which facilitates binding to the target cells. However, we do not know whether this domain has sufficient immunogenicity to resist B. bronchiseptica damage and thereby to develop a subunit vaccine for the swine industry. In this study, we prokaryotically expressed the recombinant N-terminal of DNT from B. bronchiseptica (named DNT-N) and prepared it for the subunit vaccine to evaluate its immunogenicity. Taishan Pinus massoniana pollen polysaccharide (TPPPS), a known immunomodulator, was used as the adjuvant to examine its immune-conditioning effects. At 49 d after inoculation, 10 mice from each group were challenged with B. bronchiseptica, and another 10 mice were intradermally challenged with native DNT, to examine the protection imparted by the vaccines. The immune parameters (T-lymphocyte counts, cytokine secretions, serum antibody titers, and survival rates) and skin lesions were determined. The results showed that pure DNT-N vaccine significantly induced immune responses and had limited ability to resist the B. bronchiseptica and DNT challenge, whereas the mice administered with TPPPS or Freund's incomplete adjuvant vaccine could induce higher levels of the above immune parameters. Remarkably, the DNT-N vaccine combined with TPPPS adjuvant protected the mice effectively to prevent B. bronchiseptica infection. Our findings indicated that DNT-N has potential for development as an effective subunit vaccine to counteract the damage of B. bronchiseptica infection, especially when used conjointly with TPPPS. PMID:26337750

  19. Immunological and protective effects of Bordetella bronchiseptica subunit vaccines based on the recombinant N-terminal domain of dermonecrotic toxin.

    PubMed

    Wang, Chuanwen; Liu, Liping; Zhang, Zhen; Yan, Zhengui; Yu, Cuilian; Shao, Mingxu; Jiang, Xiaodong; Chi, Shanshan; Wei, Kai; Zhu, Ruiliang

    2015-10-01

    Dermonecrotic toxin (DNT) produced by Bordetella bronchiseptica (B. bronchiseptica) can cause clinical turbinate atrophy in swine and induce dermonecrotic lesions in model mice. We know that the N-terminal of DNT molecule contains the receptor-binding domain, which facilitates binding to the target cells. However, we do not know whether this domain has sufficient immunogenicity to resist B. bronchiseptica damage and thereby to develop a subunit vaccine for the swine industry. In this study, we prokaryotically expressed the recombinant N-terminal of DNT from B. bronchiseptica (named DNT-N) and prepared it for the subunit vaccine to evaluate its immunogenicity. Taishan Pinus massoniana pollen polysaccharide (TPPPS), a known immunomodulator, was used as the adjuvant to examine its immune-conditioning effects. At 49 d after inoculation, 10 mice from each group were challenged with B. bronchiseptica, and another 10 mice were intradermally challenged with native DNT, to examine the protection imparted by the vaccines. The immune parameters (T-lymphocyte counts, cytokine secretions, serum antibody titers, and survival rates) and skin lesions were determined. The results showed that pure DNT-N vaccine significantly induced immune responses and had limited ability to resist the B. bronchiseptica and DNT challenge, whereas the mice administered with TPPPS or Freund's incomplete adjuvant vaccine could induce higher levels of the above immune parameters. Remarkably, the DNT-N vaccine combined with TPPPS adjuvant protected the mice effectively to prevent B. bronchiseptica infection. Our findings indicated that DNT-N has potential for development as an effective subunit vaccine to counteract the damage of B. bronchiseptica infection, especially when used conjointly with TPPPS.

  20. Structural characterization of the DAXX N-terminal helical bundle domain and its complex with Rassf1C.

    PubMed

    Escobar-Cabrera, Eric; Lau, Desmond K W; Giovinazzi, Serena; Ishov, Alexander M; McIntosh, Lawrence P

    2010-12-01

    DAXX is a scaffold protein with diverse roles including transcription and cell cycle regulation. Using NMR spectroscopy, we demonstrate that the C-terminal half of DAXX is intrinsically disordered, whereas a folded domain is present near its N terminus. This domain forms a left-handed four-helix bundle (H1, H2, H4, H5). However, due to a crossover helix (H3), this topology differs from that of the Sin3 PAH domain, which to date has been used as a model for DAXX. The N-terminal residues of the tumor suppressor Rassf1C fold into an amphipathic α helix upon binding this DAXX domain via a shallow cleft along the flexible helices H2 and H5 (K(D) ∼60 μM). Based on a proposed DAXX recognition motif as hydrophobic residues preceded by negatively charged groups, we found that peptide models of p53 and Mdm2 also bound the helical bundle. These data provide a structural foundation for understanding the diverse functions of DAXX.

  1. The N-terminal domain of GluR6-subtype glutamate receptor ion channels

    SciTech Connect

    Kumar, Janesh; Schuck, Peter; Jin, Rongsheng; Mayer, Mark L.

    2009-09-25

    The amino-terminal domain (ATD) of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand-bound complexes of LIVBP and G protein-coupled glutamate receptors (mGluRs), and the dimer assembly has a markedly different conformation from that found in mGluRs. This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain that are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.

  2. Crystallization and preliminary X-ray crystallographic studies of the N-terminal domain of FadD28, a fatty-acyl AMP ligase from Mycobacterium tuberculosis

    SciTech Connect

    Goyal, Aneesh; Yousuf, Malikmohamed; Rajakumara, Eerappa; Arora, Pooja; Gokhale, Rajesh S.; Sankaranarayanan, Rajan

    2006-04-01

    The crystallization and preliminary X-ray crystallographic studies of the N-terminal domain of FadD28, a fatty-acyl AMP ligase from M. tuberculosis, are reported. FadD28 from Mycobacterium tuberculosis belongs to the fatty-acyl AMP ligase (FAAL) family of proteins. It is essential for the biosynthesis of a virulent phthiocerol dimycocerosate (PDIM) lipid that is only found in the cell wall of pathogenic mycobacteria. The N-terminal domain, comprising of the first 460 residues, was crystallized by the hanging-drop vapour-diffusion method at 295 K. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 50.97, b = 60.74, c = 136.54 Å. The crystal structure of the N-terminal domain of FadD28 at 2.35 Å resolution has been solved using the MAD method.

  3. Atomic-Resolution Structures of the APC/C Subunits Apc4 and the Apc5 N-Terminal Domain

    PubMed Central

    Cronin, Nora B.; Yang, Jing; Zhang, Ziguo; Kulkarni, Kiran; Chang, Leifu; Yamano, Hiroyuki; Barford, David

    2015-01-01

    Many essential biological processes are mediated by complex molecular machines comprising multiple subunits. Knowledge on the architecture of individual subunits and their positions within the overall multimeric complex is key to understanding the molecular mechanisms of macromolecular assemblies. The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit complex that regulates cell cycle progression by ubiquitinating cell cycle proteins for proteolysis by the proteasome. The holo-complex is composed of 15 different proteins that assemble to generate a complex of 20 subunits. Here, we describe the crystal structures of Apc4 and the N-terminal domain of Apc5 (Apc5N). Apc4 comprises a WD40 domain split by a long α-helical domain, whereas Apc5N has an α-helical fold. In a separate study, we had fitted these atomic models to a 3.6-Å-resolution cryo-electron microscopy map of the APC/C. We describe how, in the context of the APC/C, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, whereas Apc5N shows small conformational changes relative to its crystal structure. We discuss the complementary approaches of high-resolution electron microscopy and protein crystallography to the structure determination of subunits of multimeric complexes. PMID:26343760

  4. Structure and dynamics of the N-terminal domain of the Cu(I) binding protein CusB.

    PubMed

    Ucisik, Melek N; Chakravorty, Dhruva K; Merz, Kenneth M

    2013-10-01

    CusCFBA is one of the metal efflux systems in Escherichia coli that is highly specific for its substrates, Cu(I) and Ag(I). It serves to protect the bacteria in environments that have lethal concentrations of these metals. The membrane fusion protein CusB is the periplasmic piece of CusCFBA, which has not been fully characterized by crystallography because of its extremely disordered N-terminal region. This region has both structural and functional importance because it has been experimentally proven to transfer the metal by itself from the metallochaperone CusF and to induce a structural change in the rest of CusB to increase Cu(I)/Ag(I) resistance. Understanding metal uptake from the periplasm is critical to gain insight into the mechanism of the whole CusCFBA pump, which makes resolving a structure for the N-terminal region necessary because it contains the metal binding site. We ran extensive molecular dynamics simulations to reveal the structural and dynamic properties of both the apo and Cu(I)-bound versions of the CusB N-terminal region. In contrast to its functional companion CusF, Cu(I) binding to the N-terminus of CusB causes only a slight, local stabilization around the metal site. The trajectories were analyzed in detail, revealing extensive structural disorder in both the apo and holo forms of the protein. CusB was further analyzed by breaking the protein up into three subdomains according to the extent of the observed disorder: the N- and C-terminal tails, the central beta strand motif, and the M21-M36 loop connecting the two metal-coordinating methionine residues. Most of the observed disorder was traced back to the tail regions, leading us to hypothesize that the latter two subdomains (residues 13-45) may form a functionally competent metal-binding domain because the tail regions appear to play no role in metal binding. PMID:23988152

  5. Function of the ATR N-terminal domain revealed by an ATM/ATR chimera

    SciTech Connect

    Chen Xinping; Zhao Runxiang; Glick, Gloria G.; Cortez, David . E-mail: david.cortez@vanderbilt.edu

    2007-05-01

    The ATM and ATR kinases function at the apex of checkpoint signaling pathways. These kinases share significant sequence similarity, phosphorylate many of the same substrates, and have overlapping roles in initiating cell cycle checkpoints. However, they sense DNA damage through distinct mechanisms. ATR primarily senses single stranded DNA (ssDNA) through its interaction with ATRIP, and ATM senses double strand breaks through its interaction with Nbs1. We determined that the N-terminus of ATR contains a domain that binds ATRIP. Attaching this domain to ATM allowed the fusion protein (ATM*) to bind ATRIP and associate with RPA-coated ssDNA. ATM* also gained the ability to localize efficiently to stalled replication forks as well as double strand breaks. Despite having normal kinase activity when tested in vitro and being phosphorylated on S1981 in vivo, ATM* is defective in checkpoint signaling and does not complement cellular deficiencies in either ATM or ATR. These data indicate that the N-terminus of ATR is sufficient to bind ATRIP and to promote localization to sites of replication stress.

  6. The N-terminal domain allosterically regulates cleavage and activation of the epithelial sodium channel.

    PubMed

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L; He, Hong; Garcia, Guilherme J M; Kubelka, Jan; Gentzsch, Martina; Stutts, M Jackson; Dokholyan, Nikolay V

    2014-08-15

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr(370) in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.

  7. The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel*

    PubMed Central

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L.; He, Hong; Garcia, Guilherme J. M.; Kubelka, Jan; Gentzsch, Martina; Stutts, M. Jackson; Dokholyan, Nikolay V.

    2014-01-01

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation. PMID:24973914

  8. The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity

    PubMed Central

    Jiang, Xia-Yun; Hou, Fei; Shen, Xiao-Dan; Du, Xue-Di; Xu, Hai-Li; Zou, Shu-Ming

    2016-01-01

    Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%–25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition. PMID:27251101

  9. The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity.

    PubMed

    Jiang, Xia-Yun; Hou, Fei; Shen, Xiao-Dan; Du, Xue-Di; Xu, Hai-Li; Zou, Shu-Ming

    2016-01-01

    Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%-25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition. PMID:27251101

  10. Selective heterogeneous acid catalyzed esterification of N-terminal sulfyhdryl fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our interest in thiol fatty acids lies in their antioxidative, free radical scavenging, and metal ion scavenging capabilities as applied to cosmeceutical and skin care formulations. The retail market is filled with products containing the disulfide-containing free fatty acid, lipoic acid. These pr...

  11. A novel mechanism of protein thermostability: a unique N-terminal domain confers heat resistance to Fe/Mn-SODs

    PubMed Central

    Wang, Wei; Ma, Ting; Zhang, Baoliang; Yao, Nana; Li, Mingchang; Cui, Lianlei; Li, Guoqiang; Ma, Zhenping; Cheng, Jiansong

    2014-01-01

    Superoxide dismutases (SODs), especially thermostable SODs, are widely applied in medical treatments, cosmetics, food, agriculture, and other industries given their excellent antioxidant properties. A novel thermostable cambialistic SOD from Geobacillus thermodenitrificans NG80-2 exhibits maximum activity at 70°C and high thermostability over a broad range of temperatures (20–80°C). Unlike other reported SODs, this enzyme contains an extra repeat-containing N-terminal domain (NTD) of 244 residues adjacent to the conserved functional SODA domain. Deletion of the NTD dramatically decreased its optimum active temperature (OAT) to 30°C and also impaired its thermostability. Conversely, appending the NTD to a mesophilic counterpart from Bacillus subtilis led to a moderately thermophilic enzyme (OAT changed from 30 to 55°C) with improved heat resistance. Temperature-dependant circular dichroism analysis revealed the enhanced conformational stability of SODs fused with this NTD. Furthermore, the NTD also contributes to the stress resistance of host proteins without altering their metal ion specificity or oligomerisation form except for a slight effect on their pH profile. We therefore demonstrate that the NTD confers outstanding thermostability to the host protein. To our knowledge, this is the first discovery of a peptide capable of remarkably improving protein thermostability and provides a novel strategy for bioengineering thermostable SODs. PMID:25445927

  12. Cross monomer substrate contacts reposition the Hsp90 N-terminal domain and prime the chaperone activity

    PubMed Central

    Street, Timothy O.; Lavery, Laura A.; Verba, Kliment; Lee, Chung-Tien; Mayer, Matthias P.; Agard, David A.

    2012-01-01

    The ubiquitous molecular chaperone Hsp90 plays a critical role in substrate protein folding and maintenance, but the functional mechanism has been difficult to elucidate. In previous work a model Hsp90 substrate revealed an activation process in which substrate binding accelerates a large open/closed conformational change required for ATP hydrolysis by Hsp90. While this could serve as an elegant mechanism for conserving ATP usage for productive interactions on the substrate, the structural origin of substrate catalyzed Hsp90 conformational changes are unknown. Here we find that substrate binding affects an intrinsically unfavorable rotation of the Hsp90 N-terminal domain (NTD) relative to the middle domain (MD) that is required for closure. We identify an MD substrate binding region on the interior cleft of the Hsp90 dimer and show that a secondary set of substrate contacts drive an NTD orientation change on the opposite monomer. These results suggest an Hsp90 activation mechanism in which cross-monomer contacts mediated by a partially structured substrate prime the chaperone for its functional activity. PMID:22063096

  13. The Prophage-encoded Hyaluronate Lyase Has Broad Substrate Specificity and Is Regulated by the N-terminal Domain*

    PubMed Central

    Singh, Sudhir Kumar; Bharati, Akhilendra Pratap; Singh, Neha; Pandey, Praveen; Joshi, Pankaj; Singh, Kavita; Mitra, Kalyan; Gayen, Jiaur R.; Sarkar, Jayanta; Akhtar, Md. Sohail

    2014-01-01

    Streptococcus equi is the causative agent of the highly contagious disease “strangles” in equines and zoonotic meningitis in human. Spreading of infection in host tissues is thought to be facilitated by the bacterial gene encoded extracellular hyaluronate lyase (HL), which degrades hyaluronan (HA), chondroitin 6-sulfate, and dermatan sulfate of the extracellular matrix). The clinical strain S. equi 4047 however, lacks a functional extracellular HL. The prophages of S. equi and other streptococci encode intracellular HLs which are reported to partially degrade HA and do not cleave any other glycosaminoglycans. The phage HLs are thus thought to play a role limited to the penetration of streptococcal HA capsules, facilitating bacterial lysogenization and not in the bacterial pathogenesis. Here we systematically looked into the structure-function relationship of S. equi 4047 phage HL. Although HA is the preferred substrate, this HL has weak activity toward chondroitin 6-sulfate and dermatan sulfate and can completely degrade all of them. Even though the catalytic triple-stranded β-helix domain of phage HL is functionally independent, its catalytic efficiency and specificity is influenced by the N-terminal domain. The phage HL also interacts with human transmembrane glycoprotein CD44. The above results suggest that the streptococci can use phage HLs to degrade glycosaminoglycans of the extracellular matrix for spreading virulence factors and toxins while utilizing the disaccharides as a nutrient source for proliferation at the site of infection. PMID:25378402

  14. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    SciTech Connect

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie; Cotmore, Susan F.; Tattersall, Peter; Zhao, Haiyan; Tang, Liang

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  15. The Chondroitin Sulfate A-binding Site of the VAR2CSA Protein Involves Multiple N-terminal Domains*

    PubMed Central

    Dahlbäck, Madeleine; Jørgensen, Lars M.; Nielsen, Morten A.; Clausen, Thomas M.; Ditlev, Sisse B.; Resende, Mafalda; Pinto, Vera V.; Arnot, David E.; Theander, Thor G.; Salanti, Ali

    2011-01-01

    Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDRPAM and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments. PMID:21398524

  16. The prophage-encoded hyaluronate lyase has broad substrate specificity and is regulated by the N-terminal domain.

    PubMed

    Singh, Sudhir Kumar; Bharati, Akhilendra Pratap; Singh, Neha; Pandey, Praveen; Joshi, Pankaj; Singh, Kavita; Mitra, Kalyan; Gayen, Jiaur R; Sarkar, Jayanta; Akhtar, Md Sohail

    2014-12-19

    Streptococcus equi is the causative agent of the highly contagious disease "strangles" in equines and zoonotic meningitis in human. Spreading of infection in host tissues is thought to be facilitated by the bacterial gene encoded extracellular hyaluronate lyase (HL), which degrades hyaluronan (HA), chondroitin 6-sulfate, and dermatan sulfate of the extracellular matrix). The clinical strain S. equi 4047 however, lacks a functional extracellular HL. The prophages of S. equi and other streptococci encode intracellular HLs which are reported to partially degrade HA and do not cleave any other glycosaminoglycans. The phage HLs are thus thought to play a role limited to the penetration of streptococcal HA capsules, facilitating bacterial lysogenization and not in the bacterial pathogenesis. Here we systematically looked into the structure-function relationship of S. equi 4047 phage HL. Although HA is the preferred substrate, this HL has weak activity toward chondroitin 6-sulfate and dermatan sulfate and can completely degrade all of them. Even though the catalytic triple-stranded β-helix domain of phage HL is functionally independent, its catalytic efficiency and specificity is influenced by the N-terminal domain. The phage HL also interacts with human transmembrane glycoprotein CD44. The above results suggest that the streptococci can use phage HLs to degrade glycosaminoglycans of the extracellular matrix for spreading virulence factors and toxins while utilizing the disaccharides as a nutrient source for proliferation at the site of infection.

  17. Bidirectional Modulation of Thermal and Chemical Sensitivity of TRPM8 Channels by the Initial Region of the N-terminal Domain*

    PubMed Central

    Pertusa, María; González, Alejandro; Hardy, Paulina; Madrid, Rodolfo; Viana, Félix

    2014-01-01

    TRPM8, a nonselective cation channel activated by cold, voltage, and cooling compounds such as menthol, is the principal molecular detector of cold temperatures in primary sensory neurons of the somatosensory system. The N-terminal domain of TRPM8 consists of 693 amino acids, but little is known about its contribution to channel function. Here, we identified two distinct regions within the initial N terminus of TRPM8 that contribute differentially to channel activity and proper folding and assembly. Deletion or substitution of the first 40 residues yielded channels with augmented responses to cold and menthol. The thermal threshold of activation of these mutants was shifted 2 °C to higher temperatures, and the menthol dose-response curve was displaced to lower concentrations. Site-directed mutagenesis screening revealed that single point mutations at positions Ser-26 or Ser-27 by proline caused a comparable increase in the responses to cold and menthol. Electrophysiological analysis of the S27P mutant revealed that the enhanced sensitivity to agonists is related to a leftward shift in the voltage dependence of activation, increasing the probability of channel openings at physiological membrane potentials. In addition, we found that the region encompassing positions 40–60 is a key element in the proper folding and assembly of TRPM8. Different deletions and mutations within this region rendered channels with an impaired function that are retained within the endoplasmic reticulum. Our results suggest a critical contribution of the initial region of the N-terminal domain of TRPM8 to thermal and chemical sensitivity and the proper biogenesis of this polymodal ion channel. PMID:24917670

  18. Bidirectional modulation of thermal and chemical sensitivity of TRPM8 channels by the initial region of the N-terminal domain.

    PubMed

    Pertusa, María; González, Alejandro; Hardy, Paulina; Madrid, Rodolfo; Viana, Félix

    2014-08-01

    TRPM8, a nonselective cation channel activated by cold, voltage, and cooling compounds such as menthol, is the principal molecular detector of cold temperatures in primary sensory neurons of the somatosensory system. The N-terminal domain of TRPM8 consists of 693 amino acids, but little is known about its contribution to channel function. Here, we identified two distinct regions within the initial N terminus of TRPM8 that contribute differentially to channel activity and proper folding and assembly. Deletion or substitution of the first 40 residues yielded channels with augmented responses to cold and menthol. The thermal threshold of activation of these mutants was shifted 2 °C to higher temperatures, and the menthol dose-response curve was displaced to lower concentrations. Site-directed mutagenesis screening revealed that single point mutations at positions Ser-26 or Ser-27 by proline caused a comparable increase in the responses to cold and menthol. Electrophysiological analysis of the S27P mutant revealed that the enhanced sensitivity to agonists is related to a leftward shift in the voltage dependence of activation, increasing the probability of channel openings at physiological membrane potentials. In addition, we found that the region encompassing positions 40-60 is a key element in the proper folding and assembly of TRPM8. Different deletions and mutations within this region rendered channels with an impaired function that are retained within the endoplasmic reticulum. Our results suggest a critical contribution of the initial region of the N-terminal domain of TRPM8 to thermal and chemical sensitivity and the proper biogenesis of this polymodal ion channel.

  19. A region of the N-terminal domain of meningococcal factor H-binding protein that elicits bactericidal antibody across antigenic variant groups.

    PubMed

    Beernink, Peter T; LoPasso, Carla; Angiolillo, Antonella; Felici, Franco; Granoff, Dan

    2009-05-01

    Meningococcal factor H-binding protein (fHbp) is a promising vaccine antigen. Previous studies described three fHbp antigenic variant groups and identified amino acid residues between 100 and 255 as important targets of variant-specific bactericidal antibodies. We investigated residues affecting expression of an epitope recognized by a murine IgG2a anti-fHbp mAb, designated JAR 4, which cross-reacted with fHbps in variant group 1 or 2 (95% of strains), and elicited human complement-mediated, cooperative bactericidal activity with other non-bactericidal anti-fHbp mAbs with epitopes involving residues between 121 and 216. From filamentous bacteriophage libraries containing random peptides that were recognized by JAR 4, we identified a consensus tripeptide, DHK that matched residues 25-27 in the N-terminal domain of fHbp. Since DHK was present in both JAR 4-reactive and non-reactive fHbps, the tripeptide was necessary but not sufficient for reactivity. Based on site-directed mutagenesis studies, the JAR 4 epitope could either be knocked out of a reactive variant 1 fHbp, or introduced into a non-reactive variant 3 protein. Collectively, the data indicated that the JAR 4 epitope was discontinuous and involved DHK residues beginning at position 25; YGN residues beginning at position 57; and a KDN tripeptide that was present in variant 3 proteins beginning at position 67 that negatively affected expression of the epitope. Thus, the region of fHbp encompassing residues 25-59 in the N-terminal domain is important for eliciting antibodies that can cooperate with other anti-fHbp antibodies for cross-reactive bactericidal activity against strains expressing fHbp from different antigenic variant groups.

  20. Effects of site-directed mutagenesis in the N-terminal domain of thermolysin on its stabilization

    PubMed Central

    Kawasaki, Yuichi; Yasukawa, Kiyoshi; Inouye, Kuniyo

    2013-01-01

    The thermolysin variant G8C/N60C/S65P in which the triple mutation in the N-terminal domain, Gly8→Cys/Asn60→Cys/Ser65→Pro, is undertaken increases stability [Yasukawa, K. and Inouye, K. (2007) Improving the activity and stability of thermolysin by site-directed mutagenesis. Biochim. Biophys. Acta 1774, 1281–1288] and its mechanism is examined in this study. The apparent denaturing temperatures based on ellipticity at 222 nm of the wild-type thermolysin (WT), G8C/N60C, S65P and G8C/N60C/S65P were 85, >95, 88 and >95°C, respectively. The first-order rate constants, kobs, of the thermal inactivation of WT and variants at 10 mM CaCl2 increased with increasing thermal treatment temperatures (70–95°C), and those at 80°C decreased with increasing CaCl2 concentrations (1–100 mM). The kobs values were in the order of WT > S65P > G8C/N60C≒G8C/N60C/S65P at all temperatures and CaCl2 concentrations. These results indicate that the mutational combination, Gly8→Cys/Asn60→Cys and Ser65→Pro, increases stability only as high as Gly8→Cys/Asn60→Cys does. Assuming that irreversible inactivation of thermolysin occurs only in the absence of calcium ions, the dissociation constants, Kd, to the calcium ions of WT, G8C/N60C, S65P and G8C/N60C/S65P were 47, 8.9, 17 and 7.2 mM, respectively, suggesting that Gly8→Cys/Asn60→Cys and Ser65→Pro stabilize thermolysin by improving its affinity to calcium ions, most probably the one at the Ca2+-binding site III in the N-terminal domain. PMID:23087322

  1. The N-terminal end of the catalytic domain of SRC kinase Hck is a conformational switch implicated in long-range allosteric regulation.

    PubMed

    Banavali, Nilesh K; Roux, Benoît

    2005-11-01

    Signal transduction in cell growth and proliferation involves regulation of kinases through long-range allostery between remote protein regions. Molecular dynamics free energy calculations are used to clarify the coupling between the catalytic domain of Src kinase Hck and its N-terminal end connecting to the regulatory SH2 and SH3 modules. The N-terminal end is stable in the orientation required for the regulatory modules to remain properly bound only in the inactive catalytic domain. In the active catalytic domain, the N-terminal end prefers a different conformation consistent with dissociation of the regulatory modules. The free energy surface shows that the N-terminal end acts as a reversible two-state conformational switch coupling the catalytic domain to the regulatory modules. Structural analogy with insulin receptor kinase and c-Src suggests that such reversible conformational switching in a critical hinge region could be a common mechanism in long-range allosteric regulation of protein kinase activity.

  2. Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

    SciTech Connect

    Nakayama, Yuji; Kawana, Akiko; Igarashi, Asae; Yamaguchi, Naoto . E-mail: nyama@p.chiba-u.ac.jp

    2006-07-15

    Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to {approx}200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation.

  3. Structural and biochemical characterization of an RNA/DNA binding motif in the N-terminal domain of RecQ4 helicases

    PubMed Central

    Marino, Francesca; Mojumdar, Aditya; Zucchelli, Chiara; Bhardwaj, Amit; Buratti, Emanuele; Vindigni, Alessandro; Musco, Giovanna; Onesti, Silvia

    2016-01-01

    The RecQ4 helicase belongs to the ubiquitous RecQ family but its exact role in the cell is not completely understood. In addition to the helicase domain, RecQ4 has a unique N-terminal part that is essential for viability and is constituted by a region homologous to the yeast Sld2 replication initiation factor, followed by a cysteine-rich region, predicted to fold as a Zn knuckle. We carried out a structural and biochemical analysis of both the human and Xenopus laevis RecQ4 cysteine-rich regions, and showed by NMR spectroscopy that the Xenopus fragment indeed assumes the canonical Zn knuckle fold, whereas the human sequence remains unstructured, consistent with the mutation of one of the Zn ligands. Both the human and Xenopus Zn knuckles bind to a variety of nucleic acid substrates, with a mild preference for RNA. We also investigated the effect of a segment located upstream the Zn knuckle that is highly conserved and rich in positively charged and aromatic residues, partially overlapping with the C-terminus of the Sld2-like domain. In both the human and Xenopus proteins, the presence of this region strongly enhances binding to nucleic acids. These results reveal novel possible roles of RecQ4 in DNA replication and genome stability. PMID:26888063

  4. Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain.

    PubMed

    Stroehlein, Andreas J; Young, Neil D; Korhonen, Pasi K; Chang, Bill C H; Sternberg, Paul W; La Rosa, Giuseppe; Pozio, Edoardo; Gasser, Robin B

    2016-01-01

    Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation. PMID:27412987

  5. The N-terminal Domain of NPC1L1 Protein Binds Cholesterol and Plays Essential Roles in Cholesterol Uptake*

    PubMed Central

    Zhang, Jin-Hui; Ge, Liang; Qi, Wei; Zhang, Liqing; Miao, Hong-Hua; Li, Bo-Liang; Yang, Maojun; Song, Bao-Liang

    2011-01-01

    Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the formation of NPC1L1-flotillin-cholesterol membrane microdomains, and prevents NPC1L1-mediated cholesterol uptake in culture cells and mice livers. NPC1L1-NTD specifically binds cholesterol but not plant sterols, which may account for the selective cholesterol absorption in intestine. Furthermore, 25- or 27-hydroxycholesterol competes with cholesterol to bind NPC1L1-NTD and inhibits the cholesterol induced endocytosis of NPC1L1. Together, these results demonstrate that plasma membrane-localized NPC1L1 binds exogenous cholesterol via its NTD, and facilitates the formation of NPC1L1-flotillin-cholesterol membrane microdomains that are then internalized into cells through the clathrin-AP2 pathway. Our study uncovers the mechanism of cholesterol sensing by NPC1L1 and proposes a mechanism for selective cholesterol absorption. PMID:21602275

  6. Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

    SciTech Connect

    Kwon, Hyock Joo; Abi-Mosleh, Lina; Wang, Michael L.; Deisenhofer, Johann; Goldstein, Joseph L.; Brown, Michael S.; Infante, Rodney E.

    2010-09-21

    LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3{beta}-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3{beta}-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.

  7. Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain

    PubMed Central

    Stroehlein, Andreas J.; Young, Neil D.; Korhonen, Pasi K.; Chang, Bill C. H.; Sternberg, Paul W.; La Rosa, Giuseppe; Pozio, Edoardo; Gasser, Robin B.

    2016-01-01

    Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation. PMID:27412987

  8. PrP N-terminal domain triggers PrP{sup Sc}-like aggregation of Dpl

    SciTech Connect

    Erlich, Paul; Cesbron, Jean-Yves; Lemaire-Vieille, Catherine; Curt, Aurelie; Andrieu, Jean-Pierre; Schoehn, Guy; Jamin, Marc; Gagnon, Jean

    2008-01-18

    Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP{sup C}, for 'cellular prion protein') into an abnormal state (PrP{sup Sc}, for 'scrapie prion protein'). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP{sup C}. In contrast to its homologue PrP{sup C}, Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP{sup Sc}-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP{sup C} (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the {alpha}-helical monomer forms soluble {beta}-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy.

  9. Role of N-terminal domain of HMW 1Dx5 in the functional and structural properties of wheat dough.

    PubMed

    Wang, Jing Jing; Liu, Guang; Huang, Yan-Bo; Zeng, Qiao-Hui; Song, Guo-Sheng; Hou, Yi; Li, Lin; Hu, Song-Qing

    2016-12-15

    Effects of N-terminal domain of high molecular weight glutenin subunit (HMW-GS) 1Dx5 (1Dx5-N) on functional and structural properties of wheat dough were determined by farinographic and rheological analysis, size exclusion chromatography, non-reducing/reducing SDS-PAGE, total free sulfhydryl determination, scanning electron microscopy and Fourier transform infrared spectroscopy. Results showed that 1Dx5-N improved the quality of dough with the increased water absorption, dough stability time, elastic and viscous modulus, and the decreased degree of softening, loss tangent. These improvements could be attributed to the formation of the macro-molecular weight aggregates and massive protein networks, which were favored by 1Dx5-N through disulfide bonds and hydrophobic interactions. Additionally, 1Dx5-N drove the transition of α-helix and random coil conformations to β-sheet and β-turn conformations, further demonstrating the formation of HMW-GS polymers and the enhancement of dough strength. Moreover, all the positive effects of 1Dx5-N were reinforced by edible salt NaCl. PMID:27451235

  10. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    NASA Technical Reports Server (NTRS)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  11. The N-terminal domain of the mammalian nucleoporin p62 interacts with other nucleoporins of the FXFG family during interphase

    SciTech Connect

    Stochaj, Ursula . E-mail: ursula.stochaj@mcgill.ca; Banski, Piotr; Kodiha, Mohamed; Matusiewicz, Neola

    2006-08-01

    Nuclear pore complexes (NPCs) provide the only sites for macromolecular transport between nucleus and cytoplasm. The nucleoporin p62, a component of higher eukaryotic NPCs, is located at the central gated channel and involved in nuclear trafficking of various cargos. p62 is organized into an N-terminal segment that contains FXFG repeats and binds the soluble transport factor NTF2, whereas the C-terminal portion associates with other nucleoporins and importin-{beta}1. We have now identified new components that interact specifically with the p62 N-terminal domain. Using the p62 N-terminal segment as bait, we affinity-purified nucleoporins Nup358, Nup214 and Nup153 from crude cell extracts. In ligand binding assays, the N-terminal p62 segment associated with Nup358 and p62, suggesting their direct binding to the p62 N-terminal portion. Furthermore, p62 was isolated in complex with Nup358, Nup214 and Nup153 from growing HeLa cells, indicating that the interactions Nup358/p62, Nup214/p62 and p62/Nup153 also occur in vivo. The formation of Nup358/p62 and p62/Nup153 complexes was restricted to interphase cells, whereas Nup214/p62 binding was detected in interphase as well as during mitosis. Our results support a model of complex interactions between FXFG containing nucleoporins, and we propose that some of these interactions may contribute to the movement of cargo across the NPC.

  12. The cardiac-specific N-terminal region of troponin I positions the regulatory domain of troponin C.

    PubMed

    Hwang, Peter M; Cai, Fangze; Pineda-Sanabria, Sandra E; Corson, David C; Sykes, Brian D

    2014-10-01

    The cardiac isoform of troponin I (cTnI) has a unique 31-residue N-terminal region that binds cardiac troponin C (cTnC) to increase the calcium sensitivity of the sarcomere. The interaction can be abolished by cTnI phosphorylation at Ser22 and Ser23, an important mechanism for regulating cardiac contractility. cTnC contains two EF-hand domains (the N and C domain of cTnC, cNTnC and cCTnC) connected by a flexible linker. Calcium binding to either domain favors an "open" conformation, exposing a large hydrophobic surface that is stabilized by target binding, cTnI[148-158] for cNTnC and cTnI[39-60] for cCTnC. We used multinuclear multidimensional solution NMR spectroscopy to study cTnI[1-73] in complex with cTnC. cTnI[39-60] binds to the hydrophobic face of cCTnC, stabilizing an alpha helix in cTnI[41-67] and a type VIII turn in cTnI[38-41]. In contrast, cTnI[1-37] remains disordered, although cTnI[19-37] is electrostatically tethered to the negatively charged surface of cNTnC (opposite its hydrophobic surface). The interaction does not directly affect the calcium binding affinity of cNTnC. However, it does fix the positioning of cNTnC relative to the rest of the troponin complex, similar to what was previously observed in an X-ray structure [Takeda S, et al. (2003) Nature 424(6944):35-41]. Domain positioning impacts the effective concentration of cTnI[148-158] presented to cNTnC, and this is how cTnI[19-37] indirectly modulates the calcium affinity of cNTnC within the context of the cardiac thin filament. Phosphorylation of cTnI at Ser22/23 disrupts domain positioning, explaining how it impacts many other cardiac regulatory mechanisms, like the Frank-Starling law of the heart. PMID:25246568

  13. Ribonucleocapsid Formation of SARS-COV Through Molecular Action of the N-Terminal Domain of N Protein

    SciTech Connect

    Saikatendu, K.S.; Joseph, J.S.; Subramanian, V.; Neuman, B.W.; Buchmeier, M.J.; Stevens, R.C.; Kuhn, P.; /Scripps Res. Inst.

    2007-07-12

    Conserved amongst all coronaviruses are four structural proteins, the matrix (M), small envelope (E) and spike (S) that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in their lumen. The N terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C-terminus of N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17A (monoclinic) and 1.85 A (cubic) respectively, solved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core and is oriented similar to that in the IBV N-NTD and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggest a common mode of RNA recognition, but probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs hints that they employ different modes of both RNA recognition as well as oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

  14. N-terminal domain of the V-ATPase a2-subunit displays integral membrane protein properties.

    PubMed

    Merkulova, Maria; McKee, Mary; Dip, Phat Vinh; Grüber, Gerhard; Marshansky, Vladimir

    2010-10-01

    V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N(1-402)) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N(1-402) recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINE®12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N(1-402) in NDSB-256 was successfully used in pull-down and BIAcore™ protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N(1-402) and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called "cytosolic" tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N(1-402) should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.

  15. Role of leucine zipper motif in apoE3 N-terminal domain lipid binding activity.

    PubMed

    Yamamoto, Taichi; Ryan, Robert O

    2006-09-01

    The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant "leucine to alanine" (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity.

  16. The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain

    PubMed Central

    Nagel, Claus-Henning; Binz, Anne; Sodeik, Beate; Bauerfeind, Rudolf; Bailer, Susanne M.

    2015-01-01

    Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+)Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment. PMID:26083367

  17. The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain.

    PubMed

    Funk, Christina; Ott, Melanie; Raschbichler, Verena; Nagel, Claus-Henning; Binz, Anne; Sodeik, Beate; Bauerfeind, Rudolf; Bailer, Susanne M

    2015-06-01

    Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+)Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment. PMID:26083367

  18. Diversified Structural Basis of a Conserved Molecular Mechanism for pH-Dependent Dimerization in Spider Silk N-Terminal Domains.

    PubMed

    Otikovs, Martins; Chen, Gefei; Nordling, Kerstin; Landreh, Michael; Meng, Qing; Jörnvall, Hans; Kronqvist, Nina; Rising, Anna; Johansson, Jan; Jaudzems, Kristaps

    2015-08-17

    Conversion of spider silk proteins from soluble dope to insoluble fibers involves pH-dependent dimerization of the N-terminal domain (NT). This conversion is tightly regulated to prevent premature precipitation and enable rapid silk formation at the end of the duct. Three glutamic acid residues that mediate this process in the NT from Euprosthenops australis major ampullate spidroin 1 are well conserved among spidroins. However, NTs of minor ampullate spidroins from several species, including Araneus ventricosus ((Av)MiSp NT), lack one of the glutamic acids. Here we investigate the pH-dependent structural changes of (Av)MiSp NT, revealing that it uses the same mechanism but involves a non-conserved glutamic acid residue instead. Homology modeling of the structures of other MiSp NTs suggests that these harbor different compensatory residues. This indicates that, despite sequence variations, the molecular mechanism underlying pH-dependent dimerization of NT is conserved among different silk types. PMID:26033527

  19. Diversified Structural Basis of a Conserved Molecular Mechanism for pH-Dependent Dimerization in Spider Silk N-Terminal Domains.

    PubMed

    Otikovs, Martins; Chen, Gefei; Nordling, Kerstin; Landreh, Michael; Meng, Qing; Jörnvall, Hans; Kronqvist, Nina; Rising, Anna; Johansson, Jan; Jaudzems, Kristaps

    2015-08-17

    Conversion of spider silk proteins from soluble dope to insoluble fibers involves pH-dependent dimerization of the N-terminal domain (NT). This conversion is tightly regulated to prevent premature precipitation and enable rapid silk formation at the end of the duct. Three glutamic acid residues that mediate this process in the NT from Euprosthenops australis major ampullate spidroin 1 are well conserved among spidroins. However, NTs of minor ampullate spidroins from several species, including Araneus ventricosus ((Av)MiSp NT), lack one of the glutamic acids. Here we investigate the pH-dependent structural changes of (Av)MiSp NT, revealing that it uses the same mechanism but involves a non-conserved glutamic acid residue instead. Homology modeling of the structures of other MiSp NTs suggests that these harbor different compensatory residues. This indicates that, despite sequence variations, the molecular mechanism underlying pH-dependent dimerization of NT is conserved among different silk types.

  20. The thermostability of two kinds of recombinant ∆6-fatty acid desaturase with different N-terminal sequence lengths in low temperature.

    PubMed

    Lu, He; Zhu, Yu

    2013-09-01

    Two recombinant Rhizopus stolonifer ∆6-fatty acid desaturase enzymes with different-length N-termini were cloned and expressed in Saccharomyces cerevisiae strain INVScl: LRsD6D begins with the sequence of the N-terminal of the R. stolonifer ∆6-fatty acid desaturase native, encoding a deduced polypeptide of 459 amino acids (M-S-T-L-D-R-Q-S-I-F-T-I-K-E-L-E-S-I-S-Q-R-I-H-D-G-D-E-E-A-M-K-F), whereas SRsD6D begins with the amino acid sequence of the predicted ORF, encoding a deduced polypeptide of 430 amino acids (M-K-F) and LRsD6D is longer than SRsD6D by 29 amino acids (M-S-T-L-D-R-Q-S-I-F-T-I-K-E-L-E-S-I-S-Q-R-I-H-D-G-D-E-E-A). Bioinformatic analysis characterized the two recombinant ∆6-fatty acid desaturase enzymes with different-length N-termini, including three conserved histidine-rich motifs, hydropathy profile, and a cytochrome b5-like domain in the N-terminus. When the coding sequence was expressed in S. cerevisiae strain INVScl, the coding produced ∆6-fatty acid desaturase activity exhibited by RsD6D, leading to a novel peak corresponding to γ-linolenic acid methyl ester standards, which was detected with the same retention time. The residual activity of LRsD6D was 74 % at 15 °C for 4 h and that of SRsD6D was 43 %. Purified recombinant LRsD6D was more stable than SRsD6D, indicating that the N-terminal extension, containing mostly hydrophobic residues, affected the overall stability of recombinant LRsD6D.

  1. Activation of c-Jun transcription factor by substitution of a charged residue in its N-terminal domain.

    PubMed Central

    Hoeffler, W K; Levinson, A D; Bauer, E A

    1994-01-01

    C-Jun is a cellular transcription factor that can control gene expression in response to treatment of cells with phorbol esters, growth factors, and expression of some oncogenes. The ability of c-Jun to catalyze the transcription of certain genes is controlled, in part, by changes in the phosphorylation state of specific amino acids in c-Jun. One of the major sites that is phosphorylated during signal response is Ser73. Here we show that substitution of a negatively charged aspartic acid residue at 73 constitutively increased transcriptional activity of c-Jun. The Asp73 substitution also enhanced its availability to bind to DNA in a whole cell extract without altering its intrinsic DNA binding activity since the intrinsic activity was unaltered for the c-Jun mutant proteins expressed in a bacterial system. The negatively charged Asp substitution may mimic the negative charge of a phosphorylated serine at 73. The substitution of an uncharged alanine at 73 resulted in lowered activities. The N-terminal end of c-Jun containing these substitutions was fused to the DNA-binding region of the bovine papilloma virus E2 protein, and was able to confer the same activation properties to the fusion protein at the heterologous E2 DNA-binding site. Ser73 lies in a region of c-Jun previously proposed to bind an uncharacterized inhibitor, perhaps related to a protein of approximately 17.5 kD that coprecipitates along with our c-Jun or the JunE2 fusion products. Images PMID:8165146

  2. N-terminal GNBP homology domain of Gram-negative binding protein 3 functions as a beta-1,3-glucan binding motif in Tenebrio molitor.

    PubMed

    Lee, Hanna; Kwon, Hyun-Mi; Park, Ji-Won; Kurokawa, Kenji; Lee, Bok Luel

    2009-08-31

    The Toll signalling pathway in invertebrates is responsible for defense against Gram-positive bacteria and fungi, leading to the expression of antimicrobial peptides via NF-kappaB-like transcription factors. Gram-negative binding protein 3 (GNBP3) detects beta-1,3-glucan, a fungal cell wall component, and activates a three step serine protease cascade for activation of the Toll signalling pathway. Here, we showed that the recombinant N-terminal domain of Tenebrio molitor GNBP3 bound to beta-1,3-glucan, but did not activate down-stream serine protease cascade in vitro. Reversely, the N-terminal domain blocked GNBP3-mediated serine protease cascade activation in vitro and also inhibited beta-1,3-glucan-mediated antimicrobial peptide induction in Tenebrio molitor larvae. These results suggest that the N-terminal GNBP homology domain of GNBP3 functions as a beta-1,3-glucan binding domain and the C-terminal domain of GNBP3 may be required for the recruitment of immediate down-stream serine protease zymogen during Toll signalling pathway activation. PMID:19712587

  3. Structure, activity, and stability of metagenome-derived glycoside hydrolase family 9 endoglucanase with an N-terminal Ig-like domain.

    PubMed

    Okano, Hiroyuki; Kanaya, Eiko; Ozaki, Masashi; Angkawidjaja, Clement; Kanaya, Shigenori

    2015-03-01

    A metagenome-derived glycoside hydrolase family 9 enzyme with an N-terminal immunoglobulin-like (Ig-like) domain, leaf-branch compost (LC)-CelG, was characterized and its crystal structure was determined. LC-CelG did not hydrolyze p-nitrophenyl cellobioside but hydrolyzed CM-cellulose, indicating that it is endoglucanase. LC-CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5-9. Its denaturation temperature was 81.4°C, indicating that LC-CelG is a thermostable enzyme. The structure of LC-CelG resembles those of CelD from Clostridium thermocellum (CtCelD), Cel9A from Alicyclobacillus acidocaldarius (AaCel9A), and cellobiohydrolase CbhA from C. thermocellum (CtCbhA), which show relatively low (29-31%) amino acid sequence identities to LC-CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC-CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC-CelG. Removal of the Ig-like domain reduced the activity and stability of LC-CelG by 100-fold and 6.3°C, respectively. Removal of the Gln40- and Asp99-mediated interactions between the Ig-like and catalytic domains destabilized LC-CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig-like domain contributes to the stabilization of LC-CelG mainly due to the Gln40- and Asp99-mediated interactions. Because the LC-CelG derivative lacking the Ig-like domain accumulated in Escherichia coli cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig-like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain. PMID:25545469

  4. Structure, activity, and stability of metagenome-derived glycoside hydrolase family 9 endoglucanase with an N-terminal Ig-like domain

    PubMed Central

    Okano, Hiroyuki; Kanaya, Eiko; Ozaki, Masashi; Angkawidjaja, Clement; Kanaya, Shigenori

    2015-01-01

    A metagenome-derived glycoside hydrolase family 9 enzyme with an N-terminal immunoglobulin-like (Ig-like) domain, leaf-branch compost (LC)-CelG, was characterized and its crystal structure was determined. LC-CelG did not hydrolyze p-nitrophenyl cellobioside but hydrolyzed CM-cellulose, indicating that it is endoglucanase. LC-CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5–9. Its denaturation temperature was 81.4°C, indicating that LC-CelG is a thermostable enzyme. The structure of LC-CelG resembles those of CelD from Clostridium thermocellum (CtCelD), Cel9A from Alicyclobacillus acidocaldarius (AaCel9A), and cellobiohydrolase CbhA from C. thermocellum (CtCbhA), which show relatively low (29–31%) amino acid sequence identities to LC-CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC-CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC-CelG. Removal of the Ig-like domain reduced the activity and stability of LC-CelG by 100-fold and 6.3°C, respectively. Removal of the Gln40- and Asp99-mediated interactions between the Ig-like and catalytic domains destabilized LC-CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig-like domain contributes to the stabilization of LC-CelG mainly due to the Gln40- and Asp99-mediated interactions. Because the LC-CelG derivative lacking the Ig-like domain accumulated in Escherichia coli cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig-like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain. PMID:25545469

  5. Structure of the N-terminal oligomerization domain of DnaD reveals a unique tetramerization motif and provides insights into scaffold formation.

    PubMed

    Schneider, S; Zhang, W; Soultanas, P; Paoli, M

    2008-03-01

    DnaD is a primosomal protein that remodels supercoiled plasmids. It binds to supercoiled forms and converts them to open forms without nicking. During this remodeling process, all the writhe is converted to twist and the plasmids are held around the periphery of large scaffolds made up of DnaD molecules. This DNA-remodeling function is the sum of a scaffold-forming activity on the N-terminal domain and a DNA-dependent oligomerization activity on the C-terminal domain. We have determined the crystal structure of the scaffold-forming N-terminal domain, which reveals a winged-helix architecture, with additional structural elements extending from both N- and C-termini. Four monomers form dimers that join into a tetramer. The N-terminal extension mediates dimerization and tetramerization, with extensive interactions and distinct interfaces. The wings and helices of the winged-helix domains remain exposed on the surface of the tetramer. Structure-guided mutagenesis and atomic force microscopy imaging indicate that these elements, together with the C-terminal extension, are involved in scaffold formation. Based upon our data, we propose a model for the DnaD-mediated scaffold formation. PMID:18206906

  6. N-Terminal Truncation of an Isolated Human IgG1 CH2 Domain Significantly Increases its Stability and Aggregation Resistance

    PubMed Central

    Gong, Rui; Wang, Yanping; Ying, Tianlei; Feng, Yang; Streaker, Emily; Prabakaran, Ponraj; Dimitrov, Dimiter S.

    2013-01-01

    Isolated human immunoglobulin G (IgG) CH2 domains are promising scaffolds for novel candidate therapeutics. Unlike other human IgG domains, CH2 is not involved in strong interchain interactions and isolated CH2 is relatively stable. However, isolated single CH2 is prone to aggregation. In native IgG and Fc molecules, the N-terminal residues of CH2 from the two heavy chains interact with each other and form hinge regions. By contrast, the N-terminal residues are highly disordered in isolated CH2. We have hypothesized that removal of the CH2 N-terminal residues may not only increase its stability but also its aggregation resistance. To test this hypothesis we constructed a shortened variant of IgG1 CH2 (CH2s) where the first seven residues of the N-terminus were deleted. We found that the thermal stability of CH2s was increased by 5°C compared to CH2. Importantly, we demonstrated that CH2s is significantly less prone to aggregation than CH2 as measured by Thioflavin T (ThT) fluorescence, turbidity and light scattering. We also found that the CH2s exhibited pH-dependent binding to a soluble single-chain human neonatal Fc receptor (shFcRn) which was significantly stronger than the very weak shFcRn binding to CH2 as measured by flow cytometry. Computer modeling suggested a possible mode of CH2 aggregation involving its N-terminal residues. Therefore, deletion of the N-terminal residues could increase drugability of CH2-based therapeutic candidates. This strategy to increase stability and aggregation resistance could also be applicable to other Ig-related proteins. PMID:23641816

  7. ADAMTS13 and 15 are not regulated by the full length and N-terminal domain forms of TIMP-1, -2, -3 and -4

    PubMed Central

    GUO, CENQI; TSIGKOU, ANASTASIA; LEE, MENG HUEE

    2016-01-01

    A disintegrin and metalloproteinase with thombospondin motifs (ADAMTS) 13 and 15 are secreted zinc proteinases involved in the turnover of von Willebrand factor and cancer suppression. In the present study, ADAMTS13 and 15 were subjected to inhibition studies with the full-length and N-terminal domain forms of tissue inhibitor of metalloproteinases (TIMPs)-1 to −4. TIMPs have no ability to inhibit the ADAMTS proteinases in the full-length or N-terminal domain form. While ADAMTS13 is also not sensitive to the hydroxamate inhibitors, batimastat and ilomastat, ADAMTS15 can be effectively inhibited by batimastat (Kiapp 299 nM). In conclusion, the present results indicate that TIMPs are not the regulators of these two ADAMTS proteinases. PMID:26870338

  8. Differential Inhibition of Macrophage Activation by Lymphocytic Choriomeningitis Virus and Pichinde Virus Is Mediated by the Z Protein N-Terminal Domain

    PubMed Central

    Xing, Junji; Chai, Zheng; Ly, Hinh

    2015-01-01

    Several arenavirus pathogens, such as Lassa and Junin viruses, inhibit macrophage activation, the molecular mechanism of which is unclear. We show that lymphocytic choriomeningitis virus (LCMV) can also inhibit macrophage activation, in contrast to Pichinde and Tacaribe viruses, which are not known to naturally cause human diseases. Using a recombinant Pichinde virus system, we show that the LCMV Z N-terminal domain (NTD) mediates the inhibition of macrophage activation and immune functions. PMID:26423945

  9. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase

    PubMed Central

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C.; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M.

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1–50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1. PMID:27375570

  10. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase.

    PubMed

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1-50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1. PMID:27375570

  11. Structural and functional insights into the role of the N-terminal Mps1 TPR domain in the SAC (spindle assembly checkpoint).

    PubMed

    Thebault, Philippe; Chirgadze, Dimitri Y; Dou, Zhen; Blundell, Tom L; Elowe, Sabine; Bolanos-Garcia, Victor M

    2012-12-15

    The SAC (spindle assembly checkpoint) is a surveillance system that ensures the timely and accurate transmission of the genetic material to offspring. The process implies kinetochore targeting of the mitotic kinases Bub1 (budding uninhibited by benzamidine 1), BubR1 (Bub1 related) and Mps1 (monopolar spindle 1), which is mediated by the N-terminus of each kinase. In the present study we report the 1.8 Å (1 Å=0.1 nm) crystal structure of the TPR (tetratricopeptide repeat) domain in the N-terminal region of human Mps1. The structure reveals an overall high similarity to the TPR motif of the mitotic checkpoint kinases Bub1 and BubR1, and a number of unique features that include the absence of the binding site for the kinetochore structural component KNL1 (kinetochore-null 1; blinkin), and determinants of dimerization. Moreover, we show that a stretch of amino acids at the very N-terminus of Mps1 is required for dimer formation, and that interfering with dimerization results in mislocalization and misregulation of kinase activity. The results of the present study provide an important insight into the molecular details of the mitotic functions of Mps1 including features that dictate substrate selectivity and kinetochore docking.

  12. Identification of key residues for the binding of glucagon to the N-terminal domain of its receptor: an alanine scan and modeling study.

    PubMed

    Prévost, M; Vertongen, P; Waelbroeck, M

    2012-10-01

    Glucagon plays an essential role in the glycemia maintenance during fasting, but also aggravates hyperglycemia in diabetic patients. A series of analogues of glucagon were synthesized replacing each amino acid of the C-terminal region (residues 15-29) with alanine. The residues affecting the binding to the glucagon receptor are found to be located on one face of the glucagon helix. Several 3-dimensional models of the N-terminal domain of the glucagon receptor in complex with its ligand peptide were built and used to analyze the peptide-receptor interface in terms of the nature of the peptide residues and the interactions they form with the receptor. The models suggest that glucagon keeps its native helical structure upon binding, and that a large part of the interface formed with the receptor is hydrophobic. We find that in the C-terminal region, F22, V23, M27, and D15 are the most important residues for peptide binding. They bury a large portion of their solvent accessible surface area and make numerous interactions with the receptor mainly of the hydrophobic type.

  13. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface.

    PubMed

    Atkison, James H; Parnham, Stuart; Marcotte, William R; Olsen, Shaun K

    2016-09-01

    Spider dragline silk is a natural polymer harboring unique physical and biochemical properties that make it an ideal biomaterial. Artificial silk production requires an understanding of the in vivo mechanisms spiders use to convert soluble proteins, called spidroins, into insoluble fibers. Controlled dimerization of the spidroin N-terminal domain (NTD) is crucial to this process. Here, we report the crystal structure of the Nephila clavipes major ampullate spidroin NTD dimer. Comparison of our N. clavipes NTD structure with previously determined Euprosthenops australis NTD structures reveals subtle conformational alterations that lead to differences in how the subunits are arranged at the dimer interface. We observe a subset of contacts that are specific to each ortholog, as well as a substantial increase in asymmetry in the interactions observed at the N. clavipes NTD dimer interface. These asymmetric interactions include novel intermolecular salt bridges that provide new insights into the mechanism of NTD dimerization. We also observe a unique intramolecular "handshake" interaction between two conserved acidic residues that our data suggest adds an additional layer of complexity to the pH-sensitive relay mechanism for NTD dimerization. The results of a panel of tryptophan fluorescence dimerization assays probing the importance of these interactions support our structural observations. Based on our findings, we propose that conformational selectivity and plasticity at the NTD dimer interface play a role in the pH-dependent transition of the NTD from monomer to stably associated dimer as the spidroin progresses through the silk extrusion duct. PMID:27445329

  14. Development and Identification of a Novel Anti-HIV-1 Peptide Derived by Modification of the N-Terminal Domain of HIV-1 Integrase.

    PubMed

    Sala, Marina; Spensiero, Antonia; Esposito, Francesca; Scala, Maria C; Vernieri, Ermelinda; Bertamino, Alessia; Manfra, Michele; Carotenuto, Alfonso; Grieco, Paolo; Novellino, Ettore; Cadeddu, Marta; Tramontano, Enzo; Schols, Dominique; Campiglia, Pietro; Gomez-Monterrey, Isabel M

    2016-01-01

    The viral enzyme integrase (IN) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) and represents an important target for the development of new antiretroviral drugs. In this study, we focused on the N-terminal domain (NTD), which is mainly involved into protein oligomerization process, for the development and synthesis of a library of overlapping peptide sequences, with specific length and specific offset covering the entire native protein sequence NTD IN 1-50. The most potent fragment, VVAKEIVAH (peptide 18), which includes a His residue instead of the natural Ser at position 39, inhibits the HIV-1 IN activity with an IC50 value of 4.5 μM. Amino acid substitution analysis on this peptide revealed essential residues for activity and allowed us to identify two nonapeptides (peptides 24 and 25), that show a potency of inhibition similar to the one of peptide 18. Interestingly, peptide 18 does not interfere with the dynamic interplay between IN subunits, while peptides 24 and 25 modulated these interactions in different manners. In fact, peptide 24 inhibited the IN-IN dimerization, while peptide 25 promoted IN multimerization, with IC50 values of 32 and 4.8 μM, respectively. In addition, peptide 25 has shown to have selective anti-infective cell activity for HIV-1. These results confirmed peptide 25 as a hit for further development of new chemotherapeutic agents against HIV-1.

  15. Functional substitution of the transient membrane-anchor domain in Escherichia coli FtsY with an N-terminal hydrophobic segment of Streptomyces lividans FtsY.

    PubMed

    Maeda, Isamu; Hirata, Asumi; Shoji, Miki; Ueda, Shunsaku; Yoshida, Kazuyuki

    2008-10-01

    FtsY is a signal recognition particle receptor in Escherichia coli that mediates the targeting of integral membrane proteins to translocons by interacting with both signal recognition particle (SRP)-nascent polypeptide-ribosome complexes and the cytoplasmic membrane. Genes encoding the N-terminal segments of Streptomyces lividans FtsY were fused to a gene encoding the E. coli FtsY NG domain (truncated versions of FtsY lacking the transient membrane-anchor domain at the N-terminus), introduced into a conditional ftsY-deletion mutant of E. coli, and expressed in trans to produce chimeric FtsY proteins. Under FtsY-depleted conditions, strains producing chimeric proteins including 34 N-terminal hydrophobic residues grew whereas strains producing chimeric proteins without these 34 residues did not. A strain producing the chimeric protein comprising the 34 residues and NG domain processed beta-lactamase, suggesting that the SRP-dependent membrane integration of leader peptidase was restored in this strain. These results suggest that the N-terminal hydrophobic segment of FtsY in this Gram-positive bacterium is responsible for its interaction with the cytoplasmic membrane.

  16. PEX5 protein binds monomeric catalase blocking its tetramerization and releases it upon binding the N-terminal domain of PEX14.

    PubMed

    Freitas, Marta O; Francisco, Tânia; Rodrigues, Tony A; Alencastre, Inês S; Pinto, Manuel P; Grou, Cláudia P; Carvalho, Andreia F; Fransen, Marc; Sá-Miranda, Clara; Azevedo, Jorge E

    2011-11-25

    Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5. PEX5 has a dual role in this process. First, it acts as a soluble receptor recognizing these proteins in the cytosol. Subsequently, at the peroxisomal docking/translocation machinery, PEX5 promotes their translocation across the organelle membrane. Despite significant advances made in recent years, several aspects of this pathway remain unclear. Two important ones regard the formation and disruption of the PEX5-cargo protein interaction in the cytosol and at the docking/translocation machinery, respectively. Here, we provide data on the interaction of PEX5 with catalase, a homotetrameric enzyme in its native state. We found that PEX5 interacts with monomeric catalase yielding a stable protein complex; no such complex was detected with tetrameric catalase. Binding of PEX5 to monomeric catalase potently inhibits its tetramerization, a property that depends on domains present in both the N- and C-terminal halves of PEX5. Interestingly, the PEX5-catalase interaction is disrupted by the N-terminal domain of PEX14, a component of the docking/translocation machinery. One or two of the seven PEX14-binding diaromatic motifs present in the N-terminal half of PEX5 are probably involved in this phenomenon. These results suggest the following: 1) catalase domain(s) involved in the interaction with PEX5 are no longer accessible upon tetramerization of the enzyme; 2) the catalase-binding interface in PEX5 is not restricted to its C-terminal peroxisomal targeting sequence type 1-binding domain and also involves PEX5 N-terminal domain(s); and 3) PEX14 participates in the cargo protein release step.

  17. Mouse Hepatitis Virus Strain A59 and Blocking Antireceptor Monoclonal Antibody Bind to the N-Terminal Domain of Cellular Receptor

    NASA Astrophysics Data System (ADS)

    Dveksler, Gabriela S.; Pensiero, Michael N.; Dieffenbach, Carl W.; Cardellichio, Christine B.; Basile, Alexis A.; Elia, Patrick E.; Holmes, Kathryn V.

    1993-03-01

    Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

  18. N-terminal amino acid sequence of the deep-sea tube worm haemoglobin remarkably resembles that of annelid haemoglobin.

    PubMed Central

    Suzuki, T; Takagi, T; Ohta, S

    1988-01-01

    The deep-sea giant tube worm Lamellibrachia, belonging to the phylum Vestimentifera, contains two extracellular haemoglobins, an Mr 3,000,000 haemoglobin and an Mr 440,000 haemoglobin. The former has a hexagonal bilayer structure and consists of six polypeptide chains (AI-VI); a study of its haem content shows that not all of the chains contain haem. The Mr 440,000 haemoglobin consists of four haem-containing chains (BI-IV). We isolated most of the chains by reverse-phase chromatography and determined the amino acid sequences of the 21-45 N-terminal residues. Eight chains (AI-IV and BI-IV) showed significant homology with haem-containing chains of annelid giant haemoglobin. The highest homology was found between Lamellibrachia chain AI and Tylorrhynchus chain I; surprisingly, 18 out of the 20 N-terminal residues are identical. On the other hand, chain AV, with an unusual Mr of 32,000, showed a rather different sequence and is likely to be a non-haem chain which might act as a linker protein in the assembly of the haem-containing chains. From these results, we conclude that the tube worm Mr 3,000,000 haemoglobin is highly homologous with annelid haemoglobin. Images Fig. 2. PMID:3202832

  19. Structure of the N-terminal domain of the protein Expansion: an ‘Expansion’ to the Smad MH2 fold

    SciTech Connect

    Beich-Frandsen, Mads; Aragón, Eric; Llimargas, Marta; Benach, Jordi; Riera, Antoni; Pous, Joan; Macias, Maria J.

    2015-04-01

    Expansion is a modular protein that is conserved in protostomes. The first structure of the N-terminal domain of Expansion has been determined at 1.6 Å resolution and the new Nα-MH2 domain was found to belong to the Smad/FHA superfamily of structures. Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.

  20. DNA and Protein Footprinting Analysis of the Modulation of DNA Binding by the N-Terminal Domain of the Saccharomyces cervisiae TATA Binding Protein

    SciTech Connect

    Gupta,S.; Cheng, H.; Mollah, A.; Jamison, E.; Morris, S.; Chance, M.; Khrapunov, S.; Brenowitz, M.

    2007-01-01

    Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by 'protein footprinting' with hydroxyl radical ({center_dot}OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.

  1. Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit.

    PubMed

    Vendel, Andrew C; Rithner, Christopher D; Lyons, Barbara A; Horne, William A

    2006-02-01

    Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating. PMID:16385006

  2. The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity.

    PubMed

    Iseli, B; Boller, T; Neuhaus, J M

    1993-09-01

    The vacuolar chitinases of class I possess an N-terminal cysteine-rich domain homologous to hevein and chitin-binding lectins such as wheat germ agglutinin and Urtica dioica lectin. To investigate the significance of this domain for the biochemical and functional characteristics of chitinase, chimeric genes encoding the basic chitinase A of tobacco (Nicotiana tabacum) with and without this domain were constructed and constitutively expressed in transgenic Nicotiana sylvestris. The chitinases were subsequently isolated and purified to homogeneity from the transgenic plants. Chromatography on colloidal chitin revealed that only the form with the N-terminal domain, and not the one without it, had chitin-binding properties, demonstrating directly that the domain is a chitin-binding domain (CBD). Under standard assay conditions with radioactive colloidal chitin, both forms of chitinase had approximately the same catalytic activity. However, kinetic analysis demonstrated that the enzyme without CBD had a considerably lower apparent affinity for its substrate. The pH and temperature optima of the two chitinases were similar, but the form with the CBD had an approximately 3-fold higher activation energy and retained a higher activity at low pH values. Both chitinases were capable of inhibiting growth of Trichoderma viride, although the form with the CBD was about three times more effective than the one without it. Thus, the CBD is not necessary for catalytic or antifungal activity of chitinase. PMID:8208848

  3. The N-terminal domain of thrombospondin-1: a key for the dual effect of TSP-1 in angiogenesis and cancer progression?

    PubMed

    Morandi, Verônica

    2009-01-01

    The data presented in this article suggest that intact TSP-1 may act, in normal vessel homeostasis, as an angiostatic factor favoring vessel quiescence, or even vessel regression, but this activity would be largely impaired in the presence of an excess of angiogenic stimuli and proteases. The fact that the N-terminal domain of TSP-1 (heparin-binding domain or HBD) is recognized by a plethora of cell receptors, all of them engaged in proangiogenic responses, strongly suggests that the proteolytic cleavage of HBD may be relevant in certain pathophysiological conditions. PMID:19219378

  4. N-terminal domains of ARC1 are essential for interaction with the N-terminal region of Exo70A1 in transducing self-incompatibility of Brassica oleracea.

    PubMed

    Shi, Songmei; Gao, Qiguo; Zeng, Jing; Liu, Xiaohuan; Pu, Quanming; Liu, Guixi; Zhang, Hecui; Yang, Xiaohong; Zhu, Liquan

    2016-09-01

    Self-incompatibility (SI) is an important mating system to prevent inbreeding and promote outcrossing. ARC1 and Exo70A1 function as the downstream targets of the S-locus receptor kinase and play conservative roles in Brassica SI signaling. Based on the sequence homology, Exo70A1 is divided into four subdomains: leucine zipper (Leu(128)-Leu(149)), hypervariable region (Ser(172)-Leu(197)), SUMO modification motif (Glu(260)-Ile(275)), and pfamExo70 domain (His(271)-Phe(627)). ARC1 contains four domains as follows: leucine zipper (Leu(116)-Leu(137)), coiled-coil domain (Thr(210)-Val(236)), U-box (Asp(282)-Trp(347)) motif, and ARM (Ala(415)-Thr(611)) domain. Bioinformatics analysis, yeast two-hybrid screening and pull-down assays show that leucine zipper and coiled-coil motifs of ARC1116-236 are required for the interaction with Exo70A1, while the addition of ARM motif results in loss of the interaction with Exo70A1. Meanwhile, the N-terminal of Exo70A1 without any domains shows a weak interaction with ARC1, and the level of LacZ expression increases with addition of leucine zipper and reaches the maximum value with hypervariable region and SUMO modification motif, indicating that hypervariable region and SUMO modification motif of Exo70A1172-275 is mainly responsible for the binding with ARC1, whereas pfamExo70 domain has little affinity for ARC1. Lys(181) located in the Exo70A1 hypervariable region may be the ubiquitination site mediating the interaction between ARC1 and Exo70A1. Therefore, both the leucine zipper with coiled-coil structure of ARC1116-236, and the hypervariable region and SUMO modification motif of Exo70A1172-275 are the core interaction domains between ARC1 and Exo70A1. Any factors affecting these core domains would be the regulators of ARC1 mediating ubiquitin degradation in self-incompatible system. PMID:27590064

  5. N-terminal domains of ARC1 are essential for interaction with the N-terminal region of Exo70A1 in transducing self-incompatibility of Brassica oleracea.

    PubMed

    Shi, Songmei; Gao, Qiguo; Zeng, Jing; Liu, Xiaohuan; Pu, Quanming; Liu, Guixi; Zhang, Hecui; Yang, Xiaohong; Zhu, Liquan

    2016-09-01

    Self-incompatibility (SI) is an important mating system to prevent inbreeding and promote outcrossing. ARC1 and Exo70A1 function as the downstream targets of the S-locus receptor kinase and play conservative roles in Brassica SI signaling. Based on the sequence homology, Exo70A1 is divided into four subdomains: leucine zipper (Leu(128)-Leu(149)), hypervariable region (Ser(172)-Leu(197)), SUMO modification motif (Glu(260)-Ile(275)), and pfamExo70 domain (His(271)-Phe(627)). ARC1 contains four domains as follows: leucine zipper (Leu(116)-Leu(137)), coiled-coil domain (Thr(210)-Val(236)), U-box (Asp(282)-Trp(347)) motif, and ARM (Ala(415)-Thr(611)) domain. Bioinformatics analysis, yeast two-hybrid screening and pull-down assays show that leucine zipper and coiled-coil motifs of ARC1116-236 are required for the interaction with Exo70A1, while the addition of ARM motif results in loss of the interaction with Exo70A1. Meanwhile, the N-terminal of Exo70A1 without any domains shows a weak interaction with ARC1, and the level of LacZ expression increases with addition of leucine zipper and reaches the maximum value with hypervariable region and SUMO modification motif, indicating that hypervariable region and SUMO modification motif of Exo70A1172-275 is mainly responsible for the binding with ARC1, whereas pfamExo70 domain has little affinity for ARC1. Lys(181) located in the Exo70A1 hypervariable region may be the ubiquitination site mediating the interaction between ARC1 and Exo70A1. Therefore, both the leucine zipper with coiled-coil structure of ARC1116-236, and the hypervariable region and SUMO modification motif of Exo70A1172-275 are the core interaction domains between ARC1 and Exo70A1. Any factors affecting these core domains would be the regulators of ARC1 mediating ubiquitin degradation in self-incompatible system.

  6. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

    SciTech Connect

    Berbís, M. Álvaro; André, Sabine; Cañada, F. Javier; Pipkorn, Rüdiger; Ippel, Hans; Mayo, Kevin H.; Kübler, Dieter; Gabius, Hans-Joachim; Jiménez-Barbero, Jesús

    2014-01-03

    Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.

  7. Evidence for an Interaction between the SH3 Domain and the N-terminal Extension of the Essential Light Chain in Class II Myosins

    PubMed Central

    Lowey, Susan; Saraswat, Lakshmi D.; Liu, HongJun; Volkmann, Niels; Hanein, Dorit

    2009-01-01

    SUMMARY The function of the src-homology 3 (SH3) domain in class II myosins, a distinct β-barrel structure, remains unknown. Here we provide evidence, using electron cryomicroscopy, in conjunction with light scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41-residue extension contains four conserved lysines followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the ~9 nm distance between the myosin lever arm and the thin filament. In order to localize the N-terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle. PMID:17597155

  8. The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function

    PubMed Central

    Nitharwal, Ram Gopal; Paul, Subhankar; Dar, Ashraf; Choudhury, Nirupam Roy; Soni, Rajesh K; Prusty, Dhaneswar; Sinha, Sukrat; Kashav, Tara; Mukhopadhyay, Gauranga; Chaudhuri, Tapan Kumar; Gourinath, Samudrala; Dhar, Suman Kumar

    2007-01-01

    Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function. PMID:17430964

  9. The N-terminal domain of antithrombin-III is essential for heparin binding and complex-formation with, but not cleavage by, alpha-thrombin.

    PubMed Central

    Austin, R C; Sheffield, W P; Rachubinski, R A; Blajchman, M A

    1992-01-01

    Normal and mutant forms of human antithrombin-III (AT-III) were synthesized in a cell-free system in order to identify putative functional domains required for heparin binding and complex-formation with alpha-thrombin. Heparin-Sepharose chromatography resulted in the elution of approx. 70% of cell-free-derived normal AT-III-(1-432)-polypeptide as a peak between 0.2 M- and 0.7 M-NaCl. The cell-free-derived normal AT-III also reacted with alpha-thrombin. Approx. 15% of this AT-III formed covalent complexes with alpha-thrombin in 2 min. Unfractionated heparin accelerated the rate of formation of such complexes. Two truncated forms of AT-III (amino acid residues 219-432 and 251-432), containing only the putative thrombin-binding domain, were synthesized independently in this cell-free system. These truncated AT-III polypeptides did not bind heparin and were unable to form stable covalent complexes with alpha-thrombin. However, both of these AT-III polypeptides were cleaved by alpha-thrombin, presumably at the reactive centre Arg-393-Ser-394. The formation of the disulphide bond between Cys-247 and Cys-430 in AT-III-(219-432)-polypeptide had no effect on the results obtained. Mutations in full-length AT-III at Cys-430 had no effect on the ability of AT-III to bind heparin. There was, however, a slight decrease in the formation of stable inhibitory complexes with alpha-thrombin. A cell-free-derived AT-III mutant, devoid of amino acid residues 41-49, which comprise heparin-binding region 1 of AT-III, had slightly decreased heparin binding compared with cell-free-derived normal AT-III-(1-432)-polypeptide. This mutant AT-III polypeptide was unable, however, to form a stable complex with alpha-thrombin. We conclude therefore that the N-terminal domain of AT-III is essential for both heparin binding and complex-formation with alpha-thrombin, but not for the cleavage of AT-III at its reactive centre by alpha-thrombin. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID

  10. An Intrabody Based on a Llama Single-domain Antibody Targeting the N-terminal α-Helical Multimerization Domain of HIV-1 Rev Prevents Viral Production*

    PubMed Central

    Vercruysse, Thomas; Pardon, Els; Vanstreels, Els; Steyaert, Jan; Daelemans, Dirk

    2010-01-01

    The human immunodeficiency virus, type 1 (HIV-1)-encoded Rev protein is essential for the expression of late viral mRNAs. Rev forms a large organized multimeric protein-protein complex on the Rev response element of these viral mRNA species and transports them from the nucleus to the cytoplasm, exploiting the CRM1-mediated cellular machinery. Here we report the selection of a nanobody, derived from a llama heavy-chain only antibody, that efficiently blocks the assembly of Rev multimers. The nanobody inhibits HIV-1 replication in cells and specifically suppresses the Rev-dependent expression of partially spliced and unspliced HIV-1 RNA. In HIV-susceptible cells, this nanobody thus has potential as an effective anti-HIV agent using genetic immunization strategies. Its binding site was mapped to Rev residues Lys-20 and Tyr-23 located in the N-terminal α-helical multimerization domain. In the presence of this nanobody, we observed an accumulation of dimeric Rev species, supporting a head-to-head/tail-to-tail molecular model for Rev assembly. The results indicate that the oligomeric assembly of Rev follows an ordered stepwise process and identify a new epitope within Rev that could guide strategies for the development of novel HIV inhibitors. PMID:20406803

  11. The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets

    PubMed Central

    Guzmán Prieto, Ana M.; Urbanus, Rolf T.; Zhang, Xinglin; Bierschenk, Damien; Koekman, C. Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P.; Pape, Marieke; Paganelli, Fernanda L.; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P. A.; Bonten, Marc J. M.; Willems, Rob J. L.; van Schaik, Willem

    2015-01-01

    Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets. PMID:26675410

  12. The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

    PubMed

    Guzmán Prieto, Ana M; Urbanus, Rolf T; Zhang, Xinglin; Bierschenk, Damien; Koekman, C Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P; Pape, Marieke; Paganelli, Fernanda L; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P A; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

    2015-12-17

    Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

  13. The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain.

    PubMed Central

    Seemann, J; Weber, K; Osborn, M; Parton, R G; Gerke, V

    1996-01-01

    Annexin I is a member of a multigene family of Ca2+/phospholipid-binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase, which has been implicated in membrane-related events along the endocytotic pathway, in particular in the sorting of internalized EGF receptors occurring in the multivesicular body. We analyzed in detail the intracellular distribution of this annexin by cell fractionation and immunoelectron microscopy. These studies used polyclonal as well as a set of species-specific monoclonal antibodies, whose epitopes were mapped to the lateral surface of the molecule next to a region thought to be involved in vesicle aggregation. Unexpectedly, the majority of annexin I was identified on early and not on multivesicular endosomes in a form that required micromolar levels of Ca2+ for the association. The specific cofractionation with early endosomes was also observed in transfected baby hamster kidney cells when the intracellular fate of ectopically expressed porcine annexin I was analyzed by using the species-specific monoclonal antibodies in Western blots of subcellular fractions. Interestingly, a truncation of the N-terminal 26, but not the N-terminal 13 residues of annexin I altered its intracellular distribution, shifting it from fractions containing early to those containing late and multivesicular endosomes. These findings underscore the regulatory importance of the N-terminal domain and provide evidence for an involvement of annexin I in early endocytotic processes. Images PMID:8885232

  14. Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.

    PubMed

    Laurent, Clémentine; Lekeux, Gilles; Ukuwela, Ashwinie A; Xiao, Zhiguang; Charlier, Jean-Benoit; Bosman, Bernard; Carnol, Monique; Motte, Patrick; Damblon, Christian; Galleni, Moreno; Hanikenne, Marc

    2016-03-01

    PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases. PMID:26797794

  15. Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.

    PubMed

    Laurent, Clémentine; Lekeux, Gilles; Ukuwela, Ashwinie A; Xiao, Zhiguang; Charlier, Jean-Benoit; Bosman, Bernard; Carnol, Monique; Motte, Patrick; Damblon, Christian; Galleni, Moreno; Hanikenne, Marc

    2016-03-01

    PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases.

  16. The structure of S. lividans acetoacetyl-CoA synthetase shows a novel interaction between the C-terminal extension and the N-terminal domain.

    PubMed

    Mitchell, Carter A; Tucker, Alex C; Escalante-Semerena, Jorge C; Gulick, Andrew M

    2015-03-01

    The adenosine monoposphate-forming acyl-CoA synthetase enzymes catalyze a two-step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ∼ 110 residue C-terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl-CoA synthetase (AacS) is presented that illustrates a novel aspect of this C-terminal domain. Specifically, several acetyl- and acetoacetyl-CoA synthetases contain a 30-residue extension on the C-terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N-terminal domain.

  17. The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly*

    PubMed Central

    Askenasy, Isabel; Pennington, Joseph M.; Tao, Yeqing; Marshall, Alan G.; Young, Nicolas L.; Shang, Weifeng; Stroupe, M. Elizabeth

    2015-01-01

    Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8β4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme. PMID:26088143

  18. The N-terminal domain tethers the voltage-gated calcium channel β2e-subunit to the plasma membrane via electrostatic and hydrophobic interactions.

    PubMed

    Miranda-Laferte, Erick; Ewers, David; Guzman, Raul E; Jordan, Nadine; Schmidt, Silke; Hidalgo, Patricia

    2014-04-11

    The β-subunit associates with the α1 pore-forming subunit of high voltage-activated calcium channels and modulates several aspects of ion conduction. Four β-subunits are encoded by four different genes with multiple splice variants. Only two members of this family, β2a and β2e, associate with the plasma membrane in the absence of the α1-subunit. Palmitoylation on a di-cysteine motif located at the N terminus of β2a promotes membrane targeting and correlates with the unique ability of this protein to slow down inactivation. In contrast, the mechanism by which β2e anchors to the plasma membrane remains elusive. Here, we identified an N-terminal segment in β2e encompassing a cluster of positively charged residues, which is strictly required for membrane anchoring, and when transferred to the cytoplasmic β1b isoform it confers membrane localization to the latter. In the presence of negatively charged phospholipid vesicles, this segment binds to acidic liposomes dependently on the ionic strength, and the intrinsic fluorescence emission maxima of its single tryptophan blue shifts considerably. Simultaneous substitution of more than two basic residues impairs membrane targeting. Coexpression of the fast inactivating R-type calcium channels with wild-type β2e, but not with a β2e membrane association-deficient mutant, slows down inactivation. We propose that a predicted α-helix within this domain orienting parallel to the membrane tethers the β2e-subunit to the lipid bilayer via electrostatic interactions. Penetration of the tryptophan side chain into the lipidic core stabilizes the membrane-bound conformation. This constitutes a new mechanism for membrane anchoring among the β-subunit family that also sustains slowed inactivation.

  19. Electrostatics analysis of the mutational and pH effects of the N-terminal domain self-association of the major ampullate spidroin.

    PubMed

    Barroso da Silva, Fernando Luís; Pasquali, Samuela; Derreumaux, Philippe; Dias, Luis Gustavo

    2016-07-01

    Spider silk is a fascinating material combining mechanical properties such as maximum strength and high toughness comparable or better than man-made materials, with biocompatible degradability characteristics. Experimental measurements have shown that pH triggers the dimer formation of the N-terminal domain (NTD) of the major ampullate spidroin 1 (MaSp 1). A coarse-grained model accounting for electrostatics, van der Waals and pH-dependent charge-fluctuation interactions, by means of Monte Carlo simulations, gave us a more comprehensive view of the NTD dimerization process. A detailed analysis of the electrostatic properties and free energy derivatives for the NTD homoassociation was carried out at different pH values and salt concentrations for the protein wild type and for several mutants. We observed an enhancement of dipole-dipole interactions at pH 6 due to the ionization of key amino acids, a process identified as the main driving force for dimerization. Analytical estimates based on the DVLO theory framework corroborate our findings. Molecular dynamics simulations using the OPEP coarse-grained force field for proteins show that the mutant E17Q is subject to larger structural fluctuations when compared to the wild type. Estimates of the association rate constants for this mutant were evaluated by the Debye-Smoluchowski theory and are in agreement with the experimental data when thermally relaxed structures are used instead of the crystallographic data. Our results can contribute to the design of new mutants with specific association properties. PMID:27250106

  20. Immunization with the DNA-encoding N-terminal domain of proteophosphoglycan of Leishmania donovani generates Th1-type immunoprotective response against experimental visceral leishmaniasis.

    PubMed

    Samant, Mukesh; Gupta, Reema; Kumari, Shraddha; Misra, Pragya; Khare, Prashant; Kushawaha, Pramod Kumar; Sahasrabuddhe, Amogh Anant; Dube, Anuradha

    2009-07-01

    Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) which exist as secretory as well as surface-bound forms in both promastigotes and amastigotes. The structure and function of PPGs have been reported to be species and stage specific as in the case of Leishmania major and Leishmania mexicana; there has been no such information available for Leishmania donovani. We have recently demonstrated that PPG is differentially expressed in sodium stibogluconate-sensitive and -resistant clinical isolates of L. donovani. To further elucidate the structure and function of the ppg gene of L. donovani, a partial sequence of its N-terminal domain of 1.6 kb containing the majority of antigenic determinants, was successfully cloned and expressed in prokaryotic as well as mammalian cells. We further evaluated the DNA-encoding N-terminal domain of the ppg gene as a vaccine in golden hamsters (Mesocricetus auratus) against the L. donovani challenge. The prophylactic efficacy to the tune of approximately 80% was observed in vaccinated hamsters and all of them could survive beyond 6 mo after challenge. The efficacy was supported by a surge in inducible NO synthase, IFN-gamma, TNF-alpha, and IL-12 mRNA levels along with extreme down-regulation of TGF-beta, IL-4, and IL-10. A rise in the level of Leishmania-specific IgG2 was also observed which was indicative of enhanced cellular immune response. The results suggest the N-terminal domain of L. donovani ppg as a potential DNA vaccine against visceral leishmaniasis.

  1. Lysozyme Mutants Accumulate in Cells while Associated at their N-terminal Alpha-domain with the Endoplasmic Reticulum Chaperone GRP78/BiP

    PubMed Central

    Kamada, Yoshiki; Nawata, Yusuke; Sugimoto, Yasushi

    2016-01-01

    Amyloidogenic human lysozyme variants deposit in cells and cause systemic amyloidosis. We recently observed that such lysozymes accumulate in the endoplasmic reticulum (ER) with the ER chaperone GRP78/BiP, accompanying the ER stress response. Here we investigated the region of lysozyme that is critical to its association with GRP78/BiP. In addition to the above-mentioned variants of lysozyme, we constructed lysozyme truncation or substitution mutants. These were co-expressed with GRP78/BiP (tagged with FLAG) in cultured human embryonic kidney cells, which were analyzed by western blotting and immunocytochemistry using anti-lysozyme and anti-FLAG antibodies. The amyloidogenic variants were confirmed to be strongly associated with GRP78/BiP as revealed by the co-immunoprecipitation assay, whereas N-terminal mutants pruned of 1-41 or 1-51 residues were found not to be associated with the chaperone. Single amino acid substitutions for the leucine array along the α-helices in the N-terminal region resulted in wild-type lysozyme remaining attached to GRP78/BiP. These mutations also tended to show lowered secretion ability. We conclude that the N-terminal α-helices region of the lysozyme is pivotal for its strong adhesion to GRP78/BiP. We suspect that wild-type lysozyme interacts with the GRP at this region as a step in the proper folding monitored by the ER chaperone. PMID:26884716

  2. A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

    PubMed

    Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H J; Snel, Berend; Perrakis, Anastassis; Kops, Geert J P L

    2013-04-15

    The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

  3. Implication of the oligomeric state of the N-terminal PTX3 domain in cumulus matrix assembly

    PubMed Central

    Ievoli, Elena; Lindstedt, Ragnar; Inforzato, Antonio; Camaioni, Antonella; Palone, Francesca; Day, Anthony J.; Mantovani, Alberto; Salvatori, Giovanni; Salustri, Antonietta

    2011-01-01

    Pentraxin 3 (PTX3) plays a key role in the formation of the hyaluronan-rich matrix of the cumulus oophorus surrounding ovulated eggs that is required for successful fertilization and female fertility. PTX3 is a multimeric protein consisting of eight identical protomers held together by a combination of non-covalent interactions and disulfide bonds. Recent findings suggest that the oligomeric status of PTX3 is important for stabilizing the cumulus matrix. Because the role of PTX3 in the cumulus resides in the unique N-terminal sequence of the protomer, we investigated further this issue by testing the ability of distinct Cys/Ser mutants of recombinant N-terminal region of PTX3 (N_PTX3) with different oligomeric arrangement to promote in vitro normal expansion in cumuli from Ptx3-null mice. Here we report that the dimer of the N_PTX3 is unable to rescue cumulus matrix organization, and that the tetrameric assembly of the protein is the minimal oligomeric state required for accomplishing this function. We have previously demonstrated that PTX3 binds to HCs of IαI and TSG-6, which are essential for cumulus matrix formation and able to interact with hyaluronan. Interestingly, here we show by solid-phase binding experiments that the dimer of the N_PTX3 retains the ability to bind to both IαI and TSG-6, suggesting that the octameric structure of PTX3 provides multiple binding sites for each of these ligands. These findings support the hypothesis that PTX3 contributes to cumulus matrix organization by cross-linking HA polymers through interactions with multiple HCs of IαI and/or TSG-6. The N-terminal PTX3 tetrameric oligomerization was recently reported to be also required for recognition and inhibition of FGF2. Given that this growth factor has been detected in the mammalian preovulatory follicle, we wondered whether FGF2 negatively influences cumulus expansion and PTX3 may also serve in vivo to antagonize its activity. We found that a molar excess of FGF2, above PTX3

  4. The SWI/SNF Subunit INI1 Contains an N-Terminal Winged Helix DNA Binding Domain that Is a Target for Mutations in Schwannomatosis

    PubMed Central

    Allen, Mark D.; Freund, Stefan M.V.; Zinzalla, Giovanna; Bycroft, Mark

    2015-01-01

    Summary SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromatin. In mammals they play a central role in regulating gene expression during differentiation and proliferation. Mutations in SWI/SNF subunits are among the most frequent gene alterations in cancer. The INI1/hSNF5/SMARCB1 subunit is mutated in both malignant rhabdoid tumor, a highly aggressive childhood cancer, and schwannomatosis, a tumor-predisposing syndrome characterized by mostly benign tumors of the CNS. Here, we show that mutations in INI1 that cause schwannomatosis target a hitherto unidentified N-terminal winged helix DNA binding domain that is also present in the BAF45a/PHF10 subunit of the SWI/SNF complex. The domain is structurally related to the SKI/SNO/DAC domain, which is found in a number of metazoan chromatin-associated proteins. PMID:26073604

  5. The SWI/SNF Subunit INI1 Contains an N-Terminal Winged Helix DNA Binding Domain that Is a Target for Mutations in Schwannomatosis.

    PubMed

    Allen, Mark D; Freund, Stefan M V; Zinzalla, Giovanna; Bycroft, Mark

    2015-07-01

    SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromatin. In mammals they play a central role in regulating gene expression during differentiation and proliferation. Mutations in SWI/SNF subunits are among the most frequent gene alterations in cancer. The INI1/hSNF5/SMARCB1 subunit is mutated in both malignant rhabdoid tumor, a highly aggressive childhood cancer, and schwannomatosis, a tumor-predisposing syndrome characterized by mostly benign tumors of the CNS. Here, we show that mutations in INI1 that cause schwannomatosis target a hitherto unidentified N-terminal winged helix DNA binding domain that is also present in the BAF45a/PHF10 subunit of the SWI/SNF complex. The domain is structurally related to the SKI/SNO/DAC domain, which is found in a number of metazoan chromatin-associated proteins.

  6. Solution behavior of the intrinsically disordered N-terminal domain of the Retinoid X Receptor alpha in the context of full-length protein

    PubMed Central

    Peluso-Iltis, Carole; Kieffer, Bruno; Svergun, Dmitri I.; Rochel, Natacha

    2016-01-01

    Retinoid X receptors (RXRs) are transcription factors with important functions in embryonic development, metabolic processes, differentiation and apoptosis. A particular feature of RXRs is their ability to act as obligatory heterodimerisation partners of class II nuclear receptors. At the same time, these receptors are also able to form homodimers that bind to direct repeat (DR1) hormone response elements. Since the discovery of RXRs, most of the studies focused on its ligand binding and DNA-binding domains, while its N-terminal domain (NTD) harboring a ligand-independent activation function remained poorly characterized. Here, we investigated the solution properties of the NTD domain of RXRα alone and in the context of the full-length receptor using small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy. We report the solution structure of the full-length homodimeric RXRα on DNA and show that the NTD remains highly flexible within this complex. PMID:26937780

  7. Structure of the TbBILBO1 Protein N-terminal Domain from Trypanosoma brucei Reveals an Essential Requirement for a Conserved Surface Patch*

    PubMed Central

    Vidilaseris, Keni; Morriswood, Brooke; Kontaxis, Georg; Dong, Gang

    2014-01-01

    TbBILBO1 is the only known component of the flagellar pocket collar, a cytoskeletal barrier element found in trypanosomes. The N-terminal domain (NTD) of TbBILBO1 was found to be dispensable for targeting of the protein in vivo. However, overexpression of constructs lacking the NTD caused complete growth inhibition, implying an essential requirement for this domain. A high resolution structure of the NTD of TbBILBO1 showed that it forms a ubiquitin-like fold with a conserved surface patch. Mutagenesis of this patch recapitulated the phenotypic effects of deleting the entire domain and was found to cause cell death. The surface patch on the NTD of TbBILBO1 is therefore a potential drug target. PMID:24362019

  8. Evaluation of combined B cell specific N-terminal immunogenic domains of LipL21 for diagnosis of leptospirosis.

    PubMed

    Anita, Kumari; Premlatha, Mallela Martha; Kanagavel, Murugesan; Akino Mercy, Charles Solomon; Raja, Veerapandian; Shanmughapriya, Santhanam; Natarajaseenivasan, Kalimuthusamy

    2016-10-01

    Leptospiral outer membrane protein LipL21 and its truncated N-terminal immunogenic region (I-LipL21) were evaluated for diagnosis of leptospirosis. The complete coding sequence of LipL21 nucleotide sequence was subjected to BCPred and VaxiJen analysis for determination of B cell specific immunogenic epitopes. Epitope1 ACS STD TGQ KDA TTV GDG (1.8837), Epitope2 WGG PPE QRN DGK TPR DTN (0.9483), Epitope3 VKG VGV YEC KAT GSG SDP (1.4077) and Epitope4 NEW ECQ CVI YAK FPG GKD (0.4462) were predicted. LipL21 and N-terminal fragment having B-cell specific epitopes with higher VaxiJen score >0.9 as truncated I-LipL21 were cloned independently in pET15b and expressed in Escherichia coli. IgM ELISA and dot blot assay was performed for sera samples collected from Delhi-NCR for leptospiral whole cell lysate (WCL), recombinant LipL21 and I-LipL21. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were found to be 92.5%, 92.8%, 83.3%, and 97% respectively for recombinant I-LipL21 by IgM-ELISA. 11-14.8% increased sensitivity was observed over LipL21 and WCL. The I-LipL21 dot blot assay showed a further increased sensitivity of 3.8% over the IgM-ELISA. Therefore I-LipL21 may be the ideal candidate protein for diagnosis of leptospirosis.

  9. Evaluation of combined B cell specific N-terminal immunogenic domains of LipL21 for diagnosis of leptospirosis.

    PubMed

    Anita, Kumari; Premlatha, Mallela Martha; Kanagavel, Murugesan; Akino Mercy, Charles Solomon; Raja, Veerapandian; Shanmughapriya, Santhanam; Natarajaseenivasan, Kalimuthusamy

    2016-10-01

    Leptospiral outer membrane protein LipL21 and its truncated N-terminal immunogenic region (I-LipL21) were evaluated for diagnosis of leptospirosis. The complete coding sequence of LipL21 nucleotide sequence was subjected to BCPred and VaxiJen analysis for determination of B cell specific immunogenic epitopes. Epitope1 ACS STD TGQ KDA TTV GDG (1.8837), Epitope2 WGG PPE QRN DGK TPR DTN (0.9483), Epitope3 VKG VGV YEC KAT GSG SDP (1.4077) and Epitope4 NEW ECQ CVI YAK FPG GKD (0.4462) were predicted. LipL21 and N-terminal fragment having B-cell specific epitopes with higher VaxiJen score >0.9 as truncated I-LipL21 were cloned independently in pET15b and expressed in Escherichia coli. IgM ELISA and dot blot assay was performed for sera samples collected from Delhi-NCR for leptospiral whole cell lysate (WCL), recombinant LipL21 and I-LipL21. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were found to be 92.5%, 92.8%, 83.3%, and 97% respectively for recombinant I-LipL21 by IgM-ELISA. 11-14.8% increased sensitivity was observed over LipL21 and WCL. The I-LipL21 dot blot assay showed a further increased sensitivity of 3.8% over the IgM-ELISA. Therefore I-LipL21 may be the ideal candidate protein for diagnosis of leptospirosis. PMID:27259643

  10. The structure of the BIR3 domain of cIAP1 in complex with the N-terminal peptides of SMAC and caspase-9

    SciTech Connect

    Kulathila, Raviraj; Vash, Brian; Sage, David; Cornell-Kennon, Susan; Wright, Kirk; Koehn, James; Stams, Travis; Clark, Kirk; Price, Allen ); )

    2009-06-24

    The inhibitor of apoptosis protein (IAP) family of molecules inhibit apoptosis through the suppression of caspase activity. It is known that the XIAP protein regulates both caspase-3 and caspase-9 through direct protein-protein interactions. Specifically, the BIR3 domain of XIAP binds to caspase-9 via a 'hotspot' interaction in which the N-terminal residues of caspase-9 bind in a shallow groove on the surface of XIAP. This interaction is regulated via SMAC, the N-terminus of which binds in the same groove, thus displacing caspase-9. The mechanism of suppression of apoptosis by cIAP1 is less clear. The structure of the BIR3 domain of cIAP1 (cIAP1-BIR3) in complex with N-terminal peptides from both SMAC and caspase-9 has been determined. The binding constants of these peptides to cIAP1-BIR3 have also been determined using the surface plasmon resonance technique. The structures show that the peptides interact with cIAP1 in the same way that they interact with XIAP: both peptides bind in a similar shallow groove in the BIR3 surface, anchored at the N-terminus by a charge-stabilized hydrogen bond. The binding data show that the SMAC and caspase-9 peptides bind with comparable affinities (85 and 48 nM, respectively).

  11. Pushing the limits of sulfur SAD phasing: de novo structure solution of the N-terminal domain of the ectodomain of HCV E1

    SciTech Connect

    El Omari, Kamel; Iourin, Oleg; Kadlec, Jan; Fearn, Richard; Hall, David R.; Harlos, Karl; Grimes, Jonathan M.; Stuart, David I.

    2014-08-01

    The sulfur SAD phasing method was successfully used to determine the structure of the N-terminal domain of HCV E1 from low-resolution diffracting crystals by combining data from 32 crystals. Single-wavelength anomalous dispersion of S atoms (S-SAD) is an elegant phasing method to determine crystal structures that does not require heavy-atom incorporation or selenomethionine derivatization. Nevertheless, this technique has been limited by the paucity of the signal at the usual X-ray wavelengths, requiring very accurate measurement of the anomalous differences. Here, the data collection and structure solution of the N-terminal domain of the ectodomain of HCV E1 from crystals that diffracted very weakly is reported. By combining the data from 32 crystals, it was possible to solve the sulfur substructure and calculate initial maps at 7 Å resolution, and after density modication and phase extension using a higher resolution native data set to 3.5 Å resolution model building was achievable.

  12. Sequence dependent N-terminal rearrangement and degradation of peptide nucleic acid (PNA) in aqueous solution

    NASA Technical Reports Server (NTRS)

    Eriksson, M.; Christensen, L.; Schmidt, J.; Haaima, G.; Orgel, L.; Nielsen, P. E.

    1998-01-01

    The stability of the PNA (peptide nucleic acid) thymine monomer inverted question markN-[2-(thymin-1-ylacetyl)]-N-(2-aminoaminoethyl)glycine inverted question mark and those of various PNA oligomers (5-8-mers) have been measured at room temperature (20 degrees C) as a function of pH. The thymine monomer undergoes N-acyl transfer rearrangement with a half-life of 34 days at pH 11 as analyzed by 1H NMR; and two reactions, the N-acyl transfer and a sequential degradation, are found by HPLC analysis to occur at measurable rates for the oligomers at pH 9 or above. Dependent on the amino-terminal sequence, half-lives of 350 h to 163 days were found at pH 9. At pH 12 the half-lives ranged from 1.5 h to 21 days. The results are discussed in terms of PNA as a gene therapeutic drug as well as a possible prebiotic genetic material.

  13. The N-terminal shuttle domain of Erv1 determines the affinity for Mia40 and mediates electron transfer to the catalytic Erv1 core in yeast mitochondria.

    PubMed

    Lionaki, Eirini; Aivaliotis, Michalis; Pozidis, Charalambos; Tokatlidis, Kostas

    2010-11-01

    Erv1 and Mia40 constitute the two important components of the disulfide relay system that mediates oxidative protein folding in the mitochondrial intermembrane space. Mia40 is the import receptor that recognizes the substrates introducing disulfide bonds while it is reduced. A key function of Erv1 is to recycle Mia40 to its active oxidative state. Our aims here were to dissect the domain of Erv1 that mediates the protein-protein interaction with Mia40 and to investigate the interactions between the shuttle domain of Erv1 and its catalytic core and their relevance for the interaction with Mia40. We purified these domains separately as well as cysteine mutants in the shuttle and the active core domains. The noncovalent interaction of Mia40 with Erv1 was measured by isothermal titration calorimetry, whereas their covalent mixed disulfide intermediate was analyzed in reconstitution experiments in vitro and in organello. We established that the N-terminal shuttle domain of Erv1 is necessary and sufficient for interaction to occur. Furthermore, we provide direct evidence for the intramolecular electron transfer from the shuttle cysteine pair of Erv1 to the core domain. Finally, we reconstituted the system by adding in trans the N- and C- terminal domains of Erv1 together with its substrate Mia40.

  14. 1H, 15N and 13C assignments of the N-terminal domain of the Mediator complex subunit MED26.

    PubMed

    Peruzzini, Riccardo; Lens, Zoé; Verger, Alexis; Dewitte, Frédérique; Ferreira, Elisabeth; Baert, Jean-Luc; Villeret, Vincent; Landrieu, Isabelle; Cantrelle, François-Xavier

    2016-04-01

    MED26 is a subunit of the Mediator, a very large complex involved in regulation of gene transcription by RNA Polymerase II. MED26 regulates the switch between initiation and elongation phases of the transcription. This function requires interaction of its N-terminal domain (NTD) with several protein partners implicated in transcriptional regulation. Molecular details of the structure and interaction mode of MED26 NTD would improve understanding of this complex regulation. As a first step towards structural characterization, sequence specific (1)H, (13)C and (15)N assignments for MED26 NTD was performed based on Nuclear Magnetic Resonance spectroscopy. TALOS+ analysis of the chemical shifts data revealed a domain solely composed of helices. Assignments will be further used to solve NMR structure and dynamics of MED26 NTD and investigate the molecular details of its interaction with protein partners.

  15. The N-terminal Domain of SIRT1 Is a Positive Regulator of Endogenous SIRT1-dependent Deacetylation and Transcriptional Outputs

    PubMed Central

    Ghisays, Fiorella; Brace, Cynthia S.; Yackly, Shawn M.; Kwon, Hyock Joo; Mills, Kathryn F.; Kashentseva, Elena; Dimitriev, Igor P.; Curiel, David T.; Imai, Shin-ichiro; Ellenberger, Tom

    2015-01-01

    SUMMARY The NAD+-dependent protein deacetylase SIRT1 regulates energy metabolism, responses to stress, and aging by deacetylating many different proteins, including histones and transcription factors. The mechanisms controlling SIRT1 enzymatic activity are complex and incompletely characterized, yet essential for understanding how to develop therapeutics that target SIRT1. Here we demonstrate that the N-terminal domain of SIRT1 (NTERM) can trans-activate deacetylation activity by physically interacting with endogenous SIRT1 and promoting its association with the deacetylation substrate NF-κB p65. Two motifs within the NTERM domain contribute to activation of SIRT1-dependent activities, and expression of one of these motifs in mice is sufficient to lower fasting glucose levels and improve glucose tolerance in a manner similar to overexpression of SIRT1. Our results provide new insights into the regulation of SIRT1 activity and a rationale for pharmacological control of SIRT1-dependent activities. PMID:25772354

  16. N-terminal domain of PB1-F2 protein of influenza A virus can fold into amyloid-like oligomers and damage cholesterol and cardiolipid containing membranes.

    PubMed

    Ajjaji, Dalila; Richard, Charles-Adrien; Mazerat, Sandra; Chevalier, Christophe; Vidic, Jasmina

    2016-08-12

    PB1-F2 protein is a factor of virulence of influenza A viruses which increases the mortality and morbidity associated with infection. Most seasonal H1N1 Influenza A viruses express nowadays a truncated version of PB1-F2. Here we show that truncation of PB1-F2 modified supramolecular organization of the protein in a membrane-mimicking environment. In addition, full-length PB1-F2(1-90) and C-terminal PB1-F2 domain (53-90), efficiently permeabilized various anionic liposomes while N-terminal domain PB1-F2(1-52) only lysed cholesterol and cardiolipin containing lipid bilayers. These findings suggest that the truncation of PB1-F2 may impact the pathogenicity of a given virus strain.

  17. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    PubMed Central

    Lübker, Carolin; Dove, Stefan; Tang, Wei-Jen; Urbauer, Ramona J. Bieber; Moskovitz, Jackob; Urbauer, Jeffrey L.; Seifert, Roland

    2015-01-01

    Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils. PMID:26184312

  18. Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression

    PubMed Central

    Oda, Shun-ichiro; Noda, Takeshi; Wijesinghe, Kaveesha J.; Halfmann, Peter; Bornholdt, Zachary A.; Abelson, Dafna M.; Armbrust, Tammy; Stahelin, Robert V.; Kawaoka, Yoshihiro

    2015-01-01

    ABSTRACT Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly. IMPORTANCE Marburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function. PMID:26656687

  19. Biosynthesis, glycosylation, and partial N-terminal amino acid sequence of the T-cell-activating protein TAP.

    PubMed Central

    Reiser, H; Coligan, J; Benacerraf, B; Rock, K L

    1987-01-01

    We have characterized the TAP molecule, an Ly-6 linked T-cell-activating glycoprotein. The three TAP bands that are precipitated from metabolically labeled cells display a common migration pattern in isoelectric focusing/NaDodSO4/PAGE gels and have common N-terminal sequences. This sequence is rich in cysteine and is homologous to that previously reported for the Ly-6.1E antigen. We, therefore, compared TAP and Ly-6.1E biochemically and found them to be structurally distinct. Given the role of TAP in T-cell activation, we further studied whether the molecule was phosphorylated. We have not found evidence for phosphorylation of the TAP protein. The carbohydrates present on the TAP molecule are resistant to peptide N-glycosidase F in vitro and tunicamycin in vivo. The upper band of the TAP triplet is susceptible to treatment with trifluoromethanesulfonic acid and thus seems to be of the O-linked rather than of the N-linked variety. The biosynthetic processing of TAP was studied in pulse-chase experiments. The middle band of the TAP triplet appears to be the earliest detectable species. Its conversion to the O-linked high molecular weight species can be blocked by monensin. Images PMID:3033645

  20. Purification, characterization, and N-terminal amino acid sequence of the adenylyl cyclase-activating protease from bovine sperm.

    PubMed

    Adeniran, A J; Shoshani, I; Minuth, M; Awad, J A; Elce, J S; Johnson, R A

    1995-03-01

    We previously reported the extraction of a factor from bovine sperm that activated adenylyl cyclases of rat brain and human platelets, and identified it as a trypsin-like protease that was referred to as "ninhibin." This proteolytic activity was purified to near homogeneity from an alkaline extract of washed sperm particles by sequential chromatography on p-aminobenzamidine agarose and CM-Sephadex. Purification was greater than 100-fold with nearly 30% recovery of protease activity exhibiting a major band of approximately 40 kDa. An approximately 45-kDa form of the protease was also evident in crude extracts and was preferentially isolated when the enzyme was prepared in the presence of a mixture of protease inhibitors. The larger form of the protease was substantially less effective in stimulating adenylyl cyclase than was the smaller form; it is likely to be a zymogen form from which the smaller, more active form is derived. Purified forms of acrosin and ninhibin exhibited similar mobilities on PAGE, similar capacities for activating adenylyl cyclase, similar patterns of proteolytic fragmentation, and similar immunoblot patterns obtained with an antibody against purified bovine acrosin. More importantly, the N-terminal amino acid sequence of bovine ninhibin was found to be identical with that of bovine acrosin and caprine acrosin and more than 75% identical with porcine acrosin. The data support the conclusion that the adenylyl cyclase-activating protease previously referred to as ninhibin is, in fact, acrosin. PMID:7756444

  1. Characterization of Mutants of Human Small Heat Shock Protein HspB1 Carrying Replacements in the N-Terminal Domain and Associated with Hereditary Motor Neuron Diseases

    PubMed Central

    Muranova, Lydia K.; Weeks, Stephen D.; Strelkov, Sergei V.; Gusev, Nikolai B.

    2015-01-01

    Physico-chemical properties of the mutations G34R, P39L and E41K in the N-terminal domain of human heat shock protein B1 (HspB1), which have been associated with hereditary motor neuron neuropathy, were analyzed. Heat-induced aggregation of all mutants started at lower temperatures than for the wild type protein. All mutations decreased susceptibility of the N- and C-terminal parts of HspB1 to chymotrypsinolysis. All mutants formed stable homooligomers with a slightly larger apparent molecular weight compared to the wild type protein. All mutations analyzed decreased or completely prevented phosphorylation-induced dissociation of HspB1 oligomers. When mixed with HspB6 and heated, all mutants yielded heterooligomers with apparent molecular weights close to ~400 kDa. Finally, the three HspB1 mutants possessed lower chaperone-like activity towards model substrates (lysozyme, malate dehydrogenase and insulin) compared to the wild type protein, conversely the environmental probe bis-ANS yielded higher fluorescence with the mutants than with the wild type protein. Thus, in vitro the analyzed N-terminal mutations increase stability of large HspB1 homooligomers, prevent their phosphorylation-dependent dissociation, modulate their interaction with HspB6 and decrease their chaperoning capacity, preventing normal functioning of HspB1. PMID:25965061

  2. Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding

    PubMed Central

    Batt, Sarah M.; Bingle, Lewis E.H.; Dafforn, Tim R.; Thomas, Christopher M.

    2009-01-01

    ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning. PMID:19109978

  3. The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity.

    PubMed

    Jongruja, Nujarin; You, Dong-Ju; Kanaya, Eiko; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2010-11-01

    Thermotoga maritima ribonuclease H (RNase H) I (Tma-RNase HI) contains a hybrid binding domain (HBD) at the N-terminal region. To analyze the role of this HBD, Tma-RNase HI, Tma-W22A with the single mutation at the HBD, the C-terminal RNase H domain (Tma-CD) and the N-terminal domain containing the HBD (Tma-ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma-RNase HI prefers Mg(2+) to Mn(2+) for activity, and specifically loses most of the Mg(2+)-dependent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma-CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage-site specificities of Tma-RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma-RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg(2+)-dependent activity and substrate binding affinity. Tma-CD was as stable as Tma-RNase HI, indicating that the HBD is not important for stability. The HBD of Tma-RNase HI is important not only for substrate binding, but also for Mg(2+)-dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg(2+) ion are arranged ideally.

  4. Localization of Daucus carota NMCP1 to the nuclear periphery: the role of the N-terminal region and an NLS-linked sequence motif, RYNLRR, in the tail domain.

    PubMed

    Kimura, Yuta; Fujino, Kaien; Ogawa, Kana; Masuda, Kiyoshi

    2014-01-01

    Recent ultrastructural studies revealed that a structure similar to the vertebrate nuclear lamina exists in the nuclei of higher plants. However, plant genomes lack genes for lamins and intermediate-type filament proteins, and this suggests that plant-specific nuclear coiled-coil proteins make up the lamina-like structure in plants. NMCP1 is a protein, first identified in Daucus carota cells, that localizes exclusively to the nuclear periphery in interphase cells. It has a tripartite structure comprised of head, rod, and tail domains, and includes putative nuclear localization signal (NLS) motifs. We identified the functional NLS of DcNMCP1 (carrot NMCP1) and determined the protein regions required for localizing to the nuclear periphery using EGFP-fused constructs transiently expressed in Apium graveolens epidermal cells. Transcription was driven under a CaMV35S promoter, and the genes were introduced into the epidermal cells by a DNA-coated microprojectile delivery system. Of the NLS motifs, KRRRK and RRHK in the tail domain were highly functional for nuclear localization. Addition of the N-terminal 141 amino acids from DcNMCP1 shifted the localization of a region including these NLSs from the entire nucleus to the nuclear periphery. Using this same construct, the replacement of amino acids in RRHK or its preceding sequence, YNL, with alanine residues abolished localization to the nuclear periphery, while replacement of KRRRK did not affect localization. The sequence R/Q/HYNLRR/H, including YNL and the first part of the sequence of RRHK, is evolutionarily conserved in a subclass of NMCP1 sequences from many plant species. These results show that NMCP1 localizes to the nuclear periphery by a combined action of a sequence composed of R/Q/HYNLRR/H, NLS, and the N-terminal region including the head and a portion of the rod domain, suggesting that more than one binding site is implicated in localization of NMCP1.

  5. Functional roles of the non-catalytic calcium-binding sites in the N-terminal domain of human peptidylarginine deiminase 4.

    PubMed

    Liu, Yi-Liang; Tsai, I-Chen; Chang, Chia-Wei; Liao, Ya-Fan; Liu, Guang-Yaw; Hung, Hui-Chih

    2013-01-01

    This study investigated the functional roles of the N-terminal Ca(2+) ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca(2+)-binding site of PAD4 were mutated to disrupt the binding of Ca(2+) ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k(cat)/K(m,BAEE) values were 0.02, 0.63 and 0.01 s(-1)mM(-1) (20.8 s(-1)mM(-1) for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k(cat) value of 0.3 s(-1) (13.3 s(-1) for wild-type), whereas D176A retained some catalytic power with a k(cat) of 9.7 s(-1). Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k(cat)/K(m,BAEE) values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca(2+) indicated that the conformational stability of the enzyme is highly dependent on Ca(2+) ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca(2+) ions in the N-terminal Ca(2+)-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca(2+) ions play critical roles in the full activation of the PAD4 enzyme.

  6. Crystal Structure of the Cytoplasmic N-Terminal Domain of Subunit I, a Homolog of Subunit a, of V-ATPase

    SciTech Connect

    Srinivasan, Sankaranarayanan; Vyas, Nand K.; Baker, Matthew L.; Quiocho, Florante A.

    2012-02-27

    Subunit 'a' is associated with the membrane-bound (VO) complex of eukaryotic vacuolar H{sup +}-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit 'I' homolog of subunit a. The structure is composed of a curved long central {alpha}-helix bundle capped on both ends by two lobes with similar {alpha}/{beta} architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H{sup +}-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.

  7. Expression and Purification of the Cytoplasmic N-Terminal Domain of the Na/HCO3 Cotransporter NBCe1-A: Structural Insights from the a Generalized Approach

    SciTech Connect

    Gill,H.; Boron, W.

    2006-01-01

    The cytoplasmic, N-terminal domain (Nt) of the electrogenic sodium/bicarbonate cotransporter -- NBCe1 -- over-expresses in Escherichia coli and yields a large amount of soluble protein. A novel purification strategy, which involves a streptomycin precipitation, overcomes obstacles of instability and copurifying proteins, and leads to the first seen Nt-NBCe1 crystals. The purification procedure generally lends itself to the purification of Nts from other classes of the SLC4 family. Size-exclusion chromatography suggests that the Nt of NBCe1 as well as the Nt of other SLC4 members form dimers. A comparison of Nt-NBCe1 to SLC4 member Nt-AE1, based on purification properties and predicted secondary-structure sequence alignments, suggests a similar mechanism for dimer stabilization.

  8. Substitution of NS5 N-terminal domain of dengue virus type 2 RNA with type 4 domain caused impaired replication and emergence of adaptive mutants with enhanced fitness.

    PubMed

    Teramoto, Tadahisa; Boonyasuppayakorn, Siwaporn; Handley, Misty; Choi, Kyung H; Padmanabhan, Radhakrishnan

    2014-08-01

    Flavivirus NS3 and NS5 are required in viral replication and 5'-capping. NS3 has NS2B-dependent protease, RNA helicase, and 5'-RNA triphosphatase activities. NS5 has 5'-RNA methyltransferase (MT)/guanylyltransferase (GT) activities within the N-terminal 270 amino acids and the RNA-dependent RNA polymerase (POL) activity within amino acids 271-900. A chimeric NS5 containing the D4MT/D4GT and the D2POL domains in the context of wild-type (WT) D2 RNA was constructed. RNAs synthesized in vitro were transfected into baby hamster kidney cells. The viral replication was analyzed by an indirect immunofluorescence assay to monitor NS1 expression and by quantitative real-time PCR. WT D2 RNA-transfected cells were NS1- positive by day 5, whereas the chimeric RNA-transfected cells became NS1-positive ∼30 days post-transfection in three independent experiments. Sequence analysis covering the entire genome revealed the appearance of a single K74I mutation within the D4MT domain ∼16 days post-transfection in two experiments. In the third, D290N mutation in the conserved NS3 Walker B motif appeared ≥16 days post-transfection. A time course study of serial passages revealed that the 30-day supernatant had gradually evolved to gain replication fitness. Trans-complementation by co-expression of WT D2 NS5 accelerated viral replication of chimeric RNA without changing the K74I mutation. However, the MT and POL activities of NS5 WT D2 and the chimeric NS5 proteins with or without the K74I mutation are similar. Taken together, our results suggest that evolution of the functional interactions involving the chimeric NS5 protein encoded by the viral genome species is essential for gain of viral replication fitness. PMID:24904061

  9. Interactions between Copper-binding Sites Determine the Redox Status and Conformation of the Regulatory N-terminal Domain of ATP7B*

    PubMed Central

    LeShane, Erik S.; Shinde, Ujwal; Walker, Joel M.; Barry, Amanda N.; Blackburn, Ninian J.; Ralle, Martina; Lutsenko, Svetlana

    2010-01-01

    Copper-transporting ATPase ATP7B is essential for human copper homeostasis and normal liver function. ATP7B has six N-terminal metal-binding domains (MBDs) that sense cytosolic copper levels and regulate ATP7B. The mechanism of copper sensing and signal integration from multiple MBDs is poorly understood. We show that MBDs communicate and that this communication determines the oxidation state and conformation of the entire N-terminal domain of ATP7B (N-ATP7B). Mutations of copper-coordinating Cys to Ala in any MBD (2, 3, 4, or 6) change the N-ATP7B conformation and have distinct functional consequences. Mutating MBD2 or MBD3 causes Cys oxidation in other MBDs and loss of copper binding. In contrast, mutation of MBD4 and MBD6 does not alter the redox status and function of other sites. Our results suggest that MBD2 and MBD3 work together to regulate access to other metal-binding sites, whereas MBD4 and MBD6 receive copper independently, downstream of MBD2 and MBD3. Unlike Ala substitutions, the Cys-to-Ser mutation in MBD2 preserves the conformation and reduced state of N-ATP7B, suggesting that hydrogen bonds contribute to interdomain communications. Tight coupling between MBDs suggests a mechanism by which small changes in individual sites (induced by copper binding or mutation) result in stabilization of distinct conformations of the entire N-ATP7B and altered exposure of sites for interactions with regulatory proteins. PMID:20032459

  10. Self-association and DNA binding of lambda cI repressor N-terminal domains reveal linkage between sequence-specific binding and the C-terminal cooperativity domain.

    PubMed

    Bain, D L; Ackers, G K

    1994-12-13

    The effects of temperature, protons, and KCl on self-assembly and site-specific binding of lambda cI N-terminal domains with operator sites OR were studied to assess the roles of these domains in DNA binding and cooperativity of the natural system. Domain self-assembly was studied using sedimentation equilibrium while domain-OR interactions were analyzed by quantitative DNase footprint titration. The self-assembly reactions were modeled best as a monomer-dimer-tetramer stoichiometry. Compared with intact cI, the monomer-dimer assembly is energetically weak and is largely independent of pH and KCl. The van't Hoff enthalpy of dimerization was found to be large and positive (+ 10.8 kcal/mol), in sharp contrast to that of intact cI (i.e., -16.1 kcal/mol; Koblan & Ackers, 1991a), indicating that different driving forces dominate the respective assembly processes. The interactions of OR with N-terminal domains were noncooperative under all conditions studied. Binding at each site is accompanied by a negative enthalpy (large at site 1, small at sites 2 and 3). Identical values for salt release and proton absorption were found for the three sites. Comparisons with the analogous thermodynamic parameters from our previous studies indicate that N-terminal domains exhibit different linkages to pH, KCl, and T from those of intact cI-OR interactions. This implies that the domains do not act independently within the intact repressor. Since the linkage differences are dependent upon which site the proteins are binding, the C-terminal domain must play a role in repressor discrimination between specific sites.

  11. Preliminary X-ray crystallographic analysis of an engineered variant of human chimera-type galectin-3 with a shortened N-terminal domain.

    PubMed

    Flores-Ibarra, Andrea; Ruiz, Federico M; Vértesy, Sabine; André, Sabine; Gabius, Hans-Joachim; Romero, Antonio

    2015-02-01

    How lectins translate sugar-encoded information into cellular effects not only depends on glycan recognition. Other domains of the protein can contribute to the functional profile of a lectin. Human galectin-3 (Gal-3), an adhesion/growth-regulatory galectin, is composed of three different domains and is thus called a chimera-type protein. In addition to the carbohydrate-recognition domain, this lectin encompasses an N-terminal domain consisting of a peptide harbouring two phosphorylation sites and nine non-triple-helical collagen-like repeats. This region plays an as yet structurally undefined role in Gal-3 aggregation and ligand recognition. To date, crystallization of full-length Gal-3 has not been achieved. With the aim of providing structural insights into this modular organization, a Gal-3 variant was crystallized maintaining the terminal peptide and three of the nine collagen-like repeats. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 94.04, b = 97.96, c = 236.20 Å, and diffracted to a resolution of 3.3 Å.

  12. A sting in the tail: the N-terminal domain of the androgen receptor as a drug target

    PubMed Central

    Monaghan, Amy E; McEwan, Iain J

    2016-01-01

    The role of androgen receptor (AR) in the initiation and progression of prostate cancer (PCa) is well established. Competitive inhibition of the AR ligand-binding domain (LBD) has been the staple of antiandrogen therapies employed to combat the disease in recent years. However, their efficacy has often been limited by the emergence of resistance, mediated through point mutations, and receptor truncations. As a result, the prognosis for patients with malignant castrate resistant disease remains poor. The amino-terminal domain (NTD) of the AR has been shown to be critical for AR function. Its modular activation function (AF-1) is important for both gene regulation and participation in protein-protein interactions. However, due to the intrinsically disordered structure of the domain, its potential as a candidate for therapeutic intervention has been dismissed in the past. The recent emergence of the small molecule EPI-001 has provided evidence that AR-NTD can be targeted therapeutically, independent of the LBD. Targeting of AR-NTD has the potential to disrupt multiple intermolecular interactions between AR and its coregulatory binding partners, in addition to intramolecular cross-talk between the domains of the AR. Therapeutics targeting these protein-protein interactions or NTD directly should also have efficacy against emerging AR splice variants which may play a role in PCa progression. This review will discuss the role of intrinsic disorder in AR function and illustrate how emerging therapies might target NTD in PCa. PMID:27212126

  13. A multilayered regulatory mechanism for the autoinhibition and activation of a plant CC-NB-LRR resistance protein with an extra N-terminal domain.

    PubMed

    Chen, Xiaojiao; Zhu, Min; Jiang, Lei; Zhao, Wenyang; Li, Jia; Wu, Jianyan; Li, Chun; Bai, Baohui; Lu, Gang; Chen, Hongyu; Moffett, Peter; Tao, Xiaorong

    2016-10-01

    The tomato resistance protein Sw-5b differs from the classical coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) resistance proteins by having an extra N-terminal domain (NTD). To understand how NTD, CC and NB-LRR regulate autoinhibition and activation of Sw-5b, we dissected the function(s) of each domain. When viral elicitor was absent, Sw-5b LRR suppressed the central NB-ARC to maintain autoinhibition of the NB-LRR segment. The CC and NTD domains independently and additively enhanced the autoinhibition of NB-LRR. When viral elicitor was present, the NB-LRR segment of Sw-5b was specifically activated to trigger a hypersensitive response. Surprisingly, Sw-5b CC suppressed the activation of NB-LRR, whereas the extra NTD of Sw-5b became a positive regulator and fully activated the resistance protein, probably by relieving the inhibitory effects of the CC. In infection assays of transgenic plants, the NB-LRR segment alone was insufficient to confer resistance against Tomato spotted wilt tospovirus; the layers of NTD and CC regulation on NB-LRR were required for Sw-5b to confer resistance. Based on these findings, we propose that, to counter the negative regulation of the CC on NB-LRR, Sw-5b evolved an extra NTD to coordinate with the CC, thus developing a multilayered regulatory mechanism to control autoinhibition and activation.

  14. Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein

    PubMed Central

    Kiss, Katalin; Kucsma, Nora; Brozik, Anna; Tusnady, Gabor E.; Bergam, Ptissam; vanNiel, Guillaume; Szakacs, Gergely

    2015-01-01

    ATP-binding cassette, subfamily B (ABCB) 6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in the intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities, including confocal microscopy and EM, we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide-binding domain (NBD) and transmembrane domain (TMD), ABCB6 contains a unique N-terminal TMD (TMD0), which does not show sequence homology to known proteins. We investigated the functional role of these domains through the molecular dissection of ABCB6. We find that the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core–ABCB6 complex devoid of TMD0 are preserved. However, in contrast with the full-length transporter, the core–ABCB6 construct is retained at the plasma membrane and does not appear in Rab5-positive endosomes. TMD0 is directly targeted to the lysosomes, without passage to the plasma membrane. Collectively, our results reveal that TMD0 represents an independently folding unit, which is dispensable for catalysis, but has a crucial role in the lysosomal targeting of ABCB6. PMID:25627919

  15. Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703.

    PubMed

    Lu, Zhenghui; Wang, Qinhong; Jiang, Sijing; Zhang, Guimin; Ma, Yanhe

    2016-01-01

    High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase's properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca(2+) were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center. PMID:26926401

  16. Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703

    PubMed Central

    Lu, Zhenghui; Wang, Qinhong; Jiang, Sijing; Zhang, Guimin; Ma, Yanhe

    2016-01-01

    High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase’s properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca2+ were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center. PMID:26926401

  17. Binding Mechanism of the N-Terminal SH3 Domain of CrkII and Proline-Rich Motifs in cAbl.

    PubMed

    Bhatt, Veer S; Zeng, Danyun; Krieger, Inna; Sacchettini, James C; Cho, Jae-Hyun

    2016-06-21

    The N-terminal Src homology 3 (nSH3) domain of a signaling adaptor protein, CT-10 regulator of kinase II (CrkII), recognizes proline-rich motifs (PRMs) of binding partners, such as cAbl kinase. The interaction between CrkII and cAbl kinase is involved in the regulation of cell spreading, microbial pathogenesis, and cancer metastasis. Here, we report the detailed biophysical characterizations of the interactions between the nSH3 domain of CrkII and PRMs in cAbl. We identified that the nSH3 domain of CrkII binds to three PRMs in cAbl with virtually identical affinities. Structural studies, by using x-ray crystallography and NMR spectroscopy, revealed that the binding modes of all three nSH3:PRM complexes are highly similar to each other. Van 't Hoff analysis revealed that nSH3:PRM interaction is associated with favorable enthalpy and unfavorable entropy change. The combination of experimentally determined thermodynamic parameters, structure-based calculations, and (15)N NMR relaxation analysis highlights the energetic contribution of conformational entropy change upon the complex formation, and water molecules structured in the binding interface of the nSH3:PRM complex. Understanding the molecular basis of nSH3:PRM interaction will provide, to our knowledge, new insights for the rational design of small molecules targeting the interaction between CrkII and cAbl. PMID:27332121

  18. Different Functions of the Paralogs to the N-Terminal Domain of the Orange Carotenoid Protein in the Cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    López-Igual, Rocío; Wilson, Adjélé; Leverenz, Ryan L; Melnicki, Matthew R; Bourcier de Carbon, Céline; Sutter, Markus; Turmo, Aiko; Perreau, François; Kerfeld, Cheryl A; Kirilovsky, Diana

    2016-07-01

    The photoactive Orange Carotenoid Protein (OCP) is involved in cyanobacterial photoprotection. Its N-terminal domain (NTD) is responsible for interaction with the antenna and induction of excitation energy quenching, while the C-terminal domain is the regulatory domain that senses light and induces photoactivation. In most nitrogen-fixing cyanobacterial strains, there are one to four paralogous genes coding for homologs to the NTD of the OCP. The functions of these proteins are unknown. Here, we study the expression, localization, and function of these genes in Anabaena sp. PCC 7120. We show that the four genes present in the genome are expressed in both vegetative cells and heterocysts but do not seem to have an essential role in heterocyst formation. This study establishes that all four Anabaena NTD-like proteins can bind a carotenoid and the different paralogs have distinct functions. Surprisingly, only one paralog (All4941) was able to interact with the antenna and to induce permanent thermal energy dissipation. Two of the other Anabaena paralogs (All3221 and Alr4783) were shown to be very good singlet oxygen quenchers. The fourth paralog (All1123) does not seem to be involved in photoprotection. Structural homology modeling allowed us to propose specific features responsible for the different functions of these soluble carotenoid-binding proteins. PMID:27208286

  19. A multilayered regulatory mechanism for the autoinhibition and activation of a plant CC-NB-LRR resistance protein with an extra N-terminal domain.

    PubMed

    Chen, Xiaojiao; Zhu, Min; Jiang, Lei; Zhao, Wenyang; Li, Jia; Wu, Jianyan; Li, Chun; Bai, Baohui; Lu, Gang; Chen, Hongyu; Moffett, Peter; Tao, Xiaorong

    2016-10-01

    The tomato resistance protein Sw-5b differs from the classical coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) resistance proteins by having an extra N-terminal domain (NTD). To understand how NTD, CC and NB-LRR regulate autoinhibition and activation of Sw-5b, we dissected the function(s) of each domain. When viral elicitor was absent, Sw-5b LRR suppressed the central NB-ARC to maintain autoinhibition of the NB-LRR segment. The CC and NTD domains independently and additively enhanced the autoinhibition of NB-LRR. When viral elicitor was present, the NB-LRR segment of Sw-5b was specifically activated to trigger a hypersensitive response. Surprisingly, Sw-5b CC suppressed the activation of NB-LRR, whereas the extra NTD of Sw-5b became a positive regulator and fully activated the resistance protein, probably by relieving the inhibitory effects of the CC. In infection assays of transgenic plants, the NB-LRR segment alone was insufficient to confer resistance against Tomato spotted wilt tospovirus; the layers of NTD and CC regulation on NB-LRR were required for Sw-5b to confer resistance. Based on these findings, we propose that, to counter the negative regulation of the CC on NB-LRR, Sw-5b evolved an extra NTD to coordinate with the CC, thus developing a multilayered regulatory mechanism to control autoinhibition and activation. PMID:27558751

  20. Different Functions of the Paralogs to the N-Terminal Domain of the Orange Carotenoid Protein in the Cyanobacterium Anabaena sp. PCC 71201[OPEN

    PubMed Central

    López-Igual, Rocío; Wilson, Adjélé; Bourcier de Carbon, Céline; Sutter, Markus; Turmo, Aiko

    2016-01-01

    The photoactive Orange Carotenoid Protein (OCP) is involved in cyanobacterial photoprotection. Its N-terminal domain (NTD) is responsible for interaction with the antenna and induction of excitation energy quenching, while the C-terminal domain is the regulatory domain that senses light and induces photoactivation. In most nitrogen-fixing cyanobacterial strains, there are one to four paralogous genes coding for homologs to the NTD of the OCP. The functions of these proteins are unknown. Here, we study the expression, localization, and function of these genes in Anabaena sp. PCC 7120. We show that the four genes present in the genome are expressed in both vegetative cells and heterocysts but do not seem to have an essential role in heterocyst formation. This study establishes that all four Anabaena NTD-like proteins can bind a carotenoid and the different paralogs have distinct functions. Surprisingly, only one paralog (All4941) was able to interact with the antenna and to induce permanent thermal energy dissipation. Two of the other Anabaena paralogs (All3221 and Alr4783) were shown to be very good singlet oxygen quenchers. The fourth paralog (All1123) does not seem to be involved in photoprotection. Structural homology modeling allowed us to propose specific features responsible for the different functions of these soluble carotenoid-binding proteins. PMID:27208286

  1. Syndecan-4 contributes to endothelial tubulogenesis through interactions with two motifs inside the pro-angiogenic N-terminal domain of thrombospondin-1.

    PubMed

    Nunes, Sara Santana; Outeiro-Bernstein, Marianna A Ferrari do; Juliano, Luiz; Vardiero, Francisco; Nader, Helena B; Woods, Anne; Legrand, Chantal; Morandi, Verônica

    2008-03-01

    Thrombospondin-1 (TSP-1) is an extracellular matrix protein that modulates focal adhesion in mammalian cells and exhibits dual roles in angiogenesis. In a previous work, we showed that a recombinant 18 kDa protein encompassing the N-terminal residues 1-174 of human TSP-1 (TSP18) induced tubulogenesis of human umbilical vein endothelial cells and protected them from apoptosis. Our results indicated that these effects were possibly mediated by syndecan-4 proteoglycan, since binding of TSP18 to endothelial extracts was inhibited by anti-syndecan-4 antibody. Syndecan-4 is a heparan-sulfate proteoglycan that regulates cell-matrix interactions and is the only member of its family present in focal adhesions. In this report, we demonstrate that a monoclonal antibody against syndecan-4 blocks TSP18-induced tubulogenesis. Furthermore, through 2D adhesion and 3D angiogenic assays, we demonstrate that two sequences, TSP Hep I and II, retain the major pro-angiogenic activity of TSP18. These TSP-1 motifs also compete with the fibronectin Hep II domain for binding to syndecan-4 on endothelial cell surface, indicating that they may exert their effects by interfering with the recognition of fibronectin by syndecan-4. Additionally, TSP18 and its derived peptides activate the PKC-dependent Akt-PKB signaling pathway. Blockage of PKC activation prevented HUVEC spreading when seeded on TSP18 fragment, and on TSP Hep I and TSP Hep II peptides, but not on gelatin-coated substrates. Our results identify syndecan-4 as a novel receptor for the N-terminus of TSP-1 and suggest that TSP-1 N-terminal pro-angiogenic activity is linked to its capacity of interfering with syndecan-4 functions in the course of cell adhesion. PMID:17879962

  2. Domain swapping reveals that the C- and N-terminal domains of DnaG and DnaB, respectively, are functional homologues.

    PubMed

    Chintakayala, Kiran; Larson, Marilynn A; Grainger, William H; Scott, David J; Griep, Mark A; Hinrichs, Steven H; Soultanas, Panos

    2007-03-01

    The bacterial primase (DnaG)-helicase (DnaB) interaction is mediated by the C-terminal domain of DnaG (p16) and a linker that joins the N- and C-terminal domains (p17 and p33 respectively) of DnaB. The crystal and nuclear magnetic resonance structures of p16 from Escherichia coli and Bacillus stearothermophilus DnaG proteins revealed a unique structural homology with p17, despite the lack of amino acid sequence similarity. The functional significance of this is not clear. Here, we have employed a 'domain swapping' approach to replace p17 with its structural homologue p16 to create chimeras. p33 alone hydrolyses ATP but exhibits no helicase activity. Fusing p16 (p16-p33) or DnaG (G-p33) to the N-terminus of p33 produced chimeras with partially restored helicase activities. Neither chimera interacted with DnaG. The p16-p33 chimera formed hexamers while G-p33 assembled into tetramers. Furthermore, G-p33 and DnaB formed mixed oligomers with ATPase activity better than that of the DnaB/DnaG complex and helicase activity better than the sum of the individual DnaB and G-p33 activities but worse than that of the DnaB/DnaG complex. Our combined data provide direct evidence that p16 and p17 are not only structural but also functional homologues, albeit their amino acid composition differences are likely to influence their precise roles. PMID:17367384

  3. In vitro catalytic activity of N-terminal and C-terminal domains in NukM, the post-translational modification enzyme of nukacin ISK-1.

    PubMed

    Shimafuji, Chinatsu; Noguchi, Megumi; Nishie, Mami; Nagao, Jun-Ichi; Shioya, Kouki; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

    2015-12-01

    Lantibiotics are antibacterial peptides containing unique thioether cross-links termed lanthionine and methyllanthionine. NukM, the modifying enzyme of nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the dehydration of specific Ser/Thr residues in a precursor peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor peptide of nisin is modified by 2 enzymes, NisB and NisC, which mediate dehydration and cyclization, respectively. While the C-terminal domain of NukM is homologous to NisC, the N-terminal domain has no homology with other known proteins. We expressed and characterized the N- and C-terminal domains of NukM, NukMN, and NukMC, separately. In vitro reconstitution revealed that full-length NukM fully modified the substrate peptide NukA. NukMN partially phosphorylated, dehydrated, and cyclized NukA. By contrast, NukMC did not catalyze dehydration, phosphorylation, or cyclization reactions. Interaction studies using surface plasmon resonance analysis indicated that NukM and NukMN can bind NukA with high affinity, whereas NukMC has low substrate-recognition activity. These results suggest that NukMN is mainly responsible for substrate recognition and dehydration and that the whole NukM structure, including the C-terminal domain, is required for the complete modification of NukA. To the best of our knowledge, this is the first report providing insights into the in vitro catalytic activity of individual domains of a LanM-type modification enzyme. PMID:25971839

  4. Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating

    PubMed Central

    Cais, Ondrej; Herguedas, Beatriz; Krol, Karolina; Cull-Candy, Stuart G.; Farrant, Mark; Greger, Ingo H.

    2014-01-01

    Summary AMPA-type glutamate receptors (AMPARs) mediate fast neurotransmission at excitatory synapses. The extent and fidelity of postsynaptic depolarization triggered by AMPAR activation are shaped by AMPAR auxiliary subunits, including the transmembrane AMPAR regulatory proteins (TARPs). TARPs profoundly influence gating, an effect thought to be mediated by an interaction with the AMPAR ion channel and ligand binding domain (LBD). Here, we show that the distal N-terminal domain (NTD) contributes to TARP modulation. Alterations in the NTD-LBD linker result in TARP-dependent and TARP-selective changes in AMPAR gating. Using peptide arrays, we identify a TARP interaction region on the NTD and define the path of TARP contacts along the LBD surface. Moreover, we map key binding sites on the TARP itself and show that mutation of these residues mediates gating modulation. Our data reveal a TARP-dependent allosteric role for the AMPAR NTD and suggest that TARP binding triggers a drastic reorganization of the AMPAR complex. PMID:25373908

  5. Preliminary X-ray Diffraction Analysis of the Cytoplasmic N-terminal Domain of the Na/HCO3 Cotransporter NBCe1-A

    SciTech Connect

    Gill,H.; Boron, W.

    2006-01-01

    The N-terminal cytoplasmic domain of the Na{sup +}-coupled HCO{sub 3}{sup -} cotransporter NBCe1-A (NtNBCe1) has been linked with proximal renal tubular acidosis. In a previous purification study of recombinant NtNBCe1, crystal growth at a suboptimal protein concentration (<1 mg ml{sup -1}) yielded small single diamond-shaped crystals that diffracted poorly. In the present study, by increasing the protein concentration 50-fold, the crystal size was doubled and robustness was also improved. Crystal annealing made the crystals suitable for X-ray diffraction. The crystals either belong to space group P3121 or P31 with pseudo P3121 symmetry, with unit-cell parameters a = 51.7, b = 51.7, c = 200.6 Angstroms, {alpha} = {beta} = 90, {gamma} = 120 deg, and diffract X-rays to 3.0 Angstroms resolution. The calculated Matthews number is 1.9 Angstroms{sup 3} Da{sup -1}, with two monomers of molecular weight {approx}83 kDa in the asymmetric unit. The molecular- replacement packing solution shows that the molecules form dimers by a domain-swapping mechanism.

  6. Crystal structure of the human CD4 N-terminal two-domain fragment complexed to a class II MHC molecule.

    SciTech Connect

    Wang, J.-H.; Meijers, R.; Xiong, Y.; Liu, J.-H.; Sakihama, T.; Zhang, R.-G.; Joachimiak, A.; Reinherz, E. L.; Biosciences Division; Dana-Farber Cancer Inst.; Harvard Medical School

    2001-09-11

    The structural basis of the interaction between the CD4 coreceptor and a class II major histocompatibility complex (MHC) is described. The crystal structure of a complex containing the human CD4 N-terminal two-domain fragment and the murine I-A{sup k }class II MHC molecule with associated peptide (pMHCII) shows that only the 'top corner' of the CD4 molecule directly contacts pMHCII. The CD4 Phe-43 side chain extends into a hydrophobic concavity formed by MHC residues from both {alpha}2 and {beta}2 domains. A ternary model of the CD4-pMHCII-T-cell receptor (TCR) reveals that the complex appears V-shaped with the membrane-proximal pMHCII at the apex. This configuration excludes a direct TCR-CD4 interaction and suggests how TCR and CD4 signaling is coordinated around the antigenic pMHCII complex. Human CD4 binds to HIV gp120 in a manner strikingly similar to the way in which CD4 interacts with pMHCII. Additional contacts between gp120 and CD4 give the CD4-gp120 complex a greater affinity. Thus, ligation of the viral envelope glycoprotein to CD4 occludes the pMHCII-binding site on CD4, contributing to immunodeficiency.

  7. A Linear Epitope in the N-Terminal Domain of CCR5 and Its Interaction with Antibody

    PubMed Central

    Chain, Benny; Arnold, Jack; Akthar, Samia; Brandt, Michael; Davis, David; Noursadeghi, Mahdad; Lapp, Thabo; Ji, Changhua; Sankuratri, Surya; Zhang, Yanjing; Govada, Lata; Saridakis, Emmanuel; Chayen, Naomi

    2015-01-01

    The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively), and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution. PMID:26030924

  8. Left-handed helical preference in an achiral peptide chain is induced by an L-amino acid in an N-terminal type II β-turn.

    PubMed

    De Poli, Matteo; De Zotti, Marta; Raftery, James; Aguilar, Juan A; Morris, Gareth A; Clayden, Jonathan

    2013-03-15

    Oligomers of the achiral amino acid Aib adopt helical conformations in which the screw-sense may be controlled by a single N-terminal residue. Using crystallographic and NMR techniques, we show that the left- or right-handed sense of helical induction arises from the nature of the β-turn at the N terminus: the tertiary amino acid L-Val induces a left-handed type II β-turn in both the solid state and in solution, while the corresponding quaternary amino acid L-α-methylvaline induces a right-handed type III β-turn.

  9. Thermodynamics of the protonation equilibria of two fragments of N-terminal β-hairpin of FPB28 WW domain.

    PubMed

    Makowska, Joanna; Uber, Dorota; Chmurzyński, Lech

    2012-01-12

    The pK(a) values of two peptides derived from the formin-binding protein 28 WW domain [Ac-Lys-Thr-Ala-Asp-Gly-Lys-Thr-NH(2) (D7), Ac-Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH(2) (D9)] were determined by potentiometric titration in the temperature range from 25 to 60 °C, and their heat capacities were determined, by differential scanning calorimetry, in the temperature range from 10 to 90 °C. For both peptides, heat capacity has a maximum at t ≈ 50 °C, with height about 0.1 kcal/(mol × deg), suggesting that a modest unfolding transition occurs. The first two pK(a)'s are low at temperatures below 50 °C, suggesting that the two lysine residues are close to each other and the peptides have bent shapes at lower temperatures; this effect is greater for D7 compared with D9. With increasing temperature beyond 50 °C (i.e., that of the thermodynamic unfolding transition), pK(a1) and pK(a2) increase rapidly for D9, whereas their temperature variation is less significant for D7. This observation, and the fact that the enthalpies and entropies of the dissociation of the two first protons (determined from the temperature dependence of the respective pK(a)'s) decrease significantly near the transition temperature, suggest that the peptide undergoes a transition from a bent to an amorphous shape and that the presence of charged lysine residues stabilizes the folded state.

  10. Thermodynamics of the protonation equilibria of two fragments of N-terminal β-hairpin of FPB28 WW domain.

    PubMed

    Makowska, Joanna; Uber, Dorota; Chmurzyński, Lech

    2012-01-12

    The pK(a) values of two peptides derived from the formin-binding protein 28 WW domain [Ac-Lys-Thr-Ala-Asp-Gly-Lys-Thr-NH(2) (D7), Ac-Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH(2) (D9)] were determined by potentiometric titration in the temperature range from 25 to 60 °C, and their heat capacities were determined, by differential scanning calorimetry, in the temperature range from 10 to 90 °C. For both peptides, heat capacity has a maximum at t ≈ 50 °C, with height about 0.1 kcal/(mol × deg), suggesting that a modest unfolding transition occurs. The first two pK(a)'s are low at temperatures below 50 °C, suggesting that the two lysine residues are close to each other and the peptides have bent shapes at lower temperatures; this effect is greater for D7 compared with D9. With increasing temperature beyond 50 °C (i.e., that of the thermodynamic unfolding transition), pK(a1) and pK(a2) increase rapidly for D9, whereas their temperature variation is less significant for D7. This observation, and the fact that the enthalpies and entropies of the dissociation of the two first protons (determined from the temperature dependence of the respective pK(a)'s) decrease significantly near the transition temperature, suggest that the peptide undergoes a transition from a bent to an amorphous shape and that the presence of charged lysine residues stabilizes the folded state. PMID:22128840

  11. [Preparation of recombinant polypeptide of N-terminal heparin-binding domain of fibronectin and its effect on disseminated intravascular coagulation in rats].

    PubMed

    Zou, Qi-Lian; Guo, Jiang-Rui; Chen, Xiao-Fang; Huang, Mei-Juan; Wu, Yong; Chen, Yuan-Zhong

    2010-06-01

    This study was aimed to prepare the polypeptide of N-terminal heparin-binding domain of fibronectin(rhFNHN-29 polypeptide) with pichia expression system, to detect biological activity of recombinant polypeptide and investigate its effect on disseminated intravascular coagulation (DIC) in rats. The sequence of N-terminal heparin-binding domain of fibronectin was amplified from FNcDNA by PCR. The aim gene was cloned into T vector for selection. Then it was cloned into pAo815SM and pPIC9K vectors.Lined pPIC9K vectors were transformed into GS115 Pichia cells so as to express the aim polypeptide in Pichia expression system. The fermentation liquid were precipitated by 80% ammonium sulfate, and the further dissolved sediment were purified using S-100 column and SP column. Its activity of binding with heparin were detected by Western-blot. The established DIC rats (40 rats) were randomly divided into two groups. One group was treated with rhFNHN-29 polypeptide, and the other was treated with normal saline. The rats in the former group were injected with rhFNHN-29 polypeptide (10 mg/kg) through tail vein at 0.5 hour before, 2 hours and 4 hours after injection of LPS respectively. The rats in latter group were injected with equal volume saline. In addition, 20 normal rats injected with normal saline were as normal controls. 500 microl blood was taken from the rat vein, at 6 hours after the injection of LPS. White blood cell (WBC), hemoglobin (Hb) and platelets were tested from 50 microl blood. The rest 450 microl blood was used to isolate plasma for detecting TNFa level and coagulogram. The rats were killed at 24 hours after injection with LPS. Their livers, lungs, hearts, kidneys, and brain tissues were taken for histopathologic examination. The results showed that the aim polypeptide was successfully expressed in Pichia expression system. The expression level reached approximately 30 mg/L. The polypeptide had activity of binding with heparin antibody. In the experiment

  12. A novel intermediate mucolipidosis II/IIIαβ caused by GNPTAB mutation in the cytosolic N-terminal domain.

    PubMed

    Leroy, Jules G; Sillence, David; Wood, Tim; Barnes, Jarrod; Lebel, Robert Roger; Friez, Michael J; Stevenson, Roger E; Steet, Richard; Cathey, Sara S

    2014-05-01

    Mucolipidosis (ML) II and ML IIIα/β are allelic autosomal recessive metabolic disorders due to mutations in GNPTAB. The gene encodes the enzyme UDP-GlcNAc-1-phosphotransferase (GNPT), which is critical to proper trafficking of lysosomal acid hydrolases. The ML phenotypic spectrum is dichotomous. Criteria set for defining ML II and ML IIIα/β are inclusive for all but the few patients with phenotypes that span the archetypes. Clinical and biochemical findings of the 'intermediate' ML in eight patients with the c.10A>C missense mutation in GNPTAB are presented to define this intermediate ML and provide a broader insight into ML pathogenesis. Extensive clinical information, including radiographic examinations at various ages, was obtained from a detailed study of all patients. GNPTAB was sequenced in probands and parents. GNPT activity was measured and cathepsin D sorting assays were performed in fibroblasts. Intermediate ML patients who share the c.10A>C/p.K4Q mutation in GNPTAB demonstrate a distinct, consistent phenotype similar to ML II in physical and radiographic features and to ML IIIα/β in psychomotor development and life expectancy. GNPT activity is reduced to 7-12% but the majority of newly synthesized cathepsin D remains intracellular. The GNPTAB c.10A>C/p.K4Q missense allele results in an intermediate ML II/III with distinct clinical and biochemical characteristics. This delineation strengthens the utility of the discontinuous genotype-phenotype correlation in ML II and ML IIIα/β and prompts additional studies on the tissue-specific pathogenesis in GNPT-deficient ML. PMID:24045841

  13. A protein interaction node at the neurotransmitter release site: domains of Aczonin/Piccolo, Bassoon, CAST, and rim converge on the N-terminal domain of Munc13-1.

    PubMed

    Wang, Xiaolu; Hu, Bin; Zieba, Agata; Neumann, Nicole G; Kasper-Sonnenberg, Monika; Honsbein, Annegret; Hultqvist, Greta; Conze, Tim; Witt, Wolfgang; Limbach, Christoph; Geitmann, Matthis; Danielson, Helena; Kolarow, Richard; Niemann, Gesa; Lessmann, Volkmar; Kilimann, Manfred W

    2009-10-01

    Multidomain scaffolding proteins organize the molecular machinery of neurotransmitter vesicle dynamics during synaptogenesis and synaptic activity. We find that domains of five active zone proteins converge on an interaction node that centers on the N-terminal region of Munc13-1 and includes the zinc-finger domain of Rim1, the C-terminal region of Bassoon, a segment of CAST1/ELKS2, and the third coiled-coil domain (CC3) of either Aczonin/Piccolo or Bassoon. This multidomain complex may constitute a center for the physical and functional integration of the protein machinery at the active zone. An additional connection between Aczonin and Bassoon is mediated by the second coiled-coil domain of Aczonin. Recombinant Aczonin-CC3, expressed in cultured neurons as a green fluorescent protein fusion protein, is targeted to synapses and suppresses vesicle turnover, suggesting involvements in synaptic assembly as well as activity. Our findings show that Aczonin, Bassoon, CAST1, Munc13, and Rim are closely and multiply interconnected, they indicate that Aczonin-CC3 can actively participate in neurotransmitter vesicle dynamics, and they highlight the N-terminal region of Munc13-1 as a hub of protein interactions by adding three new binding partners to its mechanistic potential in the control of synaptic vesicle priming.

  14. C terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain

    SciTech Connect

    South, T.L.; Blake, P.R. ); Hare, D.R.; Summers, M.F. )

    1991-06-25

    Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retriviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 (Zn(HIV1-F2)). Unlike results obtained for the first retroviral-type zinc finger peptide, Zn (HIV1-F1) broad signals indicative of confomational lability were observed in the {sup 1}H NMR spectrum of An(HIV1-F2) at 25 C. The NMR signals narrowed upon cooling to {minus}2 C, enabling complete {sup 1}H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were sued to generate 30 distance geometry (DG) structures with penalties in the range 0.02-0.03 {angstrom}{sup 2}. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. These results indicate that the r.t. zinc finger sequences observed in retroviral NCPs, simple plant virus coat proteins, and in a human single-stranded nucleic acid binding protein share a common structural motif.

  15. Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus.

    PubMed

    Arai, Toshinobu; Noguchi, Atsushi; Takano, Eriko; Kino, Kuniki

    2013-04-01

    Dipeptides exhibit unique physiological functions and physical properties, e.g., l-aspartyl-l-phenylalanine-methyl ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an l-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, l-asparagine; Gln, l-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, l-aspartic acid; and Glu, l-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated l-asparaginyl-l-valine (Asn-Val) and l-glutaminyl-glycine (Gln-Gly), but did not deamidate l-valyl-l-asparagine and l-alanyl-l-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 ± 30 nmol min(-1) mg(-1)·protein and 136 ± 6 nmol min(-1) mg(-1)·protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa.

  16. Solution NMR structure of the Ca2+-bound N-terminal domain of CaBP7: a regulator of golgi trafficking.

    PubMed

    McCue, Hannah V; Patel, Pryank; Herbert, Andrew P; Lian, Lu-Yun; Burgoyne, Robert D; Haynes, Lee P

    2012-11-01

    Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-β (PI4KIIIβ) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIβ. CaBP7 NTD encompasses the two high affinity Ca(2+) binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca(2+) but not Mg(2+) and undergoes significant conformational changes in both secondary and tertiary structure upon Ca(2+) binding. The Ca(2+)-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca(2+)-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIβ. PMID:22989873

  17. Purification, crystallization and preliminary X-ray diffraction of the N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/P{sub i} carrier SCaMC1

    SciTech Connect

    Yang, Qin Brüschweiler, Sven; Chou, James J.

    2013-12-24

    The N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/P{sub i} carrier SCaMC1 was crystallized in the presence of Ca{sup 2+}. X-ray diffraction data were collected to 2.9 Å resolution from crystals which belonged to space group P6{sub 2}22.

  18. A Rapid Diagnostic Test for Toxoplasmosis using Recombinant Antigenic N-terminal Half of SAG1 Linked with Intrinsically Unstructured Domain of GRA2 Protein

    PubMed Central

    Song, Kyoung Ju; Yang, Zhaoshou; Chong, Chom-Kyu; Kim, Jin-Soo; Lee, Kyung Chan; Kim, Tong-Soo

    2013-01-01

    Toxoplasma gondii is an apicomplexan parasite with a broad host range of most warm-blooded mammals including humans, of which one-thirds of the human population has been infected worldwide which can cause congenital defects, abortion, and neonatal complications. Here, we developed a rapid diagnostic test (RDT) for T. gondii infection. Antigenic N-terminal half of the major surface antigen (SAG1) was linked with intrinsically unstructured domain (IUD) of dense granule protein 2 (GRA2). The recombinant GST-GRA2-SAG1A protein was successfully expressed and purified as 51 kDa of molecular weight. Furthermore, antigenicity and solubility of the rGST-GRA2-SAG1A protein were significantly increased. The overall specificity and sensitivity of GST-GRA2-SAG1A loaded RDT (TgRDT) were estimated as 100% and 97.1% by comparing with ELISA result which uses T. gondii whole cell lysates as the antigen. The TgRDT tested with Uganda people sera for field trial and showed 31.9% of seroprevalence against T. gondii antibody. The TgRDT is proved to be a kit for rapid and easy to use with high accuracy, which would be a suitable serodiagnostic tool for toxoplasmosis. PMID:24327774

  19. Three new structures of left-handed RADA helical filaments: structural flexibility of N-terminal domain is critical for recombinase activity.

    PubMed

    Chang, Yu-Wei; Ko, Tzu-Ping; Lee, Chien-Der; Chang, Yuan-Chih; Lin, Kuei-Ann; Chang, Chia-Seng; Wang, Andrew H-J; Wang, Ting-Fang

    2009-01-01

    RecA family proteins, including bacterial RecA, archaeal RadA, and eukaryotic Dmc1 and Rad51, mediate homologous recombination, a reaction essential for maintaining genome integrity. In the presence of ATP, these proteins bind a single-strand DNA to form a right-handed nucleoprotein filament, which catalyzes pairing and strand exchange with a homologous double-stranded DNA (dsDNA), by as-yet unknown mechanisms. We recently reported a structure of RadA left-handed helical filament, and here present three new structures of RadA left-handed helical filaments. Comparative structural analysis between different RadA/Rad51 helical filaments reveals that the N-terminal domain (NTD) of RadA/Rad51, implicated in dsDNA binding, is highly flexible. We identify a hinge region between NTD and polymerization motif as responsible for rigid body movement of NTD. Mutant analysis further confirms that structural flexibility of NTD is essential for RadA's recombinase activity. These results support our previous hypothesis that ATP-dependent axial rotation of RadA nucleoprotein helical filament promotes homologous recombination.

  20. The Identification and Structure of an N-Terminal PR Domain Show that FOG1 Is a Member of the PRDM Family of Proteins

    PubMed Central

    Clifton, Molly K.; Westman, Belinda J.; Thong, Sock Yue; O’Connell, Mitchell R.; Webster, Michael W.; Shepherd, Nicholas E.; Quinlan, Kate G.; Crossley, Merlin; Blobel, Gerd A.; Mackay, Joel P.

    2014-01-01

    FOG1 is a transcriptional regulator that acts in concert with the hematopoietic master regulator GATA1 to coordinate the differentiation of platelets and erythrocytes. Despite considerable effort, however, the mechanisms through which FOG1 regulates gene expression are only partially understood. Here we report the discovery of a previously unrecognized domain in FOG1: a PR (PRD-BF1 and RIZ) domain that is distantly related in sequence to the SET domains that are found in many histone methyltransferases. We have used NMR spectroscopy to determine the solution structure of this domain, revealing that the domain shares close structural similarity with SET domains. Titration with S-adenosyl-L-homocysteine, the cofactor product synonymous with SET domain methyltransferase activity, indicated that the FOG PR domain is not, however, likely to function as a methyltransferase in the same fashion. We also sought to define the function of this domain using both pulldown experiments and gel shift assays. However, neither pulldowns from mammalian nuclear extracts nor yeast two-hybrid assays reproducibly revealed binding partners, and we were unable to detect nucleic-acid-binding activity in this domain using our high-diversity Pentaprobe oligonucleotides. Overall, our data demonstrate that FOG1 is a member of the PRDM (PR domain containing proteins, with zinc fingers) family of transcriptional regulators. The function of many PR domains, however, remains somewhat enigmatic for the time being. PMID:25162672

  1. A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells.

    PubMed

    Dias, Juliana Vieira; Benslimane-Ahmim, Zahia; Egot, Marion; Lokajczyk, Anna; Grelac, Françoise; Galy-Fauroux, Isabelle; Juliano, Luiz; Le-Bonniec, Bernard; Takiya, Cristina Maeda; Fischer, Anne-Marie; Blanc-Brude, Olivier; Morandi, Verônica; Boisson-Vidal, Catherine

    2012-10-15

    Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 μg/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC.

  2. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    PubMed

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  3. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation

    PubMed Central

    Harris, Samantha P.; Belknap, Betty; Van Sciver, Robert E.; White, Howard D.; Galkin, Vitold E.

    2016-01-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure–function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the “open” structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca2+ have been achieved. We suggest that Ca2+ modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  4. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    PubMed

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

  5. Crystallization and preliminary X-ray diffraction analysis of mouse galectin-4 N-terminal carbohydrate recognition domain in complex with lactose

    SciTech Connect

    Krejčiříková, Veronika; Fábry, Milan; Marková, Vladimíra; Malý, Petr; Řezáčová, Pavlína; Brynda, Jiří

    2008-07-01

    Mouse galectin-4 carbohydrate binding domain was overexpressed in E. coli and crystallized in the presence of lactose. The crystals belong to tetragonal space group P42{sub 1}2 and diffraction data were collected to 2.1 Å resolution. Galectin-4 is thought to play a role in the process of tumour conversion of cells of the alimentary tract and the breast tissue; however, its exact function remains unknown. With the aim of elucidating the structural basis of mouse galectin-4 (mGal-4) binding specificity, we have undertaken X-ray analysis of the N-terminal domain, CRD1, of mGal-4 in complex with lactose (the basic building block of known galectin-4 carbohydrate ligands). Crystals of CRD1 in complex with lactose were obtained using vapour-diffusion techniques. The crystals belong to tetragonal space group P42{sub 1}2 with unit-cell parameters a = 91.1, b = 91.16, c = 57.10 Å and preliminary X-ray diffraction data were collected to 3.2 Å resolution. An optimized crystallization procedure and cryocooling protocol allowed us to extend resolution to 2.1 Å. Structure refinement is currently under way; the initial electron-density maps clearly show non-protein electron density in the vicinity of the carbohydrate binding site, indicating the presence of one lactose molecule. The structure will help to improve understanding of the binding specificity and function of the potential colon cancer marker galectin-4.

  6. Chaperone-like activities of different molecular forms of beta-casein. Importance of polarity of N-terminal hydrophilic domain.

    PubMed

    Yousefi, Reza; Shchutskaya, Yulia Y; Zimny, Jaroslaw; Gaudin, Jean-Charles; Moosavi-Movahedi, Ali A; Muronetz, Vladimir I; Zuev, Yuriy F; Chobert, Jean-Marc; Haertlé, Thomas

    2009-08-01

    As a member of intrinsically unstructured protein family, beta-casein (beta-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native beta-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant beta-CNs wild type (WT) beta-CN, C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native beta-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (beta-CND) of C4-beta-CN and C208 beta-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT beta-CN, C208 beta-CN, C4 beta-CN and C4 beta-CND exhibited significantly lower chaperone-like activities than native beta-CN. Dimerization of C208 beta-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native beta-CN. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 623-632, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com. PMID:19322774

  7. Recombinant expression, in vitro refolding, and biophysical characterization of the N-terminal domain of T1R3 taste receptor.

    PubMed

    Maîtrepierre, Elodie; Sigoillot, Maud; Le Pessot, Laurence; Briand, Loïc

    2012-05-01

    The sweet taste receptor is a heterodimeric receptor composed of the T1R2 and T1R3 subunits, while T1R1 and T1R3 assemble to form the umami taste receptor. T1R receptors belong to the family of class C G-protein coupled receptors (GPCRs). In addition to a transmembrane heptahelical domain, class C GPCRs have a large extracellular N-terminal domain (NTD), which is the primary ligand-binding site. The T1R2 and T1R1 subunits have been shown to be responsible for ligand binding, via their NTDs. However, little is known about the contribution of T1R3-NTD to receptor functions. To enable biophysical characterization, we overexpressed the human NTD of T1R3 (hT1R3-NTD) using Escherichia coli in the form of inclusion bodies. Using a fractional factorial screen coupled to a functional assay, conditions were determined for the refolding of hT1R3-NTD. Far-UV circular dichroism spectroscopic studies revealed that hT1R3-NTD was well refolded. Using size-exclusion chromatography, we found that the refolded protein behaves as a dimer. Ligand binding quantified by tryptophan fluorescence quenching and microcalorimetry showed that hT1R3-NTD is functional and capable of binding sucralose with an affinity in the millimolar range. This study also provides a strategy to produce functional hT1R3-NTD by heterologous expression in E. coli; this is a prerequisite for structural determination and functional analysis of ligand-binding regions of other class C GPCRs. PMID:22450161

  8. A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells.

    PubMed

    Dias, Juliana Vieira; Benslimane-Ahmim, Zahia; Egot, Marion; Lokajczyk, Anna; Grelac, Françoise; Galy-Fauroux, Isabelle; Juliano, Luiz; Le-Bonniec, Bernard; Takiya, Cristina Maeda; Fischer, Anne-Marie; Blanc-Brude, Olivier; Morandi, Verônica; Boisson-Vidal, Catherine

    2012-10-15

    Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 μg/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC. PMID:22796565

  9. A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: design, synthesis, activity against multidrug-resistant bacteria and Candida.

    PubMed

    Mishra, Biswajit; Leishangthem, Geeta Devi; Gill, Kamaldeep; Singh, Abhay K; Das, Swagata; Singh, Kusum; Xess, Immaculata; Dinda, Amit; Kapil, Arti; Patro, Ishan K; Dey, Sharmistha

    2013-02-01

    Lactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8 μg/mL and 6.5 μg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800 μg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials. PMID:23026014

  10. High-resolution differentiation of transmissible spongiform encephalopathy strains by quantitative N-terminal amino acid profiling (N-TAAP) of PK-digested abnormal prion protein.

    PubMed

    Gielbert, Adriana; Davis, Linda A; Sayers, A Robin; Hope, James; Gill, Andrew C; Sauer, Maurice J

    2009-03-01

    New forms of transmissible spongiform encephalopathy (TSE) continue to be identified, and consequently sensitive differential diagnosis is increasingly important both for the management of disease in humans and livestock and in providing confidence in the safety of the food chain. TSE diseases are associated with accumulation of protease-resistant prion protein (PrP(Sc)) and detection of this marker protein is central to diagnosis. Proteolysis by proteinase K (PK) generates protease-resistant products (PrP(res)) with partially variable N-termini. The conformation(s) of PrP(Sc) and thus the points of PK cleavage are thought to be dependent on the strain of prion disease. Western blot (WB) analysis of PrP(res) gives characteristic migration patterns that can be used to diagnose TSEs, but the relatively low resolution of this technique limits its ability to differentiate certain disease strains. Mass spectrometry (MS) has the capability to resolve these various PK cleavage sites to the level of individual amino acid residues. In the present study multiple selected reaction monitoring (mSRM) was used to detect and quantify PrP(res) N-terminal tryptic peptides by MS and thus to define the N-terminal amino acid profiles (N-TAAPs) of PrP(res) characteristic for various TSEs in sheep. The fragmentation behaviour of the N-terminal tryptic peptides was studied to allow selection of the transitions specific for each peptide. Different PrP(res) preparation methods were evaluated and the most effective approach applied to differentiate the N-TAAPs corresponding to various sheep TSE isolates. Marked differences were identified between the N-TAAPs of bovine spongiform encephalopathy (BSE) and classical scrapie, and between classical scrapie and the experimental strains SSBP/1 and CH1641, thereby validating this approach as a means of TSE-strain specific diagnosis.

  11. Enhancing the Secretion Efficiency and Thermostability of a Bacillus deramificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

    PubMed Central

    Duan, Xuguo

    2015-01-01

    Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072–4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8- and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 × 108 and 449.3 × 108 U · mol−1, respectively, which are 0.94- and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7- and 1.3-fold that of the DM enzyme. Kinetic studies showed that the Km values of DM-T1 and DM-T2 were 1.5- and 2.7-fold higher and the Kcat/Km values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced d-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes. PMID:25556190

  12. The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

    PubMed Central

    Xue, Xianli; Wang, Rong; Tu, Tao; Shi, Pengjun; Ma, Rui; Luo, Huiying

    2015-01-01

    The genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical C-terminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley β-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/β-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages. PMID:25819971

  13. A prime-boost immunization with Tc52 N-terminal domain DNA and the recombinant protein expressed in Pichia pastoris protects against Trypanosoma cruzi infection.

    PubMed

    Matos, Marina N; Sánchez Alberti, Andrés; Morales, Celina; Cazorla, Silvia I; Malchiodi, Emilio L

    2016-06-14

    We have previously reported that the N-terminal domain of the antigen Tc52 (NTc52) is the section of the protein that confers the strongest protection against Trypanosoma cruzi infection. To improve vaccine efficacy, we conducted here a prime-boost strategy (NTc52PB) by inoculating two doses of pcDNA3.1 encoding the NTc52 DNA carried by attenuated Salmonella (SNTc52), followed by two doses of recombinant NTc52 expressed in Picchia pastoris plus ODN-CpG as adjuvant. This strategy was comparatively analyzed with the following protocols: (1) two doses of NTc52+ODN-CpG by intranasal route followed by two doses of NTc52+ODN-CpG by intradermal route (NTc52CpG); (2) four doses of SNTc52; and (3) a control group with four doses of Salmonella carrying the empty plasmid. All immunized groups developed a predominant Th1 cellular immune response but with important differences in antibody development and protection against infection. Thus, immunization with just SNTc52 induces a strong specific cellular response, a specific systemic antibody response that is weak yet functional (considering lysis of trypomastigotes and inhibition of cell invasion), and IgA mucosal immunity, protecting in both the acute and chronic stages of infection. The group that received only recombinant protein (NTc52CpG) developed a strong antibody immune response but weaker cellular immunity than the other groups, and the protection against infection was clear in the acute phase of infection but not in chronicity. The prime-boost strategy, which combines DNA and protein vaccine and both mucosal and systemic immunizations routes, was the best assayed protocol, inducing strong cellular and humoral responses as well as specific mucosal IgA, thus conferring better protection in the acute and chronic stages of infection.

  14. A prime-boost immunization with Tc52 N-terminal domain DNA and the recombinant protein expressed in Pichia pastoris protects against Trypanosoma cruzi infection.

    PubMed

    Matos, Marina N; Sánchez Alberti, Andrés; Morales, Celina; Cazorla, Silvia I; Malchiodi, Emilio L

    2016-06-14

    We have previously reported that the N-terminal domain of the antigen Tc52 (NTc52) is the section of the protein that confers the strongest protection against Trypanosoma cruzi infection. To improve vaccine efficacy, we conducted here a prime-boost strategy (NTc52PB) by inoculating two doses of pcDNA3.1 encoding the NTc52 DNA carried by attenuated Salmonella (SNTc52), followed by two doses of recombinant NTc52 expressed in Picchia pastoris plus ODN-CpG as adjuvant. This strategy was comparatively analyzed with the following protocols: (1) two doses of NTc52+ODN-CpG by intranasal route followed by two doses of NTc52+ODN-CpG by intradermal route (NTc52CpG); (2) four doses of SNTc52; and (3) a control group with four doses of Salmonella carrying the empty plasmid. All immunized groups developed a predominant Th1 cellular immune response but with important differences in antibody development and protection against infection. Thus, immunization with just SNTc52 induces a strong specific cellular response, a specific systemic antibody response that is weak yet functional (considering lysis of trypomastigotes and inhibition of cell invasion), and IgA mucosal immunity, protecting in both the acute and chronic stages of infection. The group that received only recombinant protein (NTc52CpG) developed a strong antibody immune response but weaker cellular immunity than the other groups, and the protection against infection was clear in the acute phase of infection but not in chronicity. The prime-boost strategy, which combines DNA and protein vaccine and both mucosal and systemic immunizations routes, was the best assayed protocol, inducing strong cellular and humoral responses as well as specific mucosal IgA, thus conferring better protection in the acute and chronic stages of infection. PMID:27177947

  15. In silico study of effects of polymorphisms on biophysical chemical properties of oxidized N-terminal domain of X-ray cross-complementing group 1 protein.

    PubMed

    Mehrzad, J; Monajjemi, M; Hashemi, M

    2014-01-01

    Base excision repair (BER) is the major pathway involved in removal of endogenous and mutagen-induced DNA damage. The X-ray cross-complementing group 1 protein (XRCC1), which participates in BER, is a scaffolding protein. The oxidized XRCC1 N-terminal domain (NTD) forms additional interactions with DNA polymerase β (Pol β). Any change in the residues of a protein (XRCC1, XRCC4, etc.) may alter its stability and function. Many coding regions of genes have single nucleotide polymorphisms (SNPs) that change the conformation of their products, and they are probably involved in some diseases. The R7L and R107H mutations are located in the XRCC1-NTD. In the present study, biophysical chemical properties of oxidized XRCC1-NTD (wild type or mutants) were investigated at different temperatures (290, 295, 298, 301, 304, 309, 310, 311, and 312 K) in water using in silico molecular mechanic computational methods. Comparison of the average calculated potential energies of oxidized XRCC1-NTD reveals that the R7L mutation increases stability, but the R107H and R7L&R107H mutations are destabilizing. Therefore, mutant types of this protein (R107H or R7L&R107H) may not function correctly. Furthermore, quantitative structure-activity relationship (QSAR) of oxidized XRCC1-NTD and docking assay showed that the R7L mutation is advantageous but the R107H and R7L&R107H mutations are disadvantageous for XRCC1-NTD, and in the latter cases it cannot interact with Pol β as well as the wild type does. Hence, DNA repair may be defective. Also, using the equation dE = ∂Ε/(∂Τ)V·dT + ∂Ε/(∂V)T·dV, it was determined that the best temperature for normal activity of oxidized XRCC1-NTD is exactly the natural body temperature (310 K).

  16. Structural characterisation of human galectin-4 N-terminal carbohydrate recognition domain in complex with glycerol, lactose, 3′-sulfo-lactose, and 2′-fucosyllactose

    PubMed Central

    Bum-Erdene, Khuchtumur; Leffler, Hakon; Nilsson, Ulf J.; Blanchard, Helen

    2016-01-01

    Galectin-4 is a tandem-repeat galectin with two distinct carbohydrate recognition domains (CRD). Galectin-4 is expressed mainly in the alimentary tract and is proposed to function as a lipid raft and adherens junction stabilizer by its glycan cross-linking capacity. Galectin-4 plays divergent roles in cancer and inflammatory conditions, either promoting or inhibiting each disease progression, depending on the specific pathological condition. The study of galectin-4’s ligand-binding profile may help decipher its roles under specific conditions. Here we present the X-ray structures of human galectin-4 N-terminal CRD (galectin-4N) bound to different saccharide ligands. Galectin-4’s overall fold and its core interactions to lactose are similar to other galectin CRDs. Galectin-4N recognises the sulfate cap of 3′-sulfated glycans by a weak interaction through Arg45 and two water-mediated hydrogen bonds via Trp84 and Asn49. When galectin-4N interacts with the H-antigen mimic, 2′-fucosyllactose, an interaction is formed between the ring oxygen of fucose and Arg45. The extended binding site of galectin-4N may not be well suited to the A/B-antigen determinants, α-GalNAc/α-Gal, specifically due to clashes with residue Phe47. Overall, galectin-4N favours sulfated glycans whilst galectin-4C prefers blood group determinants. However, the two CRDs of galectin-4 can, to a less extent, recognise each other’s ligands. PMID:26828567

  17. N-terminal groups of buffalo thyroglobulin.

    PubMed

    Deshpande, V; Ramachandran, L K

    1990-04-01

    N-Terminal analysis of purified buffalo thyroglobulin by the fluorodinitrobenzene method of Sanger yielded about 1.5 moles of DNP-glutamic acid per mole of buffalo thyroglobulin. No water-soluble DNP-amino acid was detectable as N-terminal. The presence of glutamic acid has been confirmed by Edman degradation and characterization of the PTH-amino acid in different solvent systems, and also after regeneration of free amino acid from PTH-amino acid in butanol-acetic acid-water (4:1:5, v/v) system. This is in contrast to the occurrence of aspartic acid or asparagine as N-terminals for several other mammalian thyroglobulins.

  18. NMR structure determination of the Escherichia coli DnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2-108) containing the highly conserved J domain.

    PubMed Central

    Szyperski, T; Pellecchia, M; Wall, D; Georgopoulos, C; Wüthrich, K

    1994-01-01

    DnaJ from Escherichia coli is a 376-amino acid protein that functions in conjunction with DnaK and GrpE as a chaperone machine. The N-terminal fragment of residues 2-108, DnaJ-(2-108), retains many of the activities of the full-length protein and contains a structural motif, the J domain of residues 2-72, which is highly conserved in a superfamily of proteins. In this paper, NMR spectroscopy was used to determine the secondary structure and the three-dimensional polypeptide backbone fold of DnaJ-(2-108). By using 13C/15N doubly labeled DnaJ-(2-108), nearly complete sequence-specific assignments were obtained for 1H, 15N, 13C alpha, and 13C beta, and about 40% of the peripheral aliphatic carbon resonances were also assigned. Four alpha-helices in polypeptide segments of residues 6-11, 18-31, 41-55, and 61-68 in the J domain were identified by sequential and medium-range nuclear Overhauser effects. For the J domain, the three-dimensional structure was calculated with the program DIANA from an input of 536 nuclear Overhauser effect upper-distance constraints and 52 spin-spin coupling constants. The polypeptide backbone fold is characterized by the formation of an antiparallel bundle of two long helices, residues 18-31 and 41-55, which is stabilized by a hydrophobic core of side chains that are highly conserved in homologous J domain sequences. The Gly/Phe-rich region from residues 77 to 108 is flexibly disordered in solution. Images PMID:7972061

  19. Studies on N-terminal glycation of peptides in hypoallergenic infant formulas: quantification of alpha-N-(2-furoylmethyl) amino acids.

    PubMed

    Penndorf, Ilka; Biedermann, Daniela; Maurer, Sarah V; Henle, Thomas

    2007-02-01

    To obtain information about the extent of the early Maillard reaction between the N-termini of peptides and lactose, alpha-N-(2-furoylmethyl) amino acids (FMAAs) were quantified together with epsilon-N-(2-furoylmethyl)lysine (furosine) in acid hydrolyzates of hypoallergenic infant formulas, conventional infant formulas, and human milk samples using RP-HPLC with UV-detection. FMAAs are formed during acid hydrolysis of peptide-bound N-terminal Amadori products (APs), and furosine is formed from the Amadori products of peptide-bound lysine. Unambiguous identification was achieved by means of LC/MS and UV-spectroscopy using independently prepared reference material. The extent of acid-induced conversion of APs to FMAAs was studied by RP-HPLC with chemiluminescent nitrogen detection (CLND). Depending on the corresponding alpha-N-lactulosyl amino acid, between 6.0% and 18.1% of FMAAs were formed during hydrolysis for 23 h at 110 degrees C in 8 N HCl. From epsilon-N-lactulosyllysine, 50% furosine is formed under these conditions. Whereas furosine was detectable in all assayed samples, five different FMAAs, alpha-FM-Lys, alpha-FM-Ala, alpha-FM-Val, alpha-FM-Ile, and alpha-FM-Leu, were exclusively detected in acid hydrolyzates of hypoallergenic infant formulas in amounts ranging from 35 to 396 mumol/100 g protein. Taking the conversion factors into account, modification of N-terminal amino acids in peptides by reducing carbohydrates was between 0.3% and 8.4%. This has to be considered within the discussion concerning the nutritional quality of peptide-containing foods.

  20. Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas.

    PubMed

    Osatomi, K; Masuda, Y; Hara, K; Ishihara, T

    2001-04-01

    Cu, Zn-superoxide dismutase (SOD) has been purified to homogeneity from Japanese flounder Paralichthys olivaceus hepato-pancreas. The purification of the enzyme was carried out by an ethanol/chloroform treatment and acetone precipitation, and then followed by column chromatographies on Q-Sepharose, S-Sepharose and Ultrogel AcA 54. On SDS-PAGE, the purified enzyme gave a single protein band with molecular mass of 17.8 kDa under reducing conditions, and showed approximately equal proportions of 17.8 and 36 kDa molecular mass under non-reducing conditions. Three bands were obtained when the purified enzyme was subjected to native-PAGE, both on protein and activity staining, but the electrophoretic mobility of the purified enzyme differed from that of bovine erythrocyte Cu, Zn-SOD. Isoelectric point values of 5.9, 6.0 and 6.2, respectively, were obtained for the three components. The N-terminal amino acid sequence of the purified enzyme was determined for 25 amino acid residues, and the sequence was compared with other Cu, Zn-SODs. The N-terminal alanine residue was unacetylated, as in the case of swordfish SOD. Above 60 degrees C, the thermostability of the enzyme was much lower than that of bovine Cu, Zn-SOD. PMID:11290457

  1. N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells

    PubMed Central

    2010-01-01

    Background Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved. Methods The MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively. Results Both of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity. Conclusions Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents. PMID:20939933

  2. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells.

    PubMed

    Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

    2016-01-01

    The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb St

  3. Distinct Effects of Two HIV-1 Capsid Assembly Inhibitor Families That Bind the Same Site within the N-Terminal Domain of the Viral CA Protein

    PubMed Central

    Titolo, Steve; von Schwedler, Uta; Goudreau, Nathalie; Mercier, Jean-François; Wardrop, Elizabeth; Faucher, Anne-Marie; Coulombe, René; Banik, Soma S. R.; Fader, Lee; Gagnon, Alexandre; Kawai, Stephen H.; Rancourt, Jean; Tremblay, Martin; Yoakim, Christiane; Simoneau, Bruno; Archambault, Jacques; Sundquist, Wesley I.

    2012-01-01

    The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall

  4. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells

    PubMed Central

    Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

    2016-01-01

    The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb St

  5. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells.

    PubMed

    Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

    2016-01-01

    The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb St

  6. Quantification of glycated N-terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry.

    PubMed

    Sakaguchi, Yohei; Kinumi, Tomoya; Yamazaki, Taichi; Takatsu, Akiko

    2016-02-01

    A novel method of amino acid analysis using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups) was applied to measure glycated amino acids in order to quantify glycated peptides and evaluate the degree of glycation of peptide. Amino and carboxyl groups of amino acids were derivatized with 1-bromobutane so that the hydrophobicities and basicities of the amino acids, including glycated amino acids, were improved. These derivatized amino acids could be detected with high sensitivity using LC-MS/MS. In this study, 1-deoxyfructosyl-VHLTPE and VHLTPE, which are N-terminal peptides of the β-chains of hemoglobin, were selected as target compounds. After reducing the peptide sample solution with sodium borohydride, the obtained peptides were hydrolyzed with hydrochloric acid. The released amino acids were then derivatized with 1-bromobutane and analyzed with LC-MS/MS. The derivatized amino acids, including glycated amino acids, could be separated using an octadecyl silylated silica column and good sharp peaks were detected. We show a confirmatory experiment that the proposed method can be applied to evaluate the degree of glycation of peptides, using mixtures of glycated and non-glycated peptide.

  7. The dark and bright sides of an enzyme: a three dimensional structure of the N-terminal domain of Zophobas morio luciferase-like enzyme, inferences on the biological function and origin of oxygenase/luciferase activity.

    PubMed

    Prado, R A; Santos, C R; Kato, D I; Murakami, M T; Viviani, V R

    2016-05-11

    Beetle luciferases, the enzymes responsible for bioluminescence, are special cases of CoA-ligases which have acquired a novel oxygenase activity, offering elegant models to investigate the structural origin of novel catalytic functions in enzymes. What the original function of their ancestors was, and how the new oxygenase function emerged leading to bioluminescence remains unclear. To address these questions, we solved the crystal structure of a recently cloned Malpighian luciferase-like enzyme of unknown function from Zophobas morio mealworms, which displays weak luminescence with ATP and the xenobiotic firefly d-luciferin. The three dimensional structure of the N-terminal domain showed the expected general fold of CoA-ligases, with a unique carboxylic substrate binding pocket, permitting the binding and CoA-thioesterification activity with a broad range of carboxylic substrates, including short-, medium-chain and aromatic acids, indicating a generalist function consistent with a xenobiotic-ligase. The thioesterification activity with l-luciferin, but not with the d-enantiomer, confirms that the oxygenase activity emerged from a stereoselective impediment of the thioesterification reaction with the latter, favoring the alternative chemiluminescence oxidative reaction. The structure and site-directed mutagenesis support the involvement of the main-chain amide carbonyl of the invariant glycine G323 as the catalytic base for luciferin C4 proton abstraction during the oxygenase activity in this enzyme and in beetle luciferases (G343).

  8. The dark and bright sides of an enzyme: a three dimensional structure of the N-terminal domain of Zophobas morio luciferase-like enzyme, inferences on the biological function and origin of oxygenase/luciferase activity.

    PubMed

    Prado, R A; Santos, C R; Kato, D I; Murakami, M T; Viviani, V R

    2016-05-11

    Beetle luciferases, the enzymes responsible for bioluminescence, are special cases of CoA-ligases which have acquired a novel oxygenase activity, offering elegant models to investigate the structural origin of novel catalytic functions in enzymes. What the original function of their ancestors was, and how the new oxygenase function emerged leading to bioluminescence remains unclear. To address these questions, we solved the crystal structure of a recently cloned Malpighian luciferase-like enzyme of unknown function from Zophobas morio mealworms, which displays weak luminescence with ATP and the xenobiotic firefly d-luciferin. The three dimensional structure of the N-terminal domain showed the expected general fold of CoA-ligases, with a unique carboxylic substrate binding pocket, permitting the binding and CoA-thioesterification activity with a broad range of carboxylic substrates, including short-, medium-chain and aromatic acids, indicating a generalist function consistent with a xenobiotic-ligase. The thioesterification activity with l-luciferin, but not with the d-enantiomer, confirms that the oxygenase activity emerged from a stereoselective impediment of the thioesterification reaction with the latter, favoring the alternative chemiluminescence oxidative reaction. The structure and site-directed mutagenesis support the involvement of the main-chain amide carbonyl of the invariant glycine G323 as the catalytic base for luciferin C4 proton abstraction during the oxygenase activity in this enzyme and in beetle luciferases (G343). PMID:27101527

  9. N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65.

    PubMed Central

    Toledano, M B; Ghosh, D; Trinh, F; Leonard, W J

    1993-01-01

    We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed. Images PMID:8423807

  10. UmuDAb: An Error-Prone Polymerase Accessory Homolog Whose N-Terminal Domain Is Required for Repression of DNA Damage Inducible Gene Expression in Acinetobacter baylyi

    PubMed Central

    Stinnett, DeAnna B.; Wells, Whitney K.; Peterson, Megan A.; Hare, Janelle M.

    2016-01-01

    In many bacteria, the DNA damage response induces genes (SOS genes) that were repressed by LexA. LexA represses transcription by binding to SOS promoters via a helix-turn-helix motif in its N-terminal domain (NTD). Upon DNA damage, LexA cleaves itself and allows induction of transcription. In Acinetobacter baumannii and Acinetobacter baylyi, multiple genes are induced by DNA damage, and although the Acinetobacter genus lacks LexA, a homolog of the error-prone polymerase subunit UmuD, called UmuDAb, regulates some DNA damage-induced genes. The mechanism of UmuDAb regulation has not been determined. We constructed UmuDAb mutant strains of A. baylyi to test whether UmuDAb mediates gene regulation through LexA-like repressor actions consisting of relief of repression through self-cleavage after DNA damage. Real-time quantitative PCR experiments in both a null umuDAb mutant and an NTD mutant showed that the DNA damage-inducible, UmuDAb-regulated gene ddrR was highly expressed even in the absence of DNA damage. Protein modeling identified a potential LexA-like helix-turn-helix structure in the UmuDAb NTD, which when disrupted, also relieved ddrR and umuDAb repression under non-inducing conditions. Mutations in a putative SOS box in the shared umuDAb-ddrR promoter region similarly relieved these genes’ repression under non-inducing conditions. Conversely, cells possessing a cleavage-deficient UmuDAb were unable to induce gene expression after MMC-mediated DNA damage. This evidence of a UmuDAb repressor mechanism was contrasted with the failure of umuDAb to complement an Escherichia coli umuD mutant for UmuD error-prone DNA replication activity. Similarly, A. baumannii null umuDAb mutant cells did not have a reduced UmuDˊ2UmuC-mediated mutation rate after DNA damage, suggesting that although this UmuDAb protein may have evolved from a umuDC operon in this genus, it now performs a LexA-like repressor function for a sub-set of DNA damage-induced genes. PMID:27010837

  11. c-Jun N-terminal Kinase (JNK) induces phosphorylation of amyloid precursor protein (APP) at Thr668, in okadaic acid-induced neurodegeneration

    PubMed Central

    Ahn, Ji-Hwan; So, Sang-Pil; Kim, Na-Young; Kim, Hyun-Ju; Yoon, Seung-Yong; Kim, Dong-Hou

    2016-01-01

    Several lines of evidence have revealed that phosphorylation of amyloid precursor protein (APP) at Thr668 is involved in the pathogenesis of Alzheimer’s disease (AD). Okadaic acid (OA), a protein phosphatase-2A inhibitor, has been used in AD research models to increase tau phosphorylation and induce neuronal death. We previously showed that OA increased levels of APP and induced accumulation of APP in axonal swellings. In this study, we found that in OA-treated neurons, phosphorylation of APP at Thr668 increased and accumulated in axonal swellings by c-jun N-terminal kinase (JNK), and not by Cdk5 or ERK/MAPK. These results suggest that JNK may be one of therapeutic targets for the treatment of AD. [BMB Reports 2016; 49(7): 376-381] PMID:26839154

  12. Characterization of amino acid residues within the N-terminal region of Ubc9 that play a role in Ubc9 nuclear localization

    SciTech Connect

    Sekhri, Palak; Tao, Tao; Kaplan, Feige; Zhang, Xiang-Dong

    2015-02-27

    As the sole E2 enzyme for SUMOylation, Ubc9 is predominantly nuclear. However, the underlying mechanisms of Ubc9 nuclear localization are still not well understood. Here we show that RNAi-depletion of Imp13, an importin known to mediate Ubc9 nuclear import, reduces both Ubc9 nuclear accumulation and global SUMOylation. Furthermore, Ubc9-R13A or Ubc9-H20D mutation previously shown to interrupt the interaction of Ubc9 with nucleus-enriched SUMOs reduces the nuclear enrichment of Ubc9, suggesting that the interaction of Ubc9 with the nuclear SUMOs may enhance Ubc9 nuclear retention. Moreover, Ubc9-R17E mutation, which is known to disrupt the interaction of Ubc9 with both SUMOs and Imp13, causes a greater decrease in Ubc9 nuclear accumulation than Ubc9-R13A or Ubc9-H20D mutation. Lastly, Ubc9-K74A/S89D mutations that perturb the interaction of Ubc9 with nucleus-enriched SUMOylation-consensus motifs has no effect on Ubc9 nuclear localization. Altogether, our results have elucidated that the amino acid residues within the N-terminal region of Ubc9 play a pivotal role in regulation of Ubc9 nuclear localization. - Highlights: • Imp13-mediated nuclear import of Ubc9 is critical for global SUMOylation. • Ubc9 mutations disrupting Ubc9-SUMO interaction decrease Ubc9 nuclear accumulation. • N-terminal amino acid residues of Ubc9 are critical for Ubc9 nuclear enrichment.

  13. The N-terminal repeat and the ligand binding domain A of SdrI protein is involved in hydrophobicity of S. saprophyticus.

    PubMed

    Kleine, Britta; Ali, Liaqat; Wobser, Dominique; Sakιnç, Türkân

    2015-03-01

    Staphylococcus saprophyticus is an important cause of urinary tract infection, and its cell surface hydrophobicity may contribute to virulence by facilitating adherence of the organism to uroepithelia. S. saprophyticus expresses the surface protein SdrI, a member of the serine-aspartate repeat (SD) protein family, which has multifunctional properties. The SdrI knock out mutant has a reduced hydrophobicity index (HPI) of 25%, and expressed in the non-hydrophobic Staphylococcus carnosus strain TM300 causes hydrophobicity. Using hydrophobic interaction chromatography (HIC), we confined the hydrophobic site of SdrI to the N-terminal repeat region. S. saprophyticus strains carrying different plasmid constructs lacking either the N-terminal repeats, both B or SD-repeats were less hydrophobic than wild type and fully complemented SdrI mutant (HPI: 51%). The surface hydrophobicity and HPI of both wild type and the complemented strain were also influenced by calcium (Ca(2+)) and were reduced from 81.3% and 82.4% to 10.9% and 12.3%, respectively. This study confirms that the SdrI protein of S. saprophyticus is a crucial factor for surface hydrophobicity and also gives a first significant functional description of the N-terminal repeats, which in conjunction with the B-repeats form an optimal hydrophobic conformation.

  14. Functional roles of N-terminal and C-terminal domains in the overall activity of a novel single-stranded DNA binding protein of Deinococcus radiodurans

    PubMed Central

    Ujaoney, Aman K.; Basu, Bhakti; Muniyappa, K.; Apte, Shree K.

    2015-01-01

    Single-stranded DNA binding protein (Ssb) of Deinococcus radiodurans comprises N- and C-terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: SsbN (N-terminal without connector), SsbNC (N-terminal with connector) and SsbC (C-terminal), each harboring one OB fold. Both SsbN and SsbNC displayed weak single-stranded DNA (ssDNA) binding activity, compared to the full-length Ssb (SsbFL). The level of ssDNA binding activity displayed by SsbC was intermediate between SsbFL and SsbN. SsbC and SsbFL predominantly existed as homo-dimers while SsbNC/SsbN formed different oligomeric forms. In vitro, SsbNC or SsbN formed a binary complex with SsbC that displayed enhanced ssDNA binding activity. Unlike SsbFL, Ssb variants were able to differentially modulate topoisomerase-I activity, but failed to stimulate Deinococcal RecA-promoted DNA strand exchange. The results suggest that the C-terminal OB fold is primarily responsible for ssDNA binding. The N-terminal OB fold binds weakly to ssDNA but is involved in multimerization. PMID:25973364

  15. The N-terminal repeat and the ligand binding domain A of SdrI protein is involved in hydrophobicity of S. saprophyticus.

    PubMed

    Kleine, Britta; Ali, Liaqat; Wobser, Dominique; Sakιnç, Türkân

    2015-03-01

    Staphylococcus saprophyticus is an important cause of urinary tract infection, and its cell surface hydrophobicity may contribute to virulence by facilitating adherence of the organism to uroepithelia. S. saprophyticus expresses the surface protein SdrI, a member of the serine-aspartate repeat (SD) protein family, which has multifunctional properties. The SdrI knock out mutant has a reduced hydrophobicity index (HPI) of 25%, and expressed in the non-hydrophobic Staphylococcus carnosus strain TM300 causes hydrophobicity. Using hydrophobic interaction chromatography (HIC), we confined the hydrophobic site of SdrI to the N-terminal repeat region. S. saprophyticus strains carrying different plasmid constructs lacking either the N-terminal repeats, both B or SD-repeats were less hydrophobic than wild type and fully complemented SdrI mutant (HPI: 51%). The surface hydrophobicity and HPI of both wild type and the complemented strain were also influenced by calcium (Ca(2+)) and were reduced from 81.3% and 82.4% to 10.9% and 12.3%, respectively. This study confirms that the SdrI protein of S. saprophyticus is a crucial factor for surface hydrophobicity and also gives a first significant functional description of the N-terminal repeats, which in conjunction with the B-repeats form an optimal hydrophobic conformation. PMID:25497915

  16. Mitotic lifecycle of chromosomal 3xHMG-box proteins and the role of their N-terminal domain in the association with rDNA loci and proteolysis.

    PubMed

    Antosch, Martin; Schubert, Veit; Holzinger, Philipp; Houben, Andreas; Grasser, Klaus D

    2015-12-01

    The high mobility group (HMG)-box is a DNA-binding domain characteristic of various eukaryotic DNA-binding proteins. 3xHMG-box proteins (containing three copies of the HMG-box domain and a unique basic N-terminal domain) are specific for plants and the Arabidopsis genome encodes two versions termed 3xHMG-box1 and 3xHMG-box2, whose expression is cell cycle-dependent, peaking during mitosis. Here, we analysed in detail the spatiotemporal expression, subcellular localisation and chromosome association of the Arabidopsis thaliana 3xHMG-box proteins. Live cell imaging and structured illumination microscopy revealed that the expression of the 3xHMG-box proteins is induced in late G2 phase of the cell cycle and upon nuclear envelope breakdown in prophase they rapidly associate with the chromosomes. 3xHMG-box1 associates preferentially with 45S rDNA loci and the basic N-terminal domain is involved in the targeting of rDNA loci. Shortly after mitosis the 3xHMG-box proteins are degraded and an N-terminal destruction-box mediates the proteolysis. Ectopic expression/localisation of 3xHMG-box1 in interphase nuclei results in reduced plant growth and various developmental defects including early bolting and abnormal flower morphology. The remarkable conservation of 3xHMG-box proteins within the plant kingdom, their characteristic expression during mitosis, and their striking association with chromosomes, suggest that they play a role in the organisation of plant mitotic chromosomes. PMID:26213803

  17. Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange.

    PubMed

    Sharadamma, N; Khan, Krishnendu; Kumar, Sandeep; Patil, K Neelakanteshwar; Hasnain, Seyed E; Muniyappa, K

    2011-09-01

    The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN) , HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.

  18. Purification and characterization of recombinant sugarcane sucrose phosphate synthase expressed in E. coli and insect Sf9 cells: an importance of the N-terminal domain for an allosteric regulatory property.

    PubMed

    Sawitri, Widhi Dyah; Narita, Hirotaka; Ishizaka-Ikeda, Etsuko; Sugiharto, Bambang; Hase, Toshiharu; Nakagawa, Atsushi

    2016-06-01

    Sucrose phosphate synthase (SPS) catalyses the transfer of glycosyl group of uridine diphosphate glucose to fructose-6-phosphate to form sucrose-6-phosphate. Plant SPS plays a key role in photosynthetic carbon metabolisms, which activity is modulated by an allosteric activator glucose-6-phosphate (G6P). We produced recombinant sugarcane SPS using Escherichia coli and Sf9 insect cells to investigate its structure-function relationship. When expressed in E. coli, two forms of SPS with different sizes appeared; the larger was comparable in size with the authentic plant enzyme and the shorter was trimmed the N-terminal 20 kDa region off. In the insect cells, only enzyme with the authentic size was produced. We purified the trimmed SPS and the full size enzyme from insect cells and found their enzymatic properties differed significantly; the full size enzyme was activated allosterically by G6P, while the trimmed one showed a high activity even without G6P. We further introduced a series of N-terminal truncations up to 171 residue and found G6P-independent activity was enhanced by the truncation. These combined results indicated that the N-terminal region of sugarcane SPS is crucial for the allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity. PMID:26826371

  19. Genomic analysis of 16 Colorado human NL63 coronaviruses identifies a new genotype, high sequence diversity in the N-terminal domain of the spike gene and evidence of recombination

    PubMed Central

    Sims, Gregory E.; Wentworth, David E.; Halpin, Rebecca A.; Robinson, Christine C.; Town, Christopher D.; Holmes, Kathryn V.

    2012-01-01

    This study compared the complete genome sequences of 16 NL63 strain human coronaviruses (hCoVs) from respiratory specimens of paediatric patients with respiratory disease in Colorado, USA, and characterized the epidemiology and clinical characteristics associated with circulating NL63 viruses over a 3-year period. From 1 January 2009 to 31 December 2011, 92 of 9380 respiratory specimens were found to be positive for NL63 RNA by PCR, an overall prevalence of 1 %. NL63 viruses were circulating during all 3 years, but there was considerable yearly variation in prevalence and the month of peak incidence. Phylogenetic analysis comparing the genome sequences of the 16 Colorado NL63 viruses with those of the prototypical hCoV-NL63 and three other NL63 viruses from the Netherlands demonstrated that there were three genotypes (A, B and C) circulating in Colorado from 2005 to 2010, and evidence of recombination between virus strains was found. Genotypes B and C co-circulated in Colorado in 2005, 2009 and 2010, but genotype A circulated only in 2005 when it was the predominant NL63 strain. Genotype C represents a new lineage that has not been described previously. The greatest variability in the NL63 virus genomes was found in the N-terminal domain (NTD) of the spike gene (nt 1–600, aa 1–200). Ten different amino acid sequences were found in the NTD of the spike protein among these NL63 strains and the 75 partial published sequences of NTDs from strains found at different times throughout the world. PMID:22837419

  20. Genomic analysis of 16 Colorado human NL63 coronaviruses identifies a new genotype, high sequence diversity in the N-terminal domain of the spike gene and evidence of recombination.

    PubMed

    Dominguez, Samuel R; Sims, Gregory E; Wentworth, David E; Halpin, Rebecca A; Robinson, Christine C; Town, Christopher D; Holmes, Kathryn V

    2012-11-01

    This study compared the complete genome sequences of 16 NL63 strain human coronaviruses (hCoVs) from respiratory specimens of paediatric patients with respiratory disease in Colorado, USA, and characterized the epidemiology and clinical characteristics associated with circulating NL63 viruses over a 3-year period. From 1 January 2009 to 31 December 2011, 92 of 9380 respiratory specimens were found to be positive for NL63 RNA by PCR, an overall prevalence of 1 %. NL63 viruses were circulating during all 3 years, but there was considerable yearly variation in prevalence and the month of peak incidence. Phylogenetic analysis comparing the genome sequences of the 16 Colorado NL63 viruses with those of the prototypical hCoV-NL63 and three other NL63 viruses from the Netherlands demonstrated that there were three genotypes (A, B and C) circulating in Colorado from 2005 to 2010, and evidence of recombination between virus strains was found. Genotypes B and C co-circulated in Colorado in 2005, 2009 and 2010, but genotype A circulated only in 2005 when it was the predominant NL63 strain. Genotype C represents a new lineage that has not been described previously. The greatest variability in the NL63 virus genomes was found in the N-terminal domain (NTD) of the spike gene (nt 1-600, aa 1-200). Ten different amino acid sequences were found in the NTD of the spike protein among these NL63 strains and the 75 partial published sequences of NTDs from strains found at different times throughout the world.

  1. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry.

    PubMed Central

    Sonnenberg, M G; Belisle, J T

    1997-01-01

    A number of the culture filtrate proteins secreted by Mycobacterium tuberculosis are known to contribute to the immunology of tuberculosis and to possess enzymatic activities associated with pathogenicity. However, a complete analysis of the protein composition of this fraction has been lacking. By using two-dimensional polyacrylamide gel electrophoresis, detailed maps of the culture filtrate proteins of M. tuberculosis H37Rv were generated. In total, 205 protein spots were observed. The coupling of this electrophoretic technique with Western blot analysis allowed the identification and mapping of 32 proteins. Further molecular characterization of abundant proteins within this fraction was achieved by N-terminal amino acid sequencing and liquid chromatography-mass spectrometry. Eighteen proteins were subjected to N-group analysis; of these, only 10 could be sequenced by Edman degradation. Among the most interesting were a novel 52-kDa protein demonstrating significant homology to an alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708, a 25-kDa protein corresponding to open reading frame 28 of the M. tuberculosis cosmid MTCY1A11, and a 31-kDa protein exhibiting an amino acid sequence identical to that of antigen 85A and 85B. This latter product migrated with an isoelectric point between those of antigen 85A and 85C but did not react with the antibody specific for this complex, suggesting that there is a fourth member of the antigen 85 complex. Novel N-terminal amino acid sequences were obtained for three additional culture filtrate proteins; however, these did not yield significant homology to known protein sequences. A protein cluster of 85 to 88 kDa, recognized by the monoclonal antibodies IT-57 and IT-42 and known to react with sera from a large proportion of tuberculosis patients, was refractory to N-group analysis. Nevertheless, mass spectrometry of peptides obtained from one member of this complex identified it as the M. tuberculosis Kat

  2. β-Catenin Binds to the Activation Function 2 Region of the Androgen Receptor and Modulates the Effects of the N-Terminal Domain and TIF2 on Ligand-Dependent Transcription

    PubMed Central

    Song, Liang-Nian; Herrell, Roger; Byers, Stephen; Shah, Salimuddin; Wilson, Elizabeth M.; Gelmann, Edward P.

    2003-01-01

    β-Catenin is a multifunctional molecule that is activated by signaling through WNT receptors. β-Catenin can also enhance the transcriptional activity of some steroid hormone receptors such as the androgen receptor and retinoic acid receptor α. Androgens can affect nuclear translocation of β-catenin and influence its subcellular distribution. Using mammalian two-hybrid binding assays, analysis of reporter gene transcription, and coimmunoprecipitation, we now show that β-catenin binds to the androgen receptor ligand-binding domain (LBD) and modulates the transcriptional effects of TIF2 and the androgen receptor N-terminal domain (NTD). In functional assays, β-catenin bound to androgen receptor only in the presence of ligand agonists, not antagonists. β-Catenin binding to the androgen receptor LBD was independent of and cooperative with the androgen receptor NTD and the p160 coactivator TIF2, both of which bind to the activation function 2 (AF-2) region of the androgen receptor. Different mutations of androgen receptor helix 3 amino acids disrupted binding of androgen receptor NTD and β-catenin. β-Catenin, androgen receptor NTD, and TIF2 binding to the androgen receptor LBD were affected similarly by a subset of helix 12 mutations, but disruption of two sites on helix 12 affected only binding of β-catenin and not of TIF2 or the androgen receptor NTD. Mutational disruption of each of five LXXLL peptide motifs in the β-catenin armadillo repeats did not disrupt either binding to androgen receptor or transcriptional coactivation. ICAT, an inhibitor of T-cell factor 4 (TCF-4), and E-cadherin binding to β-catenin also blocked binding of the androgen receptor LBD. We also demonstrated cross talk between the WNT and androgen receptor signaling pathways because excess androgen receptor could interfere with WNT signaling and excess TCF-4 inhibited the interaction of β-catenin and androgen receptor. Taken together, the data show that β-catenin can bind to the

  3. An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium.

    PubMed

    Myre, Michael A; O'Day, Danton H

    2005-06-24

    Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD (171EDVSRFIKGKLLQKQQKIYKDLERF195) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues 48KKSYQDPEIIAHSRPRK64 that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to 48EF49 abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the 48EF49 construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium. PMID:15896312

  4. Sequence-dependent nucleosome structural and dynamic polymorphism. Potential involvement of histone H2B N-terminal tail proximal domain.

    PubMed

    Sivolob, Andrei; Lavelle, Christophe; Prunell, Ariel

    2003-02-01

    Relaxation of nucleosomes on an homologous series (pBR) of ca 350-370 bp DNA minicircles originating from plasmid pBR322 was recently used as a tool to study their structure and dynamics. These nucleosomes thermally fluctuated between three distinct DNA conformations within a histone N-terminal tail-modulated equilibrium: one conformation was canonical, with 1.75 turn wrapping and negatively crossed entering and exiting DNAs; another was also "closed", but with these DNAs positively crossed; and the third was "open", with a lower than 1.5 turn wrapping and uncrossed DNAs. In this work, a new minicircle series (5S) of similar size was used, which contained the 5S nucleosome positioning sequence. Results showed that DNA in pBR nucleosomes was untwisted by approximately 0.2 turn relative to 5S nucleosomes, which DNase I footprinting confirmed in revealing a approximately 1 bp untwisting at each of the two dyad-distal sites where H2B N-terminal tails pass between the two gyres. In contrast, both nucleosomes showed untwistings at the dyad-proximal sites, i.e. on the other gyre, which were also observed in the high-resolution crystal structure. 5S nucleosomes also differ with respect to their dynamics: they hardly accessed the positively crossed conformation, but had an easier access to the negatively crossed conformation. Simulation showed that such reverse effects on the conformational free energies could be simply achieved by slightly altering the trajectories of entering and exiting DNAs. We propose that this is accomplished by H2B tail untwisting at the distal sites through action at a distance ( approximately 20 bp) on H3-tail interactions with the small groove at the nucleosome entry-exit. These results may help to gain a first glimpse into the two perhaps most intriguing features of the high-resolution structure: the alignment of the grooves on the two gyres and the passage of H2B and H3 N-terminal tails between them. PMID:12547190

  5. Association of the formiminotransferase N-terminal sub-domain containing gene and thrombospondin, type 1, domain-containing 7A gene with the prevalence of vertebral fracture in 2427 consecutive autopsy cases.

    PubMed

    Zhou, Heying; Mori, Seijiro; Kou, Ikuyo; Fuku, Noriyuki; Naka Mieno, Makiko; Honma, Naoko; Arai, Tomio; Sawabe, Motoji; Tanaka, Masashi; Ikegawa, Shiro; Ito, Hideki

    2013-02-01

    We previously reported 2 osteoporosis-susceptibility genes--formiminotransferase N-terminal sub-domain containing gene (FONG) and thrombospondin, type 1, domain-containing 7A (THSD7A)--in which we identified two common single-nucleotide polymorphisms, rs7605378 (FONG) and rs12673692 (THSD7A). The former was associated with a predisposition to osteoporosis and the latter with bone mineral density. To further elucidate the importance of these polymorphisms in the pathogenesis of osteoporosis, we examined their association with the incidence of vertebral fracture. DNA extracted from the renal cortex of 2427 consecutive Japanese autopsies (1331 men, mean age: 79 years; 1096 women, mean age: 82 years) were examined in this study. The presence or absence of vertebral fracture during each subject's lifetime was determined by a thorough examination of the clinical records, as well as autopsy reports. After adjustments for sex and age at autopsy, logistic regression analysis revealed that homozygotes for the risk alleles of rs7605378 (A-allele) or rs12673629 (A-allele) possess an increased risk of vertebral fracture. The subjects simultaneously homozygous for both the risk alleles of rs7605378 (AA genotype) and rs12673629 (AA genotype) showed significantly higher risk of vertebral fracture (odds ratio 2.401, 95% confidence interval 1.305-4.416, P = 0.0048) than those who had at least one non-risk allele of either rs7605378 (AC/CC genotypes) or rs12673629 (AG/GG genotypes). The results suggest that Japanese subjects homozygous for the risk alleles of rs7605378 and rs12673629 have a higher risk of vertebral fracture.

  6. Functional display of triphenylmethane reductase for dye removal on the surface of Escherichia coli using N-terminal domain of ice nucleation protein.

    PubMed

    Gao, Fen; Ding, Haitao; Feng, Zhuo; Liu, Danfeng; Zhao, Yuhua

    2014-10-01

    Traditional biological treatment for triphenylmethane dye effluent is stuck with the inaccessibility of dye molecules to intracellular dye-degrading enzyme, thus a high-efficiency and low-cost method for dye decolorization is highly desirable. Here we established a bioremediation approach to display triphenylmethane reductase (TMR) on the surface of Escherichia coli (E. coli) using N-terminal of ice nucleation protein as anchoring motif for triphenylmethane dye decolorization for the first time. Approximately 85% of recombinant protein positioning on the surface of E. coil cells exhibited high activity and stability. The optimal temperature and pH of the surface-displayed TMR are 50 °C and 8.5, respectively. Comparing with other reported microorganisms, the decolorization rate for malachite green of this engineered strain is the highest so far, reaching 640 μmol min(-1) g(-1) dry weight cells. These results indicate that this engineered E. coli strain is a very promising candidate for synthetic dye removal. PMID:25058292

  7. Functional display of triphenylmethane reductase for dye removal on the surface of Escherichia coli using N-terminal domain of ice nucleation protein.

    PubMed

    Gao, Fen; Ding, Haitao; Feng, Zhuo; Liu, Danfeng; Zhao, Yuhua

    2014-10-01

    Traditional biological treatment for triphenylmethane dye effluent is stuck with the inaccessibility of dye molecules to intracellular dye-degrading enzyme, thus a high-efficiency and low-cost method for dye decolorization is highly desirable. Here we established a bioremediation approach to display triphenylmethane reductase (TMR) on the surface of Escherichia coli (E. coli) using N-terminal of ice nucleation protein as anchoring motif for triphenylmethane dye decolorization for the first time. Approximately 85% of recombinant protein positioning on the surface of E. coil cells exhibited high activity and stability. The optimal temperature and pH of the surface-displayed TMR are 50 °C and 8.5, respectively. Comparing with other reported microorganisms, the decolorization rate for malachite green of this engineered strain is the highest so far, reaching 640 μmol min(-1) g(-1) dry weight cells. These results indicate that this engineered E. coli strain is a very promising candidate for synthetic dye removal.

  8. Functional Role of the N-Terminal Domain of ΔFosB in Response to Stress and Drugs of Abuse

    PubMed Central

    Ohnishi, Yoshinori N.; Ohnishi, Yoko H.; Vialou, Vincent; Mouzon, Ezekiell; LaPlant, Quincey; Nishi, Akinori; Nestler, Eric J.

    2014-01-01

    Previous work has implicated the transcription factor, ΔFosB, acting in the nucleus accumbens, in mediating the pro-rewarding effects of drugs of abuse such as cocaine as well as in mediating resilience to chronic social stress. However, the transgenic and viral gene transfer models used to establish these ΔFosB phenotypes express, in addition to ΔFosB, an alternative translation product of ΔFosB mRNA, termed Δ2ΔFosB, which lacks the N-terminal 78 aa present in ΔFosB. To study the possible contribution of Δ2ΔFosB to these drug and stress phenotypes, we prepared a viral vector that overexpresses a point mutant form of ΔFosB mRNA which cannot undergo alternative translation as well as a vector that overexpresses Δ2ΔFosB alone. Our results show that the mutant form of ΔFosB, when overexpressed in the nucleus accumbens, reproduces the enhancement of reward and of resilience seen with our earlier models, with no effects seen for Δ2ΔFosB. Overexpression of full length FosB, the other major product of the FosB gene, also has no effect. These findings confirm the unique role of ΔFosB in nucleus accumbens in controlling responses to drugs of abuse and stress. PMID:25313003

  9. Epstein-Barr virus nuclear protein 3C binds to the N-terminal (NTD) and beta trefoil domains (BTD) of RBP/CSL; Only the NTD interaction is essential for lymphoblastoid cell growth

    SciTech Connect

    Calderwood, Michael A.; Lee, Sungwook; Holthaus, Amy M.; Blacklow, Stephen C.; Kieff, Elliott; Johannsen, Eric

    2011-05-25

    Association of EBV nuclear proteins EBNA2, EBNA3A and EBNA3C with RBP/CSL, is essential for lymphoblastoid cell line (LCL) proliferation. Conserved residues in the EBNA3 homology domain, required for RBP/CSL interaction, lack the W{Phi}P motif that mediates EBNA2 and Notch binding to the RBP/CSL beta-trefoil domain (BTD). We map RBP/CSL interacting residues within EBNA3A(aa128-204) and EBNA3C(aa211-233). The EBNA3A results are consistent with an earlier report (aa125-222), but the EBNA3C domain is unexpectedly small and includes a 'WTP' sequence. This EBNA3C WTP motif confers RBP/CSL binding in vitro, in yeast, and in mammalian cells. Further, an EBNA3C WTP {yields} STP(W227S) mutation impaired BTD binding whereas EBNA3 homology domain mutations disrupted RBP/CSL N-terminal domain (NTD) binding. WTP was not essential for EBNA3C repression of EBNA2 in reporter assays or for maintenance of LCL growth. Our results indicate that EBNA3 proteins interact with multiple RBP/CSL domains, but only NTD interactions are required for LCL growth.

  10. The acidic domains of the Toc159 chloroplast preprotein receptor family are intrinsically disordered protein domains

    PubMed Central

    2009-01-01

    Background The Toc159 family of proteins serve as receptors for chloroplast-destined preproteins. They directly bind to transit peptides, and exhibit preprotein substrate selectivity conferred by an unknown mechanism. The Toc159 receptors each include three domains: C-terminal membrane, central GTPase, and N-terminal acidic (A-) domains. Although the function(s) of the A-domain remains largely unknown, the amino acid sequences are most variable within these domains, suggesting they may contribute to the functional specificity of the receptors. Results The physicochemical properties of the A-domains are characteristic of intrinsically disordered proteins (IDPs). Using CD spectroscopy we show that the A-domains of two Arabidopsis Toc159 family members (atToc132 and atToc159) are disordered at physiological pH and temperature and undergo conformational changes at temperature and pH extremes that are characteristic of IDPs. Conclusions Identification of the A-domains as IDPs will be important for determining their precise function(s), and suggests a role in protein-protein interactions, which may explain how these proteins serve as receptors for such a wide variety of preprotein substrates. PMID:20042108

  11. Factor D of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site.

    PubMed Central

    Johnson, D M; Gagnon, J; Reid, K B

    1980-01-01

    The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat 'group-specific protease' [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined. Images Fig. 1. Fig. 2. PMID:6821372

  12. Identification of the Cu2+ Binding Sites in the N-Terminal Domain of the Prion Protein by EPR and CD Spectroscopy†

    PubMed Central

    Aronoff-Spencer, Eliah; Burns, Colin S.; Avdievich, Nikolai I.; Gerfen, Gary J.; Peisach, Jack; Antholine, William E.; Ball, Haydn L.; Cohen, Fred E.; Prusiner, Stanley B.; Millhauser, Glenn L.

    2010-01-01

    Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60–91. This region selectively binds divalent copper ions (Cu2+) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu2+–peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu2+ binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu2+/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu2+ is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu2+ is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides. PMID:11076515

  13. Hydrogen-Rich Saline Attenuates Lipopolysaccharide-Induced Heart Dysfunction by Restoring Fatty Acid Oxidation in Rats by Mitigating C-Jun N-Terminal Kinase Activation.

    PubMed

    Tao, Bingdong; Liu, Lidan; Wang, Ni; Tong, Dongyi; Wang, Wei; Zhang, Jin

    2015-12-01

    Sepsis is common in intensive care units (ICU) and is associated with high mortality. Cardiac dysfunction complicating sepsis is one of the most important causes of this mortality. This dysfunction is due to myocardial inflammation and reduced production of energy by the heart. A number of studies have shown that hydrogen-rich saline (HRS) has a beneficial effect on sepsis. Therefore, we tested whether HRS prevents cardiac dysfunction by increasing cardiac energy. Four groups of rats received intraperitoneal injections of one of the following solutions: normal saline (NS), HRS, lipopolysaccharide (LPS), and LPS plus HRS. Cardiac function was measured by echocardiography 8 h after the injections. Gene and protein expression related to fatty acid oxidation (FAO) were measured by quantitative polymerase chain reaction (PCR) and Western blot analysis. The injection of LPS compromised heart function through decreased fractional shortening (FS) and increased left ventricular diameter (LVD). The addition of HRS increased FS, palmitate triphosphate, and the ratio of phosphocreatinine (PCr) to adenosine triphosphate (ATP) as well as decreasing LVD. The LPS challenge reduced the expression of genes related to FAO, including perioxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), perioxisome proliferator-activated receptor alpha (PPARα), Estrogen-related receptor alpha (ERRα), and their downstream targets, in mRNA and protein level, which were attenuated by HRS. However, HRS had little effect on glucose metabolism. Furthermore, HRS inhibited c-Jun N-terminal kinase (JNK) activation in the rat heart. Inhibition of JNK by HRS showed beneficial effects on LPS-challenged rats, at least in part, by restoring cardiac FAO.

  14. The adenoviral E1A N-terminal domain represses MYC transcription in human cancer cells by targeting both p300 and TRRAP and inhibiting MYC promoter acetylation of H3K18 and H4K16

    PubMed Central

    Zhao, Ling-Jun; Loewenstein, Paul M.; Green, Maurice

    2016-01-01

    Human cancers frequently arise from increased expression of proto-oncogenes, such as MYC and HER2. Understanding the cellular pathways regulating the transcription and expression of proto-oncogenes is important for targeted therapies for cancer treatment. Adenoviral (Ad) E1A 243R (243 aa residues) is a viral oncoprotein that interacts with key regulators of gene transcription and cell proliferation. We have shown previously that the 80 amino acid N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) can target the histone acetyltransferase (HAT) p300 and repress HER2 in the HER2-overexpressing human breast cancer cell line SKBR3. Expression of E1A 1-80 induces death of SKBR3 and other cancer cell lines. In this study, we performed total cell RNA sequence analysis and identified MYC as the regulatory gene for cellular proliferation most strongly repressed by E1A 1-80. By RT-quantitative PCR analysis we show that repression of MYC in SKBR3 cells occurs early after expression of E1A 1-80, suggesting that MYC may be an early responder of E1A 1-80-mediated transcriptional repression. Of interest, while E1A 1-80 repression of MYC occurs in all eight human cancer cell lines examined, repression of HER2 is cell-type dependent. We demonstrate by ChIP analysis that MYC transcriptional repression by E1A 1-80 is associated with inhibition of acetylation of H3K18 and H4K16 on the MYC promoter, as well as inhibition of RNA Pol II binding to the MYC promoter. Deletion mutant analysis of E1A 1-80 suggests that both p300/CBP and TRRAP are involved in E1A 1-80 repression of MYC transcription. Further, E1A 1-80 interaction with p300/CBP and TRRAP is correlated with inhibition of H3K18 and H4K16 acetylation on the MYC promoter, respectively. Our results indicate that E1A 1-80 may target two important pathways for histone modification to repress transcription in human cancer cells. PMID:27382434

  15. Structural characterization of the N-terminal part of the MERS-CoV nucleocapsid by X-ray diffraction and small-angle X-ray scattering.

    PubMed

    Papageorgiou, Nicolas; Lichière, Julie; Baklouti, Amal; Ferron, François; Sévajol, Marion; Canard, Bruno; Coutard, Bruno

    2016-02-01

    The N protein of coronaviruses is a multifunctional protein that is organized into several domains. The N-terminal part is composed of an intrinsically disordered region (IDR) followed by a structured domain called the N-terminal domain (NTD). In this study, the structure determination of the N-terminal region of the MERS-CoV N protein via X-ray diffraction measurements is reported at a resolution of 2.4 Å. Since the first 30 amino acids were not resolved by X-ray diffraction, the structural study was completed by a SAXS experiment to propose a structural model including the IDR. This model presents the N-terminal region of the MERS-CoV as a monomer that displays structural features in common with other coronavirus NTDs. PMID:26894667

  16. Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an N-terminal chorismate mutase-like regulatory domain

    SciTech Connect

    Light, Samuel H.; Halavaty, Andrei S.; Minasov, George; Shuvalova, Ludmilla; Anderson, Wayne F.

    2012-06-27

    3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase - an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis - and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn{sup 2+} and Mn{sup 2+} + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.

  17. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    SciTech Connect

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-15

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Angstrom-Sign resolution.

  18. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    NASA Astrophysics Data System (ADS)

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-01

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

  19. The N-terminal actin-binding tandem calponin-homology (CH) domain of dystrophin is in a closed conformation in solution and when bound to F-actin.

    PubMed

    Singh, Surinder M; Mallela, Krishna M G

    2012-11-01

    Deficiency of the vital muscle protein dystrophin triggers Duchenne/Becker muscular dystrophy, but the structure-function relationship of dystrophin is poorly understood. To date, molecular structures of three dystrophin domains have been determined, of which the N-terminal actin-binding domain (N-ABD or ABD1) is of particular interest. This domain is composed of two calponin-homology (CH) domains, which form an important class of ABDs in muscle proteins. A previously determined x-ray structure indicates that the dystrophin N-ABD is a domain-swapped dimer, with each monomer adopting an extended, open conformation in which the two CH domains do not interact. This structure is controversial because it contradicts functional studies and known structures of similar ABDs from other muscle proteins. Here, we investigated the solution conformation of the dystrophin N-ABD using a very simple and elegant technique of pyrene excimer fluorescence. Using the wild-type protein, which contains two cysteines, and the corresponding single-cysteine mutants, we show that the protein is a monomer in solution and is in a closed conformation in which the two CH domains seem to interact, as observed from the excimer fluorescence of pyrene-labeled wild-type protein. Excimer fluorescence was also observed in its actin-bound form, indicating that the dystrophin N-ABD binds to F-actin in a closed conformation. Comparison of the dystrophin N-ABD conformation with other ABDs indicates that the tandem CH domains in general may be monomeric in solution and predominantly occur in closed conformation, whereas their actin-bound conformations may differ.

  20. Identification of quercitrin as an inhibitor of the p90 S6 ribosomal kinase (RSK): structure of its complex with the N-terminal domain of RSK2 at 1.8 Å resolution

    SciTech Connect

    Derewenda, Urszula; Artamonov, Mykhaylo; Szukalska, Gabriela; Utepbergenov, Darkhan; Olekhnovich, Natalya; Parikh, Hardik I.; Kellogg, Glen E.; Somlyo, Avril V.; Derewenda, Zygmunt S.

    2013-02-01

    The crystal structure of quercitrin, a naturally occurring flavonol glycoside, has been determined in a complex with the N-terminal kinase domain of murine RSK2. The structure revealed that quercitrin inhibits the RSK2 kinase in the same fashion as another known inhibitor, SL0101. Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45–346) of mouse RSK2, or RSK2{sup NTKD}, has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3′′,4′′-di-O-acetyl-α-l-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-O-α-l-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2{sup NTKD} with a dissociation constant (K{sub d}) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K{sub d} for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca{sup 2+}.

  1. The Lys1010-Lys1325 fragment of the Wilson's disease protein binds nucleotides and interacts with the N-terminal domain of this protein in a copper-dependent manner.

    PubMed

    Tsivkovskii, R; MacArthur, B C; Lutsenko, S

    2001-01-19

    Wilson's disease, an autosomal disorder associated with vast accumulation of copper in tissues, is caused by mutations in a gene encoding a copper-transporting ATPase (Wilson's disease protein, WNDP). Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys(1010)-Lys(1325) segment; however, the biochemical properties and molecular mechanism of WNDP remain poorly characterized. Here, the Lys(1010)-Lys(1325) fragment of WNDP was overexpressed, purified, and shown to form an independently folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP analogue trinitrophenyl-ATP with high affinity, and ATP competes with trinitrophenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at the site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts specifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Strikingly, copper binding to N-WNDP diminishes these interactions, suggesting that the copper-dependent change in domain-domain contact may represent the mechanism of WNDP regulation. In agreement with this hypothesis, N-WNDP induces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP. Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical. The implications of these results for the WNDP function are discussed.

  2. Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage

    SciTech Connect

    Fokine, Andrei; Islam, Mohammad Z.; Zhang, Zhihong; Bowman, Valorie D.; Rao, Venigalla B.; Rossmann, Michael G.

    2011-09-16

    The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage.

  3. Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage▿

    PubMed Central

    Fokine, Andrei; Islam, Mohammad Z.; Zhang, Zhihong; Bowman, Valorie D.; Rao, Venigalla B.; Rossmann, Michael G.

    2011-01-01

    The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage. PMID:21632759

  4. Structure-function dissection of Myxococcus xanthus CarD N-terminal domain, a defining member of the CarD_CdnL_TRCF family of RNA polymerase interacting proteins.

    PubMed

    Bernal-Bernal, Diego; Gallego-García, Aránzazu; García-Martínez, Gema; García-Heras, Francisco; Jiménez, María Angeles; Padmanabhan, S; Elías-Arnanz, Montserrat

    2015-01-01

    Two prototypes of the large CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-binding proteins, Myxococcus xanthus CarD and CdnL, have distinct functions whose molecular basis remain elusive. CarD, a global regulator linked to the action of several extracytoplasmic function (ECF) σ-factors, binds to the RNAP β subunit (RNAP-β) and to protein CarG via an N-terminal domain, CarDNt, and to DNA via an intrinsically unfolded C-terminal domain resembling eukaryotic high-mobility-group A (HMGA) proteins. CdnL, a CarDNt-like protein that is essential for cell viability, is implicated in σA-dependent rRNA promoter activation and interacts with RNAP-β but not with CarG. While the HMGA-like domain of CarD by itself is inactive, we find that CarDNt has low but observable ability to activate ECF σ-dependent promoters in vivo, indicating that the C-terminal DNA-binding domain is required to maximize activity. Our structure-function dissection of CarDNt reveals an N-terminal, five-stranded β -sheet Tudor-like domain, CarD1-72, whose structure and contacts with RNAP-β mimic those of CdnL. Intriguingly, and in marked contrast to CdnL, CarD mutations that disrupt its interaction with RNAP-β did not annul activity. Our data suggest that the CarDNt C-terminal segment, CarD61-179, may be structurally distinct from its CdnL counterpart, and that it houses at least two distinct and crucial function determinants: (a) CarG-binding, which is specific to CarD; and (b) a basic residue stretch, which is also conserved and functionally required in CdnL. This study highlights the evolution of shared and divergent interactions in similar protein modules that enable the distinct activities of two related members of a functionally important and widespread bacterial protein family.

  5. Structure-Function Dissection of Myxococcus xanthus CarD N-Terminal Domain, a Defining Member of the CarD_CdnL_TRCF Family of RNA Polymerase Interacting Proteins

    PubMed Central

    Bernal-Bernal, Diego; Gallego-García, Aránzazu; García-Martínez, Gema; García-Heras, Francisco; Jiménez, María Angeles; Padmanabhan, S.; Elías-Arnanz, Montserrat

    2015-01-01

    Two prototypes of the large CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-binding proteins, Myxococcus xanthus CarD and CdnL, have distinct functions whose molecular basis remain elusive. CarD, a global regulator linked to the action of several extracytoplasmic function (ECF) σ-factors, binds to the RNAP β subunit (RNAP-β) and to protein CarG via an N-terminal domain, CarDNt, and to DNA via an intrinsically unfolded C-terminal domain resembling eukaryotic high-mobility-group A (HMGA) proteins. CdnL, a CarDNt-like protein that is essential for cell viability, is implicated in σA-dependent rRNA promoter activation and interacts with RNAP-β but not with CarG. While the HMGA-like domain of CarD by itself is inactive, we find that CarDNt has low but observable ability to activate ECF σ-dependent promoters in vivo, indicating that the C-terminal DNA-binding domain is required to maximize activity. Our structure-function dissection of CarDNt reveals an N-terminal, five-stranded β -sheet Tudor-like domain, CarD1–72, whose structure and contacts with RNAP-β mimic those of CdnL. Intriguingly, and in marked contrast to CdnL, CarD mutations that disrupt its interaction with RNAP-β did not annul activity. Our data suggest that the CarDNt C-terminal segment, CarD61–179, may be structurally distinct from its CdnL counterpart, and that it houses at least two distinct and crucial function determinants: (a) CarG-binding, which is specific to CarD; and (b) a basic residue stretch, which is also conserved and functionally required in CdnL. This study highlights the evolution of shared and divergent interactions in similar protein modules that enable the distinct activities of two related members of a functionally important and widespread bacterial protein family. PMID:25811865

  6. Preliminary crystallographic analysis of the N-terminal PDZ-like domain of periaxin, an abundant peripheral nerve protein linked to human neuropathies

    PubMed Central

    Han, Huijong; Kursula, Petri

    2013-01-01

    Periaxin (PRX) is an abundant protein in peripheral nerves and contains a predicted PDZ-like domain at its N-terminus. The large isoform, L-PRX, is required for the maintenance of myelin in the peripheral nervous system and its defects cause neurological disease. Here, the human periaxin PDZ-like domain was crystallized and X-ray diffraction data were collected to 2.85 Å resolution using synchrotron radiation. The crystal belonged to the primitive hexagonal space group P3121 or P3221, with unit-cell parameters a = b = 80.6, c = 81.0 Å, γ = 120° and either two or three molecules in the asymmetric unit. The structure of PRX will shed light on its poorly characterized function in the nervous system. PMID:23832213

  7. Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning: Arabidopsis h-Type Thioredoxins as a Case Study[C][W

    PubMed Central

    Traverso, José A.; Micalella, Chiara; Martinez, Aude; Brown, Spencer C.; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-01-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX–green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane. PMID:23543785

  8. Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study.

    PubMed

    Traverso, José A; Micalella, Chiara; Martinez, Aude; Brown, Spencer C; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-03-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

  9. Conserved Negative Charges in the N-terminal Tetramerization Domain Mediate Efficient Assembly of Kv2.1 and Kv2.1/Kv6.4 Channels*

    PubMed Central

    Bocksteins, Elke; Labro, Alain J.; Mayeur, Evy; Bruyns, Tine; Timmermans, Jean-Pierre; Adriaensen, Dirk; Snyders, Dirk J.

    2009-01-01

    Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual α-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 α-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels. PMID:19717558

  10. Binding of the N-terminal domain of the lactococcal bacteriophage TP901-1 CI repressor to its target DNA: a crystallography, small angle scattering, and nuclear magnetic resonance study.

    PubMed

    Frandsen, Kristian H; Rasmussen, Kim K; Jensen, Malene Ringkjøbing; Hammer, Karin; Pedersen, Margit; Poulsen, Jens-Christian N; Arleth, Lise; Lo Leggio, Leila

    2013-10-01

    In most temperate bacteriophages, regulation of the choice of lysogenic or lytic life cycle is controlled by a CI repressor protein. Inhibition of transcription is dependent on a helix-turn-helix motif, often located in the N-terminal domain (NTD), which binds to specific DNA sequences (operator sites). Here the crystal structure of the NTD of the CI repressor from phage TP901-1 has been determined at 1.6 Å resolution, and at 2.6 Å resolution in complex with a 9 bp double-stranded DNA fragment that constitutes a half-site of the OL operator. This N-terminal construct, comprising residues 2-74 of the CI repressor, is monomeric in solution as shown by nuclear magnetic resonance (NMR), small angle X-ray scattering, and gel filtration and is monomeric in the crystal structures. The binding interface between the NTD and the half-site in the crystal is very similar to the interface that can be mapped by NMR in solution with a full palindromic site. The interactions seen in the complexes (in the crystal and in solution) explain the observed affinity for the OR site that is lower than that for the OL site and the specificity for the recognized DNA sequence in comparison to that for other repressors. Compared with many well-studied phage repressor systems, the NTD from TP901-1 CI has a longer extended scaffolding helix that, interestingly, is strongly conserved in putative repressors of Gram-positive pathogens. On the basis of sequence comparisons, we suggest that these bacteria also possess repressor/antirepressor systems similar to that found in phage TP901-1. PMID:24047404

  11. The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains

    SciTech Connect

    Ghoshal, Kankana; Theilmann, Jane; Reade, Ron; Sanfacon, Helene; Rochon, D’Ann

    2014-11-15

    Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication. - Highlights: • The CLSV p25 auxiliary replicase targets the endoplasmic reticulum (ER). • Targeting of CLSV p25 is associated with ER restructuring. • Restructuring of the ER occurs during CLSV infection. • CLSV p25 contains 3 predicted transmembrane domains 2 of which are required for ER targeting. • Vesicles derived from the ER may be sites of CLSV replication.

  12. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

    SciTech Connect

    Carr, Stephen B.; Makris, George; Phillips, Simon E. V.; Thomas, Christopher D.

    2006-11-01

    The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2{sub 1}, diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism.

  13. Cd2+ and the N-terminal metal-binding domain protect the putative membranous CPC motif of the Cd2+-ATPase of Listeria monocytogenes.

    PubMed Central

    Bal, Nathalie; Wu, Chen Chou; Catty, Patrice; Guillain, Florent; Mintz, Elisabeth

    2003-01-01

    CadA, the Cd(2+)-ATPase of Listeria monocytogenes, contains four cysteine residues: two in the CTNC (Cys-Thr-Asn-Cys) sequence in the cytoplasmic metal-binding domain (MBD), and two in the CPC (Cys-Pro-Cys) sequence in the membrane domain. Taking advantage of DeltaMBD, a truncated version of CadA that lacks the MBD but which still acts as a functional Cd(2+)-ATPase [Bal, Mintz, Guillain and Catty (2001) FEBS Lett. 506, 249-252], we analysed the role of the membrane cysteine residues (studied using DeltaMBD) separately from that of the cysteine residues of the MBD, which were studied using full-length CadA. The role of the cysteines was assessed by reacting DeltaMBD and CadA with N -ethylmaleimide (NEM), an SH-specific reagent, in the presence or absence of Cd(2+). We show here that (i) in both DeltaMBD and CadA, the cysteine residues in the CPC motif are essential for phosphorylation; (ii) in both proteins, Cd(2+) protects against alkylation by NEM; and (iii) in the absence of Cd(2+), the MBD of CadA also protects against alkylation by NEM. Our results suggest that the CPC motif is present in the membrane Cd(2+) transport site(s) and that the MBD protects these site(s). PMID:12383056

  14. Structure and Dynamics of Adeno-Associated Virus Serotype 1 VP1-Unique N-Terminal Domain and Its Role in Capsid Trafficking

    PubMed Central

    Venkatakrishnan, Balasubramanian; Yarbrough, Joseph; Domsic, John; Bennett, Antonette; Bothner, Brian; Kozyreva, Olga G.; Samulski, R. Jude; Muzyczka, Nicholas

    2013-01-01

    The importance of the phospholipase A2 domain located within the unique N terminus of the capsid viral protein VP1 (VP1u) in parvovirus infection has been reported. This study used computational methods to characterize the VP1 sequence for adeno-associated virus (AAV) serotypes 1 to 12 and circular dichroism and electron microscopy to monitor conformational changes in the AAV1 capsid induced by temperature and the pHs encountered during trafficking through the endocytic pathway. Circular dichroism was also used to monitor conformational changes in AAV6 capsids assembled from VP2 and VP3 or VP1, VP2, and VP3 at pH 7.5. VP1u was predicted (computationally) and confirmed (in solution) to be structurally ordered. This VP domain was observed to undergo a reversible pH-induced unfolding/refolding process, a loss/gain of α-helical structure, which did not disrupt the capsid integrity and is likely facilitated by its difference in isoelectric point compared to the other VP sequences assembling the capsid. This study is the first to physically document conformational changes in the VP1u region that likely facilitate its externalization from the capsid interior during infection and establishes the order of events in the escape of the AAV capsid from the endosome en route to the nucleus. PMID:23427155

  15. Structure and dynamics of adeno-associated virus serotype 1 VP1-unique N-terminal domain and its role in capsid trafficking.

    PubMed

    Venkatakrishnan, Balasubramanian; Yarbrough, Joseph; Domsic, John; Bennett, Antonette; Bothner, Brian; Kozyreva, Olga G; Samulski, R Jude; Muzyczka, Nicholas; McKenna, Robert; Agbandje-McKenna, Mavis

    2013-05-01

    The importance of the phospholipase A2 domain located within the unique N terminus of the capsid viral protein VP1 (VP1u) in parvovirus infection has been reported. This study used computational methods to characterize the VP1 sequence for adeno-associated virus (AAV) serotypes 1 to 12 and circular dichroism and electron microscopy to monitor conformational changes in the AAV1 capsid induced by temperature and the pHs encountered during trafficking through the endocytic pathway. Circular dichroism was also used to monitor conformational changes in AAV6 capsids assembled from VP2 and VP3 or VP1, VP2, and VP3 at pH 7.5. VP1u was predicted (computationally) and confirmed (in solution) to be structurally ordered. This VP domain was observed to undergo a reversible pH-induced unfolding/refolding process, a loss/gain of α-helical structure, which did not disrupt the capsid integrity and is likely facilitated by its difference in isoelectric point compared to the other VP sequences assembling the capsid. This study is the first to physically document conformational changes in the VP1u region that likely facilitate its externalization from the capsid interior during infection and establishes the order of events in the escape of the AAV capsid from the endosome en route to the nucleus. PMID:23427155

  16. Influence of N-terminal hydrophobicity of cationic peptides on thermodynamics of their interaction with plasmid DNA.

    PubMed

    Goparaju, Geetha N; Bruist, Michael F; Chandran, C Satish; Gupta, Pardeep K

    2009-05-01

    There is a need to understand the thermodynamics of interaction of cationic peptides with DNA to design better peptide based non-viral gene delivery vectors. The main aim of this study was to understand the influence of N-terminal hydrophobicity of cationic amphiphilic peptides on thermodynamics of interaction with plasmid DNA. The model peptides used were TATPTD and TATPTDs modified at the N-terminal with hydrophobic amino acids. The thermodynamic binding data from isothermal titration calorimetry were compared with ethidium bromide analysis and ultrafiltration to correlate the binding parameters with the structural features of the various peptides used. It was observed that peptides having a smaller hydrophobic domain at the N-terminal have good DNA condensing ability compared with the ones with a longer hydrophobic domain. Calorimetry of peptides that reached saturation binding indicated that enthalpy and entropy are favorable for the interaction. Moreover, the interaction of these peptides with DNA appears to be predominantly electrostatic.

  17. Design, synthesis and evaluation of antimicrobial activity of N-terminal modified Leucocin A analogues.

    PubMed

    Bodapati, Krishna Chaitanya; Soudy, Rania; Etayash, Hashem; Stiles, Michael; Kaur, Kamaljit

    2013-07-01

    Class IIa bacteriocins are potent antimicrobial peptides produced by lactic acid bacteria to destroy competing microorganisms. The N-terminal domain of these peptides consists of a conserved YGNGV sequence and a disulphide bond. The YGNGV motif is essential for activity, whereas, the two cysteines involved in the disulphide bond can be replaced with hydrophobic residues. The C-terminal region has variable sequences, and folds into a conserved amphipathic α-helical structure. To elucidate the structure-activity relationship in the N-terminal domain of these peptides, three analogues (1-3) of a class IIa bacteriocin, Leucocin A (LeuA), were designed and synthesized by replacing the N-terminal β-sheet residues of the native peptide with shorter β-turn motifs. Such replacement abolished the antibacterial activity in the analogues, however, analogue 1 was able to competitively inhibit the activity of native LeuA. Native LeuA (37-mer) was synthesized using native chemical ligation method in high yield. Solution conformation study using circular dichroism spectroscopy and molecular dynamics simulations suggested that the C-terminal region of analogue 1 adopts helical folding as found in LeuA, while the N-terminal region did not fold into β-sheet conformation. These structure-activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins.

  18. The three-dimensional structure of the N-terminal domain of corticotropin-releasing factor receptors: sushi domains and the B1 family of G protein-coupled receptors.

    PubMed

    Perrin, Marilyn H; Grace, Christy R R; Riek, Roland; Vale, Wylie W

    2006-07-01

    The corticotropin-releasing factor (CRF) receptors, CRF-R1 and CRF-R2, belong to the B1 subfamily of G protein-coupled Receptors (GPCRs), including receptors for secretin, growth hormone-releasing hormone (GHRH), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), calcitonin, parathyroid hormone (PTH), glucagon, and glucagon-like peptide-1 (GLP-1). The peptide ligand family comprises CRF, Ucn 1, 2, and 3. CRF plays the major role in integrating the response to stress. Additionally, the ligands exhibit many effects on muscle, pancreas, heart, and the GI, reproductive, and immune systems. CRF-R1 has higher affinity for CRF than does CRF-R2 while both receptors bind Ucn 1 equally. CRF-R2 shows specificity for Ucns 2 and 3. A major binding domain of the CRFRs is the N terminus/first extracellular domain (ECD1). Soluble proteins corresponding to the ECD1s of each receptor bind CRF ligands with nanomolar affinities. Our three-dimensional (3D) nuclear magnetic resonance (NMR) structure of a soluble protein corresponding to the ECD1 of CRF-R2beta (1) identified its structural fold as a Sushi domain/short consensus repeat (SCR), stabilized by three disulfide bridges, two tryptophan residues, and an internal salt bridge (Asp65-Arg101). Disruption of the bridge by D65A mutation abrogates ligand recognition and results in loss of the well-defined disulfide pattern and Sushi domain structure. NMR analysis of the ECD1 in complex with astressin identified key amino acids involved in ligand recognition. Mutation of some of these residues in the full-length receptor reduces its affinity for CRF ligands. A structure-based sequence comparison shows conservation of key amino acids in all the B1 subfamily receptors, suggesting a corresponding conservation of a Sushi domain structural fold of their ECD1s.

  19. The structural and functional basis of the p97/valosin-containing protein (VCP)-interacting motif (VIM): mutually exclusive binding of cofactors to the N-terminal domain of p97.

    PubMed

    Hänzelmann, Petra; Schindelin, Hermann

    2011-11-01

    The AAA (ATPase associated with various cellular activities) ATPase p97, also referred to as valosin-containing protein (VCP), mediates essential cellular processes, including ubiquitin-dependent protein degradation, and has been linked to several human proteinopathies. p97 interacts with multiple cofactors via its N-terminal (p97N) domain, a subset of which contain the VCP-interacting motif (VIM). We have determined the crystal structure of the p97N domain in complex with the VIM of the ubiquitin E3 ligase gp78 at 1.8 Å resolution. The α-helical VIM peptide binds into a groove located in between the two subdomains of the p97N domain. Interaction studies of several VIM proteins reveal that these cofactors display dramatically different affinities, ranging from high affinity interactions characterized by dissociation constants of ∼20 nm for gp78 and ANKZF1 to only weak binding in our assays. The contribution of individual p97 residues to VIM binding was analyzed, revealing that identical substitutions do not affect all cofactors in the same way. Taken together, the biochemical and structural studies define the framework for recognition of VIM-containing cofactors by p97. Of particular interest to the regulation of p97 by its cofactors, our structure reveals that the bound α-helical peptides of VIM-containing cofactors overlap with the binding site for cofactors containing the ubiquitin regulatory X (UBX) domain present in the UBX protein family or the ubiquitin-like domain of NPL4 as further corroborated by biochemical data. These results extend the concept that competitive binding is a crucial determinant in p97-cofactor interactions.

  20. Biochemical and Structural Characterization of the Interaction between the Siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/Lipocalin 2) and the N-terminal Domain of Its Endocytic Receptor SLC22A17*

    PubMed Central

    Cabedo Martinez, Ana-Isabel; Weinhäupl, Katharina; Lee, Wing-Kee; Wolff, Natascha A.; Storch, Barbara; Żerko, Szymon; Konrat, Robert; Koźmiński, Wiktor; Breuker, Kathrin; Thévenod, Frank; Coudevylle, Nicolas

    2016-01-01

    The neutrophil gelatinase-associated lipocalin (NGAL, also known as LCN2) and its cellular receptor (LCN2-R, SLC22A17) are involved in many physiological and pathological processes such as cell differentiation, apoptosis, and inflammation. These pleiotropic functions mainly rely on NGAL's siderophore-mediated iron transport properties. However, the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo state to form a fuzzy complex. The relatively weak affinity (≈10 μm) between human LCN2-R-NTD and apoNGAL suggests that the N terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, human LCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL. PMID:26635366

  1. Oxidation of the N-terminal domain of the wheat metallothionein Ec -1 leads to the formation of three distinct disulfide bridges.

    PubMed

    Tarasava, Katsiaryna; Chesnov, Serge; Freisinger, Eva

    2016-05-01

    Metallothioneins (MTs) are low molecular weight proteins, characterized by a high cysteine content and the ability to coordinate large amounts of d(10) metal ions, for example, Zn(II), Cd(II), and Cu(I), in form of metal-thiolate clusters. Depending on intracellular conditions such as redox potential or metal ion concentrations, MTs can occur in various states ranging from the fully metal-loaded holo- to the metal-free apo-form. The Cys thiolate groups in the apo-form can be either reduced or be involved in disulfide bridges. Although oxidation-mediated Zn(II) release might be a possible mechanism for the regulation of Zn(II) availability by MTs, no concise information regarding the associated pathways and the structure of oxidized apo-MT forms is available. Using the well-studied Zn2 γ-Ec -1 domain of the wheat Zn6 Ec -1 MT we attempt here to answer several question regarding the structure and biophysical properties of oxidized MT forms, such as: (1) does disulfide bond formation increase the stability against proteolysis, (2) is the overall peptide backbone fold similar for the holo- and the oxidized apo-MT form, and (3) are disulfide bridges specifically or randomly formed? Our investigations show that oxidation leads to three distinct disulfide bridges independently of the applied oxidation conditions and of the initial species used for oxidation, that is, the apo- or the holo-form. In addition, the oxidized apo-form is as stable against proteolysis as Zn2 γ-Ec -1, rendering the currently assumed degradation of oxidized MTs unlikely and suggesting a role of the oxidation process for the extension of protein lifetime in absence of sufficient amounts of metal ions. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 295-308, 2016.

  2. The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

    DOE PAGES

    Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; Zhang, Ping; Heller, William T.; Taylor, Susan S.

    2014-08-11

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme ismore » much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.« less

  3. The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

    SciTech Connect

    Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; Zhang, Ping; Heller, William T.; Taylor, Susan S.

    2014-08-11

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.

  4. The preparation and partial characterization of N-terminal and C-terminal iron-binding fragments from rabbit serum transferrin.

    PubMed Central

    Heaphy, S; Williams, J

    1982-01-01

    Two iron-binding fragments of Mr 36 000 and 33 000 corresponding to the N-terminal domain of rabbit serum transferrin were prepared. One iron-binding fragment of Mr 39 000 corresponding to the C-terminal domain was prepared. The N-terminal amino acid sequence of rabbit serum transferrin is: Val-Thr-Glu-Lys-Thr-Val-Asn-Trp-?-Ala-Val-Ser. One glycan unit is presented in rabbit serum transferrin and it is located in the C-terminal domain. Images Fig. 2. Fig. 3. Fig. 4. PMID:6816218

  5. Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

    PubMed Central

    Xu, Jun-Wei; Xu, Yi-Ning

    2012-01-01

    Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway. PMID:22941092

  6. A novel Drosophila model of TDP-43 proteinopathies: N-terminal sequences combined with the Q/N domain induce protein functional loss and locomotion defects

    PubMed Central

    Romano, Giulia; Klima, Raffaella; Feiguin, Fabian; Cragnaz, Lucia; Romano, Maurizio

    2016-01-01

    ABSTRACT Transactive response DNA-binding protein 43 kDa (TDP-43, also known as TBPH in Drosophila melanogaster and TARDBP in mammals) is the main protein component of the pathological inclusions observed in neurons of patients affected by different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The number of studies investigating the molecular mechanisms underlying neurodegeneration is constantly growing; however, the role played by TDP-43 in disease onset and progression is still unclear. A fundamental shortcoming that hampers progress is the lack of animal models showing aggregation of TDP-43 without overexpression. In this manuscript, we have extended our cellular model of aggregation to a transgenic Drosophila line. Our fly model is not based on the overexpression of a wild-type TDP-43 transgene. By contrast, we engineered a construct that includes only the specific TDP-43 amino acid sequences necessary to trigger aggregate formation and capable of trapping endogenous Drosophila TDP-43 into a non-functional insoluble form. Importantly, the resulting recombinant product lacks functional RNA recognition motifs (RRMs) and, thus, does not have specific TDP-43-physiological functions (i.e. splicing regulation ability) that might affect the animal phenotype per se. This novel Drosophila model exhibits an evident degenerative phenotype with reduced lifespan and early locomotion defects. Additionally, we show that important proteins involved in neuromuscular junction function, such as syntaxin (SYX), decrease their levels as a consequence of TDP-43 loss of function implying that the degenerative phenotype is a consequence of TDP-43 sequestration into the aggregates. Our data lend further support to the role of TDP-43 loss-of-function in the pathogenesis of neurodegenerative disorders. The novel transgenic Drosophila model presented in this study will help to gain further insight into the

  7. Structure of Human Acid Sphingomyelinase Reveals the Role of the Saposin Domain in Activating Substrate Hydrolysis.

    PubMed

    Xiong, Zi-Jian; Huang, Jingjing; Poda, Gennady; Pomès, Régis; Privé, Gilbert G

    2016-07-31

    Acid sphingomyelinase (ASM) is a lysosomal phosphodiesterase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine. While other lysosomal sphingolipid hydrolases require a saposin activator protein for full activity, the ASM polypeptide incorporates a built-in N-terminal saposin domain and does not require an external activator protein. Here, we report the crystal structure of human ASM and describe the organization of the three main regions of the enzyme: the N-terminal saposin domain, the proline-rich connector, and the catalytic domain. The saposin domain is tightly associated along an edge of the large, bowl-shaped catalytic domain and adopts an open form that exposes a hydrophobic concave surface approximately 30Å from the catalytic center. The calculated electrostatic potential of the enzyme is electropositive at the acidic pH of the lysosome, consistent with the strict requirement for the presence of acidic lipids in target membranes. Docking studies indicate that sphingomyelin binds with the ceramide-phosphate group positioned at the binuclear zinc center and molecular dynamic simulations indicate that the intrinsic flexibility of the saposin domain is important for monomer-dimer exchange and for membrane interactions. Overall, ASM uses a combination of electrostatic and hydrophobic interactions to cause local disruptions of target bilayers in order to bring the lipid headgroup to the catalytic center in a membrane-bound reaction. PMID:27349982

  8. Structure of Human Acid Sphingomyelinase Reveals the Role of the Saposin Domain in Activating Substrate Hydrolysis.

    PubMed

    Xiong, Zi-Jian; Huang, Jingjing; Poda, Gennady; Pomès, Régis; Privé, Gilbert G

    2016-07-31

    Acid sphingomyelinase (ASM) is a lysosomal phosphodiesterase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine. While other lysosomal sphingolipid hydrolases require a saposin activator protein for full activity, the ASM polypeptide incorporates a built-in N-terminal saposin domain and does not require an external activator protein. Here, we report the crystal structure of human ASM and describe the organization of the three main regions of the enzyme: the N-terminal saposin domain, the proline-rich connector, and the catalytic domain. The saposin domain is tightly associated along an edge of the large, bowl-shaped catalytic domain and adopts an open form that exposes a hydrophobic concave surface approximately 30Å from the catalytic center. The calculated electrostatic potential of the enzyme is electropositive at the acidic pH of the lysosome, consistent with the strict requirement for the presence of acidic lipids in target membranes. Docking studies indicate that sphingomyelin binds with the ceramide-phosphate group positioned at the binuclear zinc center and molecular dynamic simulations indicate that the intrinsic flexibility of the saposin domain is important for monomer-dimer exchange and for membrane interactions. Overall, ASM uses a combination of electrostatic and hydrophobic interactions to cause local disruptions of target bilayers in order to bring the lipid headgroup to the catalytic center in a membrane-bound reaction.

  9. Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor

    SciTech Connect

    Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya; Szukalska, Gabriela; Banerjee, Budhaditya; Hilinski, Michael K.; Lannigan, Deborah A.; Stukenberg, P. Todd; Derewenda, Zygmunt S.

    2012-09-11

    The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-a-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 {angstrom} resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the aD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.

  10. N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy.

    PubMed

    Liu, Xudong; Wang, Chuan-En; Hong, Yan; Zhao, Ting; Wang, Guohao; Gaertig, Marta A; Sun, Miao; Li, Shihua; Li, Xiao-Jiang

    2016-05-01

    The Huntington's disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1-208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD. PMID:27203582

  11. A Novel Mutation p.A59P in N-Terminal Domain of Methyl-CpG-Binding Protein 2 Confers Phenotypic Variability in 3 Cases of Tunisian Rett Patients: Clinical Evaluations and In Silico Investigations.

    PubMed

    Kharrat, Marwa; Hsairi, Ines; Fendri-Kriaa, Nourhene; Kenoun, Houda; Othmen, Houda Ben; Ben Mahmoud, Afif; Ghorbel, Rania; Abid, Imen; Triki, Chahnez; Fakhfakh, Faiza

    2015-11-01

    Rett syndrome is a monogenic X-linked dominant neurodevelopmental disorder related to mutation in MECP2, which encodes the methyl-CpG-binding protein MeCP2. The aim of this study was to search for mutations of MECP2 gene in Tunisian Rett patients and to evaluate the impact of the found variants on structural and functional features of MeCP2. The result of mutation analysis revealed that 3 Rett patients shared the same novel heterozygous point mutation c.175G>C (p.A59P). The p.A59P mutation was located in a conserved amino acid in the N-terminal segment of MeCP2. This novel mutation confers a phenotypic variability with different clinical severity scores (3, 8, and 9) and predicted by Sift and PolyPhen to be damaging. Modeling results showed that p.A59P adds 2 hydrogen bonds and changes the structural conformation of MeCP2 with a significant root mean square deviation value (9.66 Å), suggesting that this mutation could probably affect the conformation, function and stability of MeCP2.

  12. A functional N-terminal domain in C/EBPβ-LAP* is required for interacting with SWI/SNF and to repress Ric-8B gene transcription in osteoblasts.

    PubMed

    Aguilar, Rodrigo; Grandy, Rodrigo; Meza, Daniel; Sepulveda, Hugo; Pihan, Philippe; van Wijnen, Andre J; Lian, Jane B; Stein, Gary S; Stein, Janet L; Montecino, Martin

    2014-10-01

    The chromatin remodeling complex SWI/SNF and the transcription factor C/EBPβ play critical roles in osteoblastic cells as they jointly control transcription of a number of bone-related target genes. The largest C/EBPβ isoform, LAP*, possesses a short additional N-terminal domain that has been proposed to mediate the interaction of this factor with SWI/SNF in myeloid cells. Here we examine the requirement of a functional N-terminus in C/EBPβ-LAP* for binding SWI/SNF and for recruiting this complex to the Ric-8B gene to mediate transcriptional repression. We find that both C/EBPβ-LAP* and SWI/SNF simultaneously bind to the Ric-8B promoter in differentiating osteoblasts that repress Ric-8B expression. This decreased expression of Ric-8B is not accompanied by significant changes in histone acetylation at the Ric-8B gene promoter sequence. A single aminoacid change at the C/EBPβ-LAP* N-terminus (R3L) that inhibits C/EBPβ-LAP*-SWI/SNF interaction, also prevents SWI/SNF recruitment to the Ric-8B promoter as well as C/EBPβ-LAP*-dependent repression of the Ric-8B gene. Inducible expression of the C/EBPβ-LAP*R3L protein in stably transfected osteoblastic cells demonstrates that this mutant protein binds to C/EBPβ-LAP*-target promoters and competes with the endogenous C/EBPβ factor. Together our results indicate that a functional N-terminus in C/EBPβ-LAP* is required for interacting with SWI/SNF and for Ric-8B gene repression in osteoblasts.

  13. Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions.

    PubMed

    Iwashita, Shintaro; Suzuki, Takehiro; Yasuda, Takeshi; Nakashima, Kentaro; Sakamoto, Taiichi; Kohno, Toshiyuki; Takahashi, Ichiro; Kobayashi, Takayasu; Ohno-Iwashita, Yoshiko; Imajoh-Ohmi, Shinobu; Song, Si-Young; Dohmae, Naoshi

    2015-06-12

    The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His-Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser(250), which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser(250) substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser(250) phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys(268) in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.

  14. Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions

    PubMed Central

    Iwashita, Shintaro; Suzuki, Takehiro; Yasuda, Takeshi; Nakashima, Kentaro; Sakamoto, Taiichi; Kohno, Toshiyuki; Takahashi, Ichiro; Kobayashi, Takayasu; Ohno-Iwashita, Yoshiko; Imajoh-Ohmi, Shinobu; Song, Si-Young; Dohmae, Naoshi

    2015-01-01

    The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His–Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser250, which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser250 substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser250 phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys268 in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels. PMID:26182435

  15. Spatial structure of oligopeptide PAP(248-261), the N-terminal fragment of the HIV enhancer prostatic acid phosphatase peptide PAP(248-286), in aqueous and SDS micelle solutions

    NASA Astrophysics Data System (ADS)

    Blokhin, Dmitriy S.; Filippov, Andrei V.; Antzutkin, Oleg N.; Karataeva, Farida Kh.; Klochkov, Vladimir V.

    2014-07-01

    Prostatic acid phosphatase (PAP) is an enzyme that facilitates infection of cells by HIV. Its peptide fragment PAP(248-286) forms amyloid fibrils known as SEVI, which enhance attachment of the virus by viral adhesion to the host cell prior to receptor-specific binding via reducing the electrostatic repulsion between the membranes of the virus and the target cell. The secondary structure of PAP(248-286) in aqueous and SDS solutions can be divided into an N-terminal disordered region, an α-helical central part and an α/310-helical C-terminal region (Nanga et al., 2009). In this work, we used NMR spectroscopy to study the spatial structure of the isolated N-terminal fragment of PAP(248-286), PAP(248-261) (GIHKQKEKSRLQGG), in aqueous and SDS micelle solutions. Formation of a PAP(248-261)-SDS complex was confirmed by chemical shift alterations in the 1H NMR spectra of the peptide, as well as by the signs and values of Nuclear Overhauser Effect (NOE). In addition, the PAP(248-261) peptide does not form any specified secondary structure in either aqueous or SDS solutions.

  16. c-Jun N-terminal Kinase-Dependent Endoplasmic Reticulum Stress Pathway is Critically Involved in Arjunic Acid Induced Apoptosis in Non-Small Cell Lung Cancer Cells.

    PubMed

    Joo, HyeEun; Lee, Hyun Joo; Shin, Eun Ah; Kim, Hangil; Seo, Kyeong-Hwa; Baek, Nam-In; Kim, Bonglee; Kim, Sung-Hoon

    2016-04-01

    Though arjunic acid, a triterpene isolated from Terminalia arjuna, was known to have antioxidant, antiinflammatory, and cytotoxic effects, its underlying antitumor mechanism still remains unclear so far. Thus, in the present study, the molecular antitumor mechanism of arjunic acid was examined in A549 and H460 non-small cell lung cancer (NSCLC) cells. Arjunic acid exerted cytotoxicity by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and significantly increased sub-G1 population in A549 and H460 cells by cell cycle analysis. Consistently, arjunic acid cleaved poly (ADP-ribose) polymerase (PARP), activated Bax, and phosphorylation of c-Jun N-terminal kinases (JNK), and also attenuated the expression of pro-caspase-3 and Bcl-2 in A549 and H460 cells. Furthermore, arjunic acid upregulated the expression of endoplasmic reticulum (ER) stress proteins such as IRE1 α, ATF4, p-eIF2α, and C/EBP homologous protein (CHOP) in A549 and H460 cells. Conversely, CHOP depletion attenuated the increase of sub-G1 population by arjunic acid, and also JNK inhibitor SP600125 blocked the cytotoxicity and upregulation of IRE1 α and CHOP induced by arjunic acid in A549 and H460 cells. Overall, our findings suggest that arjunic acid induces apoptosis in NSCLC cells via JNK mediated ER stress pathway as a potent chemotherapeutic agent for NSCLC. PMID:26787261

  17. Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

    SciTech Connect

    Borko, Ľubomír; Bauerová-Hlinková, Vladena Hostinová, Eva; Gašperík, Juraj; Beck, Konrad; Lai, F. Anthony; Zahradníková, Alexandra; Ševčík, Jozef

    2014-11-01

    X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix. Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C{sup α} atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.

  18. Structural basis for ubiquitin-like ISG 15 protein binding to the NS1 protein of influenza B virus: a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS1 protein of influenza A virus.

    PubMed

    Yuan, Weiming; Aramini, James M; Montelione, Gaetano T; Krug, Robert M

    2002-12-20

    The N-terminal domains of the NS1 protein of influenza B virus (NS1B protein) and the NS1 protein of influenza A virus (NS1A protein) share one function: binding double-stranded RNA (dsRNA). Here we show that the N-terminal domain of the NS1B protein possesses an additional function that is not shared by its NS1A counterpart: binding the ubiquitin-like ISG15 protein that is induced by influenza B virus infection. Homology modeling predicts that the dimeric six-helical N-terminal domain of the NS1B protein differs from its NS1A protein counterpart in containing large loops between helices 1 and 2 (loops 1 and 1') and between helices 2 and 3 (loops 2 and 2'). Mutagenesis establishes that residues located in loop 1/1' together with residues located in polypeptide segment 94-103 form the ISG15 protein-binding site of NS1B protein. Loop 1/1' is not required for dsRNA binding, which instead requires arginine residues R50, R53, R50', and R53' located in antiparallel helices 1 and 1'. Further, we demonstrate that the binding sites for RNA and protein are independent of each other. In particular, ISG15 and dsRNA can bind simultaneously; the binding of the ISG15 protein does not have a detectable effect on the binding of dsRNA, and vice versa.

  19. N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance.

    PubMed

    Takemoto, Daigo; Rafiqi, Maryam; Hurley, Ursula; Lawrence, Greg J; Bernoux, Maud; Hardham, Adrienne R; Ellis, Jeffrey G; Dodds, Peter N; Jones, David A

    2012-03-01

    To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.

  20. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

    SciTech Connect

    Pozo-Yauner, Luis del; Wall, Jonathan S.; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L.; Pérez Carreón, Julio I.; and others

    2014-01-10

    Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

  1. N-Terminal Fatty Acid Substitution Increases the Leishmanicidal Activity of CA(1-7)M(2-9), a Cecropin-Melittin Hybrid Peptide

    PubMed Central

    Chicharro, Cristina; Granata, Cesare; Lozano, Rosario; Andreu, David; Rivas, Luis

    2001-01-01

    In order to improve the leishmanicidal activity of the synthetic cecropin A-melittin hybrid peptide CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH2), a systematic study of its acylation with saturated linear fatty acids was carried out. Acylation of the Nɛ-7 lysine residue led to a drastic decrease in leishmanicidal activity, whereas acylation at lysine 1, in either the α or the ɛ NH2 group, increased up to 3 times the activity of the peptide against promastigotes and increased up to 15 times the activity of the peptide against amastigotes. Leishmanicidal activity increased with the length of the fatty acid chain, reaching a maximum for the lauroyl analogue (12 carbons). According to the fast kinetics, dissipation of membrane potential, and parasite membrane permeability to the nucleic acid binding probe SYTOX green, the lethal mechanism was directly related to plasma membrane permeabilization. PMID:11502512

  2. Activation of c-jun N-Terminal Kinase upon Influenza A Virus (IAV) Infection Is Independent of Pathogen-Related Receptors but Dependent on Amino Acid Sequence Variations of IAV NS1

    PubMed Central

    Nacken, Wolfgang; Anhlan, Darisuren; Hrincius, Eike R.; Mostafa, Ahmed; Wolff, Thorsten; Sadewasser, Anne; Pleschka, Stephan; Ehrhardt, Christina

    2014-01-01

    ABSTRACT A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. IMPORTANCE Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that

  3. Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

    SciTech Connect

    Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik; Whittaker, Gary R.

    2012-12-05

    A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

  4. Method to convert N-terminal glutamine to pyroglutamate for characterization of recombinant monoclonal antibodies.

    PubMed

    Xu, Wei; Peng, Yan; Wang, Fengqiang; Paporello, Brittany; Richardson, Daisy; Liu, Hongcheng

    2013-05-01

    Cyclization of N-terminal glutamine to pyroglutamate is a common modification of recombinant monoclonal antibodies that has often been identified by liquid chromatography mass spectrometry (LC-MS) analysis using separated fractions. An alternative approach of using glutaminyl-peptide cyclotransferase to convert the N-terminal glutamine to pyroglutamate was developed in the current study. Enzymatic conversion of the N-terminal glutamine to pyroglutamate not only provides an identification of the N-terminal amino acids without fraction collection but also can significantly simplify the chromatograms to assist fraction collections for the characterization of other antibody variants.

  5. Electrospray ionization tandem mass spectrometric study on the effect of N-terminal beta- and gamma-carbo amino acids on fragmentation of GABA-hybrid peptides.

    PubMed

    Ramesh, V; Ramesh, M; Srinivas, R; Sharma, G V M; Jayaprakash, P

    2008-11-01

    The fragmentations of protonated and deprotonated ions of a new class of N-blocked hybrid Boc-carbopeptides containing repeats of gamma-Caa/gammaAbu- and beta-Caa/gammaAbu- (Caa==C-linked carbo gamma(4)-/beta(3)- amino acids derived from D-xylose, gammaAbu = gamma-aminobutyric acid) have been studied using electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). MS/MS of a pair of these protonated diastereomers produces distinct fragmentation of the Boc group. The formation of [M + H-56](+) corresponding to loss of isobutylene is more pronounced for Boc-NH-(R)-gamma-Caa-gammaAbu-OH (2) whereas it is of low abundance for Boc-NH-(S)-gamma-Caa-gammaAbu--OH (1). Similarly, MS(2) of [M--H](-) of 2 produces an abundant [M--H--C(CH(3))(3)OH--CO(2)](-) ion, which is absent for its diastereomeric isomer 1. From this, it can be suggested that MS/MS of N-blocked Boc-protected carbopeptides may be helpful in distinguishing the stereochemistry of the N-terminus Caa. MS(3) of [M + H-Boc + H](+) ions of peptides with a gamma-amino acid (gamma-Caa/gammaAbu) at the N-terminus produces only abundant y(n) (+) ions. On the other hand, characteristic fragmentations involving the peptide backbone (b(n) (+) and y(n) (+)) and the side chain are seen when beta-Caa is at the N-terminus of the peptides. MS(3) of the [M--H--C(CH(3))(3)OH](-) ion of peptides containing gamma-Caa/gammaAbu at the N-terminus gave y(n) (-) and [M--H--C(CH(3))(3)OH--CO(2)](-) ions, whereas the presence of beta-Caa at the N-terminus yielded predominantly [M--H--C(CH(3))(3)OH--HNCO](-). Thus, on the basis of our previous study and that presented here we propose that the fragmentation of these hybrid carbopeptides is highly influenced by the type of carbo amino acid present at the N-terminus. PMID:18837002

  6. The C-Terminal Heavy-Chain Domain of Botulinum Neurotoxin A Is Not the Only Site That Binds Neurons, as the N-Terminal Heavy-Chain Domain Also Plays a Very Active Role in Toxin-Cell Binding and Interactions

    PubMed Central

    Aoki, K. Roger

    2015-01-01

    Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729–845). HN729–845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729–845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729–845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin. PMID:25624352

  7. Structure of the N-terminal segment of human retinol dehydrogenase 11 and its preferential lipid binding using model membranes.

    PubMed

    Lhor, Mustapha; Méthot, Mario; Horchani, Habib; Salesse, Christian

    2015-03-01

    Retinol dehydrogenase 11 (RDH11) has been postulated to be anchored to membranes by means of its N-terminal segment in retinal pigment epithelial (RPE) cells where it participates to the visual cycle. The analysis of the primary sequence of RDH11 revealed that its N-terminal hydrophobic segment could be involved in the anchoring of this enzyme to membranes. However, no information is yet available on the properties of this N-terminal segment to support this role. The secondary structure and membrane binding of two N-terminal peptides of RDH11 with different lengths have thus been investigated to provide this information. Online tools allowed predicting an α-helical secondary structure for both peptides. Infrared spectroscopy and circular dichroism have shown that the α-helix of the Long-peptide (35 amino acids) is longer and more rigid than that of the Short-peptide (25 amino acids) regardless of the type of solvent. Langmuir monolayers have been used as a model membrane to study lipid-peptide interactions. Values of maximum insertion pressure and synergy suggested a preferential binding of the Long-peptide to lipids with a phosphoethanolamine polar head group, which are abundant in the RPE. Furthermore, infrared spectroscopy in monolayers has shown that the α-helical structure of the Long-peptide is more stable in the presence of saturated phospholipids whereas the structure of the Short-peptide is mainly disordered. Altogether, the present data demonstrate that the α-helical hydrophobic core of the N-terminal segment of RDH11 displays properties typical of transmembrane domains, in agreement with its postulated role in the membrane anchoring of this protein.

  8. Purification, characterization and N-terminal amino acid sequence of a new major allergen from European chestnut pollen--Cas s 1.

    PubMed

    Kos, T; Hoffmann-Sommergruber, K; Ferreira, F; Hirschwehr, R; Ahorn, H; Horak, F; Jager, S; Sperr, W; Kraft, D; Scheiner, O

    1993-11-15

    Pollens from trees of the order Fagales (e.g. birch, alder, hazel, and hornbeam) all contain one major allergen--the main cause for tree pollen allergy. So far the major allergens from birch (Bet v 1), alder (Aln g 1), hazel (Cor a 1), and hornbeam (Car b 1) have been characterized, showing high sequence similarity with each other (1-4). We present the molecular and immunologic characterization of Cas s 1, the major allergen from the European chestnut (Castanea sativa). From aqueous pollen extracts from European chestnut a protein was purified to homogeneity and was subjected to further investigation. The protein revealed a Mr of 22 kDa and was shown to represent the major allergen of the European chestnut (immunoblotting, histamine release) and designated Cas s 1. Despite a marked difference in Mr, Cas s 1 shows significant amino acid sequence similarity at the N-terminus and is antigenically closely related to the major birch pollen allergen Bet v 1 (17 kDa), as shown by binding to the anti-Bet v 1 monoclonal antibody BIP-1 and by IgE-inhibition tests using recombinant Bet v 1.

  9. The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

    PubMed Central

    Xu, Cheng; Li, Ying-Chun; Yang, Hua; Long, Yan; Chen, Min-Jian; Qin, Yu-Feng; Xia, Yan-Kai; Song, Ling; Gu, Ai-Hua; Wang, Xin-Ru

    2014-01-01

    Follicle-stimulating hormone receptor (FSHR), which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein (FSHR-57aa) as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a(+)-FSHR-57aa plasmid was constructed and expressed in Escherichia coli strain BL21 Star™ (DE3) and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 (16 weeks) after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels. PMID:24713829

  10. N-Terminal Modification of Proteins with o-Aminophenols

    PubMed Central

    2015-01-01

    The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods. PMID:24963951

  11. An unusual peptide deformylase features in the human mitochondrial N-terminal methionine excision pathway.

    PubMed

    Serero, Alexandre; Giglione, Carmela; Sardini, Alessandro; Martinez-Sanz, Juan; Meinnel, Thierry

    2003-12-26

    Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated. PMID:14532271

  12. Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger of the HIV-1 nucleocapsid protein: NMR structure of the complex with the Psi-site analog, dACGCC.

    PubMed

    South, T L; Summers, M F

    1993-01-01

    The nucleic acid interactive properties of a synthetic peptide with sequence of the N-terminal CCHC zinc finger (CCHC = Cys-X2-Cys-X4-His-X4-Cys; X = variable amino acid) of the human immunodeficiency virus (HIV) nucleocapsid protein, Zn(HIV1-F1), have been studied by 1H NMR spectroscopy. Titration of Zn(HIV1-F1) with oligodeoxyribonucleic acids containing different nucleotide sequences reveals, for the first time, sequence-dependent binding that requires the presence of at least one guanosine residue for tight complex formation. The dynamics of complex formation are sensitive to the nature of the residues adjacent to guanosine, with residues on the 3' side of guanosine having the largest influence. An oligodeoxyribonucleotide with sequence corresponding to a portion of the HIV-1 psi-packaging signal, d(ACGCC), forms a relatively tight complex with Zn(HIV1-F1) (Kd = 5 x 10(-6) M). Two-dimensional nuclear Overhauser effect (NOESY) data indicate that the bound nucleic acid exists predominantly in a single-stranded, A-helical conformation, and the presence of more than a dozen intermolecular NOE cross peaks enabled three-dimensional modeling of the complex. The nucleic acid binds within a hydrophobic cleft on the peptide surface. This hydrophobic cleft is defined by the side chains of residues Val1, Phe4, Ile12, and Ala13. Backbone amide protons of Phe4 and Ala13 and the backbone carbonyl oxygen of Lys2 that lie within this cleft appear to form hydrogen bonds with the guanosine O6 and N1H atoms, respectively. In addition, the positively charged side chain of Arg14 is ideally positioned for electrostatic interactions with the phosphodiester backbone of the nucleic acid. The structural findings provide a rationalization for the general conservation of these hydrophobic and basic residues in CCHC zinc fingers, and are consistent with site-directed mutagenesis results that implicate these residues as direct participants in viral genome recognition.

  13. Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform

    PubMed Central

    Zuellig, Richard A.; Bornhauser, Beat C.; Amstutz, Ralf; Constantin, Bruno; Schaub, Marcus C.

    2011-01-01

    Utrophin and dystrophin present two large proteins that link the intracellular actin cytoskeleton to the extracellular matrix via the C-terminal-associated protein complex. Here we describe a novel short N-terminal isoform of utrophin and its protein product in various rat tissues (N-utro, 62 kDa, amino acids 1–539, comprising the actin-binding domain plus the first two spectrin repeats). Using different N-terminal recombinant utrophin fragments, we show that actin binding exhibits pronounced negative cooperativity (affinity constants K1 = ∼5 × 106 and K2 = ∼1 × 105 M−1) and is Ca2+-insensitive. Expression of the different fragments in COS7 cells and in myotubes indicates that the actin-binding domain alone binds exlusively to actin filaments. The recombinant N-utro analogue binds in vitro to actin and in the cells associates to the membranes. The results indicate that N-utro may be responsible for the anchoring of the cortical actin cytoskeleton to the membranes in muscle and other tissues. PMID:22228988

  14. Sorbitol dehydrogenase. Full-length cDNA sequencing reveals a mRNA coding for a protein containing an additional 42 amino acids at the N-terminal end.

    PubMed

    Wen, Y; Bekhor, I

    1993-10-01

    A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated from a rat testis lambda ZAP II cDNA library. The full-length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be initiated. A second ATG codon, however, was found 126 bp upstream, aligned in the same reading frame as that of the active enzyme. Therefore, the coding sequence for SDH can be translated into an additional 42-amino-acid polypeptide linked to the N-terminal amino acid of the enzyme, generating a pre-sorbitol dehydrogenase. The sequence data indicate that the nucleotide environment around this ATG codon is more favorable towards it being the actual open reading frame (ORF) for a pre-SDH than the ATG codon preceding the nucleotide sequence for SDH. Since no known SDH starts with the additional 42 amino acids, it may be that post-translational removal of this polypeptide accompanies the release of the active enzyme. Next, the 3' untranslated region of the cDNA contained a non-coding 1021 bp downstream from the TAA stop codon. The latter sequence included three putative poly(A) signals: one at nucleotides 1362-1367, the second at nucleotides 1465-1470, and the third at nucleotides 2212-2217 [17 bp away from the poly(A) tail]. In addition to the above findings we also report a variance in one of the amino acids in the SDH cDNA sequence. This variance occurs at position 957-960, where threonine is coded for instead of aspartic acid; in the rat testis SDH cDNA, we find the sequence is ACG instead of GAC, as was reported for the rat liver SDH cDNA. Northern-blot hybridization analysis showed that SDH mRNA is a doublet, one band of 4 kb and the other of 2.3-2.4 kb, in both the rat liver and the rat lens, further confirming that the isolated SDH cDNA constituted a full-length cDNA.

  15. Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25.

    PubMed

    Landrieu, Isabelle; Verger, Alexis; Baert, Jean-Luc; Rucktooa, Prakash; Cantrelle, François-Xavier; Dewitte, Frédérique; Ferreira, Elisabeth; Lens, Zoé; Villeret, Vincent; Monté, Didier

    2015-08-18

    The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38-68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM-MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator-transactivator interactions. PMID:26130716

  16. Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25

    PubMed Central

    Landrieu, Isabelle; Verger, Alexis; Baert, Jean-Luc; Rucktooa, Prakash; Cantrelle, François-Xavier; Dewitte, Frédérique; Ferreira, Elisabeth; Lens, Zoé; Villeret, Vincent; Monté, Didier

    2015-01-01

    The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38–68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM–MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator–transactivator interactions. PMID:26130716

  17. Glycyrrhetinic acid induces cytoprotective autophagy via the inositol-requiring enzyme 1α-c-Jun N-terminal kinase cascade in non-small cell lung cancer cells.

    PubMed

    Tang, Zheng-Hai; Zhang, Le-Le; Li, Ting; Lu, Jia-Hong; Ma, Dik-Lung; Leung, Chung-Hang; Chen, Xiu-Ping; Jiang, Hu-Lin; Wang, Yi-Tao; Lu, Jin-Jian

    2015-12-22

    Glycerrhetinic acid (GA), one of the main bioactive constituents of Glycyrrhiza uralensis Fisch, exerts anti-cancer effects on various cancer cells. We confirmed that GA inhibited cell proliferation and induced apoptosis in non-small cell lung cancer A549 and NCI-H1299 cells. GA also induced expression of autophagy marker phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II) and punta formation of green fluorescent protein microtubule-associated protein light-chain 3. We further proved that expression of GA-increased autophagy marker was attributed to activation instead of suppression of autophagic flux. The c-jun N-terminal kinase (JNK) pathway was activated after incubation with GA. Pretreatment with the JNK inhibitor SP600125 or silencing of the JNK pathway by siRNA of JNK or c-jun decreased GA-induced autophagy. The endoplasmic reticulum (ER) stress responses were also apparently stimulated by GA by triggering the inositol-requiring enzyme 1α (IRE1α) pathway. The GA-induced JNK pathway activation and autophagy were decreased by IRE1α knockdown, and inhibition of autophagy or the JNK cascade increased GA-stimulated IRE1α expression. In addition, GA-induced cell proliferative inhibition and apoptosis were increased by inhibition of autophagy or the JNK pathway. Our study was the first to demonstrate that GA induces cytoprotective autophagy in non-small cell lung cancer cells by activating the IRE1α-JNK/c-jun pathway. The combined treatment of autophagy inhibitors markedly enhances the anti-neoplasmic activity of GA. Such combination shows potential as a strategy for GA or GA-contained prescriptions in cancer therapy.

  18. Catalytic residues are shared between two pseudosubunits of the dehydratase domain of the animal fatty acid synthase.

    PubMed

    Pasta, Saloni; Witkowski, Andrzej; Joshi, Anil K; Smith, Stuart

    2007-12-01

    Expression, characterization, and mutagenesis of a series of N-terminal fragments of an animal fatty acid synthase, containing the beta-ketoacyl synthase, acyl transferase, and dehydratase domains, demonstrate that the dehydratase domain consists of two pseudosubunits, derived from contiguous regions of the same polypeptide, in which a single active site is formed by the cooperation of the catalytic histidine 878 residue of the first pseudosubunit with aspartate 1032 of the second pseudosubunit. Mutagenesis and modeling studies revealed an essential role for glutamine 1036 in anchoring the position of the catalytic aspartate. These findings establish that sequence elements previously assigned to a central structural core region of the type I fatty acid synthases and some modular polyketide synthase counterparts play an essential catalytic role as part of the dehydratase domain.

  19. Function of the N-terminal segment of the RecA-dependent nuclease Ref.

    PubMed

    Gruber, Angela J; Olsen, Tayla M; Dvorak, Rachel H; Cox, Michael M

    2015-02-18

    The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.

  20. Purification, crystallization and preliminary structural characterization of the N-terminal region of the human formin-homology protein FHOD1

    SciTech Connect

    Schulte, Antje Rak, Alexey; Pylypenko, Olena; Ludwig, Diana; Geyer, Matthias

    2007-10-01

    The N-terminal region (1–339) of the human FHOD1 protein has been crystallized in two different crystal forms. A crystal of the (C31S,C71S) mutant diffracted to around 2.3 Å resolution. Formins are key regulators of actin cytoskeletal dynamics that constitute a diverse protein family that is present in all eukaryotes examined. They typically consist of more than 1000 amino acids and are defined by the presence of two conserved regions, namely the formin homology 1 and 2 domains. Additional conserved domains comprise a GTPase-binding domain for activation, a C-terminal autoregulation motif and an N-terminal recognition domain. In this study, the N-terminal region (residues 1–339) of the human formin homology domain-containing protein 1 (FHOD1) was purified and crystallized from 20%(w/v) PEG 4000, 10%(v/v) glycerol, 0.3 M magnesium chloride and 0.1 M Tris–HCl pH 8.0. Native crystals belong to space group P1, with unit-cell parameters a = 35.4, b = 73.9, c = 78.7 Å, α = 78.2, β = 86.2, γ = 89.7°. They contain two monomers of FHOD1 in the asymmetric unit and diffract to a resolution of 2.3 Å using a synchrotron-radiation source.

  1. Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity

    PubMed Central

    D'Antuono, Alejandra; Loureiro, Maria Eugenia; Foscaldi, Sabrina; Marino-Buslje, Cristina

    2014-01-01

    ABSTRACT The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCE The mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be

  2. Structure of the Tropomyosin Overlap Complex from Chicken Smooth Muscle: Insight into the Diversity of N-Terminal Recognition

    SciTech Connect

    Frye, Jeremiah; Klenchin, Vadim A.; Rayment, Ivan

    2010-09-08

    Tropomyosin is a stereotypical {alpha}-helical coiled coil that polymerizes to form a filamentous macromolecular assembly that lies on the surface of F-actin. The interaction between the C-terminal and N-terminal segments on adjacent molecules is known as the overlap region. We report here two X-ray structures of the chicken smooth muscle tropomyosin overlap complex. A novel approach was used to stabilize the C-terminal and N-terminal fragments. Globular domains from both the human DNA ligase binding protein XRCC4 and bacteriophage {phi}29 scaffolding protein Gp7 were fused to 37 and 28 C-terminal amino acid residues of tropomyosin, respectively, whereas the 29 N-terminal amino acids of tropomyosin were fused to the C-terminal helix bundle of microtubule binding protein EB1. The structures of both the XRCC4 and Gp7 fusion proteins complexed with the N-terminal EB1 fusion contain a very similar helix bundle in the overlap region that encompasses {approx}15 residues. The C-terminal coiled coil opens to allow formation of the helix bundle, which is stabilized by hydrophobic interactions. These structures are similar to that observed in the NMR structure of the rat skeletal overlap complex [Greenfield, N. J., et al. (2006) J. Mol. Biol. 364, 80-96]. The interactions between the N- and C-terminal coiled coils of smooth muscle tropomyosin show significant curvature, which differs somewhat between the two structures and implies flexibility in the overlap complex, at least in solution. This is likely an important attribute that allows tropomyosin to assemble around the actin filaments. These structures provide a molecular explanation for the role of N-acetylation in the assembly of native tropomyosin.

  3. Structural dynamics and ssDNA binding activity of the three N-terminal domains of the large subunit of Replication Protein A from small angle X-ray scattering

    SciTech Connect

    Pretto, Dalyir I.; Tsutakawa, Susan; Brosey, Chris A.; Castillo, Amalchi; Chagot, Marie-Eve; Smith, Jarrod A.; Tainer, John A.; Chazin, Walter J.

    2010-03-11

    Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial inter-domain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module.

  4. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain

    PubMed Central

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas JD; Young, Stephen G; Ploug, Michael

    2016-01-01

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia. DOI: http://dx.doi.org/10.7554/eLife.12095.001 PMID:26725083

  5. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain.

    PubMed

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas Jd; Young, Stephen G; Ploug, Michael

    2016-01-01

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia. PMID:26725083

  6. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain.

    PubMed

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas Jd; Young, Stephen G; Ploug, Michael

    2016-01-03

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia.

  7. Structure of the N-terminal fragment of Escherichia coli Lon protease

    SciTech Connect

    Li, Mi; Gustchina, Alla; Rasulova, Fatima S.; Melnikov, Edward E.; Maurizi, Michael R.; Rotanova, Tatyana V.; Dauter, Zbigniew; Wlodawer, Alexander

    2010-08-01

    The medium-resolution structure of the N-terminal fragment of E. coli Lon protease shows that this part of the enzyme consists of two compact domains and a very long α-helix. The structure of a recombinant construct consisting of residues 1–245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.

  8. Myeloperoxidase Inactivates TIMP-1 by Oxidizing Its N-terminal Cysteine Residue

    PubMed Central

    Wang, Yi; Rosen, Henry; Madtes, David K.; Shao, Baohai; Martin, Thomas R.; Heinecke, Jay W.; Fu, Xiaoyun

    2016-01-01

    An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes. Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during inflammation. PMID:17726014

  9. NTMG (N-terminal Truncated Mutants Generator for cDNA): an automatic multiplex PCR assays design for generating various N-terminal truncated cDNA mutants.

    PubMed

    Chen, Yung-Fu; Chen, Rung-Ching; Tseng, Lin-Yu; Lin, Elong; Chan, Yung-Kuan; Pan, Ren-Hao

    2007-07-01

    The sequential deletion method is generally used to locate the functional domain of a protein. With this method, in order to find the various N-terminal truncated mutants, researchers have to investigate the ATG-like codons, to design various multiplex polymerase chain reaction (PCR) forward primers and to do several PCR experiments. This web server (N-terminal Truncated Mutants Generator for cDNA) will automatically generate groups of forward PCR primers and the corresponding reverse PCR primers that can be used in a single batch of a multiplex PCR experiment to extract the various N-terminal truncated mutants. This saves much time and money for those who use the sequential deletion method in their research. This server is available at http://oblab.cs.nchu.edu.tw:8080/WebSDL/. PMID:17488836

  10. Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

    PubMed Central

    Panayotou, G; Bax, B; Gout, I; Federwisch, M; Wroblowski, B; Dhand, R; Fry, M J; Blundell, T L; Wollmer, A; Waterfield, M D

    1992-01-01

    Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed. Images PMID:1330535

  11. Coupling of Conformational Transitions in the N-terminal Domain of the 51-kDa FK506-binding Protein (FKBP51) Near Its Site of Interaction with the Steroid Receptor Proteins.

    PubMed

    LeMaster, David M; Mustafi, Sourajit M; Brecher, Matthew; Zhang, Jing; Héroux, Annie; Li, Hongmin; Hernández, Griselda

    2015-06-19

    Interchanging Leu-119 for Pro-119 at the tip of the β4-β5 loop in the first FK506 binding domain (FK1) of the FKBP51 and FKBP52 proteins, respectively, has been reported to largely reverse the inhibitory (FKBP51) or stimulatory (FKBP52) effects of these co-chaperones on the transcriptional activity of glucocorticoid and androgen receptor-protein complexes. Previous NMR relaxation studies have identified exchange line broadening, indicative of submillisecond conformational motion, throughout the β4-β5 loop in the FK1 domain of FKBP51, which are suppressed by the FKBP52-like L119P substitution. This substitution also attenuates exchange line broadening in the underlying β2 and β3a strands that is centered near a bifurcated main chain hydrogen bond interaction between these two strands. The present study demonstrates that these exchange line broadening effects arise from two distinct coupled conformational transitions, and the transition within the β2 and β3a strands samples a transient conformation that resembles the crystal structures of the selectively inhibited FK1 domain of FKBP51 recently reported. Although the crystal structures for their series of inhibitors were interpreted as evidence for an induced fit mechanism of association, the presence of a similar conformation being significantly populated in the unliganded FKBP51 domain is more consistent with a conformational selection binding process. The contrastingly reduced conformational plasticity of the corresponding FK1 domain of FKBP52 is consistent with the current model in which FKBP51 binds to both the apo- and hormone-bound forms of the steroid receptor to modulate its affinity for ligand, whereas FKBP52 binds selectively to the latter state.

  12. Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.

    PubMed

    Sayanova, O; Shewry, P R; Napier, J A

    1999-10-01

    Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.

  13. Histidine-41 of the Cytochrome b5 Domain of the Borage Δ6 Fatty Acid Desaturase Is Essential for Enzyme Activity1

    PubMed Central

    Sayanova, Olga; Shewry, Peter R.; Napier, Johnathan A.

    1999-01-01

    Unlike most other plant microsomal desaturases, the Δ6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Δ6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Δ6-fatty acid desaturase resulted in the synthesis and accumulation of Δ6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Δ6-desaturase in transgenic plants failed to produce Δ6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Δ6-fatty acid desaturase is essential for enzymatic activity. PMID:10517856

  14. Five glutamic acid residues in the C-terminal domain of the ChlD subunit play a major role in conferring Mg(2+) cooperativity upon magnesium chelatase.

    PubMed

    Brindley, Amanda A; Adams, Nathan B P; Hunter, C Neil; Reid, James D

    2015-11-10

    Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg(2+) ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus. We show that five glutamic acid residues in the C-terminal domain play a major role in this process.

  15. Functional analysis of the extended N-terminal region in PLC-δ1 (MlPLC-δ1) from the mud loach, Misgurnus mizolepis.

    PubMed

    Kim, Na Young; Ahn, Sang Jung; Kim, Moo-Sang; Seo, Jung Soo; Jung, Se Hwan; Park, Sung Hwan; Lee, Hyung Ho; Chung, Joon Ki

    2014-01-01

    Mud loach phospholipase C-δ1 (MlPLC-δ1) contains all the characteristic domains found in mammalian PLC-δ isozymes (pleckstrin homology domain, EF-hands, X–Y catalytic region, and C2 domain) as well as an extended 26-amino acid (aa)-long N-terminal region that is an alternative splice form of PLC-δ1 and is novel to vertebrate PLC-δ. In the present structure-function analysis, deletion of the extended N-terminal region caused complete loss of phosphatidylinositol (PI)- and phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity in MlPLC-δ1. Additionally, recombinant full-length MlPLC-δ1 PLC activity was reduced in a dose-dependent manner by coincubation with the 26-aa protein fragment. Using a protein-lipid overlay assay, both full-length MlPLC-δ1 and the 26-aa protein fragment had substantial affinity for PIP2, whereas deletion of the 26-aa region from MlPLC-δ1 (MlPLC-δ1-deletion) resulted in lower affinity for PIP2. These results suggest that the novel N-terminal exon of MlPLC-δ1 could play an important role in the regulation of PLC-δ1.

  16. N-terminal sequences direct the autophosphorylation states of the FER tyrosine kinases in vivo.

    PubMed

    Orlovsky, K; Ben-Dor, I; Priel-Halachmi, S; Malovany, H; Nir, U

    2000-09-12

    p94(fer) and p51(ferT) are two tyrosine kinases which share identical SH2 and kinase domains but differ in their N-terminal regions. While p94(fer) is expressed in most mammalian cells, the accumulation of p51(ferT) is restricted to meiotic spermatocytes. Here we show that the different N-terminal tails of p94(fer) and p51(ferT) direct different autophosphorylation states of these two kinases in vivo. N-terminal coiled-coil domains cooperated to drive the oligomerization and autophosphorylation in trans of p94(fer). Moreover, the ectopically expressed N-terminal tail of p94(fer) could act as a dominant negative mutant and associated with the endogenous p94(fer) protein in CHO cells. This increased significantly the percentage of cells residing in the G0/G1 phase, thus suggesting a role for p94(fer) in the regulation of G1 progression. Unlike p94(fer), overexpressed p51(ferT) was not autophosphorylated in COS1 cells. However, removal of the unique N-terminal 43 aa of p51(ferT) or the replacement of this region by a parallel segment from p94(fer) endowed the modified p51(ferT) with the ability to autophosphorylate. The unique N-terminal sequences of p51(ferT) thus interfere with its ability to autophosphorylate in vivo. These experiments indicate that the N-terminal sequences of the FER tyrosine kinases direct their different cellular autophosphorylation states, thereby dictating their different cellular functions. PMID:10998246

  17. A mass-spectrometric method for the estimation of the ratio of gamma-carboxyglutamic acid to glutamic acid at specific sites in proteins. Application to the N-terminal region of bovine prothrombin.

    PubMed

    Rose, K; Priddle, J D; Offord, R E; Esnouf, M P

    1980-04-01

    When a polypeptide containing gamma-carboxyglutamic acid is decarboxylated in 2H2O, residue of (gamma gamma-2H2)glutamic acid are formed. Subsequent proteolytic digestion produces peptides which contain at each site 2H2-substituted and unsubstituted glutamic acid in the same ratio as existed for gramma-carboxy-substitution. The peptides may be identified and this ratio determined by combined gas chromatography--mass spectrometry. We also discuss decarboxylation in 3H2O followed by amino-acid analysis and Edman degradation.

  18. Molecular characterization of the interaction between the N-terminal region of Potato virus X (PVX) coat protein (CP) and Nicotiana benthamiana PVX CP-interacting protein, NbPCIP1.

    PubMed

    Park, Mi-Ri; Kim, Kook-Hyung

    2013-06-01

    Using yeast two-hybrid assays and a Nicotiana benthamiana cDNA library, we previously identified an N. benthamiana protein, NbPCIP1, that interacts with Potato virus X (PVX) coat protein (CP). We also previously determined that NbPCIP1 enhances PVX replication in plants. To determine the domains and/or amino acid residues required for PVX CP and NbPCIP1 interaction, here we used yeast two-hybrid and β-galactosidase filter assays to test the effects of deletion and site-directed mutations on the interaction. Truncation analysis revealed that the N-terminal region of PVX CP interacts with NbPCIP1. To identify which N-terminal region PVX CP amino acid(s) interact with NbPCIP1, we substituted the 12 charged amino acids on the PVX CP N-terminal region to alanine. Yeast two-hybrid, β-galactosidase filter, and bimolecular fluorescence complementation (BiFC) assays confirmed that ten of the 12 alanine-substituted mutations blocked the interaction with NbPCIP1. The results suggest that the N-terminal region of PVX CP including its helical structure is important for interaction with NbPCIP1.

  19. mRNA Decapping Enzyme 1a (Dcp1a)-induced Translational Arrest through Protein Kinase R (PKR) Activation Requires the N-terminal Enabled Vasodilator-stimulated Protein Homology 1 (EVH1) Domain*

    PubMed Central

    Dougherty, Jonathan D.; Reineke, Lucas C.; Lloyd, Richard E.

    2014-01-01

    We have shown previously that poliovirus infection disrupts cytoplasmic P-bodies in infected mammalian cells. During the infectious cycle, poliovirus causes the directed cleavage of Dcp1a and Pan3, coincident with the dispersion of P-bodies. We now show that expression of Dcp1a prior to infection, surprisingly, restricts poliovirus infection. This inhibition of infection was independent of P-body formation because expression of GFP-Dcp1a mutants that cannot enter P-bodies restricted poliovirus infection similar to wild-type GFP-Dcp1a. Expression of wild-type or mutant GFP-Dcp1a induced phosphorylation of eIF2α through the eIF2α kinase protein kinase R (PKR). Activation of PKR required the amino-terminal EVH1 domain of Dcp1a. This PKR-induced translational inhibition appears to be specific to Dcp1a because the expression of other P-body components, Pan2, Pan3, Ccr4, or Caf1, did not result in the inhibition of poliovirus gene expression or induce eIF2α phosphorylation. The translation blockade induced by Dcp1a expression suggests novel signaling linking RNA degradation/decapping and regulation of translation. PMID:24382890

  20. mRNA decapping enzyme 1a (Dcp1a)-induced translational arrest through protein kinase R (PKR) activation requires the N-terminal enabled vasodilator-stimulated protein homology 1 (EVH1) domain.

    PubMed

    Dougherty, Jonathan D; Reineke, Lucas C; Lloyd, Richard E

    2014-02-14

    We have shown previously that poliovirus infection disrupts cytoplasmic P-bodies in infected mammalian cells. During the infectious cycle, poliovirus causes the directed cleavage of Dcp1a and Pan3, coincident with the dispersion of P-bodies. We now show that expression of Dcp1a prior to infection, surprisingly, restricts poliovirus infection. This inhibition of infection was independent of P-body formation because expression of GFP-Dcp1a mutants that cannot enter P-bodies restricted poliovirus infection similar to wild-type GFP-Dcp1a. Expression of wild-type or mutant GFP-Dcp1a induced phosphorylation of eIF2α through the eIF2α kinase protein kinase R (PKR). Activation of PKR required the amino-terminal EVH1 domain of Dcp1a. This PKR-induced translational inhibition appears to be specific to Dcp1a because the expression of other P-body components, Pan2, Pan3, Ccr4, or Caf1, did not result in the inhibition of poliovirus gene expression or induce eIF2α phosphorylation. The translation blockade induced by Dcp1a expression suggests novel signaling linking RNA degradation/decapping and regulation of translation. PMID:24382890

  1. Domain-level rocking motion within a polymerase that translocates on single-stranded nucleic acid

    SciTech Connect

    Li, Huiyung; Li, Changzheng; Zhou, Sufeng; Poulos, Thomas L.; Gershon, Paul David

    2013-04-01

    An X-ray crystallographic structure is described for unliganded Vaccinia virus poly(A) polymerase monomer (VP55), showing the first domain-level structural isoforms among either VP55, it’s processivity factor VP39, or the VP55-VP39 heterodimer. The occurrence of domain-level motion specifically in monomeric VP55 is consistent with the finding that the monomeric protein undergoes saltatory translocation whereas the heterodimer does not. Vaccinia virus poly(A) polymerase (VP55) is the only known polymerase that can translocate independently with respect to single-stranded nucleic acid (ssNA). Previously, its structure has only been solved in the context of the VP39 processivity factor. Here, a crystal structure of unliganded monomeric VP55 has been solved to 2.86 Å resolution, showing the first backbone structural isoforms among either VP55 or its processivity factor (VP39). Backbone differences between the two molecules of VP55 in the asymmetric unit indicated that unliganded monomeric VP55 can undergo a ‘rocking’ motion of the N-terminal domain with respect to the other two domains, which may be ‘rigidified’ upon VP39 docking. This observation is consistent with previously demonstrated experimental molecular dynamics of the monomer during translocation with respect to nucleic acid and with different mechanisms of translocation in the presence and absence of processivity factor VP39. Side-chain conformational changes in the absence of ligand were observed at a key primer contact site and at the catalytic center of VP55. The current structure completes the trio of possible structural forms for VP55 and VP39, namely the VP39 monomer, the VP39–VP55 heterodimer and the VP55 monomer.

  2. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    PubMed Central

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

  3. Conformational analysis of the N-terminal sequence Met1 Val60 of the tyrosine hydroxylase

    NASA Astrophysics Data System (ADS)

    Alieva, Irada N.; Mustafayeva, Narmina N.; Gojayev, Niftali M.

    2006-03-01

    Molecular mechanics method and molecular dynamics (MD) simulation techniques are used to study the behavior and the effect of the amino acids substitution on structure and molecular dynamics of the specific portion of Met1-Val60 amino acid residues from N-terminal regulatory domain of the tyrosine hydroxylase (TH) and its mutants in which the positively charged arginine residues at positions 37 and 38 were replaced by electrically neutral Gly and negatively charged Glu, and serine residue at position 40 was replaced by Ala or Asp residue. Our study allowed us to make the following conclusions: (i) the higher conformational flexibility of the Met1-Arg16 sequence is revealed in comparision to other part of the N-terminus; (ii) the stretch of amino acid residues Met30-Ser40 within the N-terminus forms β-turn so that two α-helices (residues 16-29 and residues 41-60) are paralel one another; (ii) the significant differences that are observed for the Arg37→Gly37, Arg37-Arg38→Glu37-Glu38 mutant segments indicates that the positive charge of the Arg37 and Arg38 residues is one of the main factor that maintains the characteristic of the turn; (ii) no major conformational changes are observed between Ser40→Ala40, and Ser40→Asp40 mutant segments.

  4. Identification of the domains of cauliflower mosaic virus protein P6 responsible for suppression of RNA silencing and salicylic acid signalling

    PubMed Central

    Laird, Janet; McInally, Carol; Carr, Craig; Doddiah, Sowjanya; Yates, Gary; Chrysanthou, Elina; Khattab, Ahmed; Love, Andrew J.; Geri, Chiara; Sadanandom, Ari; Smith, Brian O.; Kobayashi, Kappei

    2013-01-01

    Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded. PMID:24088344

  5. The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains Is Governed by Their N-terminal and Internal Protein Regions.

    PubMed

    Park, Ji Won; Reed, James R; Backes, Wayne L

    2015-12-01

    In cellular membranes, different lipid species are heterogeneously distributed forming domains with different characteristics. Ordered domains are tightly packed with cholesterol, sphingomyelin, and saturated fatty acids, whereas disordered domains contain high levels of unsaturated fatty acids. Our laboratory has shown that membrane heterogeneity affects the organization of cytochrome P450s and their cognate redox partner, the cytochrome P450 reductase (CPR). Despite the high degree of sequence similarity, CYP1A1 was found to localize to disordered regions, whereas CYP1A2 resided in ordered domains. We hypothesized that regions of amino acid sequence variability may contain signal motifs that direct CYP1A proteins into ordered or disordered domains. Thus, chimeric constructs of CYP1A1 and CYP1A2 were created, and their localization was tested in HEK293T cells. CYP1A2, containing the N-terminal regions from CYP1A1, no longer localized in ordered domains, whereas the N terminus of CYP1A2 partially directed CYP1A1 into ordered regions. In addition, intact CYP1A2 containing a 206-302-residue peptide segment of CYP1A1 had less affinity to bind to ordered microdomains. After expression, the catalytic activity of CYP1A2 was higher than that of the CYP1A1-CYP1A2 chimera containing the N-terminal end of CYP1A1 with subsaturating CPR concentrations, but it was approximately equal with excess CPR suggesting that the localization of the CYP1A enzyme in ordered domains favored its interaction with CPR. These data demonstrate that both the N-terminal end and an internal region of CYP1A2 play roles in targeting CYP1A2 to ordered domains, and domain localization may influence P450 function under conditions that resemble those found in vivo. PMID:26468279

  6. The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains Is Governed by Their N-terminal and Internal Protein Regions.

    PubMed

    Park, Ji Won; Reed, James R; Backes, Wayne L

    2015-12-01

    In cellular membranes, different lipid species are heterogeneously distributed forming domains with different characteristics. Ordered domains are tightly packed with cholesterol, sphingomyelin, and saturated fatty acids, whereas disordered domains contain high levels of unsaturated fatty acids. Our laboratory has shown that membrane heterogeneity affects the organization of cytochrome P450s and their cognate redox partner, the cytochrome P450 reductase (CPR). Despite the high degree of sequence similarity, CYP1A1 was found to localize to disordered regions, whereas CYP1A2 resided in ordered domains. We hypothesized that regions of amino acid sequence variability may contain signal motifs that direct CYP1A proteins into ordered or disordered domains. Thus, chimeric constructs of CYP1A1 and CYP1A2 were created, and their localization was tested in HEK293T cells. CYP1A2, containing the N-terminal regions from CYP1A1, no longer localized in ordered domains, whereas the N terminus of CYP1A2 partially directed CYP1A1 into ordered regions. In addition, intact CYP1A2 containing a 206-302-residue peptide segment of CYP1A1 had less affinity to bind to ordered microdomains. After expression, the catalytic activity of CYP1A2 was higher than that of the CYP1A1-CYP1A2 chimera containing the N-terminal end of CYP1A1 with subsaturating CPR concentrations, but it was approximately equal with excess CPR suggesting that the localization of the CYP1A enzyme in ordered domains favored its interaction with CPR. These data demonstrate that both the N-terminal end and an internal region of CYP1A2 play roles in targeting CYP1A2 to ordered domains, and domain localization may influence P450 function under conditions that resemble those found in vivo.

  7. Structure of the N-terminal fragment of Escherichia coli Lon protease

    SciTech Connect

    Li, Mi; Gustchina, Alla; Rasulova, Fatima S.; Melnikov, Edward E.; Maurizi, Michael R.; Rotanova, Tatyana V.; Dauter, Zbigniew; Wlodawer, Alexander

    2010-10-22

    The structure of a recombinant construct consisting of residues 1-245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 {angstrom} resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal {alpha}-helix. The structure of the first subdomain (residues 1-117), which consists mostly of {beta}-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.

  8. Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin*

    PubMed Central

    Gifford, Stacey M.; Liu, Weizhi; Mader, Christopher C.; Halo, Tiffany L.; Machida, Kazuya; Boggon, Titus J.; Koleske, Anthony J.

    2014-01-01

    The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity “Arg-like” SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an “Abl-like” low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. PMID:24891505

  9. N-terminal cleavage of proTGFα occurs at the cell surface by a TACE-independent activity

    PubMed Central

    2005-01-01

    ProTGFα (transforming growth factor α precursor) maturation and conversion into soluble TGFα is a complex process that involves three proteolytic steps. One, that occurs co-translationally, eliminates the signal sequence. Another, occurring at the juxtamembrane domain, solubilizes TGFα. A third cleavage removes the N-terminal extension of proTGFα. This latter step has been poorly studied, mainly because of the rapid kinetics of this cleavage. In the present study, we have designed a strategy to analyse several aspects regarding this N-terminal cleavage. In vivo treatment with the hydroxamate-based metalloprotease inhibitors BB3103 or TAPI-2 (tumour necrosis factor-α protease inhibitor 2) reversibly induced accumulation of forms of proTGFα that included the N-terminal extension. N-terminal shedding was rapid, and occurred at the cell surface. However, the machinery responsible for the N-terminal cleavage was inactive in other cellular sites, such as the endoplasmic reticulum. Experiments of proTGFα expression and maturation in cells deficient in TACE (tumour-necrosis-factor-α-converting enzyme) activity indicated that this protease was dispensable for N-terminal processing of proTGFα in vivo, but was required for regulated cleavage at the C-terminus. These findings indicate that TACE is not involved in N-terminal processing of proTGFα, and suggest differences in the machineries that control the cleavage at both ends of TGFα within its precursor. PMID:15777285

  10. Effect of amino acid substitution in the hydrophobic face of amphiphilic peptides on membrane curvature and perturbation: N-terminal helix derived from adenovirus internal protein VI as a model.

    PubMed

    Murayama, Tomo; Pujals, Sílvia; Hirose, Hisaaki; Nakase, Ikuhiko; Futaki, Shiroh

    2016-11-01

    The N-terminal amphipathic helical segment of adenovirus internal protein VI (AdVpVI) plays a critical role in viral infection. Here, we report that the peptide segment corresponding to AdVpVI (positions 33-55) can induce positive membrane curvature together with membrane perturbation. The enhanced perturbation ability of the peptide was observed for membranes containing negatively charged phospholipids. Based on the liposome leakage assay, substitution of leucine at position 40 to other aliphatic (isoleucine) and aromatic (phenylalanine and tryptophan) residues yielded a similar degree of membrane perturbation by the peptides, which was considerably diminished by the substitution to glutamine. Further studies using the wild-type AdVpVI (33-55) (WT) and phenylalanine-substituted peptides (L40F) demonstrated that both peptides have positive membrane-curvature-inducing ability. These peptides showed higher binding affinity to 50-nm large unilamellar vesicles (LUVs) than to 200-nm LUVs. However, no enhanced perturbation by these peptides was observed for 50-nm LUVs compared to 200-nm LUVs, suggesting that both the original membrane curvature and the additional strain due to peptide insertion affect the membrane perturbation ability of these peptides. In the case of L40F, this peptide rather had a lower membrane perturbation ability for 50-nm LUVs than for 200-nm LUVs, which can be attributed to possible shallower binding of L40F on membranes. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 430-439, 2016.

  11. Effect of amino acid substitution in the hydrophobic face of amphiphilic peptides on membrane curvature and perturbation: N-terminal helix derived from adenovirus internal protein VI as a model.

    PubMed

    Murayama, Tomo; Pujals, Sílvia; Hirose, Hisaaki; Nakase, Ikuhiko; Futaki, Shiroh

    2016-11-01

    The N-terminal amphipathic helical segment of adenovirus internal protein VI (AdVpVI) plays a critical role in viral infection. Here, we report that the peptide segment corresponding to AdVpVI (positions 33-55) can induce positive membrane curvature together with membrane perturbation. The enhanced perturbation ability of the peptide was observed for membranes containing negatively charged phospholipids. Based on the liposome leakage assay, substitution of leucine at position 40 to other aliphatic (isoleucine) and aromatic (phenylalanine and tryptophan) residues yielded a similar degree of membrane perturbation by the peptides, which was considerably diminished by the substitution to glutamine. Further studies using the wild-type AdVpVI (33-55) (WT) and phenylalanine-substituted peptides (L40F) demonstrated that both peptides have positive membrane-curvature-inducing ability. These peptides showed higher binding affinity to 50-nm large unilamellar vesicles (LUVs) than to 200-nm LUVs. However, no enhanced perturbation by these peptides was observed for 50-nm LUVs compared to 200-nm LUVs, suggesting that both the original membrane curvature and the additional strain due to peptide insertion affect the membrane perturbation ability of these peptides. In the case of L40F, this peptide rather had a lower membrane perturbation ability for 50-nm LUVs than for 200-nm LUVs, which can be attributed to possible shallower binding of L40F on membranes. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 430-439, 2016. PMID:27271816

  12. Oxidation of the N-terminal methionine of lens alpha-A crystallin

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Horwitz, J.; Emmons, T.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Antiserum against the N-terminal peptide of bovine alpha-A crystallin has been used to monitor purification of two different seropositive peptides (i.e. T1a and T1b) from a tryptic digest of bovine lens proteins. Both these peptides have similar amino acid compositions, but peptide T1b has a molecular weight 16 atomic mass units larger than T1a, suggesting posttranslational modification. Analysis of ionization fragments of the T1b peptide by mass spectrometry demonstrates that this difference in molecular weight is due to the in vivo oxidation of the N-terminal met residue of the alpha-A crystallin molecule.

  13. Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion.

    PubMed

    Netter, Catharina; Weber, Gert; Benecke, Heike; Wahl, Markus C

    2009-07-01

    RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B'' proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the beta-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B'' proteins. However, unlike U1A and U2B'', some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an alpha-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.

  14. Jun N-terminal kinase signaling makes a face

    PubMed Central

    Hursh, Deborah A.; Stultz, Brian G.; Park, Sung Yeon

    2016-01-01

    ABSTRACT decapentaplegic (dpp), the Drosophila ortholog of BMP 2/4, directs ventral adult head morphogenesis through expression in the peripodial epithelium of the eye-antennal disc. This dpp expressing domain exerts effects both on the peripodial epithelium, and the underlying disc proper epithelium. We have uncovered a role for the Jun N-terminal kinase (JNK) pathway in dpp-mediated ventral head development. JNK activity is required for dpp's action on the disc proper, but in the absence of dpp expression, excessive JNK activity is produced, leading to specific loss of maxillary palps. In this review we outline our hypotheses on how dpp acts by both short range and longer range mechanisms to direct head morphogenesis and speculate on the dual role of JNK signaling in this process. Finally, we describe the regulatory control of dpp expression in the eye-antennal disc, and pose the problem of how the various expression domains of a secreted protein can be targeted to their specific functions. PMID:27384866

  15. Jun N-terminal kinase signaling makes a face.

    PubMed

    Hursh, Deborah A; Stultz, Brian G; Park, Sung Yeon

    2016-10-01

    decapentaplegic (dpp), the Drosophila ortholog of BMP 2/4, directs ventral adult head morphogenesis through expression in the peripodial epithelium of the eye-antennal disc. This dpp expressing domain exerts effects both on the peripodial epithelium, and the underlying disc proper epithelium. We have uncovered a role for the Jun N-terminal kinase (JNK) pathway in dpp-mediated ventral head development. JNK activity is required for dpp's action on the disc proper, but in the absence of dpp expression, excessive JNK activity is produced, leading to specific loss of maxillary palps. In this review we outline our hypotheses on how dpp acts by both short range and longer range mechanisms to direct head morphogenesis and speculate on the dual role of JNK signaling in this process. Finally, we describe the regulatory control of dpp expression in the eye-antennal disc, and pose the problem of how the various expression domains of a secreted protein can be targeted to their specific functions.

  16. A comparison of the N-terminal sequence of the leech Theromyzon tessulatum angiotensin converting-like enzyme with forms of vertebrate angiotensin converting enzymes.

    PubMed

    Laurent, V; Salzet, M

    1995-09-22

    This paper reports the purification of an angiotensing-converting like enzyme (ACE) of ca. 120 kDa from extracts of head membranes of the leech Theromyzon tessulatum. After solubilization with Triton X-114, the ACE-like enzyme contained in the detergent-poor fraction was separated using five steps of purification including gel permeation and anion exchange chromatographies followed by reverse-phase HPLC. The first 23 amino acid residues of the N-terminal part (GLDPELSPGCFSADEAGAQLFAE) of the purified S-pyridylethylated leech ACE established by automated Edman degradation revealed ca. 87% sequence identity with the N-terminal sequence of the guinea pig ACE. This enzyme cleaves the hyppuryl-His-Leu substrate with a specific activity of 5600 nmol hyppurate min-1 mg protein-1. Hydrolysis of this substrate by ACE-like enzyme is inhibited at 80% by 10 microM captopril or 10 microM lisinopril (IC50 of 200 nM and 50 nM, respectively). This enzyme is close in sequence and in activity to single domain vertebrate ACE. This is the first N-terminal sequence of an ACE-like enzyme determined in invertebrates.

  17. Analytical cation-exchange chromatography to assess the identity, purity, and N-terminal integrity of human lactoferrin.

    PubMed

    van Veen, Harrie A; Geerts, Marlieke E J; van Berkel, Patrick H C; Nuijens, Jan H

    2002-10-01

    Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the innate host defense. The positively charged N-terminal domain of hLF mediates several of its activities by interacting with ligands such as bacterial lipopolysaccharide (LPS), specific receptors, and other proteins. This cationic domain is highly susceptible to limited proteolysis, which impacts on the affinity of hLF for the ligand. An analytical method, employing cation-exchange chromatography on Mono S, was developed to assess the N-terminal integrity of hLF preparations. The method, which separates N-terminally intact hLF from hLF species lacking two (Gly(1)-Arg(2)) or three (Gly(1)-Arg(2)-Arg(3)) residues, showed that 5-58% of total hLF in commercially obtained preparations was N-terminally degraded. The elution profile of hLF on Mono S unequivocally differed from lactoferrins from other species as well as homologous and other whey proteins. Analysis of fresh human whey samples revealed two variants of N-terminally intact hLF, but not limitedly proteolyzed hLF. Mono S chromatography of 2 out of 26 individual human whey samples showed a rare polymorphic hLF variant with three N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Ser(5)-) instead of the usual variant with four N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Arg(5)-Ser(6)-). In conclusion, Mono S cation-exchange chromatography appeared a robust method to assess the identity, purity, N-terminal integrity, and the presence of polymorphic and intact hLF variants. PMID:12381362

  18. Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein.

    PubMed

    Hauser, Paul S; Ryan, Robert O

    2007-08-01

    Advances in expressed protein ligation (EPL) methods that permit specific introduction of unique modifications into proteins have facilitated protein engineering, structure-function and protein interaction studies. An EPL-generated hybrid exchangeable apolipoprotein has been constructed from recombinant fragments of apolipoprotein E (apoE) and apolipophorin III (apoLp-III). A recombinant fusion protein comprised of human apoE N-terminal residues 1-111, a modified Saccharomyces cerevisiae intein and a chitin binding domain was subjected to 2-mercaptoethanesulfonic acid (MESNA) induced cleavage to generate apoE(1-111)-MESNA. A second fusion protein was comprised of a bacterial pelB leader peptide fused to a variant form of Galleria mellonella apoLp-III residues 1-91. The N-terminal pelB leader sequence directed the newly synthesized fusion protein to the Escherichia coli perisplamic space where endogenous leader peptidase cleavage generated the desired N-terminal cysteine-containing protein fragment. The resulting apoLp-III fragment, which contained no sequence tags or tails, escaped the bacteria and accumulated in the culture medium. When cultured in M9 minimal medium, Asp1Cys apoLp-III(1-91) was produced in high yield and was the sole major protein in the culture supernatant. Ligation reactions with apoE(1-111)-MESNA yielded an engineered hybrid apolipoprotein. The results document the utility of the pelB fusion protein system for generating active N-terminal cysteine containing proteins for EPL applications.

  19. Nucleic acids encoding a cellulose binding domain

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1996-03-05

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 15 figs.

  20. Nucleic acids encoding a cellulose binding domain

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1996-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  1. The Venus Fly Trap domain of the extracellular Ca2+ -sensing receptor is required for L-amino acid sensing.

    PubMed

    Mun, Hee-Chang; Franks, Alison H; Culverston, Emma L; Krapcho, Karen; Nemeth, Edward F; Conigrave, Arthur D

    2004-12-10

    We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814-4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.

  2. New OprM structure highlighting the nature of the N-terminal anchor.

    PubMed

    Monlezun, Laura; Phan, Gilles; Benabdelhak, Houssain; Lascombe, Marie-Bernard; Enguéné, Véronique Y N; Picard, Martin; Broutin, Isabelle

    2015-01-01

    Among the different mechanisms used by bacteria to resist antibiotics, active efflux plays a major role. In Gram-negative bacteria, active efflux is carried out by tripartite efflux pumps that form a macromolecular assembly spanning both membranes of the cellular wall. At the outer membrane level, a well-conserved outer membrane factor (OMF) protein acts as an exit duct, but its sequence varies greatly among different species. The OMFs share a similar tri-dimensional structure that includes a beta-barrel pore domain that stabilizes the channel within the membrane. In addition, OMFs are often subjected to different N-terminal post-translational modifications (PTMs), such as an acylation with a lipid. The role of additional N-terminal anchors is all the more intriguing since it is not always required among the OMFs family. Understanding this optional PTM could open new research lines in the field of antibiotics resistance. In Escherichia coli, it has been shown that CusC is modified with a tri-acylated lipid, whereas TolC does not show any modification. In the case of OprM from Pseudomonas aeruginosa, the N-terminal modification remains a matter of debate, therefore, we used several approaches to investigate this issue. As definitive evidence, we present a new X-ray structure at 3.8 Å resolution that was solved in a new space group, making it possible to model the N-terminal residue as a palmitoylated cysteine.

  3. The N-terminal acetyltransferase Naa10 is essential for zebrafish development

    PubMed Central

    Ree, Rasmus; Myklebust, Line M.; Thiel, Puja; Foyn, Håvard; Fladmark, Kari E.; Arnesen, Thomas

    2015-01-01

    N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish. PMID:26251455

  4. Selecting protein N-terminal peptides by combined fractional diagonal chromatography.

    PubMed

    Staes, An; Impens, Francis; Van Damme, Petra; Ruttens, Bart; Goethals, Marc; Demol, Hans; Timmerman, Evy; Vandekerckhove, Joël; Gevaert, Kris

    2011-07-14

    In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.

  5. Terahertz time-domain spectroscopy of four hydroxycinnamic acid derivatives.

    PubMed

    Ge, Min; Zhao, Hongwei; Wang, Wenfeng; Zhang, Zengyan; Yu, Xiaohan; Li, Wenxin

    2006-11-01

    The well-resolved absorption spectra of the hydroxycinnamic acid (HCA) derivatives, caffeic acid, ferulic acid, sinapic acid and chlorogenic acid, were measured over the frequency region from 0.3 to 2.0 THz at 294 K with terahertz time-domain spectroscopy (THz-TDS). Theoretical calculation was applied to assist the analysis and assignment of the individual THz absorption spectra of the HCA derivatives with density functional theory (DFT). The distinctive spectral features were originated from the collective motion of molecules held together by hydrogen bonds. The real and imaginary parts of dielectric function of the four HCA derivatives were also obtained. PMID:19669446

  6. Terahertz Time-Domain Spectroscopy of Four Hydroxycinnamic Acid Derivatives

    PubMed Central

    Ge, Min; Zhao, Hongwei; Zhang, Zengyan; Yu, Xiaohan; Li, Wenxin

    2006-01-01

    The well-resolved absorption spectra of the hydroxycinnamic acid (HCA) derivatives, caffeic acid, ferulic acid, sinapic acid and chlorogenic acid, were measured over the frequency region from 0.3 to 2.0 THz at 294 K with terahertz time-domain spectroscopy (THz-TDS). Theoretical calculation was applied to assist the analysis and assignment of the individual THz absorption spectra of the HCA derivatives with density functional theory (DFT). The distinctive spectral features were originated from the collective motion of molecules held together by hydrogen bonds. The real and imaginary parts of dielectric function of the four HCA derivatives were also obtained. PMID:19669446

  7. The Thumb Domain Mediates Acid-sensing Ion Channel Desensitization.

    PubMed

    Krauson, Aram J; Carattino, Marcelo D

    2016-05-20

    Acid-sensing ion channels (ASICs) are cation-selective proton-gated channels expressed in neurons that participate in diverse physiological processes, including nociception, synaptic plasticity, learning, and memory. ASIC subunits contain intracellular N and C termini, two transmembrane domains that constitute the pore, and a large extracellular loop with defined domains termed the finger, β-ball, thumb, palm, and knuckle. Here we examined the contribution of the finger, β-ball, and thumb domains to activation and desensitization through the analysis of chimeras and the assessment of the effect of covalent modification of introduced Cys at the domain-domain interfaces. Our studies with ASIC1a-ASIC2a chimeras showed that swapping the thumb domain between subunits results in faster channel desensitization. Likewise, the covalent modification of Cys residues at selected positions in the β-ball-thumb interface accelerates the desensitization of the mutant channels. Studies of accessibility with thiol-reactive reagents revealed that the β-ball and thumb domains reside apart in the resting state but that they become closer to each other in response to extracellular acidification. We propose that the thumb domain moves upon continuous exposure to an acidic extracellular milieu, assisting with the closing of the pore during channel desensitization. PMID:27015804

  8. N-terminal protein processing: A comparative proteogenomic analysis

    SciTech Connect

    Bonissone, Stefano; Gupta, Nitin; Romine, Margaret F.; Bradshaw, Ralph A.; Pevzner, Pavel A.

    2013-01-01

    N-Terminal Methionine Excision (NME) is a universally conserved mechanism with the same specificity across all life forms that removes the first Methionine in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val. In spite of its necessity for proper cell functioning, the functional role of NME remains unclear. In 1988, Arfin and Bradshaw connected NME with the N-end protein degradation rule and postulated that the role of NME is to expose the stabilizing residues with the goal to resist protein degradation. While this explanation (that treats 7 stabilizing residues in the same manner) has become the de facto dogma of NME, comparative proteogenomics analysis of NME tells a different story. We suggest that the primary role of NME is to expose only two (rather than seven) amino acids Ala and Ser for post-translational modifications (e.g., acetylation) rather than to regulate protein degradation. We argue that, contrary to the existing view, NME is not crucially important for proteins with 5 other stabilizing residue at the 2nd positions that are merely bystanders (their function is not affected by NME) that become exposed to NME because their sizes are comparable or smaller than the size of Ala and Ser.

  9. The transcriptional activator GCN4 contains multiple activation domains that are critically dependent on hydrophobic amino acids.

    PubMed Central

    Drysdale, C M; Dueñas, E; Jackson, B M; Reusser, U; Braus, G H; Hinnebusch, A G

    1995-01-01

    GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. Previous work suggested that the principal activation domain of GCN4 is a highly acidic segment of approximately 40 amino acids located in the center of the protein. We conducted a mutational analysis of GCN4 with a single-copy allele expressed under the control of the native promoter and translational control elements. Our results indicate that GCN4 contains two activation domains of similar potency that can function independently to promote high-level transcription of the target genes HIS3 and HIS4. One of these domains is coincident with the acidic activation domain defined previously; the other extends over the N-terminal one-third of the protein. Both domains are partially dependent on the coactivator protein ADA2. Each domain appears to be composed of two or more small subdomains that have additive effects on transcription and that can cooperate in different combinations to promote high-level expression of HIS3 and HIS4. At least three of these subdomains are critically dependent on bulky hydrophobic amino acids for their function. Five of the important hydrophobic residues, Phe-97, Phe-98, Met-107, Tyr-110, and Leu-113, fall within a region of proposed sequence homology between GCN4 and the herpesvirus acidic activator VP16. The remaining three residues, Trp-120, Leu-123, and Phe-124, are highly conserved between GCN4 and its Neurospora counterpart, cpc-1. Because of the functional redundancy in the activation domain, mutations at positions 97 and 98 must be combined with mutations at positions 120 to 124 to observe a substantial reduction in activation by full-length GCN4, and substitution of all eight hydrophobic residues was required to inactivate full-length GCN4. These hydrophobic residues may mediate important interactions between GCN4 and one or more of its target proteins in the transcription initiation complex

  10. Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry.

    PubMed

    Ren, Da; Pipes, Gary D; Hambly, David; Bondarenko, Pavel V; Treuheit, Michael J; Gadgil, Himanshu S

    2009-01-01

    An N-terminal top-down sequencing approach was developed for IgG characterization, using high-resolution HPLC separation and collisionally activated dissociation (CAD) on a single-stage LCT Premier time of flight (TOF) mass spectrometer. Fragmentation of the IgG chains on the LCT Premier was optimized by varying the ion guide voltage values. Ion guide 1 voltage had the most significant effect on the fragmentation of the IgG chains. An ion guide 1 voltage value of 100 V was found to be optimum for the N-terminal fragmentation of IgG heavy and light chains, which are approximately 50 and 25 kDa, respectively. The most prominent ion series in this CAD experiment was the terminal b-ion series which allows N-terminal sequencing. Using this technique, we were able to confirm the sequence of up to seven N-terminal residues. Applications of this method for the identification of N-terminal pyroglutamic acid formation will be discussed. The method described could be used as a high-throughput method for the rapid N-terminal sequencing of IgG chains and for the detection of chemical modifications in the terminal residues.

  11. Downregulation of N-terminal acetylation triggers ABA-mediated drought responses in Arabidopsis

    PubMed Central

    Linster, Eric; Stephan, Iwona; Bienvenut, Willy V.; Maple-Grødem, Jodi; Myklebust, Line M.; Huber, Monika; Reichelt, Michael; Sticht, Carsten; Geir Møller, Simon; Meinnel, Thierry; Arnesen, Thomas; Giglione, Carmela; Hell, Rüdiger; Wirtz, Markus

    2015-01-01

    N-terminal acetylation (NTA) catalysed by N-terminal acetyltransferases (Nats) is among the most common protein modifications in eukaryotes, but its significance is still enigmatic. Here we characterize the plant NatA complex and reveal evolutionary conservation of NatA biochemical properties in higher eukaryotes and uncover specific and essential functions of NatA for development, biosynthetic pathways and stress responses in plants. We show that NTA decreases significantly after drought stress, and NatA abundance is rapidly downregulated by the phytohormone abscisic acid. Accordingly, transgenic downregulation of NatA induces the drought stress response and results in strikingly drought resistant plants. Thus, we propose that NTA by the NatA complex acts as a cellular surveillance mechanism during stress and that imprinting of the proteome by NatA is an important switch for the control of metabolism, development and cellular stress responses downstream of abscisic acid. PMID:26184543

  12. Monoclonal antibody against the N-terminal end of human plasma fibronectin.

    PubMed Central

    Vartio, T; Salonen, E M; De Petro, G; Barlati, S; Miggiano, V; Stähli, C; Virgallita, G; Takács, B; Vaheri, A

    1983-01-01

    Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin. Images Fig. 1. Fig. 2. PMID:6194791

  13. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

    PubMed Central

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F.; Wahl, Markus C.

    2015-01-01

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

  14. Role of N-terminal region of Escherichia coli maltodextrin glucosidase in folding and function of the protein.

    PubMed

    Pastor, Ashutosh; Singh, Amit K; Shukla, Prakash K; Equbal, Md Javed; Malik, Shikha T; Singh, Tej P; Chaudhuri, Tapan K

    2016-09-01

    Maltodextrin glucosidase (MalZ) hydrolyses short malto-oligosaccharides from the reducing end releasing glucose and maltose in Escherichia coli. MalZ is a highly aggregation prone protein and molecular chaperonins GroEL and GroES assist in the folding of this protein to a substantial level. The N-terminal region of this enzyme appears to be a unique domain as seen in sequence comparison studies with other amylases as well as through homology modelling. The sequence and homology model analysis show a probability of disorder in the N-