WorldWideScience

Sample records for amino terminal domain

  1. Interaction of tau with the neural plasma membrane mediated by tau's amino-terminal projection domain

    OpenAIRE

    1995-01-01

    The neuronal microtubule-associated protein tau is required for the development of cell polarity in cultured neurons. Using PC12 cells that stably express tau and tau amino-terminal fragments, we report that tau interacts with the neural plasma membrane through its amino-terminal projection domain. In differentiated PC12 transfectants, tau is found in growth cone-like structures in a nonmicrotubule-dependent manner. In hippocampal neurons, tau is differentially extracted by detergent and enri...

  2. The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

    International Nuclear Information System (INIS)

    Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation

  3. The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J.Y.; Yang, L.X. [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Huang, Z.F. [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Key Laboratory of Tropical Diseases Control, Sun Yat-sen University, Ministry of Education in China, Guangzhou (China)

    2013-12-02

    Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.

  4. The amino-terminal segment in the β-domain of δ-cadinene synthase is essential for catalysis.

    Science.gov (United States)

    González, Verónica; Grundy, Daniel J; Faraldos, Juan A; Allemann, Rudolf K

    2016-08-21

    Despite its distance from the active site the flexible amino-terminal segment (NTS) in the β-domain of the plant sesquiterpene cyclase δ-cadinene synthase (DCS) is essential for active site closure and desolvation events during catalysis. PMID:27431578

  5. E2 polypeptides encoded by bovine papillomavirus type 1 form dimers through the common carboxyl-terminal domain: transactivation is mediated by the conserved amino-terminal domain.

    Science.gov (United States)

    McBride, A A; Byrne, J C; Howley, P M

    1989-01-01

    The E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV-1) encodes positive- and negative-acting factors that regulate viral gene expression. The full-length ORF encodes a transactivator, and two transcriptional repressors are expressed from the 3' half of the ORF. Previous analysis has shown that a conserved C-terminal region of 101 amino acids, which is shared by E2 transactivator and repressor proteins, contains the specific DNA binding activity. Further analysis of the E2 transactivator shows that a conserved N-terminal domain of approximately 220 amino acids is crucial for the transcriptional activation function, whereas the variable internal region is not required. The E2 proteins bind to a sequence, ACCGN4CGGT, several copies of which are sufficient to constitute an E2-dependent enhancer. By using a gel retardation assay and proteins derived by in vitro transcription and translation, we were able to show that the E2 polypeptides bind as dimers to a single DNA binding site. The dimeric E2 proteins are stable in the absence of DNA and dimerization is mediated through the DNA binding domain. This may reveal an additional mechanism of repression that could potentially result from the formation of inactive heterodimers between transactivator and repressor species. PMID:2536165

  6. Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcriptional coactivator TIF2 (transcriptional intermediary factor2)

    OpenAIRE

    Berrevoets, Cor; P. Doesburg; Steketee, Karine; Trapman, Jan; Brinkmann, Albert

    1998-01-01

    textabstractPrevious studies in yeast and mammalian cells showed a functional interaction between the amino-terminal domain and the carboxy-terminal, ligand-binding domain (LBD) of the human androgen receptor (AR). In the present study, the AR subdomains involved in this in vivo interaction were determined in more detail. Cotransfection experiments in Chinese hamster ovary (CHO) cells and two-hybrid experiments in yeast revealed that two regions in the NH2-terminal domain are involved in the ...

  7. Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcriptional coactivator TIF2 (transcriptional intermediary factor2)

    NARCIS (Netherlands)

    C.A. Berrevoets (Cor); P. Doesburg (Paul); K. Steketee (Karine); J. Trapman (Jan); A.O. Brinkmann (Albert)

    1998-01-01

    textabstractPrevious studies in yeast and mammalian cells showed a functional interaction between the amino-terminal domain and the carboxy-terminal, ligand-binding domain (LBD) of the human androgen receptor (AR). In the present study, the AR subdomains involved in thi

  8. A triple-arginine motif in the amino-terminal domain and oligomerization are required for HIV-1 inhibition by human MX2.

    Science.gov (United States)

    Goujon, Caroline; Greenbury, Rebecca A; Papaioannou, Stelios; Doyle, Tomas; Malim, Michael H

    2015-04-01

    We have employed molecular genetic approaches to understand the domain organization of the HIV-1 resistance factor myxovirus resistance 2 (MX2). First, we describe an essential triple-arginine motif in the amino-terminal domain. Second, we demonstrate that this 91-residue domain mediates antiviral activity when appended to heterologous proteins, and we provide genetic evidence that protein oligomerization is required for MX2 function. These insights will facilitate future work aiming to elucidate MX2's mechanism of action.

  9. Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle.

    Science.gov (United States)

    Deng, Maoxian; Boopathi, Ettickan; Hypolite, Joseph A; Raabe, Tobias; Chang, Shaohua; Zderic, Stephen; Wein, Alan J; Chacko, Samuel

    2013-11-15

    Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

  10. Natural variation of the amino-terminal glutamine-rich domain in Drosophila argonaute2 is not associated with developmental defects.

    Directory of Open Access Journals (Sweden)

    Daniel Hain

    Full Text Available The Drosophila argonaute2 (ago2 gene plays a major role in siRNA mediated RNA silencing pathways. Unlike mammalian Argonaute proteins, the Drosophila protein has an unusual amino-terminal domain made up largely of multiple copies of glutamine-rich repeats (GRRs. We report here that the ago2 locus produces an alternative transcript that encodes a putative short isoform without this amino-terminal domain. Several ago2 mutations previously reported to be null alleles only abolish expression of the long, GRR-containing isoform. Analysis of drop out (dop mutations had previously suggested that variations in GRR copy number result in defects in RNAi and embryonic development. However, we find that dop mutations genetically complement transcript-null alleles of ago2 and that ago2 alleles with variant GRR copy numbers support normal development. In addition, we show that the assembly of the central RNAi machinery, the RISC (RNA induced silencing complex, is unimpaired in embryos when GRR copy number is altered. In fact, we find that GRR copy number is highly variable in natural D. melanogaster populations as well as in laboratory strains. Finally, while many other insects share an extensive, glutamine-rich Ago2 amino-terminal domain, its primary sequence varies drastically between species. Our data indicate that GRR variation does not modulate an essential function of Ago2 and that the amino-terminal domain of Ago2 is subject to rapid evolution.

  11. The Contributions of the Amino and Carboxy Terminal Domains of Flightin to the Biomechanical Properties of Drosophila Flight Muscle Thick Filaments.

    Science.gov (United States)

    Gasek, Nathan S; Nyland, Lori R; Vigoreaux, Jim O

    2016-01-01

    Flightin is a myosin binding protein present in Pancrustacea. In Drosophila, flightin is expressed in the indirect flight muscles (IFM), where it is required for the flexural rigidity, structural integrity, and length determination of thick filaments. Comparison of flightin sequences from multiple Drosophila species revealed a tripartite organization indicative of three functional domains subject to different evolutionary constraints. We use atomic force microscopy to investigate the functional roles of the N-terminal domain and the C-terminal domain that show different patterns of sequence conservation. Thick filaments containing a C-terminal domain truncated flightin (fln(ΔC44)) are significantly shorter (2.68 ± 0.06 μm; p thick filaments containing a full length flightin (fln⁺; 3.21 ± 0.05 μm) and thick filaments containing an N-terminal domain truncated flightin (fln(ΔN62); 3.21 ± 0.06 μm). Persistence length was significantly reduced in fln(ΔN62) (418 ± 72 μm; p thick filament bending propensity. Our results indicate that the flightin amino and carboxy terminal domains make distinct contributions to thick filament biomechanics. We propose these distinct roles arise from the interplay between natural selection and sexual selection given IFM's dual role in flight and courtship behaviors.

  12. The amino-terminal GAF domain of Azotobacter vinelandii NifA binds 2-oxoglutarate to resist inhibition by NifL under nitrogen-limiting conditions.

    Science.gov (United States)

    Little, Richard; Dixon, Ray

    2003-08-01

    The expression of genes required for the synthesis of molybdenum nitrogenase in Azotobacter vinelandii is controlled by the NifL-NifA transcriptional regulatory complex in response to nitrogen, carbon, and redox status. Activation of nif gene expression by the transcriptional activator NifA is inhibited by direct protein-protein interaction with NifL under conditions unfavorable for nitrogen fixation. We have recently shown that the NifL-NifA system responds directly to physiological concentrations of 2-oxoglutarate, resulting in relief of NifA activity from inhibition by NifL under conditions when fixed nitrogen is limiting. The inhibitory activity of NifL is restored under conditions of excess combined nitrogen through the binding of the signal transduction protein Av GlnK to the carboxyl-terminal domain of NifL. The amino-terminal domain of NifA comprises a GAF domain implicated in the regulatory response to NifL. A truncated form of NifA lacking this domain is not responsive to 2-oxoglutarate in the presence of NifL, suggesting that the GAF domain is required for the response to this ligand. Using isothermal titration calorimetry, we demonstrate stoichiometric binding of 2-oxoglutarate to both the isolated GAF domain and the full-length A. vinelandii NifA protein with a dissociation constant of approximately 60 microm. Limited proteolysis experiments indicate that the binding of 2-oxoglutarate increases the susceptibility of the GAF domain to trypsin digestion and also prevents NifL from protecting these cleavage sites. However, protection by NifL is restored when the non-modified (non-uridylylated) form of Av GlnK is also present. Our results suggest that the binding of 2-oxoglutarate to the GAF domain of NifA may induce a conformational change that prevents inhibition by NifL under conditions when fixed nitrogen is limiting. PMID:12759352

  13. Escherichia coli methionyl-tRNA formyltransferase: role of amino acids conserved in the linker region and in the C-terminal domain on the specific recognition of the initiator tRNA.

    Science.gov (United States)

    Gite, S; Li, Y; Ramesh, V; RajBhandary, U L

    2000-03-01

    The formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria. We are studying the molecular mechanisms of recognition of the initiator tRNA by Escherichia coli MTF. MTF from eubacteria contains an approximately 100-amino acid C-terminal extension that is not found in the E. coli glycinamide ribonucleotide formyltransferase, which, like MTF, use N(10)-formyltetrahydrofolate as a formyl group donor. This C-terminal extension, which forms a distinct structural domain, is attached to the N-terminal domain through a linker region. Here, we describe the effect of (i) substitution mutations on some nineteen basic, aromatic and other conserved amino acids in the linker region and in the C-terminal domain of MTF and (ii) deletion mutations from the C-terminus on enzyme activity. We show that the positive charge on two of the lysine residues in the linker region leading to the C-terminal domain are important for enzyme activity. Mutation of some of the basic amino acids in the C-terminal domain to alanine has mostly small effects on the kinetic parameters, whereas mutation to glutamic acid has large effects. However, the deletion of 18, 20, or 80 amino acids from the C-terminus has very large effects on enzyme activity. Overall, our results support the notion that the basic amino acid residues in the C-terminal domain provide a positively charged channel that is used for the nonspecific binding of tRNA, whereas some of the amino acids in the linker region play an important role in activity of MTF.

  14. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

    NARCIS (Netherlands)

    Rustgi, A.K.; Dyson, N.; Bernards, R.A.

    1991-01-01

    The proteins encoded by the myc gene family are involved is the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/

  15. Amino-terminal domains of the bovine papillomavirus type 1 E1 and E2 proteins participate in complex formation.

    OpenAIRE

    Benson, J D; Howley, P M

    1995-01-01

    Interaction between the E1 and E2 papillomavirus proteins appear to play an important role in viral DNA replication, although the exact domains of each protein involved in this interaction have not been identified. Using bovine papillomavirus type 1 (BPV-1) as a model for examining interactions between E1 and E2, we have used the two-hybrid and glutathione S-transferase (GST) fusion systems to map domains of BPV-1 E1 and E2 that interact in vivo and in vitro. In the two-hybrid system experime...

  16. The amino-terminal domain of human signal transducers and activators of transcription 1: Overexpression, purification and characterization

    Indian Academy of Sciences (India)

    Arati Prabhu; Evans Coutinho; Sudha Srivastava

    2005-12-01

    The dual functional signal transducers and activators of transcription (STAT) proteins are latent cytoplasmic transcription factors that play crucial roles in host defense. Animals that lack these proteins are highly susceptible to microbial and viral infections and chemically induced primary tumours. We have over expressed the aminoterminal domain of human STAT1 (hSTAT1) in Escherichia coli and purified it by affinity chromatography and gel filtration chromatography. The entire process has been monitored by gel electrophoresis. The pure protein has been characterized by mass spectrometry and 2-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. Our results indicate that the N-terminus of hSTAT1 exists as a dimer in solution.

  17. Expression and T cell recognition of hybrid antigens with amino-terminal domains encoded by Qa-2 region of major histocompatibility complex and carboxyl termini of transplantation antigens.

    Science.gov (United States)

    Stroynowski, I; Forman, J; Goodenow, R S; Schiffer, S G; McMillan, M; Sharrow, S O; Sachs, D H; Hood, L

    1985-05-01

    Coding potential of the Q6 gene from the Qa-2a region of BALB/c Crgl mice was analyzed by a combination of hybrid class I gene construction and DNA-mediated gene transfer. Recombinant genes were created by exon shuffling of the 5' coding region of the Q6 gene and the 3' coding region of a gene encoding a transplantation antigen (Kd, Dd, or Ld), or the inverse. Some of these hybrid class I genes were expressed in the transfected mouse fibroblasts (L cells). The hybrid class I molecules encoded by the 5' end of the Q6 gene and the 3' end of the Ld gene precipitated as 45,000 mol wt molecules associated with beta 2-microglobulin. The expression of the hybrid proteins indicates that 926 basepairs of the 5' flanking region upstream of the structural Q6 gene contain a promoter that functions as a transcription initiation site in L cells. The 3' portion of the Q6 gene appears to be responsible for the lack of cell surface expression of the intact Q6 and the hybrid Ld/Q6 genes in mouse fibroblasts. Accordingly, this portion of the Q6 class I gene may play a regulatory role in tissue-specific expression. Serological analyses of hybrid Q6 proteins suggested that Q6 may be a structural gene for CR (H-2 crossreactive) antigen found normally on subpopulations of lymphocytes. If this identification is correct, Q6 gene will define a new category of class I genes encoding approximately 40,000 mol wt molecules and carrying a characteristic truncated cytoplasmic tail. Analysis of L cells transfected with Q6 hybrid genes demonstrated also that the cytotoxic T cells specific for Qa-2a region-coded antigens recognize the amino-terminal alpha 1-alpha 2 domain of Q6 fusion products. This recognition can be blocked by anti-Qa-2a alloantiserum and monoclonal antibodies reactive with the alpha 3-beta 2-microglobulin portion of the Q6 hybrids. We propose that the structural requirements for the anti-Qa-2a cytotoxic T lymphocyte-specific epitopes on target molecules are the same as for anti

  18. The amino-terminal domain of ORF149 of koi herpesvirus is preferentially targeted by IgM from carp populations surviving infection.

    Science.gov (United States)

    Torrent, F; Villena, A; Lee, P A; Fuchs, W; Bergmann, S M; Coll, J M

    2016-10-01

    Recombinantly expressed fragments of the protein encoded by ORF149 (pORF149), a structural protein from the common- and koi-carp-infecting cyprinid herpesvirus-3 (CyHV-3) that was previously shown to be antigenic, were used to obtain evidence that its amino-terminal part contains immunodominant epitopes in fish populations that survived the infection. To obtain such evidence, nonspecific binding of carp serum tetrameric IgM had to be overcome by a novel ELISA protocol (rec2-ELISA). Rec2-ELISA involved pre-adsorption of carp sera with a heterologous recombinant fragment before incubation with pORF149 fragments and detection with anti-carp IgM monoclonal antibodies. Only in this way was it possible to distinguish between sera from uninfected and survivor carp populations. Although IgM from survivors recognised pORF149 fragments to a lesser degree than whole virus, specificity was confirmed by correlation of rec2- and CyHV-3-ELISAs, inhibition of rec2-ELISA by an excess of frgIIORF149, ELISA using IgM-capture, Western blotting, and reduction of reactivity in CyHV-3-ELISA by pre-adsorption of sera with frgIIORF149. The similarity of IgM-binding profiles between frgIORF149 (amino acid residues 42-629) and frgIIORF149 (42-159) and their reactivities with previously described anti-CyHV-3 monoclonal antibodies confirmed that most pORF149 epitopes were localised in its amino-terminal part. PMID:27383208

  19. Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA

    Energy Technology Data Exchange (ETDEWEB)

    Foster, Mark P.; Wuttke, Deborah S.; Clemens, Karen R.; Jahnke, Wolfgang; Radhakrishnan, Ishwar; Tennant, Linda; Reymond, Martine; Chung, John; Wright, Peter E. [Scripps Research Institute, Department of Molecular Biology and Skaggs Institute for Chemical Biology (United States)

    1998-07-15

    We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone{sup 1} H{sup N}, {sup 15}N,{sup 13} C{sup {alpha}} and{sup 13} C{sup {beta}} and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.

  20. Plasmodium falciparum: an epitope within a highly conserved region of the 47-kDa amino-terminal domain of the serine repeat antigen is a target of parasite-inhibitory antibodies.

    Science.gov (United States)

    Fox, B A; Xing-Li, P; Suzue, K; Horii, T; Bzik, D J

    1997-02-01

    Previously, the Plasmodium falciparum serine repeat antigen has been shown to be protective in primate models of malaria immunity and also to be a target of in vitro parasite-inhibitory antibodies. To further define parasite-inhibitory epitopes a series of deletions from the amino-terminal 47-kDa domain of the serine repeat antigen (SERA) were constructed as glutathione-S-transferase fusion proteins. Several GST-SERA fusion proteins were used to vaccinate mice with Freund's adjuvant and the resulting immune sera were used to assay for the inhibition of P. falciparum invasion of erythrocytes in vitro. The minimal epitope shown to be the target of invasion-blocking antibodies was SERA amino acids 17-165. Additional GST-SERA deletion constructs of the 47-kDa domain were developed and evaluated for reactivity, by Western immunoblot analysis, with a parasite-inhibitory murine monoclonal antibody (mAb 43E5), a parasite-inhibitory pooled goat polyclonal sera, and a pooled human Nigerian immune serum. The parasite-inhibitory epitope defined by mAb 43E5 was mapped to SERA amino acids 17-110 and, at least, part of the epitope was defined to include amino acids in the region of amino acids 59-72. The parasite-inhibitory epitope recognized by mAb 43E5 appears to be well conserved between diverse geographical isolates of P. falciparum. The results have relevance for malaria vaccine development and suggest that an appropriately designed recombinant SERA antigen produced from a synthetic gene in Escherichia coli may be an effective component of a candidate malaria vaccine. PMID:9030663

  1. Amino acid substitutions of conserved residues in the carboxyl-terminal domain of the [alpha]I(X) chain of type X collagen occur in two unrelated families with metaphyseal chondrodysplasia type Schmid

    Energy Technology Data Exchange (ETDEWEB)

    Wallis, G.A.; Rash, B.; Sweetman, W.A.; Thomas, J.T.; Grant, M.E.; Boot-Handford, R.P. (Univ. of Manchester (United Kingdom)); Super, M. (Royal Manchester Children' s Hospital, Manchester (United Kingdom)); Evans, G. (Robert Jones Orthopaedic Hospital, Oswestry (United Kingdom))

    1994-02-01

    Type X collagen is a homotrimeric, short-chain, nonfibrillar extracellular-matrix component that is specifically and transiently synthesized by hypertrophic chondrocytes at the site of endochondral ossification. The precise function of type X collagen is not known, but its specific pattern of expression suggests that mutations within the encoding gene (COL10A1) that alter the structure or synthesis of the protein may cause heritable forms of chondrodysplasia. The authors used the PCR and the SSCP techniques to analyze the coding and upstream promoter regions of the COL10A1 gene in a number of individuals with forms of chondrodysplasia. Using this approach, they identified two individuals with metaphyseal chondrodysplasia type Schmid (MCDS) with SSCP changes in the region of the gene encoding the carboxyl-terminal domain. Sequence analysis demonstrated that the individuals were heterozygous for two unique single-base-pair transitions that led to the substitution of the highly conserved amino acid residue tyrosine at position 598 by aspartic acid in one person and of leucine at position 614 by proline in the other. The substitution at residue 598 segregated with the phenotype in a family of eight (five affected and three unaffected) related persons. The substitutions at residue 614 occurred in a sporadically affected individual but not in her unaffected mother and brother. Additional members of this family were not available for further study. These results suggest that certain amino acid substitutions within the carboxyl-terminal domain of the chains of the type X collagen molecule cause MCDS. These amino acid substitutions are likely to alter either chain recognition or assembly of the type X collagen molecule, thereby depleting the amount of normal type X collagen deposited in the extracellular matrix, with consequent aberrations in bone growth and development. 36 refs., 5 figs.

  2. Sequence and expression pattern of a novel human orphan G-protein-coupled receptor, GPRC5B, a family C receptor with a short amino-terminal domain

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Krogsgaard-Larsen, P

    2000-01-01

    Query of GenBank with the amino acid sequence of human metabotropic glutamate receptor subtype 2 (mGluR2) identified a predicted gene product of unknown function on BAC clone CIT987SK-A-69G12 (located on chromosome band 16p12) as a homologous protein. The transcript, entitled GPRC5B, was cloned f...... pattern is markedly different from that of RAIG1, which shows a slightly more restricted expression pattern with highest abundance in lung tissue....

  3. Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

    OpenAIRE

    Gropp, Michal; Strausz, Yael; Gross, Miriam; Glaser, Gad

    2001-01-01

    The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inse...

  4. Amino-terminated diamond surfaces: Photoelectron emission and photocatalytic properties

    Science.gov (United States)

    Zhu, Di; Bandy, Jason A.; Li, Shuo; Hamers, Robert J.

    2016-08-01

    We report a new approach to making stable negative electron-affinity diamond surfaces by terminating diamond with amino groups (also known as amine groups, -NH2). Previous studies have shown that negative electron affinity can be induced by terminating diamond surfaces with hydrogen, creating a surface dipole favorable toward electron emission. Here, we demonstrate that covalent tethering of positive charges in the form of protonated amino groups, -NH3+, also leads to negative electron affinity (NEA) and facile electron emission into vacuum and into water. Amino-terminated diamond was prepared using a very mild plasma discharge. Valence-band photoemission studies of the amino-terminated diamond samples show a characteristic "NEA" peak, demonstrating that the amino-terminated surface has NEA. Diamond's ability to emit electrons into water was evaluated using photochemical conversion of N2 to NH3. Time-resolved surface photovoltage studies were used to characterize charge separation at the diamond interface, and Mott-Schottky measurements were performed to characterize band-bending at the diamond-water interface. XPS studies show that the amino-terminated surfaces provide increased chemical resistance to oxidation compared with H-terminated diamond when illuminated with ultraviolet light.

  5. Untangling spider silk evolution with spidroin terminal domains

    Directory of Open Access Journals (Sweden)

    Garb Jessica E

    2010-08-01

    Full Text Available Abstract Background Spidroins are a unique family of large, structural proteins that make up the bulk of spider silk fibers. Due to the highly variable nature of their repetitive sequences, spidroin evolutionary relationships have principally been determined from their non-repetitive carboxy (C-terminal domains, though they offer limited character data. The few known spidroin amino (N-terminal domains have been difficult to obtain, but potentially contain critical phylogenetic information for reconstructing the diversification of spider silks. Here we used silk gland expression data (ESTs from highly divergent species to evaluate the functional significance and phylogenetic utility of spidroin N-terminal domains. Results We report 11 additional spidroin N-termini found by sequencing ~1,900 silk gland cDNAs from nine spider species that shared a common ancestor > 240 million years ago. In contrast to their hyper-variable repetitive regions, spidroin N-terminal domains have retained striking similarities in sequence identity, predicted secondary structure, and hydrophobicity. Through separate and combined phylogenetic analyses of N-terminal domains and their corresponding C-termini, we find that combined analysis produces the most resolved trees and that N-termini contribute more support and less conflict than the C-termini. These analyses show that paralogs largely group by silk gland type, except for the major ampullate spidroins. Moreover, spidroin structural motifs associated with superior tensile strength arose early in the history of this gene family, whereas a motif conferring greater extensibility convergently evolved in two distantly related paralogs. Conclusions A non-repetitive N-terminal domain appears to be a universal attribute of spidroin proteins, likely retained from the origin of spider silk production. Since this time, spidroin N-termini have maintained several features, consistent with this domain playing a key role in silk

  6. Bacteriophage endolysin Lyt μ1/6: characterization of the C-terminal binding domain.

    Science.gov (United States)

    Tišáková, Lenka; Vidová, Barbora; Farkašovská, Jarmila; Godány, Andrej

    2014-01-01

    The gene product of orf50 from actinophage μ1/6 of Streptomyces aureofaciens is a putative endolysin, Lyt μ1/6. It has a two-domain modular structure, consisting of an N-terminal catalytic and a C-terminal cell wall binding domain (CBD). Comparative analysis of Streptomyces phage endolysins revealed that they all have a modular structure and contain functional C-terminal domains with conserved amino acids, probably associated with their binding function. A blast analysis of Lyt μ1/6 in conjunction with secondary and tertiary structure prediction disclosed the presence of a PG_binding_1 domain within the CBD. The sequence of the C-terminal domain of lyt μ1/6 and truncated forms of it were cloned and expressed in Escherichia coli. The ability of these CBD variants fused to GFP to bind to the surface of S. aureofaciens NMU was shown by specific binding assays.

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

    Directory of Open Access Journals (Sweden)

    Gajendradhar R Dwivedi

    Full Text Available 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.

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

    Science.gov (United States)

    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

  9. THE ESCHERICHIA COLI SIGNAL PEPTIDE PEPTIDASE A IS A SERINE-LYSINE PROTEASE WITH A LYSINE RECRUITED TO THE NON-CONSERVED AMINO-TERMINAL DOMAIN IN THE S49 PROTEASE FAMILY

    OpenAIRE

    Wang, Peng; Shim, Eunjung; Cravatt, Benjamin; Jacobsen, Richard; Schoeniger, Joe; Kim, Apollos C.; Paetzel, Mark; Dalbey, Ross E.

    2008-01-01

    The E. coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-biotin inhibits SppA and modifies the protein, but doe...

  10. Helix A Stabilization Precedes Amino-terminal Lobe Activation upon Calcium Binding to Calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baowei [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lowry, David [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Mayer, M. Uljana [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Squier, Thomas C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2008-08-09

    The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)-resorufin (ReAsH), which upon binding to an engineered tetracysteine binding motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the 1H-15N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe (Kd = 0.36 ± 0.04 μM), which results in a reduction in the rate of ReAsH binding from 4900 M-1 sec-1 to 370 M-1 sec-1. In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe requires calcium-occupancy of amino-terminal sites (Kd = 18 ± 3 μM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces

  11. Amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences

    Energy Technology Data Exchange (ETDEWEB)

    Wheeler, E.F.; Roussel, M.F.; Hampe, A.; Walker, M.H.; Fried, V.A.; Look, A.T.; Rettenmier, C.W.; Sherr, C.J.

    1986-08-01

    The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180/sup gag-fms/ encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180/sup gag-fms/) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120/sup v-fms/, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. The authors conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180/gag-fms/ is mediated by signal peptidase and that the amino termini of gp140/sup v-fms/ and the c-fms gene product are identical.

  12. Significance of the C-terminal domain of Erwinia uredovora ice nucleation-active protein (Ina U).

    Science.gov (United States)

    Michigami, Y; Abe, K; Obata, H; Arai, S

    1995-12-01

    Ice nucleation-active (Ina) proteins of bacterial origin comprise three distinct domains, i.e., N-terminal (N-), central repeat (R-), and C-terminal (C-) domains, among which the R-domain is essential, and its length may be correlated with the ice nucleation activity. In addition, the short C-terminal domain of about 50 amino acid residues is indispensable for the activity. Using the Ina U protein of Erwinia uredovora, we carried out precise mutational analyses of its C-terminus. The ice nucleation activity (T50) assay showed that the C-terminal 12 amino acids were not necessary, and a deletion mutant (delta C29) with a new C-terminal, Met29 (numbered from the first amino acid residue of the C-domain and corresponding to Met1022), exhibited almost the same activity as the wild-type Ina U protein did. However, deletion of the C-terminal 13 residues including Met29 resulted in almost complete loss of the activity. In the deletion mutant (delta C29), amino acid replacement of the C-terminus, Met29, showed that the activity was retained when Met29 was replaced with a neutral, aromatic, or basic amino acid (Gly, Phe, or Lys), but was lost on the replacement with an acidic amino acid (Asp or Glu). In addition, two other residues in the C-terminal region commonly present in all Ina proteins were examined as to their importance, and it was shown that one of these residues, Tyr27, is important for the activity, although it is not exclusively required; the activity was lost to a great extent when this residue was replaced with Gly or Ala, but to a lesser extent when it was replaced with Leu. These results suggest that significance of the secondary and/or tertiary structure of the C-terminal region of the Ina U protein for the ice nucleation activity. PMID:8720147

  13. Docking Studies of Binding of Ethambutol to the C-Terminal Domain of the Arabinosyltransferase from Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Guillermo Salgado-Moran

    2013-01-01

    Full Text Available The binding of ethambutol to the C-terminal domain of the arabinosyltransferase from Mycobacterium tuberculosis was studied. The analysis was performed using an in silico approach in order to find out, by docking calculations and energy descriptors, the conformer of Ethambutol that forms the most stable complex with the C-terminal domain of arabinosyltransferase. The complex shows that location of the Ethambutol coincides with the cocrystallization ligand position and that amino acid residues ASH1051, ASN740, ASP1052, and ARG1055 should be critical in the binding of Ethambutol to C-terminal domain EmbC.

  14. Protecting Terminals by Security Domain Mechanism Based on Trusted Computing

    Institute of Scientific and Technical Information of China (English)

    ZHOU Zheng; ZHANG Jun; LI Jian; LIU Yi

    2006-01-01

    Networks are composed with servers and rather larger amounts of terminals and most menace of attack and virus come from terminals. Eliminating malicious code and access or breaking the conditions only under witch attack or virus can be invoked in those terminals would be the most effective way to protect information systems. The concept of trusted computing was first introduced into terminal virus immunity. Then a model of security domain mechanism based on trusted computing to protect computers from proposed from abstracting the general information systems. The principle of attack resistant and venture limitation of the model was demonstrated by means of mathematical analysis, and the realization of the model was proposed.

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

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Pratibha; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.in

    2015-08-15

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

  16. Structure discrimination for the C-terminal domain of Escherichia coli trigger factor in solution

    International Nuclear Information System (INIS)

    NMR measurements can give important information on solution structure, without the necessity for a full-scale solution structure determination. The C-terminal protein binding domain of the ribosome-associated chaperone protein trigger factor is composed of non-contiguous parts of the polypeptide chain, with an interpolated prolyl isomerase domain. A construct of the C-terminal domain of Escherichia coli trigger factor containing residues 113-149 and 247-432, joined by a Gly-Ser-Gly-Ser linker, is well folded and gives excellent NMR spectra in solution. We have used NMR measurements on this construct, and on a longer construct that includes the prolyl isomerase domain, to distinguish between two possible structures for the C-terminal domain of trigger factor, and to assess the behavior of the trigger factor C-terminal domain in solution. Two X-ray crystal structures, of intact trigger factor from E. coli (Ferbitz et al., Nature 431:590-596, 2004), and of a truncated trigger factor from Vibrio cholerae (Ludlam et al., Proc Natl Acad Sci USA 101:13436-13441, 2004) showed significant differences in the structure of the C-terminal domain, such that the two structures could not be superimposed. We show using NMR chemical shifts and long range nuclear Overhauser effects that the secondary and tertiary structure of the E. coli C-terminal domain in solution is consistent with the crystal structure of the E. coli trigger factor and not with the V. cholerae protein. Given the similarity of the amino acid sequences of the E. coli and V. cholerae proteins, it appears likely that the structure of the V. cholerae protein has been distorted as a result of truncation of a 44-amino acid segment at the C-terminus. Analysis of residual dipolar coupling measurements shows that the overall topology of the solution structure is completely inconsistent with both structures. Dynamics analysis of the C-terminal domain using T1, T2 and heteronuclear NOE parameters show that the protein is

  17. Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain

    Directory of Open Access Journals (Sweden)

    Spaan Willy JM

    2009-05-01

    Full Text Available Abstract Background Hepatitis C virus (HCV induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD of NS4B is highly conserved in Hepaciviruses, though its function remains unknown. Results A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH. The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system. Conclusion Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

  18. THz time-domain spectroscopy of amino acids

    Institute of Scientific and Technical Information of China (English)

    WANG Weining; YUE Weiwei; YAN Haitao; ZHANG Cunlin; ZHAO Guozhong

    2005-01-01

    The optical characteristics of four kinds of amino acids (tyrosine, arginine, histidine and glutamine) filled with nitrogen at room temperature were studied by THz time-domain spectroscopy (THz-TDS). Well-resolved absorption and refractive spectrums between 0.1 and 2.8 THz were obtained based on the physical model for extracting the optical parameters of materials in THz range. The results not only fill up the spectra gap of amino acids in far-infrared range, supply data for amino acid molecular identification and conformation analysis, but also demonstrate significantly potential to promote the research and application of biological materials in bio-chemical and medical fields by THz-TDS.

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

    Science.gov (United States)

    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.

  20. Crystallization of the C-terminal domain of the bacteriophage T7 fibre protein gp17

    International Nuclear Information System (INIS)

    The C-terminal domain of the bacteriophage T7 fibre protein gp17, consisting of amino acids 371–553, has been crystallized. Diffraction data have been obtained to around 2.0 Å resolution from two different crystal forms. Multiwavelength anomalous dispersion phasing with a mercury derivative is in progress. Bacteriophage T7 attaches to its host using the C-terminal domains of its six fibres, which are trimers of the gp17 protein. A C-terminal fragment of gp17 consisting of amino acids 371–553 has been expressed, purified and crystallized. Crystals of two forms were obtained, belonging to space group P212121 (unit-cell parameters a = 61.2, b = 86.0, c = 118.4 Å) and space group C2221 (unit-cell parameters a = 68.3, b = 145.6, c = 172.1 Å). They diffracted to 1.9 and 2.0 Å resolution, respectively. Both crystals are expected to contain one trimer in the asymmetric unit. Multiwavelength anomalous dispersion phasing with a mercury derivative is in progress

  1. cDNA cloning of the Octopus dofleini hemocyanin: sequence of the carboxyl-terminal domain.

    Science.gov (United States)

    Lang, W H

    1988-09-20

    A cDNA library was constructed in pUC 19, using poly(A+) RNA purified from Octopus dofleini branchial gland, which is the site of hemocyanin biosynthesis in cephalopods. The library was screened with an oligonucleotide probe derived from a portion of the partially known sequence of the C-terminal domain of Paroctopus dofleini dofleini. The clone with the longest insert--called pHC1--was sequenced and used as a probe for Northern blotting. It hybridized to a 9.5-kb RNA species, which was also visible as a band after ethidium bromide staining. The cDNA insert (approximately 1200 bp) of pHC1 contained an open reading frame of 1071 bp coding for 357 amino acids. In this insert, a region coding for 42 amino acids from the N-terminal end of the C-terminal domain is missing. These were obtained by sequencing a cloned primer extension product. By comparing our sequence with Helix pomatia beta c-hemocyanin unit D, we found 42.9% identical and 11.5% similar residues. One putative copper binding site (site B) was identified by homology to Helix hemocyanin and arthropodan hemocyanin. The location of a second possible site was identified. PMID:3207675

  2. Conserved C-terminal nascent peptide binding domain of HYPK facilitates its chaperone-like activity

    Indian Academy of Sciences (India)

    Swasti Raychaudhuri; Rachana Banerjee; Subhasish Mukhopadhyay; Nitai P Bhattacharyya

    2014-09-01

    Human HYPK (Huntingtin Yeast-two-hybrid Protein K) is an intrinsically unstructured chaperone-like protein with no sequence homology to known chaperones. HYPK is also known to be a part of ribosome-associated protein complex and present in polysomes. The objective of the present study was to investigate the evolutionary influence on HYPK primary structure and its impact on the protein’s function. Amino acid sequence analysis revealed 105 orthologs of human HYPK from plants, lower invertebrates to mammals. C-terminal part of HYPK was found to be particularly conserved and to contain nascent polypeptide-associated alpha subunit (NPAA) domain. This region experiences highest selection pressure, signifying its importance in the structural and functional evolution. NPAA domain of human HYPK has unique amino acid composition preferring glutamic acid and happens to be more stable from a conformational point of view having higher content of -helices than the rest. Cell biology studies indicate that overexpressed C-terminal human HYPK can interact with nascent proteins, co-localizes with huntingtin, increases cell viability and decreases caspase activities in Huntington’s disease (HD) cell culture model. This domain is found to be required for the chaperone-like activity of HYPK in vivo. Our study suggested that by virtue of its flexibility and nascent peptide binding activity, HYPK may play an important role in assisting protein (re)folding.

  3. The N-terminal domain of apolipoprotein B-100: structural characterization by homology modeling

    Directory of Open Access Journals (Sweden)

    Khachfe Hassan M

    2007-07-01

    Full Text Available Abstract Background Apolipoprotein B-100 (apo B-100 stands as one of the largest proteins in humans. Its large size of 4536 amino acids hampers the production of X-ray diffraction quality crystals and hinders in-solution NMR analysis, and thus necessitates a domain-based approach for the structural characterization of the multi-domain full-length apo B. Results The structure of apo B-17 (the N-terminal 17% of apolipoprotein B-100 was predicted by homology modeling based on the structure of the N-terminal domain of lipovitellin (LV, a protein that shares not only sequence similarity with B17, but also a functional aspect of lipid binding and transport. The model structure was first induced to accommodate the six disulfide bonds found in that region, and then optimized using simulated annealing. Conclusion The content of secondary structural elements in this model structure correlates well with the reported data from other biophysical probes. The overall topology of the model conforms with the structural outline corresponding to the apo B-17 domain as seen in the EM representation of the complete LDL structure.

  4. Structure of the C-terminal domain of nsp4 from feline coronavirus

    Energy Technology Data Exchange (ETDEWEB)

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany); Snijder, Eric J.; Gorbalenya, Alexander E. [Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden (Netherlands); Berglind, Hanna; Nordlund, Pär [Division of Biophysics, Department of Medical Biochemistry and Biophysics, Scheeles väg 2, Karolinska Institute, SE-171 77 Stockholm (Sweden); Coutard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098, AFMB-CNRS-ESIL, Case 925, 163 Avenue de Luminy, 13288 Marseille (France); Tucker, Paul A., E-mail: tucker@embl-hamburg.de [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany)

    2009-08-01

    The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.

  5. Mapping C-terminal transactivation domains of the nuclear HER family receptor tyrosine kinase HER3.

    Directory of Open Access Journals (Sweden)

    Toni M Brand

    Full Text Available Nuclear localized HER family receptor tyrosine kinases (RTKs have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3 have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B₁ and 27 (B₂ amino acids in length that conferred the majority of HER3's transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B₁ and B₂ regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B₁ and B₂ regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs.

  6. Interfering amino terminal peptides and functional implications for heteromeric gap junction formation

    Directory of Open Access Journals (Sweden)

    Richard David Veenstra

    2013-05-01

    Full Text Available Connexin43 (Cx43 is widely expressed in many different tissues of the human body. In cells of some organs, Cx43 is co-expressed with other connexins (Cx, including Cx46 and Cx50 in lens, Cx40 in atrium, Purkinje fibers, and the blood vessel wall, Cx45 in heart, and Cx37 in the ovary. Interactions with the co-expressed connexins may have profound functional implications. The abilities of Cx37, Cx45, Cx46, and Cx50 to function in heteromeric gap junction combinations with Cx43 are well documented. Different studies disagree regarding the ability of Cx43 and Cx40 to produce functional heteromeric gap junctions with each other. We review previous studies regarding the heteromeric interactions of Cx43. The possibility of negative functional interactions between the cytoplasmic pore-forming amino terminal (NT domains of these connexins was assessed using pentameric connexin sequence-specific NT domain (iNT peptides applied to cells expressing homomeric Cx40, Cx37, Cx45, Cx46, and Cx50 gap junctions. A Cx43 iNT peptide corresponding to amino acids 9 to 13 (Ac-KLLDK-NH2 specifically inhibited the electrical coupling of Cx40 gap junctions in a transjunctional (Vj voltage-dependent manner without affecting the function of homologous Cx37, Cx46, Cx50, and Cx45 gap junctions. A Cx40 iNT (Ac-EFLEE-OH peptide counteracted the Vj-dependent block of Cx40 gap junctions, whereas a similarly charged Cx50 iNT (Ac-EEVNE-OH peptide did not, suggesting that these NT domain interactions are not solely based on electrostatics. These data are consistent with functional Cx43 heteromeric gap junction formation with Cx37, Cx45, Cx46, and Cx50 and suggest that Cx40 uniquely experiences functional suppressive interactions with a Cx43 NT domain sequence. These findings present unique functional implications about the heteromeric interactions between Cx43 and Cx40 that may influence cardiac conduction in atrial myocardium and the specialized conduction system.

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

    International Nuclear Information System (INIS)

    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. - 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

  8. Leishmania donovani Nucleoside Hydrolase terminal domains in cross-protective immunotherapy against Leishmania amazonensis murine infection

    Directory of Open Access Journals (Sweden)

    Dirlei eNico

    2014-06-01

    Full Text Available Nucleoside hydrolases of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. Leishmania donovani Nucleoside hydrolase (NH36 induced a main CD4+ T cell driven protective response against Leishmania chagasi infection in mice which is directed against its C-terminal domain. In this study, we used the three recombinant domains of NH36: N-terminal domain (F1, amino acids 1-103, central domain (F2 aminoacids 104-198 and C-terminal domain (F3 amino acids 199-314 in combination with saponin and assayed their immunotherapeutic effect on Balb/c mice previously infected with L. amazonensis. We identified that the F1 and F3 peptides determined strong cross-immunotherapeutic effects, reducing the size of footpad lesions to 48% and 64%, and the parasite load in footpads to 82.6% and 81%, respectively. The F3 peptide induced the strongest anti-NH36 antibody response and intradermal response (IDR against L. amazonenis and a high secretion of IFN-γ and TNF-α with reduced levels of IL-10. The F1 vaccine, induced similar increases of IgG2b antibodies and IFN-γ and TNF-α levels, but no IDR and no reduction of IL-10. The multiparameter flow cytometry analysis was used to assess the immune response after immunotherapy and disclosed that the degree of the immunotherapeutic effect is predicted by the frequencies of the CD4+ and CD8+ T cells producing IL-2 or TNF-α or both. Total frequencies and frequencies of double-cytokine CD4 T cell producers were enhanced by F1 and F3 vaccines. Collectively, our multifunctional analysis disclosed that immunotherapeutic protection improved as the CD4 responses progressed from 1+ to 2+, in the case of the F1 and F3 vaccines, and as the CD8 responses changed qualitatively from 1+ to 3+, mainly in the case of the F1 vaccine, providing new correlates of immunotherapeutic protection against cutaneous leishmaniasis in mice based on T-helper TH1 and CD8+ mediated

  9. A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; McCarthy, J B; Furcht, L T;

    1993-01-01

    of focal adhesion and stress fiber formation requires additional interactions. Heparin-binding fragments of fibronectin can provide this signal. The COOH-terminal heparin-binding domain of fibronectin contains five separate heparin-binding amino acid sequences. We show here that all five sequences...... PRARI. The biological response to this peptide and to the COOH-terminal fragment may be mediated through cell surface heparan sulfate proteoglycans because treatment of cells with heparinase II and III, or competition with heparin, reduces the response. Treatment with chondroitinase ABC or competition...

  10. Targeted amino-terminal acetylation of recombinant proteins in E. coli.

    Directory of Open Access Journals (Sweden)

    Matthew Johnson

    Full Text Available One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. By co-expressing the fission yeast NatB complex with the target protein in E.coli, we report a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.

  11. Role of PHD fingers and COOH-terminal 30 amino acids in AIRE transactivation activity.

    Science.gov (United States)

    Meloni, Alessandra; Incani, Federica; Corda, Denise; Cao, Antonio; Rosatelli, Maria Cristina

    2008-02-01

    Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomic autoimmune disease resulting from the defective function of a gene codifying for a transcription factor named autoimmune regulation (AIRE). The AIRE protein contains several domains among which two PHD fingers involved in the transcriptional activation. We investigated the function of the two PHD finger domains and the COOH terminal portion of AIRE by using several mutated constructs transfected in mammalian cells and a luciferase reporter assay. The results predict that the second PHD as well as the COOH terminal regions have marked transactivational properties. The COOH terminal region contains the fourth LXXLL and the PXXPXP motifs which play a critical role in mediating the transactivation capacity of the AIRE protein. Our study provides a definition of the role of the PHD fingers in transactivation and identifies a new transactivation domain of the AIRE protein localized in the COOH terminal region.

  12. Leptospira Immunoglobulin-Like Protein B (LigB) Binds to Both the C-Terminal 23 Amino Acids of Fibrinogen αC Domain and Factor XIII: Insight into the Mechanism of LigB-Mediated Blockage of Fibrinogen α Chain Cross-Linking.

    Science.gov (United States)

    Hsieh, Ching-Lin; Chang, Eric; Tseng, Andrew; Ptak, Christopher; Wu, Li-Chen; Su, Chun-Li; McDonough, Sean P; Lin, Yi-Pin; Chang, Yung-Fu

    2016-09-01

    The coagulation system provides a primitive but effective defense against hemorrhage. Soluble fibrinogen (Fg) monomers, composed of α, β and γ chains, are recruited to provide structural support for the formation of a hemostatic plug. Fg binds to platelets and is processed into a cross-linked fibrin polymer by the enzymatic clotting factors, thrombin and Factor XIII (FXIII). The newly formed fibrin-platelet clot can act as barrier to protect against pathogens from entering the bloodstream. Further, injuries caused by bacterial infections can be confined to the initial wound site. Many pathogenic bacteria have Fg-binding adhesins that can circumvent the coagulation pathway and allow the bacteria to sidestep containment. Fg expression is upregulated during lung infection providing an attachment surface for bacteria with the ability to produce Fg-binding adhesins. Fg binding by leptospira might play a crucial factor in Leptospira-associated pulmonary hemorrhage, the main factor contributing to lethality in severe cases of leptospirosis. The 12th domain of Leptospira immunoglobulin-like protein B (LigB12), a leptospiral adhesin, interacts with the C-terminus of FgαC (FgαCC). In this study, the binding site for LigB12 was mapped to the final 23 amino acids at the C-terminal end of FgαCC (FgαCC8). The association of FgαCC8 with LigB12 (ELISA, KD = 0.76 μM; SPR, KD = 0.96 μM) was reduced by mutations of both charged residues (R608, R611 and H614 from FgαCC8; D1061 from LigB12) and hydrophobic residues (I613 from FgαCC8; F1054 and A1065 from LigB12). Additionally, LigB12 bound strongly to FXIII and also inhibited fibrin formation, suggesting that LigB can disrupt coagulation by suppressing FXIII activity. Here, the detailed binding mechanism of a leptospiral adhesin to a host hemostatic factor is characterized for the first time and should provide better insight into the pathogenesis of leptospirosis. PMID:27622634

  13. The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting to chromatin.

    Science.gov (United States)

    Bao, Xiaomin; Cai, Weili; Deng, Huai; Zhang, Weiguo; Krencik, Robert; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M

    2008-11-21

    The JIL-1 histone H3S10 kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is enriched 2-fold on the male X chromosome. JIL-1 can be divided into four main domains including an NH(2)-terminal domain, two separate kinase domains, and a COOH-terminal domain. Our results demonstrate that the COOH-terminal domain of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes but that both COOH- and NH(2)-terminal sequences are necessary for enrichment on the male X chromosome. We furthermore show that a small 53-amino acid region within the COOH-terminal domain can interact with the tail region of histone H3, suggesting that this interaction is necessary for the correct chromatin targeting of the JIL-1 kinase. Interestingly, our data indicate that the COOH-terminal domain alone is sufficient to rescue JIL-1 null mutant polytene chromosome defects including those of the male X chromosome. Nonetheless, we also found that a truncated JIL-1 protein which was without the COOH-terminal domain but retained histone H3S10 kinase activity was able to rescue autosome as well as partially rescue male X polytene chromosome morphology. Taken together these findings indicate that JIL-1 may participate in regulating chromatin structure by multiple and partially redundant mechanisms.

  14. Downstream signaling mechanism of the C-terminal activation domain of transcriptional coactivator CoCoA

    OpenAIRE

    Kim, Jeong Hoon; Yang, Catherine K.; Stallcup, Michael R.

    2006-01-01

    The coiled-coil coactivator (CoCoA) is a transcriptional coactivator for nuclear receptors and enhances nuclear receptor function by the interaction with the bHLH-PAS domain (AD3) of p160 coactivators. The C-terminal activation domain (AD) of CoCoA possesses strong transactivation activity and is required for the coactivator function of CoCoA with nuclear receptors. To understand how CoCoA AD transmits its activating signal to the transcription machinery, we defined specific subregions, amino...

  15. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

    DEFF Research Database (Denmark)

    Behrendt, N; Rønne, E; Ploug, M;

    1990-01-01

    The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u......, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported...... amino-terminal fragment; 2) the identical electrophoretic mobilities observed for cross-linked conjugates, formed between either the purified protein or the u-PA receptor on intact U937 cells and the above ligands; 3) the identity of the apparent molecular weight of the purified protein...

  16. Crystal Structure of the C-terminal Domain of Splicing Factor Prp8 Carrying Retinitis Pigmentosa Mutants

    Energy Technology Data Exchange (ETDEWEB)

    Zhang,L.; Shen, J.; Guarnieri, M.; Heroux, A.; Yang, K.; Zhao, R.

    2007-01-01

    Prp8 is a critical pre-mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been hindered by its large size (over 2000 amino acids) and remarkably low-sequence similarity with other proteins. Here we present the crystal structure of the C-terminal domain (the last 273 residues) of Caenorhabditis elegans Prp8 (cPrp8). The core of the C-terminal domain is an / structure that forms the MPN (Mpr1, Pad1 N-terminal) fold but without Zn{sup 2+} coordination. We propose that the C-terminal domain is a protein interaction domain instead of a Zn{sup 2+}-dependent metalloenzyme as proposed for some MPN proteins. Mapping of RP13 mutants on the Prp8 structure suggests that these residues constitute a binding surface between Prp8 and other partner(s), and the disruption of this interaction provides a plausible molecular mechanism for RP13.

  17. Structure and function of the carboxyl-terminal oxygen-binding domain from the subunit of Octopus dofleini hemocyanin.

    Science.gov (United States)

    Miller, K I; van Holde, K E; Toumadje, A; Johnson, W C; Lamy, J

    1988-09-20

    The C-terminal domain, Od-1, of the 7-domain subunit of Octopus dofleini hemocyanin has been prepared by partial trypsinolysis followed by ion-exchange chromatography. It binds oxygen reversibly and is homogeneous in molecular weight. Its physical properties have been compared with those of the subunit. The domain molecular weight is found by sedimentation equilibrium to be 4.7 X 10(4), in excellent agreement with the result recently obtained in our laboratory from cDNA sequencing of this domain [Lang, W. H. (1988) Biochemistry (preceding paper in this issue)]. It has a sedimentation coefficient of 3.8 S. Both the molecular weight and sedimentation coefficient are consistent with the domain constituting approximately one-seventh of the Mr 3.5 X 10(5) subunit. Its amino acid composition and carbohydrate content differ significantly from that of the whole subunit, confirming the heterogeneity in domains previously established on an immunological basis. Circular dichroism predicts similar secondary structure for the domain and subunit. The domain does not self-associate in the presence of Mg2+ but does bind to the whole molecule in a ratio of approximately 1 domain/subunit. The oxygen affinity of this domain is quite low. It shows intrinsic magnesium and Bohr effects similar to those of the whole molecule but of greatly reduced magnitude. PMID:3207676

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

    OpenAIRE

    Dwivedi, Gajendradhar R.; Kolluru D Srikanth; Praveen Anand; Javed Naikoo; 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 ...

  19. Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

    International Nuclear Information System (INIS)

    The three-dimensional structure of the N-terminal SH3 domain (residues 583-660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies

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

    Science.gov (United States)

    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.

  1. N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

    Institute of Scientific and Technical Information of China (English)

    Yan-Chang Lei; Dong-Liang Yang; Yong-Jun Tian; Hong-Hui Ding; Bao-Ju Wang; Yan Yang; You-Hua Hao; Xi-Ping Zhao; Meng-Ji Lu; Fei-Li Gong

    2006-01-01

    AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G(APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo.METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA.The expression of hepatitis B virus core antigen (HBcAg)in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively.RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells,and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly,the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls.CONCLUSION: Our findings provide probably the first

  2. Amino-terminal p53 mutations lead to expression of apoptosis proficient p47 and prognosticate better survival, but predispose to tumorigenesis

    OpenAIRE

    Phang, Beng Hooi; Othman, Rashidah; Bougeard, Gaelle; Chia, Ren Hui; Frebourg, Thierry; Tang, Choong Leong; Cheah, Peh Yean; Sabapathy, Kanaga

    2015-01-01

    Mutations in the amino-terminal transactivation domain of the tumor-suppressor p53 are mostly insertions or deletions, and result in loss of full-length p53 expression. However, these changes concomitantly result in the expression of a truncated p47 isoform, which retains the ability to selectively transactivate some apoptotic target genes. The selectivity appears to be due to a default feature, stemming from the lack of acetylation on K382 at the carboxyl terminus, which requires the amino t...

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

    Science.gov (United States)

    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).

  4. Conformational effects of a common codon 751 polymorphism on the C-terminal domain of the xeroderma pigmentosum D protein

    Directory of Open Access Journals (Sweden)

    Monaco Regina

    2009-01-01

    Full Text Available Aim: The xeroderma pigmentosum D (XPD protein is a DNA helicase involved in the repair of DNA damage, including nucleotide excision repair (NER and transcription-coupled repair (TCR. The C-terminal domain of XPD has been implicated in interactions with other components of the TFIIH complex, and it is also the site of a common genetic polymorphism in XPD at amino acid residue 751 (Lys->Gln. Some evidence suggests that this polymorphism may alter DNA repair capacity and increase cancer risk. The aim of this study was to investigate whether these effects could be attributable to conformational changes in XPD induced by the polymorphism. Materials and Methods: Molecular dynamics techniques were used to predict the structure of the wild-type and polymorphic forms of the C-terminal domain of XPD and differences in structure produced by the polymorphic substitution were determined. Results: The results indicate that, although the general configuration of both proteins is similar, the substitution produces a significant conformational change immediately N-terminal to the site of the polymorphism. Conclusion: These results provide support for the hypothesis that this polymorphism in XPD could affect DNA repair capability, and hence cancer risk, by altering the structure of the C-terminal domain.

  5. The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

    Directory of Open Access Journals (Sweden)

    Noonan James P

    2001-07-01

    Full Text Available Abstract Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10 or PML (promyelocytic leukemia nuclear bodies, where it associates with TOPIIIα, and to the nucleolus. Results This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities. Conclusion The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies.

  6. N-terminal PDZ-like domain of chromatin organizer SATB1 contributes towards its function as transcription regulator

    Indian Academy of Sciences (India)

    Dimple Notani; Praveena L Ramanujam; P Pavan Kumar; Kamalvishnu P Gottimukkala; Chandan Kumar-Sinha; Sanjeev Galande

    2011-08-01

    The special AT-rich DNA-binding protein 1 (SATB1) is a matrix attachment region (MAR)-binding protein that acts as a global repressor via recruitment of CtBP1:HDAC1-containing co-repressors to its binding targets. The N-terminal PSD95/Dlg-A/ZO-1 (PDZ)-like domain of SATB1 mediates interactions with several chromatin proteins. In the present study, we set out to address whether the PDZ-domain-mediated interactions of SATB1 are critical for its in vivo function as a global repressor. We reasoned that since the N-terminal PDZ-like domain (amino acid residues 1–204) lacks DNA binding activity, it would fail to recruit the interacting partners of SATB1 to its genomic binding sites and hence would not repress the SATB1-regulated genes. Indeed, in vivo MAR-linked luciferase reporter assay revealed that overexpression of the PDZ-like domain resulted in de-repression, indicating that the PDZ-like domain exerts a dominant negative effect on genes regulated by SATB1. Next, we developed a stable dominant negative model in human embryonic kidney (HEK) 293T cells that conditionally expressed the N-terminal 1–204 region harbouring the PDZ-like domain of SATB1. To monitor the effect of sequestration of the interaction partners on the global gene regulation by SATB1, transcripts from the induced and uninduced clones were subjected to gene expression profiling. Clustering of expression data revealed that 600 out of 19000 genes analysed were significantly upregulated upon overexpression of the PDZ-like domain. Induced genes were found to be involved in important signalling cascades and cellular functions. These studies clearly demonstrated the role of PDZ domain of SATB1 in global gene regulation presumably through its interaction with other cellular proteins.

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

    Science.gov (United States)

    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

  8. Segments in the C-terminal folding domain of lipoprotein lipase important for binding to the low density lipoprotein receptor-related protein and to heparan sulfate proteoglycans

    DEFF Research Database (Denmark)

    Nielsen, Morten Schallburg; Brejning, Jeanette; García, R.;

    1997-01-01

    -terminal folding domain binds to alpha2MR/LRP, it remains uncertain whether it binds to heparin and to HSPG. To identify segments important for binding to alpha2MR/LRP and to clarify possible binding to heparin, we produced constructs of the human C-terminal folding domain, LpL-(313-448), and of the fragment Lp....../LRP was essentially abolished following deletion of residues 404-430, and pretreatment of CHO cells with the peptide comprising aa 402-423 inhibited the binding of LpL-(313-448). We conclude that the C-terminal folding domain of human LpL has a site for binding to heparin and to HSPG, presumably involving amino acids...... within residues 404-430. Two segments of the domain are necessary for efficient binding to alpha2MR/LRP, one comprising residues 380-384 and another overlapping the segment important for binding to heparin....

  9. Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics.

    Science.gov (United States)

    Kountz, Timothy S; Lee, Kyung-Soon; Aggarwal-Howarth, Stacey; Curran, Elizabeth; Park, Ji-Min; Harris, Dorathy-Ann; Stewart, Aaron; Hendrickson, Joseph; Camp, Nathan D; Wolf-Yadlin, Alejandro; Wang, Edith H; Scott, John D; Hague, Chris

    2016-08-26

    The α1D-adrenergic receptor (ADRA1D) is a key regulator of cardiovascular, prostate, and central nervous system functions. This clinically relevant G protein-coupled receptor has proven difficult to study, as it must form an obligate modular homodimer containing the PDZ proteins scribble and syntrophin or become retained in the endoplasmic reticulum as non-functional protein. We previously determined that targeted removal of the N-terminal (NT) 79 amino acids facilitates ADRA1D plasma membrane expression and agonist-stimulated functional responses. However, whether such an event occurs in physiological contexts was unknown. Herein, we report the ADRA1D is subjected to innate NT processing in cultured human cells. SNAP near-infrared imaging and tandem-affinity purification revealed the ADRA1D is expressed as both full-length and NT truncated forms in multiple human cell lines. Serial truncation mapping identified the cleavage site as Leu(90)/Val(91) in the 95-amino acid ADRA1D NT domain, suggesting human cells express a Δ1-91 ADRA1D species. Tandem-affinity purification MS/MS and co-immunoprecipitation analysis indicate NT processing of ADRA1D is not required to form scribble-syntrophin macromolecular complexes. Yet, label-free dynamic mass redistribution signaling assays demonstrate that Δ1-91 ADRA1D agonist responses were greater than WT ADRA1D. Mutagenesis of the cleavage site nullified the processing event, resulting in ADRA1D agonist responses less than the WT receptor. Thus, we propose that processing of the ADRA1D NT domain is a physiological mechanism employed by cells to generate a functional ADRA1D isoform with optimal pharmacodynamic properties. PMID:27382054

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

    Science.gov (United States)

    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. Rational Molecular Design of Potent PLK1 PBD Domain-binding Phosphopeptides Using Preferential Amino Acid Building Blocks.

    Science.gov (United States)

    Mao, Xin-Li; Wang, Kui-Feng; Zhu, Feng; Pan, Zhao-Hu; Wu, Guo-Min; Zhu, Hong-Yuan

    2016-08-01

    Polo-like kinase 1 (PLK1) is an important regulator in diverse aspects of the cell cycle and proliferation. The protein has a highly conserved polo-box domain (PBD) present in C-terminal noncatalytic region, which exhibits a relatively broad sequence specificity in recognizing and binding phosphorylated substrates to control substrate phosphorylation by the kinase. In order to elucidate the structural basis, thermodynamic property, and biological implication underlying PBD-substrate recognition and association, a systematic amino acid preference profile of phosphopeptide interaction with PLK1 PBD domain was established via virtual mutagenesis analysis and mutation energy calculation, from which the contribution of different amino acids at each residue position of two reference phosphopeptides to domain-peptide binding was characterized comprehensively and quantitatively. With the profile, we are able to determine the favorable, neutral, and unfavorable amino acid types for each position of PBD-binding phosphopeptides, and we also explored the molecular origin of the broad sequence specificity in PBD-substrate recognition. To practice computational findings, the profile was further employed to guide rational design of potent PBD binders; three 6-mer phosphopeptides (i.e., IQSpSPC, LQSpTPF, and LNSpTPT) were successfully developed, which can efficiently target PBD domain with high affinity (Kd = 5.7 ± 1.1, 0.75 ± 0.18, and 7.2 ± 2.6 μm, resp.) as measured by a fluorescence anisotropy assay. The complex structure of PLK1 PBD domain with a newly designed, potent phosphopeptide LQSpTPF as well as diverse noncovalent chemical forces, such as H-bonds and hydrophobic interactions at the complex interface, were examined in detail to reveal the molecular mechanism of high affinity and stability of the complex system.

  14. Functional analysis of the NH2-terminal hydrophobic region and BRICHOS domain of GKN1

    International Nuclear Information System (INIS)

    Highlights: •NH2-terminal and BRICHOS domain of GKN1 inhibited tumor cell growth. •NH2-terminal and BRICHOS domain of GKN1 regulated cell cycle. •NH2-terminal and BRICHOS domain of GKN1 inhibited epigenetic regulators. -- Abstract: Gastrokine 1 (GKN1) protects the gastric antral mucosa and promotes healing by facilitating restitution and proliferation after injury. GKN1 is down-regulated in Helicobacter pylori-infected gastric epithelial cells and loss of GKN1 expression is tightly associated with gastric carcinogenesis. However, the underlying mechanisms as a tumor suppressor are largely unknown. Presently, the hydrophobic region and BRICHOS domain of GKN1, pGKN1D13N, pGKN1Δ68–199, and pGKN1Δ1–67,165–199 were shown to suppress gastric cancer cell growth and recapitulate GKN1 functions. As well, the hydrophobic region and BRICHOS domain of GKN1 had a synergistic anti-cancer effect with 5-FU on tumor cell growth, implying that the NH2-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for tumor suppression, thereby suggesting a therapeutic intervention for gastric cancer. Also, its domain inducing endogenous miR-185 directly targeted the epigenetic effectors DNMT1 and EZH2 in gastric cancer cells. Our results suggest that the NH2-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for its tumor suppressor activities

  15. Synthesis of Novel Cellulose Carbamates Possessing Terminal Amino Groups and Their Bioactivity.

    Science.gov (United States)

    Ganske, Kristin; Wiegand, Cornelia; Hipler, Uta-Christina; Heinze, Thomas

    2016-03-01

    Cellulose phenyl carbonates are an excellent platform to synthesize a broad variety of soluble and functional cellulose carbamates. In this study, the synthesis of cellulose carbamates with terminal amino groups, namely ω-aminoethylcellulose- and ω-aminoethyl-p-aminobenzyl-cellulose carbamate, is discussed. The products are well soluble and their structures can be clearly described by NMR spectroscopy. The cellulose carbamates exhibit a bactericide and fungicide activity in vitro. The ω-aminoethylcellulose carbamate possesses a strong activity against Candida albicans and Staphylococcus aureus (IC50 of 0.02 mg mL(-1) and 0.05 mg mL(-1)). The antimicrobial activity and cytotoxicity can be improved by p-amino-benzylamine (ABA) as an additional substituent. The mixed cellulose carbamate exhibits a high biocompatibility (LC50 of 3.18 mg mL(-1)) and forms films on cotton and PES, which exhibit a strong activity against S. aureus and Klebsiella pneumoniae. PMID:26612063

  16. Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.

    Directory of Open Access Journals (Sweden)

    Jorge Fernández-Trillo

    Full Text Available A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.

  17. The cyanobacterial cell division factor Ftn6 contains an N-terminal DnaD-like domain

    Directory of Open Access Journals (Sweden)

    Saguez Cyril

    2009-08-01

    Full Text Available Abstract Background DNA replication and cell cycle as well as their relationship have been extensively studied in the two model organisms E. coli and B. subtilis. By contrast, little is known about these processes in cyanobacteria, even though they are crucial to the biosphere, in utilizing solar energy to renew the oxygenic atmosphere and in producing the biomass for the food chain. Recent studies have allowed the identification of several cell division factors that are specifics to cyanobacteria. Among them, Ftn6 has been proposed to function in the recruitment of the crucial FtsZ proteins to the septum or the subsequent Z-ring assembly and possibly in chromosome segregation. Results In this study, we identified an as yet undescribed domain located in the conserved N-terminal region of Ftn6. This 77 amino-acids-long domain, designated here as FND (Ftn6 N-Terminal Domain, exhibits striking sequence and structural similarities with the DNA-interacting module, listed in the PFAM database as the DnaD-like domain (pfam04271. We took advantage of the sequence similarities between FND and the DnaD-like domains to construct a homology 3D-model of the Ftn6 FND domain from the model cyanobacterium Synechocystis PCC6803. Mapping of the conserved residues exposed onto the FND surface allowed us to identify a highly conserved area that could be engaged in Ftn6-specific interactions. Conclusion Overall, similarities between FND and DnaD-like domains as well as previously reported observations on Ftn6 suggest that FND may function as a DNA-interacting module thereby providing an as yet missing link between DNA replication and cell division in cyanobacteria. Consistently, we also showed that Ftn6 is involved in tolerance to DNA damages generated by UV rays.

  18. The impact of the human DNA topoisomerase II C-terminal domain on activity.

    Directory of Open Access Journals (Sweden)

    Emma L Meczes

    Full Text Available BACKGROUND: Type II DNA topoisomerases (topos are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, alpha and beta, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity. METHODOLOGY/PRINCIPLE FINDINGS: We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIalpha and beta and topoIIalpha+beta-tail and topoIIbeta+alpha-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIalpha-CTD increasing activity, and the topoIIbeta-CTD decreasing activity. CONCLUSIONS/SIGNIFICANCE: In vivo complementation data show that the topoIIalpha C-terminal domain is needed for growth, but the topoIIbeta isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIbeta has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIalpha or beta C-terminal domain can affect strand passage activity. Data indicates that the topoIIbeta-CTD may be a negative regulator. This is the first report of in vitro

  19. Mg and Mc: mutations within the amino-terminal region of glycophorin A.

    Science.gov (United States)

    Furthmayr, H; Metaxas, M N; Metaxas-Bühler, M

    1981-01-01

    M and N are the two common ("normal") alleles at the MN locus of the MNSs blood group system. The antigens M and N that they determine are located within the amino-terminal region of glycophorin A. In the serologically active and glycosylated (*) fragment of glycophorin AN the sequence is Leu-Ser*-Thr*-Thr*-Glu-, and in that of glycophorin AM it is Ser-Ser*-Thr*-Thr*-Gly-. Mg and Mc are very rare ("variant") alleles of M and N; as to the corresponding antigens, Mg is serologically quite distinct from M and N, while Mc is a compound of both. Erythrocytes of genotypes MgN, MgM, MgMg, and McM, which were the object of the present study, contain normal amounts of glycophorin A in their membrane. In glycophorin AMg the amino-terminal sequence is related to that of glycophorin AN by substitution of asparagine for threonine in position 4, and it is nonglycosylated: Leu-Ser-Thr-Asn-Glu-. The corresponding structure of glycophorin AMc is Ser-Ser*-Thr*-Thr*-Glu-; it is thus closely related to that of glycophorin AN and AM, by substitution of the amino acids in positions 1 or 5, respectively. All of these substitutions can be explained by single base changes. The distinctions in chemical structure not only confirm the location of M and N in this region of glycophorin A, because they are the only differences observed, but also indicate, because they are correlated with the distinctions in antigenic specificity, that M and N are structural genes coding for amino acid sequences. The finding that Mc contains structural features of both M and N suggests that these two forms of glycophorin A have evolved from a common ancestral gene by single base substitutions at sites in the genome coding for amino acids in positions 1 and 5 of the sequence. Carbohydrate structures, however, are also necessary for full expression of antigens M and N. Glycosylation during biosynthesis of residues within the polypeptide appears to depend on a particular protein structure. PMID:6166001

  20. The carboxy-terminal domain of Dictyostelium C-module-binding factor is an independent gene regulatory entity.

    Directory of Open Access Journals (Sweden)

    Jörg Lucas

    Full Text Available The C-module-binding factor (CbfA is a multidomain protein that belongs to the family of jumonji-type (JmjC transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD. An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

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

    Science.gov (United States)

    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.

  2. NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling

    Energy Technology Data Exchange (ETDEWEB)

    Vourtsis, Dionysios J.; Chasapis, Christos T.; Pairas, George [Department of Pharmacy, University of Patras, GR-26504 Patras (Greece); Bentrop, Detlef [Institute of Physiology II, University of Freiburg, D-79104 Freiburg (Germany); Spyroulias, Georgios A., E-mail: G.A.Spyroulias@upatras.gr [Department of Pharmacy, University of Patras, GR-26504 Patras (Greece)

    2014-07-18

    Highlights: • A polypeptide, N-ALF{sub 233}, was overexpressed in E. coli and successfully isolated. • We produced {sup 2}H/{sup 15}N/{sup 13}C labeled protein samples. • Amino acid selective approaches were applied. • We acquired several heteronuclear NMR spectra, to complete the backbone assignment. • Prediction of the secondary structure was performed. - Abstract: NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successful overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.

  3. Thrombospondin-1-N-Terminal Domain Induces a Phagocytic State and Thrombospondin-1-C-Terminal Domain Induces a Tolerizing Phenotype in Dendritic Cells

    OpenAIRE

    Tabib, Adi; Krispin, Alon; Trahtemberg, Uriel; Verbovetski, Inna; Lebendiker, Mario; Danieli, Tsafi; Mevorach, Dror

    2009-01-01

    In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine ...

  4. Functional implications of C-terminus of TBX5 with high homology to C-terminal domain of yeast DNA-directed RNA polymerase Ⅱ largest subunit

    Institute of Scientific and Technical Information of China (English)

    ZHOU Zhu-ren; GONG Li-guo; GENG Wen-qing; QIU Guang-rong; SUN Kai-lai

    2008-01-01

    @@ TBX5, as a member of the T-box-containing transcription factor family, encodes a protein of 518 amino acids and is expressed in the embryonic heart and developing limb tissues.1 The coding region of TBX5 cDNA is 1.5 kb with eight exons including the N-terminal portion, the DNA binding domain and C-terminal region. We reported that the abnormality in transcription level of the TbX5 gene might be the mechanism underlying human simple congenital heart disease in the absence of TBX5 mutations.

  5. The C-terminal domain of 4-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii is an autoinhibitory domain.

    Science.gov (United States)

    Phongsak, Thanawat; Sucharitakul, Jeerus; Thotsaporn, Kittisak; Oonanant, Worrapoj; Yuvaniyama, Jirundon; Svasti, Jisnuson; Ballou, David P; Chaiyen, Pimchai

    2012-07-27

    p-Hydroxyphenylacetate (HPA) 3-hydroxylase from Acinetobacter baumannii consists of a reductase component (C(1)) and an oxygenase component (C(2)). C(1) catalyzes the reduction of FMN by NADH to provide FMNH(-) as a substrate for C(2). The rate of reduction of flavin is enhanced ∼20-fold by binding HPA. The N-terminal domain of C(1) is homologous to other flavin reductases, whereas the C-terminal domain (residues 192-315) is similar to MarR, a repressor protein involved in bacterial antibiotic resistance. In this study, three forms of truncated C(1) variants and single site mutation variants of residues Arg-21, Phe-216, Arg-217, Ile-246, and Arg-247 were constructed to investigate the role of the C-terminal domain in regulating C(1). In the absence of HPA, the C(1) variant in which residues 179-315 were removed (t178C(1)) was reduced by NADH and released FMNH(-) at the same rates as wild-type enzyme carries out these functions in the presence of HPA. In contrast, variants with residues 231-315 removed behaved similarly to the wild-type enzyme. Thus, residues 179-230 are involved in repressing the production of FMNH(-) in the absence of HPA. These results are consistent with the C-terminal domain in the wild-type enzyme being an autoinhibitory domain that upon binding the effector HPA undergoes conformational changes to allow faster flavin reduction and release. Most of the single site variants investigated had catalytic properties similar to those of the wild-type enzyme except for the F216A variant, which had a rate of reduction that was not stimulated by HPA. F216A could be involved with HPA binding or in the required conformational change for stimulation of flavin reduction by HPA. PMID:22661720

  6. Adsorption behavior of oxidized galactomannans onto amino terminated surfaces and their interaction with bovine serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Sierakowski, M.-R; Silva, Maria R.V. da [Universidade Federal do Parana, Curitiba, PR (Brazil). Dept. de Quimica. Lab. de Biopolimeros]. E-mail: mrbiopol@quimica.ufpr.br; Freitas, R.A.; Moreira, Jose S.R. [Universidade Federal do Parana, Curitiba, PR (Brazil). Dept. de Bioquimica; Fujimoto, J.; Petri, D.F.S.; Cordeiro, Paulo R.D. [Sao Paulo Univ., SP (Brazil). Inst. de Quimica]. E-mail: dfsp@quim.iq.usp.br; Andrade, Fabiana D

    2001-07-01

    A galactomannan (CF) extracted from Cassia fastuosa seeds was purified and oxidized with (2,2,6,6- tetramethylpiperidine-1-oxyl) to form a uronic acid-containing polysaccharide (CFOX) with a degree of oxidation (DO) of 0.22. The chemical structures of CF and CFOX were characterized. The adsorption behavior of CF and CFOX onto amino-terminated surfaces was studied by means of ellipsometric measurements. The influence of p H and ionic strength on the adsorption was also investigated. At p H 4, there was a maximum in the adsorbed amount caused by strong electrostatic attraction between the substrate and the oxidized galactomannans. There was no ionic strength effect on the adsorption behavior. The immobilization of bovine serum albumin onto CF and CFOX was studied as a function of p H. At the isoelectric point a maximum in the adsorbed amount was found. (author)

  7. Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

    Directory of Open Access Journals (Sweden)

    Raats Jos MH

    2009-07-01

    Full Text Available Abstract Background Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL. However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging. Results Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step. Conclusion A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

  8. Specific RNA binding by amino-terminal peptides of alfalfa mosaic virus coat protein.

    Science.gov (United States)

    Baer, M L; Houser, F; Loesch-Fries, L S; Gehrke, L

    1994-01-01

    Specific RNA-protein interactions and ribonucleoprotein complexes are essential for many biological processes, but our understanding of how ribonucleoprotein particles form and accomplish their biological functions is rudimentary. This paper describes the interaction of alfalfa mosaic virus (A1MV) coat protein or peptides with viral RNA. A1MV coat protein is necessary both for virus particle formation and for the initiation of replication of the three genomic RNAs. We have examined protein determinants required for specific RNA binding and analyzed potential structural changes elicited by complex formation. The results indicate that the amino-terminus of the viral coat protein, which lacks primary sequence homology with recognized RNA binding motifs, is both necessary and sufficient for binding to RNA. Circular dichroism spectra and electrophoretic mobility shift experiments suggest that the RNA conformation is altered when amino-terminal coat protein peptides bind to the viral RNA. The peptide--RNA interaction is functionally significant because the peptides will substitute for A1MV coat protein in initiating RNA replication. The apparent conformational change that accompanies RNA--peptide complex formation may generate a structure which, unlike the viral RNA alone, can be recognized by the viral replicase. Images PMID:8313916

  9. Tip-induced domain structures and polarization switching in ferroelectric amino acid glycine

    Science.gov (United States)

    Seyedhosseini, E.; Bdikin, I.; Ivanov, M.; Vasileva, D.; Kudryavtsev, A.; Rodriguez, B. J.; Kholkin, A. L.

    2015-08-01

    Bioorganic ferroelectrics and piezoelectrics are becoming increasingly important in view of their intrinsic compatibility with biological environment and biofunctionality combined with strong piezoelectric effect and a switchable polarization at room temperature. Here, we study tip-induced domain structures and polarization switching in the smallest amino acid β-glycine, representing a broad class of non-centrosymmetric amino acids. We show that β-glycine is indeed a room-temperature ferroelectric and polarization can be switched by applying a bias to non-polar cuts via a conducting tip of atomic force microscope (AFM). Dynamics of these in-plane domains is studied as a function of an applied voltage and pulse duration. The domain shape is dictated by polarization screening at the domain boundaries and mediated by growth defects. Thermodynamic theory is applied to explain the domain propagation induced by the AFM tip. Our findings suggest that the properties of β-glycine are controlled by the charged domain walls which in turn can be manipulated by an external bias.

  10. Tip-induced domain structures and polarization switching in ferroelectric amino acid glycine

    Energy Technology Data Exchange (ETDEWEB)

    Seyedhosseini, E., E-mail: Seyedhosseini@ua.pt; Ivanov, M. [CICECO-Aveiro Institute of Materials and Department of Physics, University of Aveiro, 3810-193 Aveiro (Portugal); Bdikin, I. [TEMA and Department of Mechanical Engineering, University of Aveiro, 3810-193 Aveiro (Portugal); Vasileva, D. [Institute of Natural Sciences, Ural Federal University, 620000 Ekaterinburg (Russian Federation); Kudryavtsev, A. [Moscow State Institute of Radioengineering, Electronics and Automation, 119454 Moscow (Russian Federation); Rodriguez, B. J. [Conway Institute of Biomolecular and Biomedical Research and School of Physics, University College Dublin, Dublin (Ireland); Kholkin, A. L. [CICECO-Aveiro Institute of Materials and Department of Physics, University of Aveiro, 3810-193 Aveiro (Portugal); Institute of Natural Sciences, Ural Federal University, 620000 Ekaterinburg (Russian Federation)

    2015-08-21

    Bioorganic ferroelectrics and piezoelectrics are becoming increasingly important in view of their intrinsic compatibility with biological environment and biofunctionality combined with strong piezoelectric effect and a switchable polarization at room temperature. Here, we study tip-induced domain structures and polarization switching in the smallest amino acid β-glycine, representing a broad class of non-centrosymmetric amino acids. We show that β-glycine is indeed a room-temperature ferroelectric and polarization can be switched by applying a bias to non-polar cuts via a conducting tip of atomic force microscope (AFM). Dynamics of these in-plane domains is studied as a function of an applied voltage and pulse duration. The domain shape is dictated by polarization screening at the domain boundaries and mediated by growth defects. Thermodynamic theory is applied to explain the domain propagation induced by the AFM tip. Our findings suggest that the properties of β-glycine are controlled by the charged domain walls which in turn can be manipulated by an external bias.

  11. Tip-induced domain structures and polarization switching in ferroelectric amino acid glycine

    International Nuclear Information System (INIS)

    Bioorganic ferroelectrics and piezoelectrics are becoming increasingly important in view of their intrinsic compatibility with biological environment and biofunctionality combined with strong piezoelectric effect and a switchable polarization at room temperature. Here, we study tip-induced domain structures and polarization switching in the smallest amino acid β-glycine, representing a broad class of non-centrosymmetric amino acids. We show that β-glycine is indeed a room-temperature ferroelectric and polarization can be switched by applying a bias to non-polar cuts via a conducting tip of atomic force microscope (AFM). Dynamics of these in-plane domains is studied as a function of an applied voltage and pulse duration. The domain shape is dictated by polarization screening at the domain boundaries and mediated by growth defects. Thermodynamic theory is applied to explain the domain propagation induced by the AFM tip. Our findings suggest that the properties of β-glycine are controlled by the charged domain walls which in turn can be manipulated by an external bias

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Kyle P Obergfell

    2016-05-01

    Full Text Available 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

  15. An amino-terminal variant of the central cannabinoid receptor resulting from alternative splicing.

    Science.gov (United States)

    Shire, D; Carillon, C; Kaghad, M; Calandra, B; Rinaldi-Carmona, M; Le Fur, G; Caput, D; Ferrara, P

    1995-02-24

    The cDNA sequences encoding the central cannabinoid receptor, CB1, are known for two species, rat and human. However, little information concerning the flanking, noncoding regions is presently available. We have isolated two overlapping clones from a human lung cDNA library with CB1 cDNA inserts. One of these, cann7, contains a short stretch of the CB1 coding region and 4 kilobase pairs (kb) of the 3'-untranslated region (UTR), including two polyadenylation signals. The other, cann6, is identical to cann7 upstream from the first polyadenylation signal, and in addition, it contains the whole coding region and extends for 1.8 kb into the 5'-UTR. Comparison of cann6 with the published sequence (Gérard, C. M., Mollereau, C., Vassart, G., and Parmentier, M. (1991) Biochem. J. 279, 129-134) shows the coding regions to be identical, but reveals important differences in the flanking regions. Notably, the cann6 sequence appears to be that of an immature transcript, containing 1.8 kb of an intronic sequence in the 5'-UTR. In addition, polymerase chain reaction amplification of the CB1 coding region in the IM-9 cell line cDNA resulted in two fragments, one containing the whole CB1 coding region and the second lacking a 167-base pair intron within the sequence encoding the amino-terminal tail of the receptor. This alternatively spliced form would translate to an NH2-terminal modified isoform (CB1A) of the receptor, shorter than CB1 by 61 amino acids. In addition, the first 28 amino acids of the putative truncated receptor are completely different from those of CB1, containing more hydrophobic residues. Rat CB1 mRNA is similarly alternatively spliced. A study of the distribution of the human CB1 and CB1A mRNAs by reverse transcription-polymerase chain reaction analysis showed the presence of both CB1 and CB1A throughout the brain and in all the peripheral tissues examined, with CB1A being present in amounts of up to 20% of CB1. PMID:7876112

  16. Avian sarcoma virus gag and env gene structural protein precursors contain a common amino-terminal sequence.

    Science.gov (United States)

    Ficht, T A; Chang, L J; Stoltzfus, C M

    1984-01-01

    The initiation site for translation of the avian sarcoma virus glycoprotein precursor, Pr63env, has been determined by analyzing the amino-terminal peptides of Pr63env and the polyprotein precursor Pr76gag encoded by the viral gag gene. The acceptor splice junction used to form the env gene mRNA has also been identified. Hybrid-selected virus-specific mRNAs were translated in vitro in the presence of either L-[35S]methionine to label at every methionine residue or L-[35S]methionine-tRNAMeti to label specifically at the amino-terminal methionine residues. Tryptic peptide maps of Pr63env labeled at every methionine residue contain all of the peptides, plus one additional peptide, present in the map of Pr57env, a nonglycosylated env-encoded polypeptide of molecular weight 57,000 immunoprecipitated from tunicamycin-treated cells. Specific amino-terminal labeling of the in vitro-synthesized polypeptides showed that the peptide missing from Pr57env corresponds to the amino-terminal tryptic peptide of Pr63env, which is removed in vivo as part of the amino-terminal signal peptide. Comparison of the amino-terminal tryptic peptides of Pr63env and Pr76gag showed that they are identical. In contrast, the chymotryptic amino-terminal peptides of Pr76gag and Pr63env are not identical. The location of the acceptor-splice junction in the env mRNA of the Prague A strain of avian sarcoma virus was determined by mung bean nuclease mapping to be at nucleotide 5,078. Fusion of the gag and env gene sequences during splicing results in use of the same AUG codon to initiate synthesis of Pr76gag and Pr63env. This sequence is contained within the 397-nucleotide 5' terminal leader that is spliced to the body of the env mRNA. The possible significance of these results for the regulation of avian sarcoma virus synthesis and translation is discussed.

  17. The N-terminal domain of the Drosophila retinoblastoma protein Rbf1 interacts with ORC and associates with chromatin in an E2F independent manner.

    Directory of Open Access Journals (Sweden)

    Joseph Ahlander

    Full Text Available BACKGROUND: The retinoblastoma (Rb tumor suppressor protein can function as a DNA replication inhibitor as well as a transcription factor. Regulation of DNA replication may occur through interaction of Rb with the origin recognition complex (ORC. PRINCIPAL FINDINGS: We characterized the interaction of Drosophila Rb, Rbf1, with ORC. Using expression of proteins in Drosophila S2 cells, we found that an N-terminal Rbf1 fragment (amino acids 1-345 is sufficient for Rbf1 association with ORC but does not bind to dE2F1. We also found that the C-terminal half of Rbf1 (amino acids 345-845 interacts with ORC. We observed that the amino-terminal domain of Rbf1 localizes to chromatin in vivo and associates with chromosomal regions implicated in replication initiation, including colocalization with Orc2 and acetylated histone H4. CONCLUSIONS/SIGNIFICANCE: Our results suggest that Rbf1 can associate with ORC and chromatin through domains independent of the E2F binding site. We infer that Rbf1 may play a role in regulating replication directly through its association with ORC and/or chromatin factors other than E2F. Our data suggest an important role for retinoblastoma family proteins in cell proliferation and tumor suppression through interaction with the replication initiation machinery.

  18. The N-terminal domain is a transcriptional activation domain required for Nanog to maintain ES cell self-renewal

    Institute of Scientific and Technical Information of China (English)

    GUO YunQian; ZHANG Juan; YE Li; CHEN Mo; YAO Dong; PAN GuangJin; ZHANG JieQiong; PEI DuanQing

    2009-01-01

    Nanog is a transcription factor identified by its ability to maintain the self-renewal of ES cells in the absence of leukemia inhibitory factor (LIF). Nanog protein contains an N-terminal domain (ND),a DNA-binding homeobox domain (HD) and a C-terminal domain (CD). We previously reported that the CD in Nanog is a transcriptional activation domain essential for the in vivo function of Nanog. Here we demonstrated that the ND in Nanog is also functionally important. Deletion of the ND reduces the transcriptional activity of Nanog on either artificial reporters or native Nanog promoters. This truncated Nanog is also less effective in regulating the endogenous Nanog target genes. Furthermore,the ND truncation disrupted the ability of Nanog to maintain ES cell self-renewal as well. We found that the ND Is not required for the nuclear localization of Nanog. These results suggest that the regulation of endogenous pluripotent genes such as oct3/4 and rex-1 is required for the in vivo function of Nanog.

  19. Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

    NARCIS (Netherlands)

    Simeonova, I.; Jaber, S.; Draskovic, I.; Bardot, B.; Fang, M.; Bouarich-Bourimi, R.; Lejour, V.; Charbonnier, L.; Soudais, C.; Bourdon, J.C.; Huerre, M.; Londono-Vallejo, A.; Toledo, F.

    2013-01-01

    Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53(Delta31), a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis,

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G. (Case Western); (Michigan)

    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.

  3. Hepatic and renal extraction of circulating type III procollagen amino-terminal propeptide and hyaluronan in pig

    DEFF Research Database (Denmark)

    Bentsen, K D; Henriksen, Jens Henrik Sahl; Boesby, S;

    1989-01-01

    Hepatic and renal clearance of the amino-terminal propeptide of type III procollagen (PIIINP) and of the glycosaminoglycan, hyaluronan (HA) were investigated in a catheterization study of seven healthy anesthetized pigs. Two assays were used, in order to distinguish between the metabolism of...

  4. N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

    OpenAIRE

    Kuhn, H; Fietzek, P P; Lampen, J. O.

    1982-01-01

    The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.

  5. Barley polyamine oxidase: Characterisation and analysis of the cofactor and the N-terminal amino acid sequence

    DEFF Research Database (Denmark)

    Radova, A.; Sebela, M.; Galuszka, P.;

    2001-01-01

    was further confirmed by measuring the fluorescence spectra, Barley PAO is an acidic protein (pI 5.4) containing 3% of neutral sugars: its molecular mass determined by SDS-PAGE was 56 kDa, whilst gel permeation chromatography revealed the higher value of 76 kDa. The N-terminal amino acid sequence of barley...

  6. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

    2016-01-22

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

  7. Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo.

    Directory of Open Access Journals (Sweden)

    Sebastien Lageix

    2015-02-01

    Full Text Available The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype, while other substitutions block kinase activation (Gcn- phenotype, in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD, previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.

  8. [Effect of mutations and modifications of amino acid residues on zinc-induced interaction of the metal-binding domain of β-amyloid with DNA].

    Science.gov (United States)

    Khmeleva, S A; Mezentsev, Y V; Kozin, S A; Mitkevich, V A; Medvedev, A E; Ivanov, A S; Bodoev, N V; Makarov, A A; Radko, S P

    2015-01-01

    Interaction of intranuclear β-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of β-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of β-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the β-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aβ facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA.

  9. Domain structures and molecular evolution of class I and class II major histocompatibility gene complex (MHC) products deduced from amino acid and nucleotide sequence homologies

    Science.gov (United States)

    Ohnishi, Koji

    1984-12-01

    Domain structures of class I and class II MHC products were analyzed from a viewpoint of amino acid and nucleotide sequence homologies. Alignment statistics revealed that class I (transplantation) antigen H chains consist of four mutually homologous domains, and that class II (HLA-DR) antigen β and α chains are both composed of three mutually homologous ones. The N-terminal three and two domains of class I and class II (both β and α) gene products, respectively, all of which being ˜90 residues long, were concluded to be homologous to β2-microglobulin (β2M). The membraneembedded C-terminal shorter domains of these MHC products were also found to be homologous to one another and to the third domain of class I H chains. Class I H chains were found to be more closely related to class II α chains than to class II β chains. Based on these findings, an exon duplication history from a common ancestral gene encoding a β2M-like primodial protein of one-domain-length up to the contemporary MHC products was proposed.

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

    Science.gov (United States)

    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. Activation of the plasma membrane Na/H antiporter salt-overly-sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain

    KAUST Repository

    Quintero, Francisco J.

    2011-01-24

    The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2-SOS3 calcium-dependent protein kinase complex upregulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2-SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2-SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.

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

    Science.gov (United States)

    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.

  13. Thrombospondin-1-N-Terminal Domain Induces a Phagocytic State and Thrombospondin-1-C-Terminal Domain Induces a Tolerizing Phenotype in Dendritic Cells

    Science.gov (United States)

    Tabib, Adi; Krispin, Alon; Trahtemberg, Uriel; Verbovetski, Inna; Lebendiker, Mario; Danieli, Tsafi; Mevorach, Dror

    2009-01-01

    In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine the role of HBD in the clearance of apoptotic cells, and whether the phagocytic and tolerizing state of DCs is mediated by the HBD itself, or whether the entire TSP-1 is needed. Therefore, we have cloned the human HBD, and compared its interactions with DC to those with TSP-1. Here we show that rHBD by itself is not directly responsible for immune paralysis and tolerizing phenotype of DCs, at least in the monomeric form, but has a significant role in rendering DCs phagocytic. Binding of TSP-1-C-terminal domain on the other hand induces a tolerizing phenotype in dendritic cells. PMID:19721725

  14. Arabidopsis MKS1 is involved in basal immunity and requires an intact N-terminal domain for proper function.

    Directory of Open Access Journals (Sweden)

    Klaus Petersen

    Full Text Available BACKGROUND: Innate immune signaling pathways in animals and plants are regulated by mitogen-activated protein kinase (MAPK cascades. MAP kinase 4 (MPK4 functions downstream of innate immune receptors via a nuclear substrate MKS1 to regulate the activity of the WRKY33 transcription factor, which in turn controls the production of anti-microbial phytoalexins. METHODOLOGY/PRINCIPAL FINDINGS: We investigate the role of MKS1 in basal resistance and the importance of its N- and C-terminal domains for MKS1 function. We used the information that mks1 loss-of-function partially suppresses the mpk4 loss-of-function phenotype, and that transgenic expression of functional MKS1 in mpk4/mks1 double mutants reverted the mpk4 dwarf phenotype. Transformation of mks1/mpk4 with mutant versions of MKS1 constructs showed that a single amino acid substitution in a putative MAP kinase docking domain, MKS1-L32A, or a truncated MKS1 version unable to interact with WRKY33, were deficient in reverting the double mutant to the mpk4 phenotype. These results demonstrate functional requirement in MKS1 for the interaction with MPK4 and WRKY33. In addition, nuclear localization of MKS1 was shown to depend on an intact N-terminal domain. Furthermore, loss-of-function mks1 mutants exhibited increased susceptibility to strains of Pseudomonas syringae and Hyaloperonospora arabidopsidis, indicating that MKS1 plays a role in basal defense responses. CONCLUSIONS: Taken together, our results indicate that MKS1 function and subcellular location requires an intact N-terminus important for both MPK4 and WRKY33 interactions.

  15. The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

    OpenAIRE

    Noonan James P; Yankiwski Victor; Neff Norma F

    2001-01-01

    Abstract Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of ...

  16. Regulation of plant plasma membrane H+- and Ca2+-ATPases by terminal domains

    DEFF Research Database (Denmark)

    Bækgaard, Lone; Fuglsang, Anja Thoe; Palmgren, Michael Gjedde

    2005-01-01

    In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H(+)-and Ca(2+)-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulatory...... mechanisms of these pumps. In plant plasma membrane H(+)- and Ca(2+)-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed....

  17. Interaction between GInB and the N-terminal domain of NifA in Azospirillum brasilense

    Institute of Scientific and Technical Information of China (English)

    ZHOU XiaoYu; ZOU XiaoXiao; LI JiLun

    2008-01-01

    Azospirillum brasilense is a diazotroph associated with many important agricultural crops and shows potential as a biofertilizer. NifA, the transcriptional activator of nitrogen fixation (nif) genes, and GInB, one of P,, signal transduction family protein, are key proteins in the regulation of nitrogen fixation in A. brasilense. It was previously reported that the regulation of NifA activity in A. brasilense depends on GInB. We report here that GInB was found to interact directly with the N-terminal domain of NifA in vivo under nitrogen-free conditions and the N-terminal mutant of NifA in which the Tyr residues at position 18 and 53 were replaced by Phe (NifA-N-Y18/53F) strengthened the interaction with GInB. Moreover, we also found that the amino acid residues 66-88 and 165-176 in N-terminus of NifA are responsible for the interaction with GInB.

  18. Small-angle X-ray scattering reveals compact domain-domain interactions in the N-terminal region of filamin C.

    Directory of Open Access Journals (Sweden)

    Ritika Sethi

    Full Text Available Filamins are multi-domain, actin cross-linking, and scaffolding proteins. In addition to the actin cross-linking function, filamins have a role in mechanosensor signaling. The mechanosensor function is mediated by domain-domain interaction in the C-terminal region of filamins. Recently, we have shown that there is a three-domain interaction module in the N-terminal region of filamins, where the neighboring domains stabilize the structure of the middle domain and thereby regulate its interaction with ligands. In this study, we have used small-angle X-ray scattering as a tool to screen for potential domain-domain interactions in the N-terminal region. We found evidence of four domain-domain interactions with varying flexibility. These results confirm our previous study showing that domains 3, 4, and 5 exist as a compact three domain module. In addition, we report interactions between domains 11-12 and 14-15, which are thus new candidate sites for mechanical regulation.

  19. Dual Thermosensitive Hydrogels Assembled from the Conserved C-Terminal Domain of Spider Dragline Silk.

    Science.gov (United States)

    Qian, Zhi-Gang; Zhou, Ming-Liang; Song, Wen-Wen; Xia, Xiao-Xia

    2015-11-01

    Stimuli-responsive hydrogels have great potentials in biomedical and biotechnological applications. Due to the advantages of precise control over molecular weight and being biodegradable, protein-based hydrogels and their applications have been extensively studied. However, protein hydrogels with dual thermosensitive properties are rarely reported. Here we present the first report of dual thermosensitive hydrogels assembled from the conserved C-terminal domain of spider dragline silk. First, we found that recombinant C-terminal domain of major ampullate spidroin 1 (MaSp1) of the spider Nephila clavipes formed hydrogels when cooled to approximately 2 °C or heated to 65 °C. The conformational changes and self-assembly of the recombinant protein were studied to understand the mechanism of the gelation processes using multiple methods. It was proposed that the gelation in the low-temperature regime was dominated by hydrogen bonding and hydrophobic interaction between folded protein molecules, whereas the gelation in the high-temperature regime was due to cross-linking of the exposed hydrophobic patches resulting from partial unfolding of the protein upon heating. More interestingly, genetic fusion of the C-terminal domain to a short repetitive region of N. clavipes MaSp1 resulted in a chimeric protein that formed a hydrogel with significantly improved mechanical properties at low temperatures between 2 and 10 °C. Furthermore, the formation of similar hydrogels was observed for the recombinant C-terminal domains of dragline silk of different spider species, thus demonstrating the conserved ability to form dual thermosensitive hydrogels. These findings may be useful in the design and construction of novel protein hydrogels with tunable multiple thermosensitivity for applications in the future.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  2. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    Science.gov (United States)

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle. PMID:26405031

  3. The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions.

    Science.gov (United States)

    Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M; Holthauzen, Luis Marcelo F; Li, Jing; Oezguen, Numan; Hilser, Vincent J; Tainer, John A; Mitra, Sankar

    2013-07-10

    NEIL1 [Nei (endonuclease VIII)-like protein 1], one of the five mammalian DNA glycosylases that excise oxidized DNA base lesions in the human genome to initiate base excision repair, contains an intrinsically disordered C-terminal domain (CTD; ~100 residues), not conserved in its Escherichia coli prototype Nei. Although dispensable for NEIL1's lesion excision and AP lyase activities, this segment is required for efficient in vivo enzymatic activity and may provide an interaction interface for many of NEIL1's interactions with other base excision repair proteins. Here, we show that the CTD interacts with the folded domain in native NEIL1 containing 389 residues. The CTD is poised for local folding in an ordered structure that is induced in the purified fragment by osmolytes. Furthermore, deletion of the disordered tail lacking both Tyr and Trp residues causes a red shift in NEIL1's intrinsic Trp-specific fluorescence, indicating a more solvent-exposed environment for the Trp residues in the truncated protein, which also exhibits reduced stability compared to the native enzyme. These observations are consistent with stabilization of the native NEIL1 structure via intramolecular, mostly electrostatic, interactions that were disrupted by mutating a positively charged (Lys-rich) cluster of residues (amino acids 355-360) near the C-terminus. Small-angle X-ray scattering (SAXS) analysis confirms the flexibility and dynamic nature of NEIL1's CTD, a feature that may be critical to providing specificity for NEIL1's multiple, functional interactions.

  4. Characterization of five new mutants in the carboxyl-terminal domain of human apolipoprotein E: No cosegregation with severe hyperlipidemia

    Energy Technology Data Exchange (ETDEWEB)

    Maagdenberg, A.M.J.M. van den; Bruijn, I.H. de; Hofker, M.H.; Frants, R.R. (Leiden Univ. (Netherlands)); Knijff, P. de; Smelt, A.H.M.; Leuven, J.A.G.; van' t Hooft, F.; Assmann, G.; Havekes, L.M. (Univ. Hospital, Leiden (Netherlands)); Weng, Wei; Funke, H. (Westfalische Wilhelms-Universitaet, Muester (Germany))

    1993-05-01

    Assessment of the apolipoprotein E (apoE) phenotype by isoelectric focusing of both hyperlipidemic and normolipidemic individuals identified five new variants. All mutations were confined to the downstream part of the APOE gene by using denaturing gradient gel electrophoresis (DGGE). Sequence analysis revealed five new mutations causing unique amino acid substitutions in the carboxyl-terminal part of the protein containing the putative lipid-binding domain. Three hyperlipoproteinemic probands were carriers of the APOE*2(Va1236[r arrow]Glu) allele, the APOE*3(Cys112-Arg; Arg251[r arrow]Gly) allele, or the APOE*1(Arg158[r arrow]Cys; Leu252[r arrow]Glu) allele. DGGE of the region encoding the receptor-binding domain was useful for haplotyping the mutations at codons 112 and 158. Family studies failed to demonstrate cosegregation between the new mutations and severe hyperlipoproteinemia, although a number of carriers for the APOE*3(Cys112[r arrow]Arg; Arg251[r arrow]Gly) allele and the APOE*1(Arg158-Cys; Leu252[r arrow]Glu) allele expressed hypertriglyceridemia and/ or hypercholesterolemia. Two other mutant alleles, APOE*4[sup [minus

  5. Cloning, purification and preliminary X-ray analysis of the C-terminal domain of Helicobacter pylori MotB

    Energy Technology Data Exchange (ETDEWEB)

    Roujeinikova, Anna, E-mail: anna.roujeinikova@manchester.ac.uk [Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom)

    2008-04-01

    The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of a putative peptidoglycan-binding domain of H. pylori MotB, a stator component of the bacterial flagellar motor, are reported. The C-terminal domain of MotB (MotB-C) contains a putative peptidoglycan-binding motif and is believed to anchor the MotA/MotB stator unit of the bacterial flagellar motor to the cell wall. Crystals of Helicobacter pylori MotB-C (138 amino-acid residues) were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 50.8, b = 89.5, c = 66.3 Å, β = 112.5°. The crystals diffract X-rays to at least 1.6 Å resolution using a synchrotron-radiation source. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains one tetramer with 222 point-group symmetry. The anomalous difference Patterson maps calculated for an ytterbium-derivative crystal using diffraction data at a wavelength of 1.38 Å showed significant peaks on the v = 1/2 Harker section, suggesting that ab initio phase information could be derived from the MAD data.

  6. The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production.

    Science.gov (United States)

    Mine, Junki; Tamura, Tomokazu; Mitsuhashi, Kazuya; Okamatsu, Masatoshi; Parchariyanon, Sujira; Pinyochon, Wasana; Ruggli, Nicolas; Tratschin, Jon-Duri; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-07-01

    The viral protein Npro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, Npro suppresses type I IFN (IFN-α/β) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the Npro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of Npro coordinating zinc by means of the amino acid residues C112, C134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-α/β induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of Npro of these four isolates were related to the lack of IRF-3-degrading activity. Restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional Npro contributed to higher stability of the reconstructed Npro compared with the Npro from the Thai isolate. This led to enhanced interaction of Npro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of Npro are involved in the stability of Npro, in interaction of Npro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-α/β production. PMID:25809915

  7. Use of amino terminal type III procollagen peptide (P3NP) assay in methotrexate therapy for psoriasis

    OpenAIRE

    Khan, S; Subedi, D; Chowdhury, M M U

    2006-01-01

    Hepatic fibrosis continues to be a risk in patients receiving methotrexate for psoriasis. Measurement of amino terminal levels of type III procollagen (P3NP) has been advocated as an effective non‐invasive test for ongoing hepatic fibrogenesis that could avoid liver biopsies. An audit was conducted to assess the practice of P3NP monitoring using guidelines produced by Manchester and whether the agreed levels correlate with histological severity. Sixty five patients with 174 P3NP assays and 30...

  8. Isolation of key amino acid residues at the N-terminal end of the core region Streptococcus downei glucansucrase, GTF-I.

    Science.gov (United States)

    Monchois, V; Vignon, M; Russell, R R

    1999-11-01

    Related streptococcal and Leuconostoc mesenteroides glucansucrases are enzymes of medical and biotechnological interest. Molecular modelling has suggested that the catalytic domain contains a circularly permuted version of the (beta/alpha)8 barrel structure found in the amylase superfamily, and site-directed mutagenesis has identified critical amino acids in this region. In this study, sequential N-terminal truncations of Streptococcus downei GTF-I showed that key amino acids are also present in the first one-third of the core domain. Mutations were introduced at Trp-344, Glu-349 and His-355, residues that are conserved in all glucansucrases and lie within a region which is a target for inhibitory antibodies. W344L, E349L and H355V substitutions were assayed for their effect on mutan synthesis and also on oligosaccharide synthesis with various acceptors. It appeared that Trp-344 and His-355 are involved in the action mechanism of GTF-I; His-355 may also play a role in a binding subsite necessary for oligosaccharide and glucan elongation. PMID:10570812

  9. Secondary structural analysis of the carboxyl-terminal domain from different connexin isoforms.

    Science.gov (United States)

    Spagnol, Gaëlle; Al-Mugotir, Mona; Kopanic, Jennifer L; Zach, Sydney; Li, Hanjun; Trease, Andrew J; Stauch, Kelly L; Grosely, Rosslyn; Cervantes, Matthew; Sorgen, Paul L

    2016-03-01

    The connexin carboxyl-terminal (CxCT) domain plays a role in the trafficking, localization, and turnover of gap junction channels, as well as the level of gap junction intercellular communication via numerous post-translational modifications and protein-protein interactions. As a key player in the regulation of gap junctions, the CT presents itself as a target for manipulation intended to modify function. Specific to intrinsically disordered proteins, identifying residues whose secondary structure can be manipulated will be critical toward unlocking the therapeutic potential of the CxCT domain. To accomplish this goal, we used biophysical methods to characterize CxCT domains attached to their fourth transmembrane domain (TM4). Circular dichroism and nuclear magnetic resonance were complementary in demonstrating the connexin isoforms that form the greatest amount of α-helical structure in their CT domain (Cx45 > Cx43 > Cx32 > Cx50 > Cx37 ≈ Cx40 ≈ Cx26). Studies compared the influence of 2,2,2-trifluoroethanol, pH, phosphorylation, and mutations (Cx32, X-linked Charcot-Marie Tooth disease; Cx26, hearing loss) on the TM4-CxCT structure. While pH modestly influences the CT structure, a major structural change was associated with phosphomimetic substitutions. Since most connexin CT domains are phosphorylated throughout their life cycle, studies of phospho-TM4-CxCT isoforms will be critical toward understanding the role that structure plays in regulating gap junction function. PMID:26542351

  10. Effect of amino-terminated substrates onto surface properties of cellulose esters and their interaction with lectins

    Energy Technology Data Exchange (ETDEWEB)

    Amim, Jorge [Instituto de Quimica, Universidade de Sao Paulo, Av. Prof. Lineu Prestes 748, 05508-000 Sao Paulo (Brazil); Instituto de Quimica, Universidade Federal do Rio de Janeiro, Campus Macae, Av. Aluizio Gomes da Silva 50, 27930-560, Macae (Brazil); Petri, Denise F.S., E-mail: dfsp@usp.br [Instituto de Quimica, Universidade de Sao Paulo, Av. Prof. Lineu Prestes 748, 05508-000 Sao Paulo (Brazil)

    2012-02-01

    Films of cellulose acetate butyrate (CAB) and carboxymethylcellulose acetate butyrate (CMCAB) were deposited from ethyl acetate solutions onto bare silicon wafers (Si/SiO{sub 2}) or amino-terminated surfaces (APS) by means of equilibrium adsorption. All surfaces were characterized by means of ellipsometry, atomic force microscopy (AFM) and contact angle measurements. The presence of amino groups on the support surface favored the adsorption of CAB and CMCAB, inducing the orientation of most polar groups to the surface and the exposition of alkyl group to the air. Such molecular orientation caused increase of the dispersive component of surface energy ({gamma}{sub s}{sup d}) and decrease of the polar component of surface energy ({gamma}{sub s}{sup p}) of cellulose esters in comparison to those values determined for films deposited onto bare Si/SiO{sub 2} wafers. Adsorption behavior of jacalin or concanavalin A onto CAB and CMCAB films was also investigated. The adsorbed amounts of lectins were more pronounced on cellulose esters with high ({gamma}{sub s}{sup p}) and total surface energy ({gamma}{sub s}{sup t}) values. - Highlights: Black-Right-Pointing-Pointer Amino groups on the substrate induce the orientation of cellulose esters polar groups. Black-Right-Pointing-Pointer Amino terminated substrate caused decrease of surface energy of cellulose ester films. Black-Right-Pointing-Pointer Lectins adsorbed preferentially onto cellulose esters with high surface energy.

  11. Feature-Based Classification of Amino Acid Substitutions outside Conserved Functional Protein Domains

    Directory of Open Access Journals (Sweden)

    Branislava Gemovic

    2013-01-01

    Full Text Available There are more than 500 amino acid substitutions in each human genome, and bioinformatics tools irreplaceably contribute to determination of their functional effects. We have developed feature-based algorithm for the detection of mutations outside conserved functional domains (CFDs and compared its classification efficacy with the most commonly used phylogeny-based tools, PolyPhen-2 and SIFT. The new algorithm is based on the informational spectrum method (ISM, a feature-based technique, and statistical analysis. Our dataset contained neutral polymorphisms and mutations associated with myeloid malignancies from epigenetic regulators ASXL1, DNMT3A, EZH2, and TET2. PolyPhen-2 and SIFT had significantly lower accuracies in predicting the effects of amino acid substitutions outside CFDs than expected, with especially low sensitivity. On the other hand, only ISM algorithm showed statistically significant classification of these sequences. It outperformed PolyPhen-2 and SIFT by 15% and 13%, respectively. These results suggest that feature-based methods, like ISM, are more suitable for the classification of amino acid substitutions outside CFDs than phylogeny-based tools.

  12. Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

    Energy Technology Data Exchange (ETDEWEB)

    Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N. (UW)

    2012-03-15

    The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

  13. The C-terminal domain of Tetrahymena thermophila telomerase holoenzyme protein p65 induces multiple structural changes in telomerase RNA

    OpenAIRE

    Akiyama, Benjamin M.; Loper, John; Najarro, Kevin; Stone, Michael D.

    2012-01-01

    The C-terminal domain of Tetrahymena thermophila telomerase holoenzyme protein p65 induces multiple structural changes in telomerase RNA. Telomerase holoenzyme proteins are required to fold telomerase RNA into its active conformation. In this study, the Stone laboratory employed a combination of single-molecule FRET and RNase protection mapping to demonstrate that the C-terminal domain of the Tetrahymena telomerase holoenzyme protein p65 is essential for its RNA folding activity. RNase probin...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Fine tuning of the catalytic activity of colicin e7 nuclease domain by systematic n-terminal mutations

    DEFF Research Database (Denmark)

    Németh, Eszter; Körtvélyesi, Tamás; Thulstrup, Peter W.;

    2014-01-01

    The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446–449NColE75KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuc...

  16. A C-terminal PDZ domain-binding sequence is required for striatal distribution of the dopamine transporter

    DEFF Research Database (Denmark)

    Rickhag, Karl Mattias; Hansen, Freja Herborg; Sørensen, Gunnar;

    2013-01-01

    The dopamine transporter mediates reuptake of dopamine from the synaptic cleft. The cellular mechanisms controlling dopamine transporter levels in striatal nerve terminals remain poorly understood. The dopamine transporters contain a C-terminal PDZ (PSD-95/Discs-large/ZO-1) domain-binding sequenc...

  17. A novel PKD2L1 C-terminal domain critical for trimerization and channel function

    OpenAIRE

    Zheng, Wang; Hussein, Shaimaa; Yang, Jungwoo; Huang, Jun; Zhang, Fan; Hernandez-Anzaldo, Samuel; Fernandez-Patron, Carlos; Cao, Ying; Zeng, Hongbo; Tang, Jingfeng; Chen, Xing-Zhen

    2015-01-01

    As a transient receptor potential (TRP) superfamily member, polycystic kidney disease 2-like-1 (PKD2L1) is also called TRPP3 and has similar membrane topology as voltage-gated cation channels. PKD2L1 is involved in hedgehog signaling, intestinal development, and sour tasting. PKD2L1 and PKD1L3 form heterotetramers with 3:1 stoichiometry. C-terminal coiled-coil-2 (CC2) domain (G699-W743) of PKD2L1 was reported to be important for its trimerization but independent studies showed that CC2 does n...

  18. Logic circuit prototypes for three-terminal magnetic tunnel junctions with mobile domain walls

    Science.gov (United States)

    Currivan-Incorvia, J. A.; Siddiqui, S.; Dutta, S.; Evarts, E. R.; Zhang, J.; Bono, D.; Ross, C. A.; Baldo, M. A.

    2016-01-01

    Spintronic computing promises superior energy efficiency and nonvolatility compared to conventional field-effect transistor logic. But, it has proven difficult to realize spintronic circuits with a versatile, scalable device design that is adaptable to emerging material physics. Here we present prototypes of a logic device that encode information in the position of a magnetic domain wall in a ferromagnetic wire. We show that a single three-terminal device can perform inverter and buffer operations. We demonstrate one device can drive two subsequent gates and logic propagation in a circuit of three inverters. This prototype demonstration shows that magnetic domain wall logic devices have the necessary characteristics for future computing, including nonlinearity, gain, cascadability, and room temperature operation.

  19. Quantitative measurements of binary amino acids mixtures in yellow foxtail millet by terahertz time domain spectroscopy.

    Science.gov (United States)

    Lu, Shaohua; Zhang, Xin; Zhang, Zhuoyong; Yang, Yuping; Xiang, Yuhong

    2016-11-15

    Terahertz time domain spectroscopy (THz-TDS) combined with chemometrics has been utilized for the qualitative and quantitative analysis of binary mixtures of l-glutamic acid and l-glutamine which have similar chemical structures and properties. The binary mixtures of amino acids were prepared with yellow foxtail millet matrix, substituted for polyethylene (PE) as previously reported. After proper pretreatment of absorption spectra, quantitative analysis was achieved by partial least squares (PLS) and interval partial least squares (iPLS) regressions. The performance of models was evaluated based on the root mean square error of prediction (RMSEP) and correlation coefficient (R(2)) of cross-validations with bootstrapped Latin partitions as criterion. The iPLS yielded better results with low RMSEP (0.39±0.02%, 0.39±0.02%), and higher R(2) values (0.9904, 0.9906) for glutamine and glutamic acid comparing to the conventional PLS models. Multivariate curve resolution alternating least squares (MCR-ALS) was successfully applied for resolution of pure THz spectra and concentration profiles of two amino acids components from mixtures. PMID:27283659

  20. C-terminal domain of hepatitis C virus core protein is essential for secretion

    Institute of Scientific and Technical Information of China (English)

    Soo-Ho Choi; Kyu-Jin Park; So-Yeon Kim; Dong-Hwa Choi; Jung-Min Park; Soon B. Hwang

    2005-01-01

    AIM: We have previously demonstrated that hepatitis C virus (HCV) core protein is efficiently released into the culture medium in insect cells. The objective of this study is to characterize the HCV core secretion in insect cells.METHODS: We constructed recombinant baculoviruses expressing various-length of mutant core proteins, expressed these proteins in insect cells, and examined core protein secretion in insect cells.RESULTS: Only wild type core was efficiently released into the culture medium, although the protein expression level of wild type core was lower than those of other mutant core proteins. We found that the shorter form of the core construct expressed the higher level of protein. However, if more than 18 amino acids of the core were truncated at the C-terminus,core proteins were no longer seareted into the culture medium.Membrane flotation data show that the secreted core proteins are associated with the cellular membrane protein, indicating that HCV core is secreted as a membrane complex.CONCLUSION: The C-terminal 18 amino acids of HCV core were crucial for core secretion into the culture media.Since HCV replication occurs on lipid raft membrane structure,these results suggest that HCV may utilize a unique core release mechanism to escape immune surveillance, thereby potentially representing the feature of HCV morphogenesis.

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

    Science.gov (United States)

    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.

  2. The role of the N-terminal loop in the function of the colicin E7 nuclease domain

    DEFF Research Database (Denmark)

    Czene, Anikó; Németh, Eszter; Zóka, István G.;

    2013-01-01

    Colicin E7 (ColE7) is a metallonuclease toxin of Escherichia coli belonging to the HNH superfamily of nucleases. It contains highly conserved amino acids in its HHX14NX8HX3H ββα-type metal ion binding C-terminal active centre. However, the proximity of the arginine at the N-terminus of the nuclea...

  3. Direct influence of C-terminally substituted amino acids in the Dmt-Tic pharmacophore on delta-opioid receptor selectivity and antagonism.

    Science.gov (United States)

    Balboni, Gianfranco; Salvadori, Severo; Guerrini, Remo; Negri, Lucia; Giannini, Elisa; Bryant, Sharon D; Jinsmaa, Yunden; Lazarus, Lawrence H

    2004-07-29

    A series of 17 analogues were developed on the basis of the general formula H-Dmt-Tic-NH-CH(R)-R' (denotes chirality; R = charged, neutral, or aromatic functional group; R' = -OH or -NH(2)). These compounds were designed to test the following hypothesis: the physicochemical properties of third-residue substitutions C-terminal to Tic in the Dmt-Tic pharmacophore modify delta-opioid receptor selectivity and delta-opioid receptor antagonism through enhanced interactions with the mu-opioid receptor. The data substantiate the following conclusions: (i) all compounds had high receptor affinity [K(i)(delta) = 0.034-1.1 nM], while that for the mu-opioid receptor fluctuated by orders of magnitude [K(i)(mu) = 15.1-3966 nM]; (ii) delta-opioid receptor selectivity [K(i)(mu)/K(i)(delta)] declined 1000-fold from 22,600 to 21; (iii) a C-terminal carboxyl group enhanced selectivity but only as a consequence of the specific residue; (iv) amidated, positive charged residues [Lys-NH(2) (6), Arg-NH(2) (7)], and a negatively charged aromatic residue [Trp-OH (11)] enhanced mu-opioid affinity [K(i)(mu) = 17.0, 15.1, and 15.7 nM, respectively], while Gly-NH(2) (8), Ser-NH(2) (10), and His-OH (12) were nearly one-tenth as active; and (v) D-isomers exhibited mixed effects on mu-opioid receptor affinity (2' 1 microM) except H-Dmt-Tic-Glu-NH(2) (3), which was a partial delta-opioid receptor agonist (IC(50) = 2.5 nM). Thus, these C-terminally extended analogues indicated that an amino acid residue containing a single charge, amino or guanidino functionality, or aromatic group substantially altered the delta-opioid receptor activity profile (selectivity and antagonism) of the Dmt-Tic pharmacophore, which suggests that the C-terminal constituent plays a major role in determining opioid receptor activity as an "address domain".

  4. Insights into the Function of the Unstructured N-Terminal Domain of Proteins 4.1R and 4.1G in Erythropoiesis

    Directory of Open Access Journals (Sweden)

    Wataru Nunomura

    2011-01-01

    Full Text Available Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N, and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80, as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. One goal in the field is to better understand the complex regulation of cell type and isoform-specific expression of 4.1 proteins. To start answering these questions, we are studying in depth the important functions of 4.1 proteins in the organization and function of the membrane skeleton in erythrocytes. We have previously reported that the binding profiles of 4.1R80 and 4.1R135 to membrane proteins and calmodulin are very different despite the similar structure of the membrane-binding domain of 4.1G and 4.1R135. We have accumulated evidence for those differences being caused by the N-terminal 209 amino acids headpiece region (HP. Interestingly, the HP region is an unstructured domain. Here we present an overview of the differences and similarities between 4.1 isoforms and paralogs. We also discuss the biological significance of unstructured domains.

  5. Method for the selective measurement of amino-terminal variants of procalcitonin.

    NARCIS (Netherlands)

    Struck, J.; Strebelow, M.; Tietz, S.; Alonso, C.; Morgenthaler, N.G.; Hoeven, J.G. van der; Pickkers, P.; Bergmann, A.

    2009-01-01

    BACKGROUND: Procalcitonin (PCT) is an established marker for diagnosing and monitoring bacterial infections. Full-length PCT [116 amino acids that make up procalcitonin (PCT1-116)] can be truncated, leading to des-Ala-Pro-PCT (des-Alanin-Prolin-Procalcitonin; PCT3-116). Current immunoassays for PCT

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

    Energy Technology Data Exchange (ETDEWEB)

    Liebschner, Dorothee [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); Brzezinski, Krzysztof [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); University of Bialystok, 15-399 Bialystok (Poland); Dauter, Miroslawa [Argonne National Laboratory, Argonne, IL 60439 (United States); Dauter, Zbigniew, E-mail: dauter@anl.gov [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); Nowak, Marta; Kur, Józef; Olszewski, Marcin, E-mail: dauter@anl.gov [Gdansk University of Technology, 80-952 Gdansk (Poland); National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States)

    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.

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

    Science.gov (United States)

    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.

  8. Importance of the terminal α-amino group of bradykinin and some kynins on capillary permeability increase

    International Nuclear Information System (INIS)

    A simple and reliable method is described for the quantitative evaluation of vascular permeability increase induced by vasoactive drugs with Evans blue labelled with iodine-125 or 131. By using this method the importance of α-amino group of bradykinin (Bk), kallidin (Kd) and methionyl-kallidin (Met-Kd) on the biological activity were studied after reacting the kinins with pyridoxal 5'-phosphate followed by reduction with sodium borohydride. Phosphopyridoxyl-kinins were formed leaving free the guanidino groups. Aminoacid analysis of phosphopyridoxyl-kinin showed that the efficiency of the reaction was extremely good in the blockage of α-amino groups [phosphopyridoxyl-bradikinin (PP-Bk) = 98,8%, phosphopyridoxyl-kallidin (PP-Kd) = 95,2%, phosphopyridoxyl-methionyl-kallidin (PP-Met-Kd) = 98,0%. Log dose-response curves were obtained for Bk, Kd, Met-Kd, acetyl-bradykinin (Ac-Bk), PP-Bk, PP-Kd and PP-Met-Kd and the relative potencies calculated through the Lineweaver-Burk plots. The relative potencies were: PP-Bk about 16% the activity of Bk, Ac-Bk about 31% the activity of Bk, PP-Kd about 17% the activity of Kd, PP-Met-Kd about 12% the activity of Met-Kd. The results show that the terminal α-amino group of kinins is important in the mechanisms of biological activity. (Author)

  9. Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

    OpenAIRE

    Melikyan, Grigory B.; Lin, Sasa; Roth, Michael G.; Cohen, Fredric S.

    1999-01-01

    The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity w...

  10. Characterization of niphatenones that inhibit androgen receptor N-terminal domain.

    Directory of Open Access Journals (Sweden)

    Carmen A Banuelos

    Full Text Available Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC. The androgen receptor (AR remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD. Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD. Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S-niphatenone had significantly better activity against the AR NTD compared to (R-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR activity and covalently bound to GR activation function-1 (AF-1 region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

  11. Structural modeling of the N-terminal signal–receiving domain of IκBα

    Directory of Open Access Journals (Sweden)

    Samira eYazdi

    2015-06-01

    Full Text Available The transcription factor nuclear factor-κB (NF-κB exerts essential roles in many biological processes including cell growth, apoptosis and innate and adaptive immunity. The NF-kB inhibitor (IκBα retains NF-κB in the cytoplasm and thus inhibits nuclear localization of NF-κB and its association with DNA. Recent protein crystal structures of the C-terminal part of IκBα in complex with NF-κB provided insights into the protein-protein interactions but could not reveal structural details about the N-terminal signal receiving domain (SRD. The SRD of IκBα contains a degron, formed following phosphorylation by IκB kinases (IKK. In current protein X-ray structures, however, the SRD is not resolved and assumed to be disordered. Here, we combined secondary structure annotation and domain threading followed by long molecular dynamics (MD simulations and showed that the SRD possesses well-defined secondary structure elements. We show that the SRD contains 3 additional stable α-helices supplementing the six ARDs present in crystallized IκBα. The IκBα/NF-κB protein-protein complex remained intact and stable during the entire simulations. Also in solution, free IκBα retains its structural integrity. Differences in structural topology and dynamics were observed by comparing the structures of NF-κB free and NF-κB bound IκBα-complex. This study paves the way for investigating the signaling properties of the SRD in the IκBα degron. A detailed atomic scale understanding of molecular mechanism of NF-κB activation, regulation and the protein-protein interactions may assist to design and develop novel chronic inflammation modulators.

  12. Functional analysis of the NH{sub 2}-terminal hydrophobic region and BRICHOS domain of GKN1

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Jung Hwan; Choi, Yoo Jin; Choi, Won Suk; Nam, Suk Woo; Lee, Jung Young; Park, Won Sang, E-mail: wonsang@catholic.ac.kr

    2013-11-01

    Highlights: •NH{sub 2}-terminal and BRICHOS domain of GKN1 inhibited tumor cell growth. •NH{sub 2}-terminal and BRICHOS domain of GKN1 regulated cell cycle. •NH{sub 2}-terminal and BRICHOS domain of GKN1 inhibited epigenetic regulators. -- Abstract: Gastrokine 1 (GKN1) protects the gastric antral mucosa and promotes healing by facilitating restitution and proliferation after injury. GKN1 is down-regulated in Helicobacter pylori-infected gastric epithelial cells and loss of GKN1 expression is tightly associated with gastric carcinogenesis. However, the underlying mechanisms as a tumor suppressor are largely unknown. Presently, the hydrophobic region and BRICHOS domain of GKN1, pGKN1{sup D13N}, pGKN1{sup Δ68–199}, and pGKN1{sup Δ1–67,165–199} were shown to suppress gastric cancer cell growth and recapitulate GKN1 functions. As well, the hydrophobic region and BRICHOS domain of GKN1 had a synergistic anti-cancer effect with 5-FU on tumor cell growth, implying that the NH{sub 2}-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for tumor suppression, thereby suggesting a therapeutic intervention for gastric cancer. Also, its domain inducing endogenous miR-185 directly targeted the epigenetic effectors DNMT1 and EZH2 in gastric cancer cells. Our results suggest that the NH{sub 2}-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for its tumor suppressor activities.

  13. Amino-terminal extension present in the methionine aminopeptidase type 1c of Mycobacterium tuberculosis is indispensible for its activity

    Directory of Open Access Journals (Sweden)

    Kumaran Sangaralingam

    2011-07-01

    Full Text Available Abstract Background Methionine aminopeptidase (MetAP is a ubiquitous enzyme in both prokaryotes and eukaryotes, which catalyzes co-translational removal of N-terminal methionine from elongating polypeptide chains during protein synthesis. It specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. The MetAP action for exclusion of N-terminal methionine is mandatory in 50-70% of nascent proteins. Such an activity is required for proper sub cellular localization, additional processing and eventually for the degradation of proteins. Results We cloned genes encoding two such metalloproteases (MtMetAP1a and MtMetAP1c present in Mycobacterium tuberculosis and expressed them as histidine-tagged proteins in Escherichia coli. Although they have different substrate preferences, for Met-Ala-Ser, we found, MtMetAP1c had significantly high enzyme turnover rate as opposed to MtMetAP1a. Circular dichroism spectroscopic studies as well as monitoring of enzyme activity indicated high temperature stability (up to 50°C of MtMetAP1a compared to that of the MtMetAP1c. Modelling of MtMetAP1a based on MtMetAP1c crystal structure revealed the distinct spatial arrangements of identical active site amino acid residues and their mutations affected the enzymatic activities of both the proteins. Strikingly, we observed that 40 amino acid long N-terminal extension of MtMetAP1c, compared to its other family members, contributes towards the activity and stability of this enzyme, which has never been reported for any methionine aminopeptidase. Furthermore, mutational analysis revealed that Val-18 and Pro-19 of MtMetAP1c are crucial for its enzymatic activity. Consistent with this observation, molecular dynamic simulation studies of wild-type and these variants strongly suggest their involvement in maintaining active site conformation of MtMetAP1c. Conclusion Our findings unequivocally emphasized that N-terminal

  14. PrP N-terminal domain triggers PrPSc-like aggregation of Dpl

    International Nuclear Information System (INIS)

    Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrPC, for 'cellular prion protein') into an abnormal state (PrPSc, for 'scrapie prion protein'). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrPC. In contrast to its homologue PrPC, Dpl is unable to participate in prion disease progression or to achieve an abnormal PrPSc-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrPC (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 α-helical monomer forms soluble β-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

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

    Science.gov (United States)

    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.

  16. A C-terminal PDZ domain binding sequence is required for striatal distribution of the dopamine transporter

    OpenAIRE

    Rickhag, Mattias; Hansen, Freja Herborg; Sørensen, Gunnar; Strandfelt, Kristine Nørgaard; Andresen, Bjørn; Gotfryd, Kamil; Madsen, Kenneth L; Vestergaard-Klewe, Ib; Ammendrup-Johnsen, Ina; Eriksen, Jacob; Füchtbauer, Ernst-Martin; Gomeza, Jesus; Woldbye, David P.D.; Wörtwein, Gitta; Gether, Ulrik

    2013-01-01

    The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft. The cellular mechanisms controlling DAT levels in striatal nerve terminals remain poorly understood. DAT contains a C-terminal PDZ (PSD-95/Discs-large/ZO-1) domain binding sequence believed to bind synaptic scaffolding proteins, but its functional significance is uncertain. Here we demonstrate that two different DAT knock-in mice with disrupted PDZ-binding motifs (DAT-AAA and DAT+Ala) are characterized by dr...

  17. Prokaryotic Expression and Purification of Human TLE1 N-terminal Q Domain Fragment and Production of its Polyclonal Antibody

    Directory of Open Access Journals (Sweden)

    Su WANG

    2010-11-01

    Full Text Available Background and objective TLE1 is an important protein in regulating Wnt, Notch and EGFR signaling pathways. The TLE1 N-terminal Q domain regulates the pathways by mediating its oligomerization and interaction with LEF1. The aim of this study is to construct the human TLE1 N-terminal Q domain fragment in prokaryotic expression system, express and purify protein TLE1 N-terminal Q domain and prepare its polyclonal antibody. Methods The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA, was cloned into the prokaryotic expression vector pGEX-4T-1 containing Glutathione S-transferase (GST. Vector pGEX-4T1-TLE1-Q was transformed into E.coli BL21 condon plus. The GST-TLE1-Q(1-136 fusion protein was induced by IPTG, digested by Thrombin, purified with glutathione-sepharose beads and FPLC, identified by SDS-PAGE. Then rabbits were immunized with the purified protein TLE1-Q(1-136 for obtaining the antiserum. The titers and specificity of antibodies were measured by ELISA and Western blot. Results The PCR identification and the sequencing of recombinant plasmid demonstrated that vector pGEX-4T1-TLE1-Q was successfully constructed. The SDS-PAGE shows target protein (14 000 Da is the interest protein TLE1-Q(1-136. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired, with an antibody titer of 1:20 000. Conclusion Expression vector pGEX-4T1-TLE1-Q is correctly constructed. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired. These work established the foundation for further biological study between TLE1 and lung cancers.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Identification of an amino-terminal fragment of apolipoprotein E4 that localizes to neurofibrillary tangles of the Alzheimer's disease brain.

    Science.gov (United States)

    Rohn, Troy T; Catlin, Lindsey W; Coonse, Kendra G; Habig, Jeffrey W

    2012-09-26

    Although the risk factor for harboring the apolipoprotein E4 (apoE4) allele in late-onset Alzheimer's disease (AD) is well known, the mechanism by which apoE4 contributes to AD pathogenesis has yet to be clarified. Preferential cleavage of the ApoE4 isoform relative to other polymorphic forms appears to be significant, as the resulting fragments are associated with hallmarks of AD. To examine the possible role of apoE4 proteolysis in AD, we designed a site-directed antibody directed at position D172, which would yield a predicted amino-terminal fragment previously identified in AD brain extracts. Western blot analysis utilizing this novel antibody, termed the amino-terminal apoE4 cleavage fragment (nApoE4CF) Ab, consistently identified the predicted amino-terminal fragment (∼18kDa) in several commercially available forms of human recombinant apoE4 purified from E. coli. Mass spectrometry confirmed the identity of this 18kDa fragment as being an amino-terminal fragment of apoE4. Immunohistochemical experiments indicated the nApoE4CF Ab specifically labeled neurofibrillary tangles (NFTs) in AD frontal cortex sections that colocalized with the mature tangle marker PHF-1. Taken together, these results suggest a novel cleavage event of apoE4, generating an amino-terminal fragment that localizes within NFTs of the AD brain.

  20. Ligand-Binding Properties of the Carboxyl-Terminal Repeat Domain of Streptococcus mutans Glucan-Binding Protein A

    OpenAIRE

    Haas, Wolfgang; Banas, Jeffrey A.

    2000-01-01

    Streptococcus mutans glucan-binding protein A (GbpA) has sequence similarity in its carboxyl-terminal domain with glucosyltransferases (GTFs), the enzymes responsible for catalyzing the synthesis of the glucans to which GbpA and GTFs can bind and which promote S. mutans attachment to and accumulation on the tooth surface. It was predicted that this C-terminal region, comprised of what have been termed YG repeats, represents the GbpA glucan-binding domain (GBD). In an effort to test this hypot...

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

    A gene encoding a special thermophilic muitifunetional amylase OPMA-N was cloned from Bacillus sp. ZW25311. OPMA-N has an additional 124-residue N-terminal domain compared with typical amylases and forms a relatively independent domain with a IS-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 OPMAN (12.82 U/mg) and its N-terminal domain-truncated AOPMA-N (12.55 U/mg) only degraded starch to produce oligosaccharides including maltose, maltotriose, isomaitotriose, and isomaitotetraose, 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 IS-pleated sheet structure. However, the N-terminal domain of OPMA-N seemed to modulate its catalytic feature, leading to the production of more isomaitotriose and less maltose, and it seemed to contribute to OPMA-N's thermostability since OPMA-N showed higher activity than AOPMA-N in a temperature range from 40 to 80~C and the halflife (tl) was 5 h for OPMA-N and 2 h for AOPMA-N at 60~C. Both OPMA-N and AOPMA-N were Ca-independent, but their activities could be influenced by Cu2+, Niz+, Zn2+, 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 AOPMA-N could exist in the forms of monomer, homodimer, and homooligomer at the same time.

  2. Identification of an Amino Acid Domain Encoded by the Capsid Protein Gene of Porcine Circovirus Type 2 that Modulates Viral Protein Distribution During Replication

    Science.gov (United States)

    Previous work showed that distinct amino acid motifs are encoded by the Rep, Cap and ORF3 genes of two subgroups of porcine circoviruses (PCV), PCV2a and PCV2b. At a specific location of the gene, a certain amino acid residue or sequence is preferred. Specifically, two amino acid domains located in ...

  3. 1H, 13C and 15N resonance assignments of a C-terminal domain of human CHD1.

    Science.gov (United States)

    Mohanty, Biswaranjan; Silva, Ana P G; Mackay, Joel P; Ryan, Daniel P

    2016-04-01

    Chromatin remodelling proteins are an essential family of eukaryotic proteins. They harness the energy from ATP hydrolysis and apply it to alter chromatin structure in order to regulate all aspects of genome biology. Chromodomain helicase DNA-binding protein 1 (CHD1) is one such remodelling protein that has specialised nucleosome organising abilities and is conserved across eukaryotes. CHD1 possesses a pair of tandem chromodomains that directly precede the core catalytic Snf2 helicase-like domain, and a C-terminal SANT-SLIDE DNA-binding domain. We have identified an additional conserved domain in the C-terminal region of CHD1. Here, we report the backbone and side chain resonance assignments for this domain from human CHD1 at pH 6.5 and 25 °C (BMRB No. 25638). PMID:26286320

  4. Quantification of glycated N-terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry.

    Science.gov (United States)

    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.

  5. The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain

    DEFF Research Database (Denmark)

    Bräuner-Osborne, H; Jensen, Anders A.; Sheppard, P O;

    1999-01-01

    inositol phosphate production when exposed to the cationic agonists Ca2+, Mg2+, and Ba2+ in transiently transfected tsA cells (a transformed HEK 293 cell line). The pharmacological profile of Ca/1a (EC50 values of 3.3, 2.6, and 3.9 mM for these cations, respectively) was very similar to that of the wild...

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

    Science.gov (United States)

    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

  7. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers.

    Science.gov (United States)

    Zhang, Shengnan; Hinck, Andrew P; Fitzpatrick, Paul F

    2015-08-25

    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10-50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains.

  8. Uniform {sup 15}N- and {sup 15}N/{sup 13}C-labeling of proteins in mammalian cells and solution structure of the amino terminal fragment of u-PA

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, A.P.; Petros, A.M.; Meadows, R.P.; Mazar, A.P.; Nettesheim, D.G.; Pederson, T.M.; Fesik, S.W. [Abbott Laboratories, Abbott Park, IL (United States)

    1994-12-01

    Urokinase-type plasminogen activator (u-PA) is a 54-kDa glycoprotein that catalyzes the conversion of plasminogen to plasmin, a broad-specificity protease responsible for the degradation of fibrin clots and extracellular matrix components. The u-PA protein consists of three individual modules: a growth factor domain (GFD), a kringle, and a serine protease domain. The amino terminal fragment (ATF) includes the GFD-responsible for u-PA binding to its receptor-and the kringle domains. This protein was expressed and uniformly {sup 15}N-and {sup 15}N/{sup 13}C-labeled in mammalian cells by methods that will be described. In addition, we present the three-dimensional structure of ATF that was derived from 1299 NOE-derived distance restraints along with the {phi} angle and hydrogen bonding restraints. Although the individual domains in the structures were highly converged, the two domains are structurally independent. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor domain of u-PA and other homologous proteins were observed in the region that has been implicated in binding the urokinase receptor. These results may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

  9. Urea Unfolding Study of E. coli Alanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

    Science.gov (United States)

    Banerjee, Baisakhi; Banerjee, Rajat

    2015-01-01

    E. coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in the dimerization process. The truncated N-terminal containing the first 700 amino acids (1–700) forms a monomeric variant possessing similar aminoacylation activity like wild type. A point mutation in the C-terminal domain (G674D) also produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild type enzyme. Urea induced denaturation of these monomeric mutants along with another alaRS variant (N461 alaRS) was studied together with the full-length enzyme using various spectroscopic techniques such as intrinsic tryptophan fluorescence, 1-anilino-8-naphthalene-sulfonic acid binding, near- and far-UV circular dichroism, and analytical ultracentrifugation. Aminoacylation activity assay after refolding from denatured state revealed that the monomeric mutants studied here were unable to regain their activity, whereas the dimeric full-length alaRS gets back similar activity as the native enzyme. This study indicates that dimerization is one of the key regulatory factors that is important in the proper folding and stability of E. coli alaRS. PMID:26617997

  10. Urea Unfolding Study of E. coli Alanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

    Directory of Open Access Journals (Sweden)

    Baisakhi Banerjee

    2015-01-01

    Full Text Available E. coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in the dimerization process. The truncated N-terminal containing the first 700 amino acids (1–700 forms a monomeric variant possessing similar aminoacylation activity like wild type. A point mutation in the C-terminal domain (G674D also produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild type enzyme. Urea induced denaturation of these monomeric mutants along with another alaRS variant (N461 alaRS was studied together with the full-length enzyme using various spectroscopic techniques such as intrinsic tryptophan fluorescence, 1-anilino-8-naphthalene-sulfonic acid binding, near- and far-UV circular dichroism, and analytical ultracentrifugation. Aminoacylation activity assay after refolding from denatured state revealed that the monomeric mutants studied here were unable to regain their activity, whereas the dimeric full-length alaRS gets back similar activity as the native enzyme. This study indicates that dimerization is one of the key regulatory factors that is important in the proper folding and stability of E. coli alaRS.

  11. Growth hormone receptor C-terminal domains required for growth hormone-induced intracellular free Ca2+ oscillations and gene transcription

    DEFF Research Database (Denmark)

    Billestrup, N; Bouchelouche, P; Allevato, G;

    1995-01-01

    of varying frequency and amplitude. GH-induced transcription of the serine protease inhibitor 2.1 gene required the same C-terminal 52-amino acid domain of the receptor as for Ca2+ signaling. Mutation of the four proline residues in the conserved box 1 region of the GHR, which is responsible for binding......The biological effects of growth hormone (GH) are initiated by its binding to the GH receptor (GHR) followed by association and activation of the tyrosine kinase JAK2. Here we report that GH can stimulate an increase in intracellular free Ca2+ concentration ([Ca2+]i) in cells expressing wild...... and activation of JAK2 kinase, completely abolished GH-induced gene transcription but did not affect the GH-induced rise in [Ca2+]i. The Ca2+ channel blocker verapamil prevented GH-induced Ca2+ signaling as well as GH-induced gene transcription in cells expressing endogenous GHRs. These findings indicate...

  12. A Short Amino-Terminal Part of Arabidopsis Phytochrome A Induces Constitutive Photomorphogenic Response

    Institute of Scientific and Technical Information of China (English)

    András Viczián; (E)va (A)dám; Iris Wolf; János Bindics; Stefan Kircher; Marc Heijde; Roman UIm; Eberhard Sch(a)fer; Ferenc Nagy

    2012-01-01

    Phytochrome A (phyA) is the dominant photoreceptor of far-red light sensing in Arabidopsis thaliana.phyA accumulates at high levels in the cytoplasm of etiolated seedlings,and light-induced phyA signaling is mediated by a complex regulatory network.This includes light- and FHY1/FHL protein-dependent translocation of native phyA into the nucleus in vivo.It has also been shown that a short N-terminal fragment of phyA (PHYA406) is sufficient to phenocopy this highly regulated cellular process in vitro.To test the biological activity of this N-terminal fragment of phyA in planta,we produced transgenic phyA-201 plants expressing the PHYA406-YFP (YELLOW FLUORESCENT PROTEIN)-DD,PHYA406-YFP-DD-NLS (nuclear localization signal),and PHYA406-YFP-DD-NES (nuclear export signal) fusion proteins.Here,we report that PHYA406-YFP-DD is imported into the nucleus and this process is partially light-dependent whereas PHYA406-YFP-DD-NLS and PHYA406-YFP-DD-NES display the expected constitutive localization patterns.Our results show that these truncated phyA proteins are light-stable,they trigger a constitutive photomorphogenic-like response when localized in the nuclei,and neither of them induces proper phyA signaling.We demonstrate that in vitro and in vivo PHYA406 Pfr and Pr bind COP1,a general repressor of photomorphogenesis,and co-localize with it in nuclear bodies.Thus,we conclude that,in planta,the truncated PHYA406 proteins inactivate COP1 in the nuclei in a light-independent fashion.

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

    Science.gov (United States)

    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.

  14. The C-Terminal RpoN Domain of sigma54 Forms an unpredictedHelix-Turn-Helix Motif Similar to domains of sigma70

    Energy Technology Data Exchange (ETDEWEB)

    Doucleff, Michaeleen; Malak, Lawrence T.; Pelton, Jeffrey G.; Wemmer, David E.

    2005-11-01

    The ''{delta}'' subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two {sigma} families have been identified, {sigma}{sup 70} and {sigma}{sup 54}, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the {sigma}{sup 70}-type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the {sigma}{sup 54}-type factors. Here we present the NMR derived structure of the C-terminal domain of {sigma}{sup 54} from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the {sigma}{sup 70}-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of {sigma}{sup 54} binds to DNA.

  15. Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction.

    Science.gov (United States)

    Subramanyam, Shyamal; Jones, William T; Spies, Maria; Spies, M Ashley

    2013-10-01

    RAD51 DNA strand exchange protein catalyzes the central step in homologous recombination, a cellular process fundamentally important for accurate repair of damaged chromosomes, preservation of the genetic integrity, restart of collapsed replication forks and telomere maintenance. BRCA2 protein, a product of the breast cancer susceptibility gene, is a key recombination mediator that interacts with RAD51 and facilitates RAD51 nucleoprotein filament formation on single-stranded DNA generated at the sites of DNA damage. An accurate atomistic level description of this interaction, however, is limited to a partial crystal structure of the RAD51 core fused to BRC4 peptide. Here, by integrating homology modeling and molecular dynamics, we generated a structure of the full-length RAD51 in complex with BRC4 peptide. Our model predicted previously unknown hydrogen bonding patterns involving the N-terminal domain (NTD) of RAD51. These interactions guide positioning of the BRC4 peptide within a cavity between the core and the NTDs; the peptide binding separates the two domains and restricts internal dynamics of RAD51 protomers. The model's depiction of the RAD51-BRC4 complex was validated by free energy calculations and in vitro functional analysis of rationally designed mutants. All generated mutants, RAD51(E42A), RAD51(E59A), RAD51(E237A), RAD51(E59A/E237A) and RAD51(E42A/E59A/E237A) maintained basic biochemical activities of the wild-type RAD51, but displayed reduced affinities for the BRC4 peptide. Strong correlation between the calculated and experimental binding energies confirmed the predicted structure of the RAD51-BRC4 complex and highlighted the importance of RAD51 NTD in RAD51-BRCA2 interaction. PMID:23935068

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

    Science.gov (United States)

    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.

  17. Comparison of amino acids physico-chemical properties and usage of late embryogenesis abundant proteins, hydrophilins and WHy domain.

    Science.gov (United States)

    Jaspard, Emmanuel; Hunault, Gilles

    2014-01-01

    Late Embryogenesis Abundant proteins (LEAPs) comprise several diverse protein families and are mostly involved in stress tolerance. Most of LEAPs are intrinsically disordered and thus poorly functionally characterized. LEAPs have been classified and a large number of their physico-chemical properties have been statistically analyzed. LEAPs were previously proposed to be a subset of a very wide family of proteins called hydrophilins, while a domain called WHy (Water stress and Hypersensitive response) was found in LEAP class 8 (according to our previous classification). Since little is known about hydrophilins and WHy domain, the cross-analysis of their amino acids physico-chemical properties and amino acids usage together with those of LEAPs helps to describe some of their structural features and to make hypothesis about their function. Physico-chemical properties of hydrophilins and WHy domain strongly suggest their role in dehydration tolerance, probably by interacting with water and small polar molecules. The computational analysis reveals that LEAP class 8 and hydrophilins are distinct protein families and that not all LEAPs are a protein subset of hydrophilins family as proposed earlier. Hydrophilins seem related to LEAP class 2 (also called dehydrins) and to Heat Shock Proteins 12 (HSP12). Hydrophilins are likely unstructured proteins while WHy domain is structured. LEAP class 2, hydrophilins and WHy domain are thus proposed to share a common physiological role by interacting with water or other polar/charged small molecules, hence contributing to dehydration tolerance. PMID:25296175

  18. Comparison of amino acids physico-chemical properties and usage of late embryogenesis abundant proteins, hydrophilins and WHy domain.

    Directory of Open Access Journals (Sweden)

    Emmanuel Jaspard

    Full Text Available Late Embryogenesis Abundant proteins (LEAPs comprise several diverse protein families and are mostly involved in stress tolerance. Most of LEAPs are intrinsically disordered and thus poorly functionally characterized. LEAPs have been classified and a large number of their physico-chemical properties have been statistically analyzed. LEAPs were previously proposed to be a subset of a very wide family of proteins called hydrophilins, while a domain called WHy (Water stress and Hypersensitive response was found in LEAP class 8 (according to our previous classification. Since little is known about hydrophilins and WHy domain, the cross-analysis of their amino acids physico-chemical properties and amino acids usage together with those of LEAPs helps to describe some of their structural features and to make hypothesis about their function. Physico-chemical properties of hydrophilins and WHy domain strongly suggest their role in dehydration tolerance, probably by interacting with water and small polar molecules. The computational analysis reveals that LEAP class 8 and hydrophilins are distinct protein families and that not all LEAPs are a protein subset of hydrophilins family as proposed earlier. Hydrophilins seem related to LEAP class 2 (also called dehydrins and to Heat Shock Proteins 12 (HSP12. Hydrophilins are likely unstructured proteins while WHy domain is structured. LEAP class 2, hydrophilins and WHy domain are thus proposed to share a common physiological role by interacting with water or other polar/charged small molecules, hence contributing to dehydration tolerance.

  19. The activation function 2 domain of hepatic nuclear factor 4 is regulated by a short C-terminal proline-rich repressor domain.

    OpenAIRE

    Iyemere, V P; Davies, N H; Brownlee, G G

    1998-01-01

    Hepatic nuclear factor 4 (HNF4) is a transcription factor whose expression is crucial for mouse embryonic development, for liver-specific gene expression and for the prevention of one form of maturity-onset diabetes of the young. Its domain structure has been defined previously and is similar to other members of the nuclear receptor superfamily. A repressor domain has now been localised to a region of 14 amino acids (residues 428-441) near the C-terminus of HNF4 and is sufficient by itself to...

  20. From start to finish: amino-terminal protein modifications as degradation signals in plants.

    Science.gov (United States)

    Gibbs, Daniel J; Bailey, Mark; Tedds, Hannah M; Holdsworth, Michael J

    2016-09-01

    Contents 1188 I. 1188 II. 1189 III. 1190 IV. 1191 V. 1192 1192 References 1192 SUMMARY: The amino- (N-) terminus (Nt) of a protein can undergo a diverse array of co- and posttranslational modifications. Many of these create degradation signals (N-degrons) that mediate protein destruction via the N-end rule pathway of ubiquitin-mediated proteolysis. In plants, the N-end rule pathway has emerged as a major system for regulated control of protein stability. Nt-arginylation-dependent degradation regulates multiple growth, development and stress responses, and recently identified functions of Nt-acetylation can also be linked to effects on the in vivo half-lives of Nt-acetylated proteins. There is also increasing evidence that N-termini could act as important protein stability determinants in plastids. Here we review recent advances in our understanding of the relationship between the nature of protein N-termini, Nt-processing events and proteolysis in plants. PMID:27439310

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

    Science.gov (United States)

    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.

  2. NMR assignments of the N-terminal domain of Ogataea polymorpha telomerase reverse transcriptase.

    Science.gov (United States)

    Polshakov, Vladimir I; Petrova, Olga A; Parfenova, Yulia Yu; Efimov, Sergey V; Klochkov, Vladimir V; Zvereva, Maria I; Dontsova, Olga A

    2016-04-01

    Telomerase is a ribonucleoprotein enzyme that adds telomeric DNA fragments to the ends of chromosomes. This enzyme is the focus of substantial attention, both because its structure and mechanism of action are still poorly studied, and because of its pivotal roles in aging and cellular proliferation. The use of telomerase as a potential target for the design of new anticancer drugs is also of great interest. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is essential for activity and processivity. Elucidation of the structure and dynamics of TEN in solution is important for understanding the molecular mechanism of telomerase activity and for the design of new telomerase inhibitors. To approach this problem, in this study we report the (1)H, (13)C, and (15)N chemical shift assignments of TEN from Ogataea polymorpha. Analysis of the assigned chemical shifts allowed us to identify secondary structures and protein regions potentially involved in interaction with other participants of the telomerase catalytic cycle. PMID:26721464

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

    Science.gov (United States)

    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.

  4. N- and C-terminal domains determine differential nucleosomal binding geometry and affinity of linker histone isotypes H1(0) and H1c.

    Science.gov (United States)

    Vyas, Payal; Brown, David T

    2012-04-01

    Eukaryotic linker or H1 histones modulate DNA compaction and gene expression in vivo. In mammals, these proteins exist as multiple isotypes with distinct properties, suggesting a functional significance to the heterogeneity. Linker histones typically have a tripartite structure composed of a conserved central globular domain flanked by a highly variable short N-terminal domain and a longer highly basic C-terminal domain. We hypothesized that the variable terminal domains of individual subtypes contribute to their functional heterogeneity by influencing chromatin binding interactions. We developed a novel dual color fluorescence recovery after photobleaching assay system in which two H1 proteins fused to spectrally separable fluorescent proteins can be co-expressed and their independent binding kinetics simultaneously monitored in a single cell. This approach was combined with domain swap and point mutagenesis to determine the roles of the terminal domains in the differential binding characteristics of the linker histone isotypes, mouse H1(0) and H1c. Exchanging the N-terminal domains between H1(0) and H1c changed their overall binding affinity to that of the other variant. In contrast, switching the C-terminal domains altered the chromatin interaction surface of the globular domain. These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes. The methods developed in this study will have broad applications in studying dynamic properties of additional histone subtypes and other mobile proteins.

  5. Interaction of p190RhoGAP with C-terminal domain of p120-catenin modulates endothelial cytoskeleton and permeability.

    Science.gov (United States)

    Zebda, Noureddine; Tian, Yufeng; Tian, Xinyong; Gawlak, Grzegorz; Higginbotham, Katherine; Reynolds, Albert B; Birukova, Anna A; Birukov, Konstantin G

    2013-06-21

    p120-catenin is a multidomain intracellular protein, which mediates a number of cellular functions, including stabilization of cell-cell transmembrane cadherin complexes as well as regulation of actin dynamics associated with barrier function, lamellipodia formation, and cell migration via modulation of the activities of small GTPAses. One mechanism involves p120 catenin interaction with Rho GTPase activating protein (p190RhoGAP), leading to p190RhoGAP recruitment to cell periphery and local inhibition of Rho activity. In this study, we have identified a stretch of 23 amino acids within the C-terminal domain of p120 catenin as the minimal sequence responsible for the recruitment of p190RhoGAP (herein referred to as CRAD; catenin-RhoGAP association domain). Expression of the p120-catenin truncated mutant lacking the CRAD in endothelial cells attenuated effects of barrier protective oxidized phospholipid, OxPAPC. This effect was accompanied by inhibition of membrane translocation of p190RhoGAP, increased Rho signaling, as well as suppressed activation of Rac1 and its cytoskeletal effectors PAK1 (p21-activated kinase 1) and cortactin. Expression of p120 catenin-truncated mutant lacking CRAD also delayed the recovery process after thrombin-induced endothelial barrier disruption. Concomitantly, RhoA activation and downstream signaling were sustained for a longer period of time, whereas Rac signaling was inhibited. These data demonstrate a critical role for p120-catenin (amino acids 820-843) domain in the p120-catenin·p190RhoGAP signaling complex assembly, membrane targeting, and stimulation of p190RhoGAP activity toward inhibition of the Rho pathway and reciprocal up-regulation of Rac signaling critical for endothelial barrier regulation. PMID:23653363

  6. The BARD1 C-Terminal Domain Structure and Interactions with Polyadenylation Factor CstF-50

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, Ross A.; Lee, Megan S.; Tsutakawa, Susan E.; Williams, R. Scott; Tainer, John A.; Glover, J. N. Mark

    2009-07-13

    The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF- 50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins.Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

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

    Directory of Open Access Journals (Sweden)

    Carolin Lübker

    2015-07-01

    Full Text Available 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.

  8. Evaluation through literature data of the amount and amino acid composition of basal endogenous crude protein at the terminal ileum of pigs

    NARCIS (Netherlands)

    Jansman, A.J.M.; Smink, W.; Leeuwen, van P.; Rademacher, M.

    2002-01-01

    The paper summarizes and evaluates data on estimates for the amount and amino acid (AA) composition of basal endogenous crude protein (CP) at the terminal ileum of pigs derived from 33 ileal digestibility studies with pigs (30-100kg BW). Different methods and approaches are used to obtain these esti

  9. High-resolution structure of the N-terminal endonuclease domain of the Lassa virus L polymerase in complex with magnesium ions.

    Directory of Open Access Journals (Sweden)

    Gregor D Wallat

    Full Text Available Lassa virus (LASV causes deadly hemorrhagic fever disease for which there are no vaccines and limited treatments. LASV-encoded L polymerase is required for viral RNA replication and transcription. The functional domains of L-a large protein of 2218 amino acid residues-are largely undefined, except for the centrally located RNA-dependent RNA polymerase (RdRP motif. Recent structural and functional analyses of the N-terminal region of the L protein from lymphocytic choriomeningitis virus (LCMV, which is in the same Arenaviridae family as LASV, have identified an endonuclease domain that presumably cleaves the cap structures of host mRNAs in order to initiate viral transcription. Here we present a high-resolution crystal structure of the N-terminal 173-aa region of the LASV L protein (LASV L173 in complex with magnesium ions at 1.72 Å. The structure is highly homologous to other known viral endonucleases of arena- (LCMV NL1, orthomyxo- (influenza virus PA, and bunyaviruses (La Crosse virus NL1. Although the catalytic residues (D89, E102 and K122 are highly conserved among the known viral endonucleases, LASV L endonuclease structure shows some notable differences. Our data collected from in vitro endonuclease assays and a reporter-based LASV minigenome transcriptional assay in mammalian cells confirm structural prediction of LASV L173 as an active endonuclease. The high-resolution structure of the LASV L endonuclease domain in complex with magnesium ions should aid the development of antivirals against lethal Lassa hemorrhagic fever.

  10. Comparison of adjuvant activity of N- and C-terminal domain of gp96 in a Her2-positive breast cancer model

    OpenAIRE

    Pakravan, Nafiseh; Hassan, Zuhair Mohammad

    2011-01-01

    It has been frequently reported that gp96 acts as a strong biologic adjuvant. Some studies have even investigated adjuvant activity of the gp96 C- or N-terminal domain. The controversy surrounding adjuvant activity of gp96 terminal domains prompted us to compare adjuvant activity of gp96 C- or N-terminal domain toward Her2/neu, as DNA vaccine in a Her2/neu-positive breast cancer model. To do so, mice were immunized with DNA vaccine consisting of transmembrane and extracellular domain (TM + EC...

  11. AN APPROACH TO SYNTHESIZE POLY(ETHYLENE GLYCOL)-b-POLY(ε-CAPROLACTONE) WITH TERMINAL AMINO GROUP via SCHIFF'S BASE AS AN INITIATOR

    Institute of Scientific and Technical Information of China (English)

    Li Chen; Xiu-li Zhuang; Guo-en Sun; Xue-si Chen; Xia-bin Jing

    2008-01-01

    A new method to synthesize a degradable terminal amino group-containing copolymer, poly(ethylene glycol)-b-poly(ε-caprolactone) (MPEG-PCL-NH2), was developed in the following three steps: (1) the ring-opening polymerization (ROP) of ε-caprolactone from the Schiff base prepared from benzaldehyde and ethanolamine (Ph-CH=NCH2CH2OH) used as an initiator to obtain heterobifunctional poly(ε-caprolactone) with one terminal Schiff base group and one hydroxyl group (HO-PCL-CH2CH2N = CH-Ph); (2) the coupling reaction of two reactive precursors, a hydroxy-terminated HO-PCL-CH2CH2N=CH-Ph and α-monocarboxy-ωmonomethoxy poly(ethylene glycol) (CMPEG) to synthesize MPEG-PCL-CH2CH2N=CH-Ph; (3) the conversion of the -N=CH-Ph end-group into NH2 end-group by acidification of acetic acid to obtain MPEG-PCL-NH2. The structures from the precursors to the terminal amino group-containing copolymer were confirmed by 1H-NMR and their molecular weights were measured by gel permeation chromatography. The amphiphilic terminal amino group-containing copolymer could self-assemble into micelles in an aqueous system with PCL block as the core and PEG block as the shell. The micelle formation of the terminal amino group-containing block copolymer was studied by fluorescent probe technique and the existence of critical micellar concentration (cmc) confirmed the amphiphilic nature of the resulting copolymer. ESEM and DLS analysis of the micelles revealed a homogeneous spherical morphology and a unimodal size distribution.

  12. The Myc Transactivation Domain Promotes Global Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain Independently of Direct DNA Binding▿ †

    Science.gov (United States)

    Cowling, Victoria H.; Cole, Michael D.

    2007-01-01

    Myc is a transcription factor which is dependent on its DNA binding domain for transcriptional regulation of target genes. Here, we report the surprising finding that Myc mutants devoid of direct DNA binding activity and Myc target gene regulation can rescue a substantial fraction of the growth defect in myc−/− fibroblasts. Expression of the Myc transactivation domain alone induces a transcription-independent elevation of the RNA polymerase II (Pol II) C-terminal domain (CTD) kinases cyclin-dependent kinase 7 (CDK7) and CDK9 and a global increase in CTD phosphorylation. The Myc transactivation domain binds to the transcription initiation sites of these promoters and stimulates TFIIH binding in an MBII-dependent manner. Expression of the Myc transactivation domain increases CDK mRNA cap methylation, polysome loading, and the rate of translation. We find that some traditional Myc transcriptional target genes are also regulated by this Myc-driven translation mechanism. We propose that Myc transactivation domain-driven RNA Pol II CTD phosphorylation has broad effects on both transcription and mRNA metabolism. PMID:17242204

  13. Solution NMR characterization of WT CXCL8 monomer and dimer binding to CXCR1 N-terminal domain

    OpenAIRE

    Joseph, Prem Raj B.; Rajarathnam, Krishna

    2014-01-01

    Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites – the N-terminal domain (Site-I) and the extracellular/transmembrane domain (Site-II). Therefore, higher monomer affinity could be due...

  14. Use of amino terminal type III procollagen peptide (P3NP) assay in methotrexate therapy for psoriasis

    Science.gov (United States)

    Khan, S; Subedi, D; Chowdhury, M M U

    2006-01-01

    Hepatic fibrosis continues to be a risk in patients receiving methotrexate for psoriasis. Measurement of amino terminal levels of type III procollagen (P3NP) has been advocated as an effective non‐invasive test for ongoing hepatic fibrogenesis that could avoid liver biopsies. An audit was conducted to assess the practice of P3NP monitoring using guidelines produced by Manchester and whether the agreed levels correlate with histological severity. Sixty five patients with 174 P3NP assays and 30 liver biopsies were reviewed between the years 1999 and 2003. Total number of patient‐methotrexate years was 278.9 and the mean cumulative dose of methotrexate received was 2000 (SD 1838) mg. A higher cumulative dose of methotrexate correlated significantly with high mean and maximum P3NP levels. Of the 30 liver biopsies, 26 (86.6%) showed normal histology or mild to moderate steatosis, three had focal fibrosis, and one had early cirrhosis. A median P3NP value of 5.8 μg/l or higher had a stronger correlation with histological severity. It is concluded that P3NP assay is a valuable adjunct to the clinical management of patients receiving long term methotrexate that can avoid or reduce unnecessary liver biopsies. PMID:16679477

  15. Structure of a double hexamer of the Pyrococcus furiosus minichromosome maintenance protein N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Meagher, Martin; Enemark, Eric J.

    2016-06-22

    The crystal structure of the N-terminal domain of thePyrococcus furiosusminichromosome maintenance (MCM) protein as a double hexamer is described. The MCM complex is a ring-shaped helicase that unwinds DNA at the replication fork of eukaryotes and archaea. Prior to replication initiation, the MCM complex assembles as an inactive double hexamer at specific sites of DNA. The presented structure is highly consistent with previous MCM double-hexamer structures and shows two MCM hexamers with a head-to-head interaction mediated by the N-terminal domain. Minor differences include a diminished head-to-head interaction and a slightly reduced inter-hexamer rotation.

  16. The carboxyl-terminal domain of large T antigen rescues SV40 host range activity in trans independent of acetylation.

    Science.gov (United States)

    Poulin, Danielle L; DeCaprio, James A

    2006-05-25

    The host range activity of SV40 has been described as the inability of mutant viruses with deletions in the C terminal region of large T Ag to replicate in certain types of African green monkey kidney cells. We constructed new mutant viruses expressing truncated T Ag proteins and found that these mutant viruses exhibited the host range phenotype. The host range phenotype was independent of acetylation of T Ag at lysine 697. Co-expression of the C terminal domain of T Ag (aa 627-708) in trans increased both T Ag and VP1 mRNA as well as protein levels for host range mutant viruses in the restrictive cell type. In addition, the T Ag 627-708 fragment promoted the productive lytic infection of host range mutant viruses in the nonpermissive cell type. The carboxyl-terminal region of T Ag contains a biological function essential for the SV40 viral life cycle. PMID:16510165

  17. A segment of 97 amino acids within the translocation domain of Clostridium difficile toxin B is essential for toxicity.

    Directory of Open Access Journals (Sweden)

    Yongrong Zhang

    Full Text Available Clostridium difficile toxin B (TcdB intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa consists of at least 4 functional domains including a glucosyltransferase domain (GTD, a cysteine protease domain (CPD, a translocation domain (TD, and a receptor binding domain (RBD. The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown. Here we show that a 97-amino-acid segment (AA1756 - 1852, designated as ?97 or D97, located in the C-terminus of the TD and adjacent to the RBD, is essential for the cellular activity of TcdB. Deletion of this segment in TcdB (designated as TxB-D97, did not adversely alter toxin enzymatic activities or its cellular binding and uptake capacity. TxB-D97 bound to and entered cells in a manner similar to TcdB holotoxin. Both wild type and mutant toxins released their GTDs similarly in the presence of inositol hexakisphosphate (InsP6, and showed a similar glucosyltransferase activity in a cell-free glucosylating assay. Despite these similarities, the cytotoxic activity of TxB-D97 was reduced by more than 5 logs compared to wild type toxin, supported by the inability of TxB-D97 to glucosylate Rac1 of target cells. Moreover, the mutant toxin failed to elicit tumor necrosis factor alpha (TNF-α in macrophages, a process dependent on the glucosyltransferase activity of the toxin. Cellular fractionation of toxin-exposed cells revealed that TxB-D97 was unable to efficiently release the GTD into cytosol. Thereby, we conclude the 97-amino-acid region of the TD C-terminus of TcdB adjacent to the RBD, is essential for the toxicity of TcdB.

  18. Solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP).

    Science.gov (United States)

    Liew, Chu Kong; Crossley, Merlin; Mackay, Joel P; Nicholas, Hannah R

    2007-02-16

    The THAP (Thanatos-associated protein) domain is a recently discovered zinc-binding domain found in proteins involved in transcriptional regulation, cell-cycle control, apoptosis and chromatin modification. It contains a single zinc atom ligated by cysteine and histidine residues within a Cys-X(2-4)-Cys-X(35-53)-Cys-X(2)-His consensus. We have determined the NMR solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP) and show that it adopts a fold containing a treble clef motif, bearing similarity to the zinc finger-associated domain (ZAD) from Drosophila Grauzone. The CtBP THAP domain contains a large, positively charged surface patch and we demonstrate that this domain can bind to double-stranded DNA in an electrophoretic mobility-shift assay. These data, together with existing reports, indicate that THAP domains might exhibit a functional diversity similar to that observed for classical and GATA-type zinc fingers. PMID:17174978

  19. Human Naa50 Protein Displays Broad Substrate Specificity for Amino-terminal Acetylation: DETAILED STRUCTURAL AND BIOCHEMICAL ANALYSIS USING TETRAPEPTIDE LIBRARY.

    Science.gov (United States)

    Reddi, Ravikumar; Saddanapu, Venkateshwarlu; Chinthapalli, Dinesh Kumar; Sankoju, Priyanka; Sripadi, Prabhakar; Addlagatta, Anthony

    2016-09-23

    Amino-terminal acetylation is a critical co-translational modification of the newly synthesized proteins in a eukaryotic cell carried out by six amino-terminal acetyltransferases (NATs). All NATs contain at least one catalytic subunit, and some contain one or two additional auxiliary subunits. For example, NatE is a complex of Naa10, Naa50, and Naa15 (auxiliary). In the present study, the crystal structure of human Naa50 suggested the presence of CoA and acetylated tetrapeptide (AcMMXX) that have co-purified with the protein. Biochemical and thermal stability studies on the tetrapeptide library with variations in the first and second positions confirm our results from the crystal structure that a peptide with Met-Met in the first two positions is the best substrate for this enzyme. In addition, Naa50 acetylated all MXAA peptides except for MPAA. Transcriptome analysis of 10 genes that make up six NATs in humans from eight different cell lines suggests that components of NatE are transcribed in all cell lines, whereas others are variable. Because Naa10 is reported to acetylate all amino termini that are devoid of methionine and Naa50 acetylates all other peptides that are followed by methionine, we believe that NatE complex can be a major contributor for amino-terminal acetylation at the ribosome exit tunnel. PMID:27484799

  20. Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

    Directory of Open Access Journals (Sweden)

    Merkel George

    2006-06-01

    Full Text Available Abstract Background To further our understanding of the structure and function of HIV-1 integrase (IN we developed and characterized a library of monoclonal antibodies (mAbs directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. Results We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A and IN (I267A/I268A, exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. Conclusion It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather

  1. Chaperone-like effect of the linker on the isolated C-terminal domain of rabbit muscle creatine kinase.

    Science.gov (United States)

    Chen, Zhe; Chen, Xiang-Jun; Xia, Mengdie; He, Hua-Wei; Wang, Sha; Liu, Huihui; Gong, Haipeng; Yan, Yong-Bin

    2012-08-01

    Intramolecular chaperones (IMCs), which are specific domains/segments encoded in the primary structure of proteins, exhibit chaperone-like activity against the aggregation of the other domains in the same molecule. In this research, we found that the truncation of the linker greatly promoted the thermal aggregation of the isolated C-terminal domain (CTD) of rabbit muscle creatine kinase (RMCK). Either the existence of the linker covalently linked to CTD or the supply of the synthetic linker peptide additionally could successfully protect the CTD of RMCK against aggregation in a concentration-dependent manner. Truncated fragments of the linker also behaved as a chaperone-like effect with lower efficiency, revealing the importance of its C-terminal half in the IMC function of the linker. The aggregation sites in the CTD of RMCK were identified by molecular dynamics simulations. Mutational analysis of the three key hydrophobic residues resulted in opposing effects on the thermal aggregation between the CTD with intact or partial linker, confirming the role of linker as a lid to protect the hydrophobic residues against exposure to solvent. These observations suggested that the linkers in multidomain proteins could act as IMCs to facilitate the correct folding of the aggregation-prone domains. Furthermore, the intactness of the IMC linker after proteolysis modulates the production of off-pathway aggregates, which may be important to the onset of some diseases caused by the toxic effects of aggregated proteolytic fragments.

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

    Science.gov (United States)

    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

  3. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part I. Importance of hydrophobic interactions in stabilization of β-hairpin structure

    OpenAIRE

    Skwierawska, Agnieszka; Makowska, Joanna; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2009-01-01

    We previously studied a 16-amino acid-residue fragment of the C-terminal β-hairpin (residues 46–61), [IG(46–61)], of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, and found that hydrophobic interactions and the turn region play an important role in stabilizing the structure. Based on these results, we carried out systematic structural studies of peptides derived from the sequence of IG(46–61) by systematically shortening the peptide by one residue at a time from bo...

  4. Alamethicin-like behaviour of new 18-residue peptaibols, trichorzins PA. Role of the C-terminal amino-alcohol in the ion channel forming activity.

    Science.gov (United States)

    Duval, D; Cosette, P; Rebuffat, S; Duclohier, H; Bodo, B; Molle, G

    1998-03-01

    The influences of peptide length, absence of a Glx (Gln/Glu) residue and the C-terminal amino alcohol on liposome permeabilization and ion-channel characteristics in planar lipid bilayers were examined with two 18-residue peptaibols, PA V and PA IX. As compared to the 20-residue alamethicin, both peptides belonging to the newly isolated trichorzin family, lack a proline in the N-terminal part and one of the two Gln/Glu residues in the C-terminal part of the sequence. The two analogues studied here differ among themselves in their C-terminal amino alcohol (tryptophanol for PA V and phenylalaninol for PA IX). These alpha-helical peptaibols modify to a similar extent the permeability of liposomes, as measured by leakage of a previously entrapped fluorescent probe. Monitoring tryptophanol fluorescence, a greater embedment of the peptide PA V is observed in cholesterol-free bilayers. Macroscopic conductance studies for PA V and PA IX display alamethicin-like current-voltage curves, with a similar voltage dependence, but a smaller mean number of monomers per conducting aggregate is estimated for the tryptophanol analogue, PA V. Single-channel recordings indicate faster current fluctuations for PA IX, while amplitude histograms show lower conductance levels for PA V. Apart from underlining the role of the mismatch between helix length and bilayer hydrophobic thickness, these results stress that the C-terminal tryptophanol favours a stabilization of the conducting aggregates. PMID:9518665

  5. Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Bin [Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, 100101 Beijing (China); Zhu, Shunyi, E-mail: Zhusy@ioz.ac.cn [Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, 100101 Beijing (China)

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer Al-M is an engineered fungal defensin with the n-loop of an insect defensin. Black-Right-Pointing-Pointer Al-M adopts a native defensin-like structure with high antibacterial potency. Black-Right-Pointing-Pointer Al-M kills bacteria through a membrane disruptive mechanism. Black-Right-Pointing-Pointer This work sheds light on the functional evolution of CS{alpha}{beta}-type defensins. -- Abstract: Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized {alpha}-helical and {beta}-sheet (CS{alpha}{beta}) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wall biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76 {mu}M. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.

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

    Science.gov (United States)

    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.

  7. Tumor-associated NH2-terminal fragments are the most stable part of the adenomatous polyposis coli protein and can be regulated by interactions with COOH-terminal domains.

    Science.gov (United States)

    Li, Zhuoyu; Näthke, Inke S

    2005-06-15

    Truncation mutations in the adenomatous polyposis coli (APC) gene are responsible for familial and sporadic colorectal cancer. APC is a large, multifunctional protein involved in cell migration, proliferation, and differentiation. Dominant effects that have been attributed to the NH2-terminal fragments of APC expressed in tumors may result from loss of functions due to lack of COOH-terminal regions or gain of functions due to fewer regulatory interactions. Resolving this issue and determining how structural changes contribute to the multiple functions of the APC protein requires knowledge about the structural organization of the APC molecule. To this end, we used limited proteolysis to distinguish regions of the molecule with limited structure from those that form well-folded domains. We discovered that the NH2-terminal region of APC was most resistant to proteolytic degradation, whereas middle and COOH-terminal regions were significantly more sensitive. Binding of APC to microtubules protected COOH-terminal regions of APC against proteolysis, consistent with the idea that this region of the molecule becomes ordered when bound to microtubules. Furthermore, interactions between the NH2- and COOH-terminal domains of APC were identified in vitro and in vivo, suggesting that NH2-terminal fragments of APC may be regulated by interactions with COOH-terminal domains. Indeed, expressing COOH-terminal APC fragments in tumor cells resulted in changes in the protein interactions of endogenous NH2-terminal fragments in these cells. Thus, the dominant function of NH2-terminal APC fragments found in tumor cells could be explained by loss of this regulation in tumors where COOH-terminal domains are missing.

  8. Correlation analysis for protein evolutionary family based on amino acid position mutations and application in PDZ domain.

    Directory of Open Access Journals (Sweden)

    Qi-Shi Du

    Full Text Available BACKGROUND: It has been widely recognized that the mutations at specific directions are caused by the functional constraints in protein family and the directional mutations at certain positions control the evolutionary direction of the protein family. The mutations at different positions, even distantly separated, are mutually coupled and form an evolutionary network. Finding the controlling mutative positions and the mutative network among residues are firstly important for protein rational design and enzyme engineering. METHODOLOGY: A computational approach, namely amino acid position conservation-mutation correlation analysis (CMCA, is developed to predict mutually mutative positions and find the evolutionary network in protein family. The amino acid position mutative function, which is the foundational equation of CMCA measuring the mutation of a residue at a position, is derived from the MSA (multiple structure alignment database of protein evolutionary family. Then the position conservation correlation matrix and position mutation correlation matrix is constructed from the amino acid position mutative equation. Unlike traditional SCA (statistical coupling analysis approach, which is based on the statistical analysis of position conservations, the CMCA focuses on the correlation analysis of position mutations. CONCLUSIONS: As an example the CMCA approach is used to study the PDZ domain of protein family, and the results well illustrate the distantly allosteric mechanism in PDZ protein family, and find the functional mutative network among residues. We expect that the CMCA approach may find applications in protein engineering study, and suggest new strategy to improve bioactivities and physicochemical properties of enzymes.

  9. Comparison of the frequency of functional SH3 domains with different limited sets of amino acids using mRNA display.

    Directory of Open Access Journals (Sweden)

    Junko Tanaka

    Full Text Available Although modern proteins consist of 20 different amino acids, it has been proposed that primordial proteins consisted of a small set of amino acids, and additional amino acids have gradually been recruited into the genetic code. This hypothesis has recently been supported by comparative genome sequence analysis, but no direct experimental approach has been reported. Here, we utilized a novel experimental approach to test a hypothesis that native-like globular proteins might be easily simplified by a set of putative primitive amino acids with retention of its structure and function than by a set of putative new amino acids. We performed in vitro selection of a functional SH3 domain as a model from partially randomized libraries with different sets of amino acids using mRNA display. Consequently, a library rich in putative primitive amino acids included a larger number of functional SH3 sequences than a library rich in putative new amino acids. Further, the functional SH3 sequences were enriched from the primitive library slightly earlier than from a randomized library with the full set of amino acids, while the function and structure of the selected SH3 proteins with the primitive alphabet were comparable with those from the 20 amino acid alphabet. Application of this approach to various combinations of codons in protein sequences may be useful not only for clarifying the precise order of the amino acid expansion in the early stages of protein evolution but also for efficiently creating novel functional proteins in the laboratory.

  10. Human dermatosparaxis: a form of Ehlers-Danlos syndrome that results from failure to remove the amino-terminal propeptide of type I procollagen.

    Science.gov (United States)

    Smith, L T; Wertelecki, W; Milstone, L M; Petty, E M; Seashore, M R; Braverman, I M; Jenkins, T G; Byers, P H

    1992-08-01

    Dermatosparaxis is a recessively inherited connective-tissue disorder that results from lack of the activity of type I procollagen N-proteinase, the enzyme that removes the amino-terminal propeptides from type I procollagen. Initially identified in cattle more than 20 years ago, the disorder was subsequently characterized in sheep, cats, and dogs. Affected animals have fragile skin, lax joints, and often die prematurely because of sepsis following avulsion of portions of skin. We recently identified two children with soft, lax, and fragile skin, which, when examined by transmission electron microscopy, contained the twisted, ribbon-like collagen fibrils characteristic of dermatosparaxis. Skin extracts from one child contained collagen precursors with amino-terminal extensions. Cultured fibroblasts from both children failed to cleave the amino-terminal propeptides from the pro alpha 1(I) and pro alpha 2(I) chains in type I procollagen molecules. Extracts of normal cells cleaved to collagen, the type I procollagen synthesized by cells from both children, demonstrating that the enzyme, not the substrate, was defective. These findings distinguish dermatosparaxis from Ehlers-Danlos syndrome type VII, which results from substrate mutations that prevent proteolytic processing of type I procollagen molecules.

  11. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC". The calpain-mediated ACT processing allows trafficking of the "soluble AC" domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools", which would play different roles in the cell pathophysiology.

  12. The carboxy-terminal domain of Erb1 is a seven-bladed ß-propeller that binds RNA.

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    Marcin Wegrecki

    Full Text Available Erb1 (Eukaryotic Ribosome Biogenesis 1 protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other β-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU, thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.

  13. Probing the Impact of the EchinT C-Terminal Domain on Structure and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    S Bardaweel; J Pace; T Chou; V Cody; C Wagner

    2011-12-31

    Histidine triad nucleotide binding protein (Hint) is considered as the ancestor of the histidine triad protein superfamily and is highly conserved from bacteria to humans. Prokaryote genomes, including a wide array of both Gram-negative bacteria and Gram-positive bacteria, typically encode one Hint gene. The cellular function of Hint and the rationale for its evolutionary conservation in bacteria have remained a mystery. Despite its ubiquity and high sequence similarity to eukaryote Hint1 [Escherichia coli Hint (echinT) is 48% identical with human Hint1], prokaryote Hint has been reported in only a few studies. Here we report the first conformational information on the full-length N-terminal and C-terminal residues of Hint from the E. coli complex with GMP. Structural analysis of the echinT-GMP complex reveals that it crystallizes in the monoclinic space group P2{sub 1} with four homodimers in the asymmetric unit. Analysis of electron density for both the N-terminal residues and the C-terminal residues of the echinT-GMP complex indicates that the loops in some monomers can adopt more than one conformation. The observation of conformational flexibility in terminal loop regions could explain the presence of multiple homodimers in the asymmetric unit of this structure. To explore the impact of the echinT C-terminus on protein structure and catalysis, we conducted a series of catalytic radiolabeling and kinetic experiments on the C-terminal deletion mutants of echinT. In this study, we show that sequential deletion of the C-terminus likely has no effect on homodimerization and a modest effect on the secondary structure of echinT. However, we observed a significant impact on the folding structure, as reflected by a significant lowering of the T{sub m} value. Kinetic analysis reveals that the C-terminal deletion mutants are within an order of magnitude less efficient in catalysis compared to wild type, while the overall kinetic mechanism that proceeds through a fast step

  14. Ligation of cancer cell surface GRP78 with antibodies directed against its COOH-terminal domain up-regulates p53 activity and promotes apoptosis.

    Science.gov (United States)

    Misra, Uma Kant; Mowery, Yvonne; Kaczowka, Steven; Pizzo, Salvatore Vincent

    2009-05-01

    Binding of activated α(2)-macroglobulin to GRP78 on the surface of human prostate cancer cells promotes proliferation by activating signaling cascades. Autoantibodies directed against the activated α(2)-macroglobulin binding site in the NH(2)-terminal domain of GRP78 are receptor agonists, and their presence in the sera of cancer patients is a poor prognostic indicator. We now show that antibodies directed against the GRP78 COOH-terminal domain inhibit [(3)H]thymidine uptake and cellular proliferation while promoting apoptosis as measured by DNA fragmentation, Annexin V assay, and clonogenic assay. These antibodies are receptor antagonists blocking autophosphorylation and activation of GRP78. Using 1-LN and DU145 prostate cancer cell lines and A375 melanoma cells, which express GRP78 on their cell surface, we show that antibodies directed against the COOH-terminal domain of GRP78 up-regulate the tumor suppressor protein p53. By contrast, antibody directed against the NH(2)-terminal domain of GRP78 shows negligible effects on p53 expression. PC-3 prostate cancer cells, which do not express GRP78 on their cell surface, are refractory to the effects of anti-GRP78 antibodies directed against either the COOH- or NH(2)-terminal domains. However, overexpression of GRP78 in PC-3 cells causes translocation of GRP78 to the cell surface and promotes apoptosis when these cells are treated with antibody directed against its COOH-terminal domain. Silencing GRP78 or p53 expression by RNA interference significantly blocked the increase in p53 induced by antibodies. Antibodies directed against the COOH-terminal domain may play a therapeutic role in cancer patients whose tumors trigger the production of autoantibodies directed against the NH(2)-terminal domain of GRP78.

  15. The functional domain of GCS1-based gamete fusion resides in the amino terminus in plant and parasite species.

    Directory of Open Access Journals (Sweden)

    Toshiyuki Mori

    Full Text Available Fertilization is one of the most important processes in all organisms utilizing sexual reproduction. In a previous study, we succeeded in identifying a novel male gametic transmembrane protein GCS1 (GENERATIVE CELL SPECIFIC 1, also called HAP2 (HAPLESS 2 in the male-sterile Arabidopsis thaliana mutants, as a factor critical to gamete fusion in flowering plants. Interestingly, GCS1 is highly conserved among various eukaryotes covering plants, protists and invertebrates. Of these organisms, Chlamydomonas (green alga and Plasmodium (malaria parasite GCS1s similarly show male gametic expression and gamete fusion function. Since it is generally believed that protein factors controlling gamete fusion have rapidly evolved and different organisms utilize species-specific gamete fusion factors, GCS1 may be an ancient fertilization factor derived from the common ancestor of those organisms above. And therefore, its molecular structure and function are important to understanding the common molecular mechanics of eukaryotic fertilization. In this study, we tried to detect the central functional domain(s of GCS1, using complementation assay of Arabidopsis GCS1 mutant lines expressing modified GCS1. As a result, the positively-charged C-terminal sequence of this protein is dispensable for gamete fusion, while the highly conserved N-terminal domain is critical to GCS1 function. In addition, in vitro fertilization assay of Plasmodium berghei (mouse malaria parasite knock-in lines expressing partly truncated GCS1 showed similar results. Those findings above indicate that the extracellular N-terminus alone is sufficient for GCS1-based gamete fusion.

  16. Jun Dimerization Protein 2 Functions as a Progesterone Receptor N-Terminal Domain Coactivator

    OpenAIRE

    Wardell, Suzanne E.; Boonyaratanakornkit, Viroj; Adelman, James S.; Aronheim, Ami; Edwards, Dean P.

    2002-01-01

    The progesterone receptor (PR) contains two transcription activation function (AF) domains, constitutive AF-1 in the N terminus and AF-2 in the C terminus. AF-2 activity is mediated by a hormone-dependent interaction with a family of steroid receptor coactivators (SRCs). SRC-1 can also stimulate AF-1 activity through a secondary domain that interacts simultaneously with the primary AF-2 interaction site. Other protein interactions and mechanisms that mediate AF-1 activity are not well defined...

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

    International Nuclear Information System (INIS)

    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

  18. Interplay of positive and negative effectors in function of the C-terminal repeat domain of RNA polymerase II.

    OpenAIRE

    Li, Y.; Kornberg, R D

    1994-01-01

    RNA polymerase II lacking a C-terminal domain (CTD) was active in transcription with purified proteins from yeast but failed to support transcription in a yeast extract. CTD dependence could be reconstituted in the purified system by addition of two fractions from the extract. An inhibitory fraction abolished transcription by both wild-type and CTD-less RNA polymerases; a stimulatory fraction restored activity of the wild-type polymerase but had a much lesser effect on the CTD-less enzyme. Pa...

  19. Anti-androgen effects of cypermethrin on the amino- and carboxyl-terminal interaction of the androgen receptor

    International Nuclear Information System (INIS)

    Graphical abstract: Both the known AR antagonist nilutamide and the pyrethroid insecticide cypermethrin inhibited DHT-induced AR N/C interaction in the mammalian two-hybrid assay. However, cypermethrin was a weaker androgen antagonist than nilutamide. Highlights: ► We have developed the mammalian two-hybrid assay. ► The assay displayed appropriate response to DHT and nilutamide. ► The N/C interaction was induced by DHT in a dose-dependent manner. ► Nilutamide inhibited DHT-induced AR N/C interaction. ► Cypermethrin exhibits inhibitory effects on DHT-induced AR N/C interaction. -- Abstract: The pyrethroid insecticide, cypermethrin has been demonstrated to be an environmental anti-androgen in the androgen receptor (AR) reporter gene assay. The amino- and carboxyl-terminal (N/C) interaction is required for transcription potential of the AR. In order to characterize the anti-androgen effects of cypermethrin involved in the N/C interaction of AR, the mammalian two-hybrid assay has been developed in the study. The fusion vectors pVP16-ARNTD, pM-ARLBD and the pG5CAT Reporter Vector were cotransfected into the CV-1 cells. The assay displayed appropriate response to the potent, classical AR agonist 5α-dihydrotestosterone (DHT) and known AR antagonist nilutamide. The N/C interaction was induced by DHT from 10−11 M to 10−5 M in a dose-dependent manner. Nilutamide did not activate N/C interaction, while inhibited DHT-induced AR N/C interaction at the concentrations from 10−7 M to 10−5 M. Treatment of CV-1 cells with cypermethrin alone did not activate the reporter CAT. Cypermethrin significantly decreased the DHT-induced reporter CAT expression at the higher concentration of 10−5 M. The mammalian two-hybrid assay provides a promising tool both for defining mechanism involved in AR N/C interaction of EDCs and for screening of chemicals with androgen agonistic and antagonistic activities. Cypermethrin exhibits inhibitory effects on the DHT-induced AR N

  20. FS23 binds to the N-terminal domain of human Hsp90:A novel small inhibitor for Hsp90

    Institute of Scientific and Technical Information of China (English)

    李健; 石峰; 陈丹琦; 曹慧玲; 熊兵; 沈竞康; 何建华

    2015-01-01

    The N-terminal domain of heat shock protein 90 (Hsp90N) is responsible for the catalytic activity of Hsp90. The reported inhibitors of Hsp90 bind to this domain and would inhibit tumor growth and progression. Here, we synthesized FS23, a small molecule inhibitor of hsp90 and collected X-ray diffraction data of the complex crystal of Hsp90-FS23. High resolution X-ray crystallography shows that FS23 interacted with Hsp90N at the nucleotide binding cleft, and this suggests that FS23 may complete with nucleotides to bind to Hsp90N. The crystal structure and the interaction between Hsp90N and FS23 suggest a rational basis for the design of novel antitumor drugs.

  1. The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains

    DEFF Research Database (Denmark)

    Dahlbäck, Madeleine; Jørgensen, Lars M; Nielsen, Morten A;

    2011-01-01

    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...... of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite...

  2. Characterization of the N-terminal domain of BteA: a Bordetella type III secreted cytotoxic effector.

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    Chen Guttman

    Full Text Available BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Currently there is limited information regarding the structure of BteA or its subdomains, and no insight as to the identity of its eukaryotic partners(s and their modes of interaction with BteA. The mechanisms that lead to BteA dependent cell death also remain elusive. The N-terminal domain of BteA is multifunctional, acting as a docking platform for its cognate chaperone (BtcA in the bacterium, and targeting the protein to lipid raft microdomains within the eukaryotic host cell. In this study we describe the biochemical and biophysical characteristics of this domain (BteA287 and determine its architecture. We characterize BteA287 as being a soluble and highly stable domain which is rich in alpha helical content. Nuclear magnetic resonance (NMR experiments combined with size exclusion and analytical ultracentrifugation measurements confirm these observations and reveal BteA287 to be monomeric in nature with a tendency to oligomerize at concentrations above 200 µM. Furthermore, diffusion-NMR demonstrated that the first 31 residues of BteA287 are responsible for the apparent aggregation behavior of BteA287. Light scattering analyses and small angle X-ray scattering experiments reveal a prolate ellipsoidal bi-pyramidal dumb-bell shape. Thus, our biophysical characterization is a first step towards structure determination of the BteA N-terminal domain.

  3. Amino substituted nitrogen heterocycle ureas as kinase insert domain containing receptor (KDR inhibitors: Performance of structure–activity relationship approaches

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    Hayriye Yilmaz

    2015-06-01

    Full Text Available A quantitative structure–activity relationship (QSAR study was performed on a set of amino-substituted nitrogen heterocyclic urea derivatives. Two novel approaches were applied: (1 the simplified molecular input-line entry systems (SMILES based optimal descriptors approach; and (2 the fragment-based simplex representation of molecular structure (SiRMS approach. Comparison with the classic scheme of building up the model and balance of correlation (BC for optimal descriptors approach shows that the BC scheme provides more robust predictions than the classic scheme for the considered pIC50 of the heterocyclic urea derivatives. Comparison of the SMILES-based optimal descriptors and SiRMS approaches has confirmed good performance of both techniques in prediction of kinase insert domain containing receptor (KDR inhibitory activity, expressed as a logarithm of inhibitory concentration (pIC50 of studied compounds.

  4. Alternate utilization of two regulatory domains within the Moloney murine sarcoma virus long terminal repeat.

    OpenAIRE

    Graves, B J; Eisenberg, S P; Coen, D M; McKnight, S L

    1985-01-01

    The Moloney murine sarcoma virus long terminal repeat (LTR) harbors two distinct positive activators of transcription, namely, a distal signal and an enhancer. In this report we demonstrate that infection by herpes simplex virus (HSV) can markedly affect the utilization of these two Moloney murine sarcoma virus transcription signals. We investigated the HSV-mediated trans-acting effects with two goals in mind: first, to gain insight into LTR function, and second, to probe the mechanisms used ...

  5. Domain mapping of Escherichia coli RecQ defines the roles of conserved N- and C-terminal regions in the RecQ family

    OpenAIRE

    Bernstein, Douglas A.; Keck, James L.

    2003-01-01

    RecQ DNA helicases function in DNA replication, recombination and repair. Although the precise cellular roles played by this family of enzymes remain elusive, the importance of RecQ proteins is clear; mutations in any of three human RecQ genes lead to genomic instability and cancer. In this report, proteolysis is used to define a two-domain structure for Escherichia coli RecQ, revealing a large (∼59 kDa) N-terminal and a small (∼9 kDa) C-terminal domain. A short N-terminal segment (7 or 21 re...

  6. Structure of the MutL C-terminal domain: a model of intact MutL and its roles in mismatch repair

    OpenAIRE

    Guarné, Alba; Ramon-Maiques, Santiago; Wolff, Erika M.; Ghirlando, Rodolfo; Hu, Xiaojian; Miller, Jeffrey H.; Yang, Wei

    2004-01-01

    MutL assists the mismatch recognition protein MutS to initiate and coordinate mismatch repair in species ranging from bacteria to humans. The MutL N-terminal ATPase domain is highly conserved, but the C-terminal region shares little sequence similarity among MutL homologs. We report here the crystal structure of the Escherichia coli MutL C-terminal dimerization domain and the likelihood of its conservation among MutL homologs. A 100-residue proline-rich linker between the ATPase and dimerizat...

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

    Science.gov (United States)

    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.

  8. Development and identification of a novel anti-HIV-1 peptide derived by modification of the N-terminal domain of HIV-1 integrase

    Directory of Open Access Journals (Sweden)

    Marina eSala

    2016-06-01

    Full Text Available 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 M. 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.

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

    Science.gov (United States)

    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. Síntesis de lreidodipeptidos a partir de 0-succinimidil carbamatos derivados de a-aminoácidos protegidos de su función amino terminal

    OpenAIRE

    Juan Manuel Lozano; Gilíes Guichard; Jean Paul Briand; José Libardo Torres-Castellanosq; Fabiolo Espejo; Manuel Elkín Patarroyo

    2010-01-01

    Una estrategia hacia el desarrollo de nuevos agentes terapéuticos e inmunoprofilácticos contra enfermedades transmisibles consiste en la alteración de la identidad química del enlace peptídico entre dos aminoácidos determinados, así como la modificación de la conformación de los carbonos alpha de tales residuos.

  11. Síntesis de lreidodipeptidos a partir de 0-succinimidil carbamatos derivados de a-aminoácidos protegidos de su función amino terminal

    Directory of Open Access Journals (Sweden)

    Juan Manuel Lozano

    2010-10-01

    Full Text Available Una estrategia hacia el desarrollo de nuevos agentes terapéuticos e inmunoprofilácticos contra enfermedades transmisibles consiste en la alteración de la identidad química del enlace peptídico entre dos aminoácidos determinados, así como la modificación de la conformación de los carbonos alpha de tales residuos.

  12. In Sup35p filaments (the [PSI+] prion), the globular C-terminal domains are widely offset from the amyloid fibril backbone

    Energy Technology Data Exchange (ETDEWEB)

    Baxa, U.; Wall, J.; Keller, P. W.; Cheng, N.; Steven, A. C.

    2011-01-01

    In yeast cells infected with the [PSI+] prion, Sup35p forms aggregates and its activity in translation termination is downregulated. Transfection experiments have shown that Sup35p filaments assembled in vitro are infectious, suggesting that they reproduce or closely resemble the prion. We have used several EM techniques to study the molecular architecture of filaments, seeking clues as to the mechanism of downregulation. Sup35p has an N-terminal 'prion' domain; a highly charged middle (M-)domain; and a C-terminal domain with the translation termination activity. By negative staining, cryo-EM and scanning transmission EM (STEM), filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse 65-nm-wide cloud of globular C-domains. In diameter ({approx}8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield -1 subunit per 0.47 nm for N-fibrils, NM-filaments and Sup35p filaments, further supporting the fibril backbone model. The 30 nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations, offering an explanation for the residual Sup35p activity in infected cells, whereby the C-domains remain free enough to interact with ribosomes.

  13. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

    Energy Technology Data Exchange (ETDEWEB)

    Serek, Robert; Forwood, Jade K. [School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072 (Australia); Hume, David A. [School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072 (Australia); Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072 (Australia); Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Queensland 4072 (Australia); Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072 (Australia); Martin, Jennifer L.; Kobe, Bostjan, E-mail: b.kobe@uq.edu.au [School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072 (Australia); Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072 (Australia); Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072 (Australia)

    2006-02-01

    The C-terminal domain of the mouse long-chain acyl-CoA thioesterase has been expressed in bacteria and crystallized by vapour diffusion. The crystals diffract to 2.4 Å resolution. The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination.

  14. Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

    Directory of Open Access Journals (Sweden)

    Marijke De Bock

    2015-01-01

    Full Text Available The coordination of tissue function is mediated by gap junctions (GJs that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury.

  15. Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    Science.gov (United States)

    Scharinger, Anja; Eckrich, Stephanie; Vandael, David H.; Schönig, Kai; Koschak, Alexandra; Hecker, Dietmar; Kaur, Gurjot; Lee, Amy; Sah, Anupam; Bartsch, Dusan; Benedetti, Bruno; Lieb, Andreas; Schick, Bernhard; Singewald, Nicolas; Sinnegger-Brauns, Martina J.; Carbone, Emilio; Engel, Jutta; Striessnig, Jörg

    2015-01-01

    Cav1.3 L-type Ca2+-channel function is regulated by a C-terminal automodulatory domain (CTM). It affects channel binding of calmodulin and thereby tunes channel activity by interfering with Ca2+- and voltage-dependent gating. Alternative splicing generates short C-terminal channel variants lacking the CTM resulting in enhanced Ca2+-dependent inactivation and stronger voltage-sensitivity upon heterologous expression. However, the role of this modulatory domain for channel function in its native environment is unkown. To determine its functional significance in vivo, we interrupted the CTM with a hemagglutinin tag in mutant mice (Cav1.3DCRDHA/HA). Using these mice we provide biochemical evidence for the existence of long (CTM-containing) and short (CTM-deficient) Cav1.3 α1-subunits in brain. The long (HA-labeled) Cav1.3 isoform was present in all ribbon synapses of cochlear inner hair cells. CTM-elimination impaired Ca2+-dependent inactivation of Ca2+-currents in hair cells but increased it in chromaffin cells, resulting in hyperpolarized resting potentials and reduced pacemaking. CTM disruption did not affect hearing thresholds. We show that the modulatory function of the CTM is affected by its native environment in different cells and thus occurs in a cell-type specific manner in vivo. It stabilizes gating properties of Cav1.3 channels required for normal electrical excitability. PMID:26379493

  16. Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    Directory of Open Access Journals (Sweden)

    Anja eScharinger

    2015-08-01

    Full Text Available Cav1.3 L-type Ca2+-channel function is regulated by a C-terminal automodulatory domain (CTM. It affects channel binding of calmodulin and thereby tunes channel activity by interfering with Ca2+- and voltage-dependent gating. Alternative splicing generates short C-terminal channel variants lacking the CTM resulting in enhanced Ca2+-dependent inactivation and stronger voltage-sensitivity upon heterologous expression. However, the role of this modulatory domain for channel function in its native environment is unkown. To determine its functional significance in vivo, we interrupted the CTM with a hemagglutinin tag in mutant mice (Cav1.3DCRDHA/HA. Using these mice we provide biochemical evidence for the existence of long (CTM-containing and short (CTM-deficient Cav1.3 α1-subunits in brain. The long (HA-labeled Cav1.3 isoform was present in all ribbon synapses of cochlear inner hair cells. CTM-elimination impaired Ca2+-dependent inactivation of Ca2+-currents in hair cells but increased it in chromaffin cells, resulting in hyperpolarized resting potentials and reduced pacemaking. CTM disruption did not affect hearing thresholds. We show that the modulatory function of the CTM is affected by its native environment in different cells and thus occurs in a cell-type specific manner in vivo. It is required to stabilize gating properties of Cav1.3 channels required for normal electrical excitability.

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

    Directory of Open Access Journals (Sweden)

    Justin Doritchamou

    Full Text Available 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.

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

    Science.gov (United States)

    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

  19. The effects of amino acid composition of glutamine-rich domains on amyloid formation and fragmentation.

    Directory of Open Access Journals (Sweden)

    Alexander I Alexandrov

    Full Text Available Fragmentation of amyloid polymers by the chaperone Hsp104 allows them to propagate as prions in yeast. The factors which determine the frequency of fragmentation are unclear, though it is often presumed to depend on the physical strength of prion polymers. Proteins with long polyglutamine stretches represent a tractable model for revealing sequence elements required for polymer fragmentation in yeast, since they form poorly fragmented amyloids. Here we show that interspersion of polyglutamine stretches with various amino acid residues differentially affects the in vivo formation and fragmentation of the respective amyloids. Aromatic residues tyrosine, tryptophan and phenylalanine strongly stimulated polymer fragmentation, leading to the appearance of oligomers as small as dimers. Alanine, methionine, cysteine, serine, threonine and histidine also enhanced fragmentation, while charged residues, proline, glycine and leucine inhibited polymerization. Our data indicate that fragmentation frequency primarily depends on the recognition of fragmentation-promoting residues by Hsp104 and/or its co-chaperones, rather than on the physical stability of polymers. This suggests that differential exposure of such residues to chaperones defines prion variant-specific differences in polymer fragmentation efficiency.

  20. The N-terminal domain of NifA determines the temperature sensitivity of Nif A in Klebsiella pneumoniae and Enterobacter cloacae

    Institute of Scientific and Technical Information of China (English)

    顾剑颖; 俞冠翘; 朱家璧; 沈善炯

    2000-01-01

    The NifA protein is the central regulator of the nitrogen fixation genes. It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor. The NifA protein from Klebsiella pneumoniae consists of the N-terminal domain of unknown function, the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain. The Kp NifA protein is sensitive to temperature, while the Enterobacter cloacae NifA protein is less sensitive to temperature than Kp NifA. Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.

  1. The N-terminal domain of NifA determines the temperature sensitivity of NifA in Klebsiella pneumoniae and Enterobacter cloacae

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The NifA protein is the central regulator of the nitrogen fixation genes.It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor.The NifA protein from Klebsiella pneumoniae consists of the N-terminal domain of unknown function,the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain.The Kp NifA protein is sensitive to temperature,while the Enterobacter cloacae NifA protein is less sensitive to temperature than Kp NifA.Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.

  2. A cholesterol recognition amino acid consensus domain in GP64 fusion protein facilitates anchoring of baculovirus to mammalian cells.

    Science.gov (United States)

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R; Sampieri, Alicia; Vaca, Luis

    2013-11-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.

  3. A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells

    Science.gov (United States)

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R.; Sampieri, Alicia

    2013-01-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV. PMID:23986592

  4. Expanding the Activity of Tissue Inhibitors of Metalloproteinase (TIMP)-1 against Surface-Anchored Metalloproteinases by the Replacement of Its C-Terminal Domain: Implications for Anti-Cancer Effects

    OpenAIRE

    Jing Xian Duan; Magdalini Rapti; Anastasia Tsigkou; Meng Huee Lee

    2015-01-01

    Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). TIMP molecules are made up of two domains: an N-terminal domain that associates with the catalytic cleft of the metalloproteinases (MP) and a smaller C-terminal domain whose role in MP association is still poorly understood. This work is aimed at investigating the role of the C-terminal domain in MP selectivity. In this study, ...

  5. Adaptive immunity against Leishmania nucleoside hydrolase maps its c-terminal domain as the target of the CD4+ T cell-driven protective response.

    Science.gov (United States)

    Nico, Dirlei; Claser, Carla; Borja-Cabrera, Gulnara P; Travassos, Luiz R; Palatnik, Marcos; Soares, Irene da Silva; Rodrigues, Mauricio Martins; Palatnik-de-Sousa, Clarisa B

    2010-01-01

    Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent

  6. Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell–Driven Protective Response

    Science.gov (United States)

    Nico, Dirlei; Claser, Carla; Borja-Cabrera, Gulnara P.; Travassos, Luiz R.; Palatnik, Marcos; da Silva Soares, Irene; Rodrigues, Mauricio Martins; Palatnik-de-Sousa, Clarisa B.

    2010-01-01

    Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199–314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5–88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for

  7. Adaptive immunity against Leishmania nucleoside hydrolase maps its c-terminal domain as the target of the CD4+ T cell-driven protective response.

    Directory of Open Access Journals (Sweden)

    Dirlei Nico

    Full Text Available Nucleoside hydrolases (NHs show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36 responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL. Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314 and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011 that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and

  8. DNA double strand break repair is enhanced by P53 following induction by DNA damage and is dependent on the C-terminal domain of P53

    International Nuclear Information System (INIS)

    Purpose: The tumor suppressor gene p53 can mediate cell cycle arrest or apoptosis in response to DNA damage. Accumulating evidence suggests that it may also directly or indirectly influence the DNA repair machinery. In the present study, we investigated whether p53, induced by DNA damage, could enhance the rejoining of double-strand DNA breaks. Materials and Methods: DNA double-strand breaks (dsb) were made by restriction enzyme digestion of a plasmid, between a promoter and a 'reporter' gene: luciferase (LUC) or chloramphenicol acetyl-transferase (CAT). Linear or circular plasmid DNA (LUC or CAT) was co-transfected with circular β-Gal plasmid (to normalize for uptake) into mouse embryonic fibroblasts genetically matched to be (+/+) or (-/-) for p53. Their ability to rejoin linearized plasmid was measured by the luciferase or CAT activity detected in rescued plasmids. The activity detected in cells transfected with linear plasmid was scored relative to the activity detected in cells transfected with circular plasmid. Results: Ionizing radiation (IR, 2 Gy) enhanced the dsb repair activity in wild type p53 cells; however, p53 null cells lose this effect, indicating that the enhancement of dsb repair was p53-dependent. REF cells with dominant-negative mutant p53 showed a similar induction compared with the parental REF cells with wild-type p53. This ala-143 mutant p53 prevents cell cycle arrest and transactivation of p21WAF1/cip1) following IR, indicating that the p53-dependent enhancement of DNA repair is distinct from transactivation. Immortalized murine embryonic fibroblasts, 10(1)VasK1 cells, which express p53 cDNA encoding a temperature-sensitive mutant in the DNA sequence specific binding domain (ala135 to val135) with an alternatively spliced C-terminal domain (ASp53: amino-acids 360-381) and, 10(1)Val5 cells, which express the normal spliced p53 (NSp53) with the same temperature-sensitive mutant were compared. It was found that 10(1)VasK1 cells showed no DNA

  9. Differential Recognition of Terminal Extracellular Plasmodium falciparum VAR2CSA Domains by Sera from Multigravid, Malaria-Exposed Malian Women

    Science.gov (United States)

    Travassos, Mark A.; Coulibaly, Drissa; Bailey, Jason A.; Niangaly, Amadou; Adams, Matthew; Nyunt, Myaing M.; Ouattara, Amed; Lyke, Kirsten E.; Laurens, Matthew B.; Pablo, Jozelyn; Jasinskas, Algis; Nakajima, Rie; Berry, Andrea A.; Takala-Harrison, Shannon; Kone, Abdoulaye K.; Kouriba, Bourema; Rowe, J. Alexandra; Doumbo, Ogobara K.; Thera, Mahamadou A.; Laufer, Miriam K.; Felgner, Philip L.; Plowe, Christopher V.

    2015-01-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates parasite sequestration in small capillaries through tissue-specific cytoadherence. The best characterized of these proteins is VAR2CSA, which is expressed on the surface of infected erythrocytes that bind to chondroitin sulfate in the placental matrix. Antibodies to VAR2CSA prevent placental cytoadherence and protect against placental malaria. The size and complexity of the VAR2CSA protein pose challenges for vaccine development, but smaller constitutive domains may be suitable for subunit vaccine development. A protein microarray was printed to include five overlapping fragments of the 3D7 VAR2CSA extracellular region. Malian women with a history of at least one pregnancy had antibody recognition of four of these fragments and had stronger reactivity against the two distal fragments than did nulliparous women, children, and men from Mali, suggesting that the C-terminal extracellular VAR2CSA domains are a potential focus of protective immunity. With carefully chosen sera from longitudinal studies of pregnant women, this approach has the potential to identify seroreactive VAR2CSA domains associated with protective immunity against pregnancy-associated malaria. PMID:25918203

  10. NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1

    Energy Technology Data Exchange (ETDEWEB)

    Verdone, Giuliana [Istituto Biochimico Italiano ' G. Lorenzini' (Italy); Corazza, Alessandra [Universita di Udine, Dipartimento di Scienze e Tecnologie Biomediche - MATI Centre of Excellence (Italy); Colebrooke, Simon A. [University of Oxford, Department of Biochemistry (United Kingdom); Cicero, Daniel; Eliseo, Tommaso [Universita di Tor Vergata, Dipartimento di Chimica (Italy); Boyd, Jonathan [University of Oxford, Department of Biochemistry (United Kingdom); Doliana, Roberto [Centro di Riferimento Oncologico di Aviano, Divisione di Oncologia Sperimentale 2 (Italy); Fogolari, Federico; Viglino, Paolo; Colombatti, Alfonso [Universita di Udine, Dipartimento di Scienze e Tecnologie Biomediche - MATI Centre of Excellence (Italy); Campbell, Iain D. [University of Oxford, Department of Biochemistry (United Kingdom); Esposito, Gennaro [Universita di Udine, Dipartimento di Scienze e Tecnologie Biomediche - MATI Centre of Excellence (Italy)], E-mail: gesposito@mail.dstb.uniud.it

    2009-02-15

    EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated {sup 15}N, {sup 13}C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded {beta} sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor {alpha}4{beta}1.

  11. NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1

    International Nuclear Information System (INIS)

    EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated 15N, 13C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded β sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor α4β1

  12. Amino- and Carboxyl-Terminal CCR5 Mutations in Brazilian HIV-1-Infected Women and Homology Model of p.L55Q CCR5 Mutant.

    Science.gov (United States)

    Costa, Giselle Calasans de Souza; Nunes, Marcio Roberto T; Jesus, Jaqueline Goes; Novaes, Thiago; Cardoso, Jedson Ferreira; Sousa Júnior, Edivaldo Costa; Santos, Edson de Souza; Galvão-Castro, Bernardo; Zanette, Dalila Luciola; Gonçalves, Marilda de Souza; Alcantara, Luiz Carlos Junior

    2015-07-01

    Genetic factors from an HIV-1 host can affect the rate of progression to AIDS and HIV infection. To investigate the frequency of mutations in the CCR5 gene, HIV-1 samples from infected women and uninfected individuals were selected for sequencing of the CCR5 gene regions encoding the N- and C-terminal protein domains. Physicochemical CCR5 modeling and potential protein domain analysis were performed in order to evaluate the impact of the mutations found in the properties and structure of CCR5. The p.L55Q mutation in the N-terminal protein domain was observed only in uninfected individuals, with an allelic frequency of 1.8%. Physicochemical analysis revealed that the p.L55Q mutation magnified the flexibility and accessibility profiles and the modeling of CCR5 structures showed resulting in a small deviation to the right, as well as a hydrophobic to hydrophilic property alteration. The p.L55Q mutation also resulted in a slight modification of the electrostatic load of this region. Additionally, three novel silent mutations were found at the C-terminal coding region among HIV-1-infected women. The results suggest that the p.L55Q mutation might alter CCR5 conformation. Further studies should be conducted to verify the role of this mutation in HIV-1 susceptibility.

  13. N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity

    Directory of Open Access Journals (Sweden)

    Barreto Filho J.B.

    2001-01-01

    Full Text Available The structure-function relationship of interferons (IFNs has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBoIFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

  14. Isolation of L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide), a sea anemone neuropeptide containing an unusual amino-terminal blocking group

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Rinehart, K L; Jacob, E;

    1990-01-01

    Using a radioimmunoassay for the carboxyl-terminal sequence Arg-Asn-NH2, we have purified a peptide from acetic acid extracts of the sea anemone Anthopleura elegantissima. By classical amino acid analyses, mass spectrometry, and 1H NMR spectroscopy, the structure of this peptide was determined as 3......-phenyllactyl-Leu-Arg-Asn-NH2. By using reversed-phase HPLC and a chiral mobile phase, it was shown that the 3-phenyllactyl group had the L configuration. Immunocytochemical staining with antiserum against Arg-Asn-NH2 showed that L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide) was localized in neurons of sea...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  17. Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles.

    Science.gov (United States)

    Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

    2014-01-01

    Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

  18. Biochemical analysis of the interactions of IQGAP1 C-terminal domain with CDC42

    Institute of Scientific and Technical Information of China (English)

    Sarah; F; Elliott; George; Allen; David; J; Timson

    2012-01-01

    AIM:To understand the interaction of human IQGAP1 and CDC42,especially the effects of phosphorylation and a cancer-associated mutation. METHODS:Recombinant CDC42 and a novel C-termi- nal fragment of IQGAP1 were expressed in,and puri- fied from,Escherichia coli.Site directed mutagenesis was used to create coding sequences for three phos- phomimicking variants(S1441E,S1443D and S1441E/ S1443D)and to recapitulate a cancer-associated mu- tation(M1231I).These variant proteins were also ex- pressed and purified.Protein-protein crosslinking using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to investigate interactions between the C-terminal fragment and CDC42.These interactions were quanti- fied using surface plasmon resonance measurements.Molecular modelling was employed to make predictions about changes to the structure and flexibility of the protein which occur in the cancer-associated variant. RESULTS:The novel,C-terminal region of human IQGAP1 (residues 877-1558)is soluble following expression and purification.It is also capable of binding to CDC42,as judged by crosslinking experiments.Interaction appears to be strongest in the presence of added GTP.The three phosphomimicking mutants had different affini- ties for CDC42.S1441E had an approximately 200-fold reduction in affinity compared to wild type.This was caused largely by a dramatic reduction in the associa- tion rate constant.In contrast,both S1443D and the double variant S1441E/S1443D had similar affinities to the wild type.The cancer-associated variant,M1231I, also had a similar affinity to wild type.However,in the case of this variant,both the association and dis- sociation rate constants were reduced approximately 10-fold.Molecular modelling of the M1231I variant, based on the published crystal structure of part of the C-terminal region,revealed no gross structural changes compared to wild type(root mean square deviation of 0.564over 5556 equivalent atoms).However,pre- dictions of the

  19. Solution structure of the calmodulin-like C-terminal domain of Entamoeba α-actinin2.

    Science.gov (United States)

    Karlsson, Göran; Persson, Cecilia; Mayzel, Maxim; Hedenström, Mattias; Backman, Lars

    2016-04-01

    Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross-links, or caps the filament ends have been identified and the actin cross-linker α-actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α-actinin is believed to be required for infection. To better understand the role of α-actinin in the infectious process we have determined the solution structure of the C-terminal calmodulin-like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium-binding EF-hand motifs, connected with a mobile linker. PMID:26800385

  20. The formation of right-handed and left-handed chiral nanopores within a single domain during amino acid self-assembly on Au(111).

    Science.gov (United States)

    Yang, Sena; Jeon, Aram; Driver, Russell W; Kim, Yeonwoo; Jeon, Eun Hee; Kim, Sehun; Lee, Hee-Seung; Lee, Hangil

    2016-05-25

    We report the formation of both right- and left-handed chiral nanopores within a single domain during the self-assembly of an amino acid derivative on an inert Au(111) surface using STM. DFT calculations employed to rationalize this unusual result identified that intermolecular interactions between chiral, windmill-shaped tetramers are crucial for self-assembly.

  1. Ligand-binding properties of the carboxyl-terminal repeat domain of Streptococcus mutans glucan-binding protein A.

    Science.gov (United States)

    Haas, W; Banas, J A

    2000-02-01

    Streptococcus mutans glucan-binding protein A (GbpA) has sequence similarity in its carboxyl-terminal domain with glucosyltransferases (GTFs), the enzymes responsible for catalyzing the synthesis of the glucans to which GbpA and GTFs can bind and which promote S. mutans attachment to and accumulation on the tooth surface. It was predicted that this C-terminal region, comprised of what have been termed YG repeats, represents the GbpA glucan-binding domain (GBD). In an effort to test this hypothesis and to quantitate the ligand-binding specificities of the GbpA GBD, several fusion proteins were generated and tested by affinity electrophoresis or by precipitation of protein-ligand complexes, allowing the determination of binding constants. It was determined that the 16 YG repeats in GbpA comprise its GBD and that GbpA has a greater affinity for dextran (a water-soluble form of glucan) than for mutan (a water-insoluble form of glucan). Placement of the GBD at the carboxyl terminus was necessary for maximum glucan binding, and deletion of as few as two YG repeats from either end of the GBD reduced the affinity for dextran by over 10-fold. Interestingly, the binding constant of GbpA for dextran was 34-fold higher than that calculated for the GBDs of two S. mutans GTFs, one of which catalyzes the synthesis of water-soluble glucan and the other of which catalyzes the synthesis of water-insoluble glucan. PMID:10633107

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. NMR determines transient structure and dynamics in the disordered C-terminal domain of WASp interacting protein.

    Science.gov (United States)

    Haba, Noam Y; Gross, Renana; Novacek, Jiri; Shaked, Hadassa; Zidek, Lukas; Barda-Saad, Mira; Chill, Jordan H

    2013-07-16

    WASp-interacting protein (WIP) is a 503-residue proline-rich polypeptide expressed in human T cells. The WIP C-terminal domain binds to Wiskott-Aldrich syndrome protein (WASp) and regulates its activation and degradation, and the WIP-WASp interaction has been shown to be critical for actin polymerization and implicated in the onset of WAS and X-linked thrombocytopenia. WIP is predicted to be an intrinsically disordered protein, a class of polypeptides that are of great interest because they violate the traditional structure-function paradigm. In this first (to our knowledge) study of WIP in its unbound state, we used NMR to investigate the biophysical behavior of WIP(C), a C-terminal domain fragment of WIP that includes residues 407-503 and contains the WASp-binding site. In light of the poor spectral dispersion exhibited by WIP(C) and the high occurrence (25%) of proline residues, we employed 5D-NMR(13)C-detected NMR experiments with nonuniform sampling to accomplish full resonance assignment. Secondary chemical-shift analysis, (15)N relaxation rates, and protection from solvent exchange all concurred in detecting transient structure located in motifs that span the WASp-binding site. Residues 446-456 exhibited a propensity for helical conformation, and an extended conformation followed by a short, capped helix was observed for residues 468-478. The (13)C-detected approach allows chemical-shift assignment in the WIP(C) polyproline stretches and thus sheds light on their conformation and dynamics. The effects of temperature on chemical shifts referenced to a denatured sample of the polypeptide demonstrate that heating reduces the structural character of WIP(C). Thus, we conclude that the disordered WIP(C) fragment is comprised of regions with latent structure connected by flexible loops, an architecture with implications for binding affinity and function.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Christina Funk

    2015-06-01

    Full Text Available 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

  7. Organoselenium-catalyzed, hydroxy-controlled regio- and stereoselective amination of terminal alkenes: efficient synthesis of 3-amino allylic alcohols.

    Science.gov (United States)

    Deng, Zhimin; Wei, Jialiang; Liao, Lihao; Huang, Haiyan; Zhao, Xiaodan

    2015-04-17

    An efficient route to prepare 3-amino allylic alcohols in excellent regio- and stereoselectivity in the presence of bases by orangoselenium catalysis has been developed. In the absence of bases α,β-unsaturated aldehydes were formed in up to 97% yield. Control experiments reveal that the hydroxy group is crucial for the direct amination. PMID:25849818

  8. End-labeled amino terminated monotelechelic glycopolymers generated by ROMP and Cu(I-catalyzed azide–alkyne cycloaddition

    Directory of Open Access Journals (Sweden)

    Ronald Okoth

    2013-03-01

    Full Text Available Functionalizable monotelechelic polymers are useful materials for chemical biology and materials science. We report here the synthesis of a capping agent that can be used to terminate polymers prepared by ring-opening metathesis polymerization of norbornenes bearing an activated ester. The terminating agent is a cis-butene derivative bearing a Teoc (2-trimethylsilylethyl carbamate protected primary amine. Post-polymerization modification of the polymer was accomplished by amidation with an azido-amine linker followed by Cu(I-catalyzed azide–alkyne cycloaddition with propargyl sugars. Subsequent Teoc deprotection and conjugation with pyrenyl isothiocyanates afforded well-defined end-labeled glycopolymers.

  9. Crystal structure of the C-terminal domain of the Salmonella type III secretion system export apparatus protein InvA.

    Science.gov (United States)

    Worrall, Liam J; Vuckovic, Marija; Strynadka, Natalie C J

    2010-05-01

    InvA is a prominent inner-membrane component of the Salmonella type III secretion system (T3SS) apparatus, which is responsible for regulating virulence protein export in pathogenic bacteria. InvA is made up of an N-terminal integral membrane domain and a C-terminal cytoplasmic domain that is proposed to form part of a docking platform for the soluble export apparatus proteins notably the T3SS ATPase InvC. Here, we report the novel crystal structure of the C-terminal domain of Salmonella InvA which shows a compact structure composed of four subdomains. The overall structure is unique although the first and second subdomains exhibit structural similarity to the peripheral stalk of the A/V-type ATPase and a ring building motif found in other T3SS proteins respectively.

  10. Transient viral DNA replication and repression of viral transcription are supported by the C-terminal domain of the bovine papillomavirus type 1 E1 protein.

    Science.gov (United States)

    Ferran, M C; McBride, A A

    1998-01-01

    The bovine papillomavirus type 1 E1 protein is important for viral DNA replication and transcriptional repression. It has been proposed that the full-length E1 protein consists of a small N-terminal and a larger C-terminal domain. In this study, it is shown that an E1 polypeptide containing residues 132 to 605 (which represents the C-terminal domain) is able to support transient viral DNA replication, although at a level lower than that supported by the wild-type protein. This domain can also repress E2-mediated transactivation from the P89 promoter as well as the wild-type E1 protein can. PMID:9420289

  11. Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas.

    Science.gov (United States)

    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

  12. The N-terminal domain of ERK1 accounts for the functional differences with ERK2.

    Directory of Open Access Journals (Sweden)

    Matilde Marchi

    Full Text Available The Extracellular Regulated Kinase 1 and 2 transduce a variety of extracellular stimuli regulating processes as diverse as proliferation, differentiation and synaptic plasticity. Once activated in the cytoplasm, ERK1 and ERK2 translocate into the nucleus and interact with nuclear substrates to induce specific programs of gene expression. ERK1/2 share 85% of aminoacid identity and all known functional domains and thence they have been considered functionally equivalent until recent studies found that the ablation of either ERK1 or ERK2 causes dramatically different phenotypes. To search a molecular justification of this dichotomy we investigated whether the different functions of ERK1 and 2 might depend on the properties of their cytoplasmic-nuclear trafficking. Since in the nucleus ERK1/2 is predominantly inactivated, the maintenance of a constant level of nuclear activity requires continuous shuttling of activated protein from the cytoplasm. For this reason, different nuclear-cytoplasmic trafficking of ERK1 and 2 would cause a differential signalling capability. We have characterised the trafficking of fluorescently tagged ERK1 and ERK2 by means of time-lapse imaging in living cells. Surprisingly, we found that ERK1 shuttles between the nucleus and cytoplasm at a much slower rate than ERK2. This difference is caused by a domain of ERK1 located at its N-terminus since the progressive deletion of these residues converted the shuttling features of ERK1 into those of ERK2. Conversely, the fusion of this ERK1 sequence at the N-terminus of ERK2 slowed down its shuttling to a similar value found for ERK1. Finally, computational, biochemical and cellular studies indicated that the reduced nuclear shuttling of ERK1 causes a strong reduction of its nuclear phosphorylation compared to ERK2, leading to a reduced capability of ERK1 to carry proliferative signals to the nucleus. This mechanism significantly contributes to the differential ability of ERK1 and

  13. The Rho Termination Factor of Clostridium botulinum contains a Prion-Like Domain with a highly Amyloidogenic Core

    Directory of Open Access Journals (Sweden)

    Irantzu ePallares

    2016-01-01

    Full Text Available Prion-like proteins can switch between a soluble intrinsically disordered conformation and a highly ordered amyloid assembly. This conformational promiscuity is encoded in specific sequence regions, known as prion domains (PrDs. Prions are best known as the causative factors of neurological diseases in mammals. However, bioinformatics analyses reveal that proteins bearing PrDs are present in all kingdoms of life, including bacteria, thus supporting the idea that they serve conserved beneficial cellular functions. Despite the proportion of predicted prion-like proteins in bacterial proteomes is generally low, pathogenic species seem to have a higher prionic load, suggesting that these malleable proteins may favor pathogenic traits. In the present work, we performed a stringent computational analysis of the Clostridium botulinum pathogen proteome in the search for prion-like proteins. A total of 54 candidates were predicted for this anaerobic bacterium, including the transcription termination Rho factor. This RNA-binding protein has been shown to play a crucial role in bacterial adaptation to changing environments. We show here that the predicted disordered PrD domain of this RNA-binding protein contains an inner, highly polar, asparagine-rich short sequence able to spontaneously self-assemble into amyloid-like structures, bearing thus the potential to induce a Rho factor conformational switch that might rewire gene expression in response to environmental conditions.

  14. The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain

    Science.gov (United States)

    Shi, Hang; Rojas, Raul; Bonifacino, Juan S.; Hurley, James H.

    2006-01-01

    The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29, and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1Å resolution reveals two curvedβ -sandwich domains connected by a polar core and a flexible linker. Vps26 has an unexpected structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235–246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a Gly in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting. PMID:16732284

  15. Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.

    Science.gov (United States)

    Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

    2013-08-01

    Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity.

  16. Carboxyl-terminal receptor domains control the differential dephosphorylation of somatostatin receptors by protein phosphatase 1 isoforms.

    Directory of Open Access Journals (Sweden)

    Andreas Lehmann

    Full Text Available We have recently identified protein phosphatase 1β (PP1β as G protein-coupled receptor (GPCR phosphatase for the sst2 somatostatin receptor using siRNA knockdown screening. By contrast, for the sst5 somatostatin receptor we identified protein phosphatase 1γ (PP1γ as GPCR phosphatase using the same approach. We have also shown that sst2 and sst5 receptors differ substantially in the temporal dynamics of their dephosphorylation and trafficking patterns. Whereas dephosphorylation and recycling of the sst2 receptor requires extended time periods of ∼30 min, dephosphorylation and recycling of the sst5 receptor is completed in less than 10 min. Here, we examined which receptor domains determine the selection of phosphatases for receptor dephosphorylation. We found that generation of tail-swap mutants between sst2 and sst5 was required and sufficient to reverse the patterns of dephosphorylation and trafficking of these two receptors. In fact, siRNA knockdown confirmed that the sst5 receptor carrying the sst2 tail is predominantly dephosphorylated by PP1β, whereas the sst2 receptor carrying the sst5 tail is predominantly dephosphorylated by PP1γ. Thus, the GPCR phosphatase responsible for dephosphorylation of individual somatostatin receptor subtypes is primarily determined by their different carboxyl-terminal receptor domains. This phosphatase specificity has in turn profound consequences for the dephosphorylation dynamics and trafficking patterns of GPCRs.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

    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.

  18. The solution structure of the N-terminal domain of human tubulin binding cofactor C reveals a platform for tubulin interaction.

    Directory of Open Access Journals (Sweden)

    Ma Flor Garcia-Mayoral

    Full Text Available Human Tubulin Binding Cofactor C (TBCC is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers.

  19. A novel N-terminal domain may dictate the glucose response of Mondo proteins.

    Directory of Open Access Journals (Sweden)

    Lisa G McFerrin

    Full Text Available Glucose is a fundamental energy source for both prokaryotes and eukaryotes. The balance between glucose utilization and storage is integral for proper energy homeostasis, and defects are associated with several diseases, e.g. type II diabetes. In vertebrates, the transcription factor ChREBP is a major component in glucose metabolism, while its ortholog MondoA is involved in glucose uptake. Both MondoA and ChREBP contain five Mondo conserved regions (MCRI-V that affect their cellular localization and transactivation ability. While phosphorylation has been shown to affect ChREBP function, the mechanisms controlling glucose response of both ChREBP and MondoA remain elusive. By incorporating sequence analysis techniques, structure predictions, and functional annotations, we synthesized data surrounding Mondo family proteins into a cohesive, accurate, and general model involving the MCRs and two additional domains that determine ChREBP and MondoA glucose response. Paramount, we identified a conserved motif within the transactivation region of Mondo family proteins and propose that this motif interacts with the phosphorylated form of glucose. In addition, we discovered a putative nuclear receptor box in non-vertebrate Mondo and vertebrate ChREBP sequences that reveals a potentially novel interaction with nuclear receptors. These interactions are likely involved in altering ChREBP and MondoA conformation to form an active complex and induce transcription of genes involved in glucose metabolism and lipogenesis.

  20. The NusA N-terminal domain is necessary and sufficient for enhancement of transcriptional pausing via interaction with the RNA exit channel of RNA polymerase

    OpenAIRE

    Ha, Kook Sun; Toulokhonov, Innokenti; Vassylyev, Dmitry G.; Landick, Robert

    2010-01-01

    NusA is a core regulator of transcript elongation that is well conserved in bacteria and archaea. NusA interacts with elongating complexes and the nascent RNA transcript in ways that stimulate pausing and termination but can be switched to anti-pausing and anti-termination by other accessory proteins. The regulatory complexity of NusA likely depends on the presence of multiple discrete NusA domains, but it remains unclear which NusA domains possess which regulatory activity and how they inter...

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

    Science.gov (United States)

    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

  2. Studies on N-terminal glycation of peptides in hypoallergenic infant formulas: quantification of alpha-N-(2-furoylmethyl) amino acids.

    Science.gov (United States)

    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.

  3. Glutamic Acid at Residue 125 of the prM Helix Domain Interacts with Positively Charged Amino Acids in E Protein Domain II for Japanese Encephalitis Virus-Like-Particle Production

    OpenAIRE

    Peng, Jia-Guan; Wu, Suh-Chin

    2014-01-01

    Interaction between E and prM proteins in flavivirus-infected cells is a major factor for virus-like particle (VLP) production. The prM helical (prM-H) domain is topologically close to and may interact with domain II of the E protein (EDII). In this study, we investigated prM-H domain amino acid residues facing Japanese encephalitis virus EDII using site-directed mutagenesis to determine their roles in prM-E interaction and VLP production. Our results indicate that negatively charged prM-E125...

  4. The N-terminal domains of Vps3 and Vps8 are critical for localization and function of the CORVET tethering complex on endosomes.

    Directory of Open Access Journals (Sweden)

    Nadine Epp

    Full Text Available Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.

  5. Identification of a natural mutant of HBV X protein truncated 27 amino acids at the COOH terminal and its effect on liver cell proliferation

    Institute of Scientific and Technical Information of China (English)

    Hang ZHANG; Xiao-dong ZHANG; Chang-liang SHAN; Nan LI; Xuan ZHANG; Xue-zhi ZHANG; Fu-qing XU; Shuai ZHANG; Li-yan QIU; Li-hong YE

    2008-01-01

    Aim:To identify mutants of the hepatitis B virus (HBV) X (HBx) gene and inves-tigate the effect of the natural mutant on liver cell proliferation. Methods:We identified natural mutants of the HBx gene from 188 sera and 48 tissues of Chinese patients infected with HBV by PCR, respectively. Based on the identification of the mutants ofHBx gene, we cloned the fragments of the mutants into the pcDNA3 vector. The biological activities of the mutants were investigated. Results:We identified a natural mutant of the HBx gene with deletion from 382 to 401 base pairs from 3 sera out of 188 patients, which resulted in the expression deletion of the HBx protein from the 128th amino acid at the COOH terminal. The similar mutant with deletion from 382 base pair at the COOH terminal was identified from 5 cases of genomes out of 48 hepatocellular carcinoma tissues. Regarding the biological activities of the mutant, we found that the mutant of the HBx protein failed to induce apoptosis by transient transfection, but promoted proliferation of human liver immortalized L-O2 cells by stable transfection, compared with the wild-type HBx protein. The data showed that the proliferation of the mutant stably-trans-fected L-O2-X-Sera cells and fragment stably-transfected L-O2-X△127 cells was enhanced by the BrdU incorporation assay and flow cytometry analysis. Lu-ciferase reporter gene assay showed that the transcriptional activities of NF-kB, survivin, and human telomerase reverse transcriptase were upregulated, and West-ern blot analysis revealed that the expression levels of c-Myc and proliferating cell nuclear antigen (PCNA) were upregulated in the cells. Conclusion:Our find-ings suggest that the natural HBx mutant truncated 27 amino acids at the COOH terminal promotes cell proliferation.

  6. Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Aquifex aeolicus

    International Nuclear Information System (INIS)

    The cytoplasmic domain of FlhB from A. aeolicus has been purified and crystallized. FlhB is a key protein in the regulation of protein export by the bacterial flagellar secretion system. It is composed of two domains: an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhBc). Here, the crystallization and preliminary crystallographic analysis of FlhBc from Aquifex aeolicus are reported. Purified protein was crystallized using the vapour-diffusion technique. The crystals diffracted to 2.3 Å resolution and belonged to space group C2, with unit-cell parameters a = 114.49, b = 33.89, c = 122.13 Å, β = 107.53°

  7. On the activation of bovine plasma factor XIII. Amino acid sequence of the peptide released by thrombin and the terminal residues of the subunit polypeptides.

    Science.gov (United States)

    Nakamura, S; Iwanaga, S; Suzuki, T

    1975-12-01

    A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII

  8. On the structure of the N-terminal domain of the MscL channel: helical bundle or membrane interface.

    Science.gov (United States)

    Iscla, Irene; Wray, Robin; Blount, Paul

    2008-09-01

    The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock and is to date the best characterized mechanosensitive channel. A well-recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein form a bundle of amphipathic helices in the closed state that functionally serves as a cytoplasmic second gate. However, a recently reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the cytoplasmic surface of the membrane. Thus, it is unclear if one structural model is correct or if they both reflect valid closed states. Here, we have systematically reevaluated this region utilizing cysteine-scanning, in vivo functional characterization, in vivo SCAM, electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not support the helical bundle and second-gate hypothesis but correlate well with the proposed structure for M. tuberculosis MscL. We propose a functional model that is consistent with the collective data. PMID:18515388

  9. Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis.

    Science.gov (United States)

    Fan, Jin-Yuan; Means, John C; Bjes, Edward S; Price, Jeffrey L

    2015-07-01

    Drosophila DBT and vertebrate CKIε/δ phosphorylate the period protein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBT(C/ala)) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIδ, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBT(C/ala) did not affect circadian behavior differently from wild-type DBT (DBT(WT)), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT(WT) protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT(C/ala) did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.

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

    Science.gov (United States)

    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

  11. Multicentric Carpotarsal Osteolysis Is Caused by Mutations Clustering in the Amino-Terminal Transcriptional Activation Domain of MAFB

    OpenAIRE

    Zankl, Andreas; Duncan, Emma L.; Leo, Paul J.; Clark, Graeme R.; Glazov, Evgeny A.; Addor, Marie-Claude; Herlin, Troels; Kim, Chong Ae; Leheup, Bruno P.; McGill, Jim; McTaggart, Steven; Mittas, Stephen; Mitchell, Anna L.; Mortier, Geert R.; Robertson, Stephen P.

    2012-01-01

    Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated s...

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

    Energy Technology Data Exchange (ETDEWEB)

    Beich-Frandsen, Mads; Aragón, Eric [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Llimargas, Marta [Institut de Biologia Molecular de Barcelona, IBMB–CSIC, Baldiri Reixac 10, 08028 Barcelona (Spain); Benach, Jordi [ALBA Synchrotron, BP 1413, km 3.3, Cerdanyola del Vallès (Spain); Riera, Antoni [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Universitat de Barcelona, Martí i Franqués 1-11, 08028 Barcelona (Spain); Pous, Joan [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Platform of Crystallography IBMB–CSIC, Baldiri Reixac 10, 08028 Barcelona (Spain); Macias, Maria J., E-mail: maria.macias@irbbarcelona.org [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Catalan Institution for Research and Advanced Studies (ICREA), Passeig Lluís Companys 23, 08010 Barcelona (Spain)

    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.

  13. Facile Gold-Catalyzed Heterocyclization of Terminal Alkynes and Cyanamides Leading to Substituted 2-Amino-1,3-Oxazoles.

    Science.gov (United States)

    Rassadin, Valentin A; Boyarskiy, Vadim P; Kukushkin, Vadim Yu

    2015-07-17

    Facile gold-catalyzed heterocyclization based upon intermolecular trapping of the generated α-oxo gold carbenes with various cyanamides R(2)R(3)NCN (R(2)/R(3) = Alk/Alk, -(CH2)2O(CH2)2-, Ar/Ar, Ar/H) has been developed. In most cases, 2-amino-1,3-oxazoles functionalized at the nitrogen atom as well as at the fifth position of the heterocyclic ring (12 examples) were isolated in good to moderate yields. PMID:26135038

  14. Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: Mapping of residues that when altered give rise to an activated enzyme

    DEFF Research Database (Denmark)

    Axelsen, K.B.; Venema, K.; Jah, T.;

    1999-01-01

    The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants, The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme....... Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H......+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP...

  15. The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer.

    Directory of Open Access Journals (Sweden)

    Sivakumar Namadurai

    Full Text Available The mismatch repair (MMR pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD of the MutL homolog of Neisseria gonorrhoeae (NgoL determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.

  16. The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer.

    Science.gov (United States)

    Namadurai, Sivakumar; Jain, Deepti; Kulkarni, Dhananjay S; Tabib, Chaitanya R; Friedhoff, Peter; Rao, Desirazu N; Nair, Deepak T

    2010-01-01

    The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage. PMID:21060849

  17. Ultrafast resonance energy transfer from a site-specifically attached fluorescent chromophore reveals the folding of the N-terminal domain of CP29

    NARCIS (Netherlands)

    Oort, van B.F.; Murali, S.; Wientjes, E.; Koehorst, R.B.M.; Spruijt, R.B.; Hoek, van A.; Croce, R.; Amerongen, van H.

    2009-01-01

    The photosynthetic minor antenna complex CP29 of higher plants was singly mutated, overexpressed in Escherichia coli, selectively labeled with the fluorescent dye TAMRA at three positions in the N-terminal domain, and reconstituted with its natural pigments. Picosecond fluorescence experiments revea

  18. An antibody against the C-terminal domain of PCSK9 lowers LDL cholesterol levels in vivo.

    Science.gov (United States)

    Schiele, Felix; Park, John; Redemann, Norbert; Luippold, Gerd; Nar, Herbert

    2014-02-20

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.

  19. Measurement of amino terminal propeptide of type III procollagen (PIIINP) employing the ADVIA Centaur platform. Validation, reference interval and comparison to UniQ RIA

    DEFF Research Database (Denmark)

    Knudsen, Cindy Soendersoe; Heickendorff, Lene; Nexo, Ebba

    2014-01-01

    ). In this article, we show that the Centaur PIIINP may be used in place of the much more labor-intensive RIA method, and we present an age stratified reference interval. Methods: We analyzed four control samples 20 times over a period of 5 days. Centaur PIIINP assay measurements were compared with the widely used......Background: Recently, measurement of amino terminal propeptide of type III procollagen (PIIINP) was introduced as a part of the hepatic cirrhotic marker enhanced liver fibrosis™ test on the automated ADVIA Centaur® immunoassay platform (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA...... PIIINP assay is suitable for routine use with our newly defined reference interval. The results obtained by Centaur correlates well with those obtained by the previously employed RIA, though the absolute values are higher....

  20. Amino-terminal pro-B-type natriuretic peptide testing to assist the diagnostic evaluation of heart failure in symptomatic primary care patients

    DEFF Research Database (Denmark)

    Hildebrandt, P.; Collinson, P.O.

    2008-01-01

    When used for the evaluation of symptomatic patients in general practice, amino-terminal pro-B-type natriuretic peptide (NT-proBNP) testing is highly sensitive, with an excellent negative predictive value for cost-effective exclusion of the diagnosis of heart failure (HF). Importantly (similar...... to other NP assays), lower values for NT-proBNP are expected among patients with HF in the primary care setting compared with patients with acute dyspnea. Among primary care patients with dyspnea, a noncardiac source of dyspnea is most likely in patients with findings below the recommended age......-stratified NT-proBNP cut points. Conversely, an NT-proBNP result above the age-stratified primary care cut points does not absolutely indicate the presence of HF; a more directed cardiovascular workup is indicated Udgivelsesdato: 2008/2/4...

  1. Crystal Structure of the C-terminal Region of Streptococcus mutans Antigen I/II and Characterization of Salivary Agglutinin Adherence Domains

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Crowley, Paula J.; Kelly, Charles; Mitchell, Tim J.; Brady, L. Jeannine; Deivanayagam, Champion (King); (Cornell); (UAB); (Glasgow); (Florida)

    2012-05-29

    The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 {angstrom} resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C{sub 1}, C{sub 2}, and C{sub 3}. Each domain adopts a DE-variant IgG fold, with two {beta}-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C{sub 1} and C{sub 2}) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C{sub 1} and C{sub 2} domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C{sub 1} and C{sub 2} domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

  2. Expression and characterization of Kunitz domain 3 and C-terminal of human tissue factor pathway inhibitor-2

    Institute of Scientific and Technical Information of China (English)

    Lina Zhu; Jiping Wang; Jingui Mu; Huijun Wang; Chenqi Zhang; Jue Wang; Xingang Liu; Xiaomin Yan; Linsen Dai; Duan Ma

    2009-01-01

    Human tissue factor pathway inhibitor-2 (hTFPI-2) is a serine protease inhibitor and its inhibitory activity is enhanced by heparin. The Kunitz domain 3 and C-terminal of hTFPI-2 (bTFPI-2/KD3C), which has the activity toward heparin calcium, have been successfully expressed in Pichia pastoris and purified by SP-Sepharose and heparin-Sepharose chromatography. The Fourier transformed infrared spectroscopy (FTIR),Raman spectroscopy, and circular dichroism (CD)experiment results implied that hTFPI-2/KD3C con-tained small contents of or-helix and β-strand, but large amounts of random coil and two kinds of disulfide bonds, gauche-gauche-gauche (ggg) and trans-gauche-trans (tgt). The interaction of hTFPI-2/KD3C with heparin calcium was investigated by CD. It was found that heparin calcium induced β-strands in hTFPI-2/KD3C to different extents depending on the ratio of hTFPI-2/KD3C and heparin calcium.

  3. Resolving hot spots in the C-terminal dimerization domain that determine the stability of the molecular chaperone Hsp90.

    Directory of Open Access Journals (Sweden)

    Emanuele Ciglia

    Full Text Available Human heat shock protein of 90 kDa (hHsp90 is a homodimer that has an essential role in facilitating malignant transformation at the molecular level. Inhibiting hHsp90 function is a validated approach for treating different types of tumors. Inhibiting the dimerization of hHsp90 via its C-terminal domain (CTD should provide a novel way to therapeutically interfere with hHsp90 function. Here, we predicted hot spot residues that cluster in the CTD dimerization interface by a structural decomposition of the effective energy of binding computed by the MM-GBSA approach and confirmed these predictions using in silico alanine scanning with DrugScore(PPI. Mutation of these residues to alanine caused a significant decrease in the melting temperature according to differential scanning fluorimetry experiments, indicating a reduced stability of the mutant hHsp90 complexes. Size exclusion chromatography and multi-angle light scattering studies demonstrate that the reduced stability of the mutant hHsp90 correlates with a lower complex stoichiometry due to the disruption of the dimerization interface. These results suggest that the identified hot spot residues can be used as a pharmacophoric template for identifying and designing small-molecule inhibitors of hHsp90 dimerization.

  4. A Superhelical Spiral in the Escherichia coli DNA Gyrase A C-terminal Domain Imparts Unidirectional Supercoiling Bias

    Energy Technology Data Exchange (ETDEWEB)

    Ruthenburg,A.; Graybosch, D.; Huetsch, J.; Verdine, G.

    2005-01-01

    DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped {beta}-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (-) DNA supercoiling.

  5. Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

    International Nuclear Information System (INIS)

    Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [32P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Andreas J. Stroehlein

    2016-09-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  9. Characterization of amino acid residues within the N-terminal region of Ubc9 that play a role in Ubc9 nuclear localization

    International Nuclear Information System (INIS)

    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

  10. Characterization of amino acid residues within the N-terminal region of Ubc9 that play a role in Ubc9 nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Sekhri, Palak [Department of Biological Sciences, Wayne State University, 5947 Gullen Mall, Detroit, MI 48202 (United States); Tao, Tao [School of Life Sciences, Xiamen University, Xiamen (China); Kaplan, Feige [Department of Human Genetics, McGill University, Montreal (Canada); Zhang, Xiang-Dong, E-mail: xzhang@wayne.edu [Department of Biological Sciences, Wayne State University, 5947 Gullen Mall, Detroit, MI 48202 (United States)

    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.

  11. Mechanism of formation of the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. I. Importance of hydrophobic interactions in stabilization of beta-hairpin structure.

    Science.gov (United States)

    Skwierawska, Agnieszka; Makowska, Joanna; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A

    2009-06-01

    We previously studied a 16-amino acid-residue fragment of the C-terminal beta-hairpin of the B3 domain (residues 46-61), [IG(46-61)] of the immunoglobulin binding protein G from Streptoccocus, and found that hydrophobic interactions and the turn region play an important role in stabilizing the structure. Based on these results, we carried out systematic structural studies of peptides derived from the sequence of IG (46-61) by systematically shortening the peptide by one residue at a time from both the C- and the N-terminus. To determine the structure and stability of two resulting 12- and 14-amino acid-residue peptides, IG(48-59) and IG(47-60), respectively, we carried out circular dichroism, NMR, and calorimetric studies of these peptides in pure water. Our results show that IG(48-59) possesses organized three-dimensional structure stabilized by hydrophobic interactions (Tyr50-Phe57 and Trp48-Val59) at T = 283 and 305 K. At T = 313 K, the structure breaks down because of increased chain entropy, but the turn region is preserved in the same position observed for the structure of the whole protein. The breakdown of structure occurs near the melting temperature of this peptide (T(m) = 310 K) measured by differential scanning calorimetry (DSC). The melting temperature of IG(47-60) determined by DSC is T(m) = 330 K and its structure is similar to that of the native beta-hairpin at all (lower) temperatures examined (283-313 K). Both of these truncated sequences are conserved in all known amino acid sequences of the B domains of the immunoglobulin binding protein G from bacteria. Thus, this study contributes to an understanding of the mechanism of folding of this whole family of proteins, and provides information about the mechanism of formation and stabilization of a beta-hairpin structural element. PMID:19089955

  12. Exploring the activity space of peptides binding to diverse SH3 domains using principal property descriptors derived from amino acid rotamers.

    Science.gov (United States)

    He, Ping; Wu, Wei; Yang, Kang; Jing, Tao; Liao, Ke-Long; Zhang, Wei; Wang, Hai-Dong; Hua, Xing

    2011-01-01

    Although there were intensive works addressed on multivariate extraction of the informative components from numerous physicochemical parameters of amino acids in isolated state, the various conformational behaviors of amino acids in complicated biological context have long been underappreciated in the field of quantitative structure-activity relationship (QSAR). In this work, the amino acid rotamers, which were derived from statistical survey of protein crystal structures, were used to reproduce the conformational variety of amino acid side-chains in real condition. In this procedure, these rotamers were superposed into a nx x ny x nz lattice and an artificial probe was employed to detect four kinds of nonbonding field potentials (i.e., electrostatic, steric, hydrophobic, and hydrogen bonds) at each lattice point using a Gaussian-type potential function; the generated massive data were then subjected to a principal component analysis (PCA) treatment to obtain a set of few, informative amino acid descriptors. We used this set of descriptors, that we named principal property descriptors derived from amino acid rotamers (PDAR), to characterize over 13,000 peptides with known binding affinities to 10 types of SH3 domains. Genetic algorithm/ partial least square regression (GA/PLS) modeling and Monte Carlo cross-validation (MCCV) demonstrated that the correlation between the PDAR descriptors and the binding affinities of peptides are comparable with or even better than previously published models. Furthermore, from the PDAR-based QSAR models we concluded that the core motif of peptides, particularly the electrostatic property, hydrophobicity, and hydrogen bond at residue positions P3, P2, and/or P0, contribute significantly to the hAmph SH3 domain-peptide binding, whereas two ends of the peptides, such as P6, P4, P-4, and P5, only play a secondary role in the binding.

  13. Phylogenetic analysis of evolutionary relationships of the planctomycete division of the domain bacteria based on amino acid sequences of elongation factor Tu.

    Science.gov (United States)

    Jenkins, C; Fuerst, J A

    2001-05-01

    Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique 11-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum-likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria. It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions. PMID:11443344

  14. The Crystal Structure of the C-Terminal Domain of the Salmonella enterica PduO Protein: An Old Fold with a New Heme-Binding Mode.

    Science.gov (United States)

    Ortiz de Orué Lucana, Darío; Hickey, Neal; Hensel, Michael; Klare, Johann P; Geremia, Silvano; Tiufiakova, Tatiana; Torda, Andrew E

    2016-01-01

    The two-domain protein PduO, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica is an ATP:Cob(I)alamin adenosyltransferase, but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) is, however, unknown. In this study, comparative growth assays with a set of Salmonella mutant strains showed that this domain is necessary for effective in vivo catabolism of 1,2-propanediol. It was also shown that isolated, recombinantly-expressed PduOC binds heme in vivo. The structure of PduOC co-crystallized with heme was solved (1.9 Å resolution) showing an octameric assembly with four heme moieities. The four heme groups are highly solvent-exposed and the heme iron is hexa-coordinated with bis-His ligation by histidines from different monomers. Static light scattering confirmed the octameric assembly in solution, but a mutation of the heme-coordinating histidine caused dissociation into dimers. Isothermal titration calorimetry using the PduOC apoprotein showed strong heme binding (K d = 1.6 × 10(-7) M). Biochemical experiments showed that the absence of the C-terminal domain in PduO did not affect adenosyltransferase activity in vitro. The evidence suggests that PduOC:heme plays an important role in the set of cobalamin transformations required for effective catabolism of 1,2-propanediol. Salmonella PduO is one of the rare proteins which binds the redox-active metabolites heme and cobalamin, and the heme-binding mode of the C-terminal domain differs from that in other members of this protein family. PMID:27446048

  15. Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A

    Institute of Scientific and Technical Information of China (English)

    袁翀; 胡红雨; 许根俊

    2001-01-01

    Lactate dehydrogenase A (LDHA) is a well-characterized tetrameric enzyme. Its N-terminal arm, comprised of an (-helix and a (-strand, was suggested to be essential for subunit interactions. To examine the critical amino acid residues in the N-terminus involved in the subunit association, two single-point mutants, Leu3Pro (L3P) and Ile8Glu (I8E), have been constructed. We compared the stability of WT-LDHA (WT) and its variants by unfolding experiments. For WT, a dimeric but inactive intermediate was observed by size-exclusion chromatography at 0.6-0.8 mol/L GdmCl. Leu3Pro exists in an active tetrameric structure in aqueous solution as WT does, but it dissociates into dimers under lower concentration of GdmCl (0.2 mol/L). In aqueous solution, the Ile8Glu variant exists predominantly in the dimeric form with increased KM and decreased kcat as compared with those of WT and L3P. However, the activity of Ile8Glu increases significantly in the presence of sodium sulfate. In conclusion, two mutants are less stable than WT in oligomer structure. Results also support the fact that some residues in the N-terminal arm, especially the Leu8 in the (-structure, contribute the important binding energies to the dimerization of dimers, which might affect the assembly of the enzyme as well as the catalytic function.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  18. A core of three amino acids at the carboxyl-terminal region of glutamine synthetase defines its regulation in cyanobacteria.

    Science.gov (United States)

    Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel

    2015-05-01

    Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein-protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site-directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C-terminus of Synechocystis GS. A protein-protein docking modeling of Synechocystis GS in complex with IF7 supports the role of the identified core for GS/IF interaction.

  19. Antimicrobial activity of pleurocidin is retained in Plc-2, a C-terminal 12-amino acid fragment.

    Science.gov (United States)

    Souza, Andre L A; Díaz-Dellavalle, Paola; Cabrera, Andrea; Larrañaga, Patricia; Dalla-Rizza, Marco; De-Simone, Salvatore G

    2013-07-01

    An analysis of a series of five peptides composed of various portions of the pleurocidin (Plc) sequence identified a l2-amino acid fragment from the C-terminus of Plc, designated Plc-2, as the smallest fragment that retained a antimicrobial activity comparable to that of the parent compound. MIC tests in vitro with low-ionic-strength medium showed that Plc-2 has potent activity against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus but not against Enterococcus faecalis. The antifungal activity of the synthetic peptides against phytopathogenic fungi, such as Fusarium oxysporum, Colletotrichum sp., Aspergillus niger and Alternaria sp., also identified Plc-2 as a biologically active peptide. Microscopy studies of fluorescently stained fungi treated with Plc-2 demonstrated that cytoplasmic and nuclear membranes were compromised in all strains of phytopathogenic fungi tested. Together, these results identify Plc-2 as a potential antimicrobial agent with similar properties to its parent compound, pleurocidin. In addition, it demonstrated that the KHVGKAALTHYL residues are critical for the antimicrobial activity described for pleurocidin.

  20. A dielectric barrier discharge terminally inactivates RNase A by oxidizing sulfur-containing amino acids and breaking structural disulfide bonds

    Science.gov (United States)

    Lackmann, J.-W.; Baldus, S.; Steinborn, E.; Edengeiser, E.; Kogelheide, F.; Langklotz, S.; Schneider, S.; Leichert, L. I. O.; Benedikt, J.; Awakowicz, P.; Bandow, J. E.

    2015-12-01

    RNases are among the most stable proteins in nature. They even refold spontaneously after heat inactivation, regaining full activity. Due to their stability and universal presence, they often pose a problem when experimenting with RNA. We investigated the capabilities of nonthermal atmospheric-pressure plasmas to inactivate RNase A and studied the inactivation mechanism on a molecular level. While prolonged heating above 90 °C is required for heat inactivating RNase A, direct plasma treatment with a dielectric barrier discharge (DBD) source caused permanent inactivation within minutes. Circular dichroism spectroscopy showed that DBD-treated RNase A unfolds rapidly. Raman spectroscopy indicated methionine modifications and formation of sulfonic acid. A mass spectrometry-based analysis of the protein modifications that occur during plasma treatment over time revealed that methionine sulfoxide formation coincides with protein inactivation. Chemical reduction of methionine sulfoxides partially restored RNase A activity confirming that sulfoxidation is causal and sufficient for RNase A inactivation. Continued plasma exposure led to over-oxidation of structural disulfide bonds. Using antibodies, disulfide bond over-oxidation was shown to be a general protein inactivation mechanism of the DBD. The antibody’s heavy and light chains linked by disulfide bonds dissociated after plasma exposure. Based on their ability to inactivate proteins by oxidation of sulfur-containing amino acids and over-oxidation of disulfide bonds, DBD devices present a viable option for inactivating undesired or hazardous proteins on heat or solvent-sensitive surfaces.

  1. A core of three amino acids at the carboxyl-terminal region of glutamine synthetase defines its regulation in cyanobacteria.

    Science.gov (United States)

    Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel

    2015-05-01

    Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein-protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site-directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C-terminus of Synechocystis GS. A protein-protein docking modeling of Synechocystis GS in complex with IF7 supports the role of the identified core for GS/IF interaction. PMID:25626767

  2. 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

    DEFF Research Database (Denmark)

    Pedersen, Lotte Bang; Birkelund, S; Holm, A;

    1996-01-01

    , 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...... 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...

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

    Science.gov (United States)

    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.

  4. The solution structure of the C-terminal domain of NfeD reveals a novel membrane-anchored OB-fold.

    Science.gov (United States)

    Kuwahara, Yohta; Ohno, Ayako; Morii, Taichi; Yokoyama, Hideshi; Matsui, Ikuo; Tochio, Hidehito; Shirakawa, Masahiro; Hiroaki, Hidekazu

    2008-11-01

    Nodulation formation efficiency D (NfeD) is a member of a class of membrane-anchored ClpP-class proteases. There is a second class of NfeD homologs that lack the ClpP domain. The genes of both NfeD classes usually are part of an operon that also contains a gene for a prokaryotic homolog of stomatin. (Stomatin is a major integral-membrane protein of mammalian erythrocytes.) Such NfeD/stomatin homolog gene pairs are present in more than 290 bacterial and archaeal genomes, and their protein products may be part of the machinery used for quality control of membrane proteins. Herein, we report the structure of the isolated C-terminal domain of PH0471, a Pyrococcus horikoshii NfeD homolog, which lacks the ClpP domain. This C-terminal domain (termed NfeDC) contains a five-strand beta-barrel, which is structurally very similar to the OB-fold (oligosaccharide/oligonucleotide-binding fold) domain. However, there is little sequence similarity between it and previously characterized OB-fold domains. The NfeDC domain lacks the conserved surface residues that are necessary for the binding of an OB-fold domain to DNA/RNA, an ion. Instead, its surface is composed of residues that are uniquely conserved in NfeD homologs and that form the structurally conserved surface turns and beta-bulges. There is also a conserved tryptophan present on the surface. We propose that, in general, NfeDC domains may interact with other spatially proximal membrane proteins and thereby regulate their activities. PMID:18687870

  5. Interactions of the C-terminal Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

    Science.gov (United States)

    Hu, Shaowen; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

    2007-01-01

    NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku(sub 70/80) complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The C-terminal domain of Ku70 (Ku70c, residues 559-609), contains an helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the Ku70c/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the subunit Ku70c and a 14 base pairs DNA duplex, whose starting structures are designed to be variable so as to mimic their different binding modes. By analyzing conformational changes and energetic properties of the complex during MD simulations, we found that interactions are preferred at DNA ends, and within the major groove, which is consistent with previous experimental investigations. In addition, the results indicate that cooperation of Ku70c with other subunits of Ku(sub 70/80) is necessary to explain the high affinity of binding as observed in experiments.

  6. Runx2-I isoform contributes to fetal bone formation even in the absence of specific N-terminal amino acids.

    Directory of Open Access Journals (Sweden)

    Hideaki Okura

    Full Text Available The Runt-related transcription factor 2 (Runx2 gene encodes the transcription factor Runx2, which is the master regulator of osteoblast development; insufficiency of this protein causes disorders of bone development such as cleidocranial dysplasia. Runx2 has two isoforms, Runx2-II and Runx2-I, and production of each isoform is controlled by a unique promoter: a distal promoter (P1 and a proximal promoter (P2, respectively. Although several studies have focused on differences and similarities between the two Runx2 isoforms, their individual roles in bone formation have not yet been determined conclusively, partly because a Runx2-I-targeted mouse model is not available. In this study, we established a novel Runx2-manipulated mouse model in which the first ATG of Runx2-I was replaced with TGA (a stop codon, and a neomycin-resistant gene (neo cassette was inserted at the first intron of Runx2-I. Homozygous Runx2-Ineo/neo mice showed severely reduced expression of Runx2-I, whereas Runx2-II expression was largely retained. Runx2-Ineo/neo mice showed neonatal lethality, and in these mice, intramembranous ossification was more severely defective than endochondral ossification, presumably because of the greater involvement of Runx2-I, compared with that of Runx2-II in intramembranous ossification. Interestingly, the depletion of neo rescued the above-described phenotypes, indicating that the isoform-specific N-terminal region of Runx2-I is not functionally essential for bone development. Taken together, our results provide a novel clue leading to a better understanding of the roles of Runx2 isoforms in osteoblast development.

  7. LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization

    Energy Technology Data Exchange (ETDEWEB)

    Rapali, Peter [Dept. Biochemistry, Eoetvoes Lorand University, Budapest (Hungary); Garcia-Mayoral, Maria Flor [Dept. Biological Physical Chemistry, IQFR, CSIC, Madrid (Spain); Martinez-Moreno, Monica [Dept. Biochemistry and Molecular Biology I, Universidad Complutense, Madrid (Spain); Tarnok, Krisztian; Schlett, Katalin [Dept. Physiology and Neurobiology, Eoetvoes Lorand University, Budapest (Hungary); Albar, Juan Pablo [Proteomics Facility, CNB, CSIC, Madrid (Spain); Bruix, Marta [Dept. Biological Physical Chemistry, IQFR, CSIC, Madrid (Spain); Nyitray, Laszlo, E-mail: nyitray@elte.hu [Dept. Biochemistry, Eoetvoes Lorand University, Budapest (Hungary); Rodriguez-Crespo, Ignacio, E-mail: nacho@bbm1.ucm.es [Dept. Biochemistry and Molecular Biology I, Universidad Complutense, Madrid (Spain)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer We have screened a human library with dynein light chain DYNLL1 (DLC8) as bait. Black-Right-Pointing-Pointer Dynein light chain DYNLL1 binds to ATM-kinase interacting protein (ATMIN). Black-Right-Pointing-Pointer ATMIN has 17 SQ/TQ motifs, a motif frequently found in DYNLL1-binding partners. Black-Right-Pointing-Pointer The two proteins interact in vitro, with ATMIN displaying at least five binding sites. Black-Right-Pointing-Pointer The interaction of ATMIN and DYNNL1 in transfected cells can also be observed. -- Abstract: LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1

  8. A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation.

    Science.gov (United States)

    Leen, Eoin N; Sorgeloos, Frédéric; Correia, Samantha; Chaudhry, Yasmin; Cannac, Fabien; Pastore, Chiara; Xu, Yingqi; Graham, Stephen C; Matthews, Stephen J; Goodfellow, Ian G; Curry, Stephen

    2016-01-01

    Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.

  9. A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation.

    Directory of Open Access Journals (Sweden)

    Eoin N Leen

    2016-01-01

    Full Text Available Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES, adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs in the host cell. For murine norovirus (MNV we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132. Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.

  10. Chemical shift assignments and secondary structure prediction of the C-terminal domain of the response regulator BfmR from Acinetobacter baumannii.

    Science.gov (United States)

    Olson, Andrew L; Thompson, Richele J; Melander, Christian; Cavanagh, John

    2014-04-01

    Acinetobacter baumannii is a Gram-negative pathogen responsible for severe nocosomial infections by forming biofilms in healthcare environments. The two-domain response regulator BfmR has been shown to be the master controller for biofilm formation. Inactivation of BfmR resulted in an abolition of pili production and consequently biofilm creation. Here we report backbone and sidechain resonance assignments and secondary structure prediction for the C-terminal domain of BfmR (residues 130-238) from A. baumannii.

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

    OpenAIRE

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

    2007-01-01

    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 ...

  12. Higher-order structure in the 3'-terminal domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus

    DEFF Research Database (Denmark)

    Garrett, R A; Christensen, A; Douthwaite, S

    1984-01-01

    ribosomes of flowering plants. The structure of domain VI within the eubacterial RNAs was probed with chemical reagents in order to establish the degree of stacking and/or accessibility of each adenosine, cytidine and guanosine residue; the double-helical segments were localized with the cobra venom...... level of structural conservation has occurred throughout the RNA domain during the evolution of the Gram negative and Gram positive bacteria although the thermophile was generally more stable at base-pairs adjacent to the terminal loops....

  13. The N-terminal cellulose-binding domain of EGXA increases thermal stability of xylanase and changes its specific activities on different substrates

    Institute of Scientific and Technical Information of China (English)

    Ming Ding; Yigang Teng; Qiuyu Yin; Jie Zhao; Fukun Zhao

    2008-01-01

    A full-length EGXA enzyme from a mollusk, Ampullaria crossean, was cloned into pFastBac vector and then heterogeneously expressed in insect Tn5 cells. Its natural N-terminal signal peptide worked well in the insect Tn5 cells.The recombinant EGXA was a 63 kDa protein and had active endo-β-1,4-glucanase (EC 3.2.1.4) and endo-β-1,4-xylanase (EC 3.2.1.8). The specific activity of endo-β-1,4-xylanase was higher than in the EGX, which was purified from the stomach tissues of Ampullaria crossen. The N-terminal cellulosebinding domain of EGXA made it bind to cellulose and xylan more efficiently. This cellulose-binding domain also increased the thermal stability of this recombinant enzyme and decreased the recombinant EGXA's specific activities on p-nitrophenyi-β-D-cellobioside and sodium carboxymethyl cellulose.

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

    Science.gov (United States)

    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. PMID:27282484

  15. Soluble forms of tumor necrosis factor receptors (TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor

    DEFF Research Database (Denmark)

    Nophar, Y; Kemper, O; Brakebusch, C;

    1990-01-01

    in the extracellular domains of the nerve growth factor receptor and the B lymphocytes surface antigen CDw40. The amino acid composition and size of the extracellular domain of the type I TNF-R closely resemble those of TBPI. The COOH-terminal amino acid sequence of the four cysteine rich repeats within...... the extracellular domain of the type I TNF-R matches the COOH-terminal sequence of TBPI. Amino acid sequences in the extracellular domain also fully match other sequences found in TBPI. On the other hand, amino acid sequences in the soluble form of the type II TNF-R (TBPII), while indicating a marked homology...

  16. Three-dimensional structure of N-terminal domain of DnaB helicase and helicase-primase interactions in Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Tara Kashav

    Full Text Available Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD of H. pylori DnaB (HpDnaB helicase at 2.2 A resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

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

    Science.gov (United States)

    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.

  18. Properties and catalytic activities of MICAL1, the flavoenzyme involved in cytoskeleton dynamics, and modulation by its CH, LIM and C-terminal domains.

    Science.gov (United States)

    Vitali, Teresa; Maffioli, Elisa; Tedeschi, Gabriella; Vanoni, Maria A

    2016-03-01

    MICAL1 is a cytoplasmic 119 kDa protein participating in cytoskeleton dynamics through the NADPH-dependent oxidase and F-actin depolymerizing activities of its N-terminal flavoprotein domain, which is followed by calponin homology (CH), LIM domains and a C-terminal region with Pro-, Glu-rich and coiled-coil motifs. MICAL1 and truncated forms lacking the C-terminal, LIM and/or CH regions have been produced and characterized. The CH, LIM and C-terminal regions cause an increase of Km,NADPH exhibited by the NADPH oxidase activity of the flavoprotein domain, paralleling changes in the overall protein charge. The C-terminus also determines a ∼ 10-fold decrease of kcat, revealing its role in establishing an inactive/active conformational equilibrium, which is at the heart of the regulation of MICAL1 in cells. F-actin lowers Km,NADPH (10-50 μM) and increases kcat (10-25 s(-1)) to similar values for all MICAL forms. The apparent Km,actin of MICAL1 is ∼ 10-fold higher than that of the other forms (3-5 μM), reflecting the fact that F-actin binds to the flavoprotein domain in the MICAL's active conformation and stabilizes it. Analyses of the reaction in the presence of F-actin indicate that actin depolymerization is mediated by H2O2 produced by the NADPH oxidase reaction, rather than due to direct hydroxylation of actin methionine residues. PMID:26845023

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

    International Nuclear Information System (INIS)

    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 15N-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

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

    Energy Technology Data Exchange (ETDEWEB)

    Berbís, M. Álvaro [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); André, Sabine [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Cañada, F. Javier [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); Pipkorn, Rüdiger [Central Peptide Synthesis Unit, German Cancer Research Center, 69120 Heidelberg (Germany); Ippel, Hans [Department of Biochemistry, CARIM, University of Maastricht, Maastricht (Netherlands); Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Mayo, Kevin H. [Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Kübler, Dieter [Biomolecular Interactions, German Cancer Research Center, 69120 Heidelberg (Germany); Gabius, Hans-Joachim [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Jiménez-Barbero, Jesús, E-mail: jjbarbero@cib.csic.es [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain)

    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.

  1. Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

    Energy Technology Data Exchange (ETDEWEB)

    Pazehoski, Kristina O., E-mail: pazehosk@pitt.edu [Division of Natural Sciences, University of Pittsburgh at Greensburg, Greensburg, PA 15601 (United States); Cobine, Paul A., E-mail: pac0006@auburn.edu [Department of Biological Sciences, 101 Rouse Life Science Building, Auburn University, AL 36849 (United States); Winzor, Donald J. [Department of Biochemistry, University of Queensland, Brisbane, Queensland 4072 (Australia); Dameron, Charles T., E-mail: cdameron@francis.edu [Department of Chemistry, Saint Francis University, Loretto, PA 15940 (United States)

    2011-03-11

    Research highlights: {yields} A metal-binding protein domain is directly involved in protein dimerization. {yields} Fusing the metal-binding domain to a monomeric protein induces dimerization. {yields} Frontal size-exclusion chromatography measures the strength of dimer interaction. {yields} Ultracentrifugation studies confirm the influence of metal binding on dimerization. -- Abstract: Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size-exclusion chromatography studies. The studies demonstrate that the attachment of these CopY residues to the immunoglobulin-binding domain of streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.

  2. Expression of the Gene Encoding the Tetraploid of Carboxyl-terminal Peptide of β-hCG Containing Thirty-seven Amino Acid Residues in E. coli

    Institute of Scientific and Technical Information of China (English)

    王健; 沈卫英; 周清平; 申庆祥

    2000-01-01

    Objective This study was carried out to investigate the possible enhancement of immunogenicity of the carboxyl-terminal peptide of β-hCG which is made up of 37 amino acid residues (109~145) and contains the specific epitope (antigenic determinant) of hCG.Materials & Methods hCGβ-CTP37 tetraploid cDNA was constructed by linking four hCGβ-CTP37 cDNAs together. The product was then subcloned into the E. coli expression vector pQE60 to construct the expression vector pQE60/ (hCGβ-CTP37)4. Recombinant (hCGβ-CTP37 ) 4 was expressed in E. coil-X-blue.Results Western blot analysis showed that the tetraploid of hCGβ-CTP37 had an apparent molecular weight of 20 kD and had relatively stronger anti-hCG antibody-binding activity compared with the diploid from.Conclusion The tetraploid of hCGβ-CTP37 may be a more potent immunogen for raising anti-hCG vaccines for fertility regulation or suppression of tumor.

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

    Science.gov (United States)

    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. PMID:26073604

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

    Science.gov (United States)

    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

  5. Bound or free: interaction of the C-terminal domain of Escherichia coli single-stranded DNA-binding protein (SSB) with the tetrameric core of SSB.

    Science.gov (United States)

    Su, Xun-Cheng; Wang, Yao; Yagi, Hiromasa; Shishmarev, Dmitry; Mason, Claire E; Smith, Paul J; Vandevenne, Marylène; Dixon, Nicholas E; Otting, Gottfried

    2014-04-01

    Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

  6. Pub1p C-terminal RRM domain interacts with Tif4631p through a conserved region neighbouring the Pab1p binding site.

    Directory of Open Access Journals (Sweden)

    Clara M Santiveri

    Full Text Available Pub1p, a highly abundant poly(A+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3 shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM. Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to β-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1-402 of yeast eIF4G1 (Tif4631p, very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role.

  7. Carboxy-terminal telopeptide (CTX) and amino-terminal propeptide (PINP) of type I collagen as markers of bone metastases in patients with non-small cell lung cancer.

    Science.gov (United States)

    Lumachi, Franco; Santeufemia, Davide A; Del Conte, Alessandro; Mazza, Francesco; Tozzoli, Renato; Chiara, Giordano B; Basso, Stefano M M

    2013-06-01

    The early diagnosis of non-small cell lung carcinoma (NSCLC) is difficult, and 30-40% of patients with NSCLC develop bone metastases (BMs) during the course of their disease. Because the delayed demonstration of skeletal involvement may seriously affect survival, there is a need for early diagnosis of BMs. Unfortunately, the sensitivity of common serum tumor markers is low and they are used mainly for monitoring the efficacy of therapy and detection of recurrence. The aim of this study was to evaluate the utility of a panel of serum biomarkers in patients with NSCLC and BMs. Sixteen patients (11 males, 5 females; median age=64 years, range 54-68 years) with NSCLC and BMs (cases), and 18 age- and stage-matched patients without BMs (controls) underwent measurement of serum carboxy-terminal telopeptide of type I collagen (CTX), tartrate-resistant acid phosphatase isoform type 5b (TRAP5b) and amino-terminal propeptide of type I collagen (PINP), carcinoembryonic antigen (CEA) and fragments of cytokeratin 19 (CYFRA 21-1. CTX (443.7 ± 945.1 vs. 402.7 ± 28.4 pg/ml, p=0.003) and PINP (75.9 ± 11.4 vs. 64.1 ± 7.5 μg/l, p=0.001) were significantly higher in patients with BMs, while the mean value of the other markers did not differ (p=NS) between cases and controls. The sensitivity, specificity and accuracy were 73.3%, 86.7% and 79.4% for CTX; 55.5%, 62.5% and 58.8% for CEA; 65.0%, 78.6% and 70.6% for CYFRA; 30.4%, 76.2% and 67.6% for TRAP5b; and 72.2%, 81.2% and 76.5% for PINP, respectively. The area under the receiver operating characteristic curve (AUC) for CTX was 0.68. In conclusion, CTX and PINP measurement can be useful in monitoring patients with NSCLC during follow-up, with the aim of detecting BMs early. PMID:23749913

  8. The structure of the RNA m5C methyltransferase YebU from Escherichia coli reveals a C-terminal RNA-recruiting PUA domain.

    Science.gov (United States)

    Hallberg, B Martin; Ericsson, Ulrika B; Johnson, Kenneth A; Andersen, Niels Møller; Douthwaite, Stephen; Nordlund, Pär; Beuscher, Albert E; Erlandsen, Heidi

    2006-07-21

    Nucleotide methylations are the most common type of rRNA modification in bacteria, and are introduced post-transcriptionally by a wide variety of site-specific enzymes. Three 5-methylcytidine (m(5)C) bases are found in the rRNAs of Escherichia coli and one of these, at nucleotide 1407 in 16 S rRNA, is the modification product of the methyltransferase (MTase) YebU (also called RsmF). YebU requires S-adenosyl-l-methionine (SAM) and methylates C1407 within assembled 30 S subunits, but not in naked 16 S rRNA or within tight-couple 70 S ribosomes. Here, we describe the three-dimensional structure of YebU determined by X-ray crystallography, and we present a molecular model for how YebU specifically recognizes, binds and methylates its ribosomal substrate. The YebU protein has an N-terminal SAM-binding catalytic domain with structural similarity to the equivalent domains in several other m(5)C RNA MTases including RsmB and PH1374. The C-terminal one-third of YebU contains a domain similar to that in pseudouridine synthases and archaeosine-specific transglycosylases (PUA-domain), which was not predicted by sequence alignments. Furthermore, YebU is predicted to contain extended regions of positive electrostatic potential that differ from other RNA-MTase structures, suggesting that YebU interacts with its RNA target in a different manner. Docking of YebU onto the 30 S subunit indicates that the PUA and MTase domains make several contacts with 16 S rRNA as well as with the ribosomal protein S12. The ribosomal protein interactions would explain why the assembled 30 S subunit, and not naked 16 S rRNA, is the preferred substrate for YebU.

  9. Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit.

    Science.gov (United States)

    Suwa, Yoshiaki; Gu, Jianyou; Baranovskiy, Andrey G; Babayeva, Nigar D; Pavlov, Youri I; Tahirov, Tahir H

    2015-06-01

    In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Å(2). Importantly, in the structure of the p180C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.

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

    Science.gov (United States)

    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.

  11. The C-terminal domains of NF-H and NF-M subunits maintain axonal neurofilament content by blocking turnover of the stationary neurofilament network.

    Directory of Open Access Journals (Sweden)

    Mala V Rao

    Full Text Available Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M(tailΔ] mice that the deletion of both of these domains selectively lowers NF levels 3-6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M(tailΔ mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M(tailΔ axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.

  12. Association of Atrial Fibrillation and Amino-terminal Pro-brain Natriuretic Peptide Concentrations in Patients After Off-Pump Coronary Artery Bypass Grafting

    Institute of Scientific and Technical Information of China (English)

    Junquan Li; Qinghua Zhang; Weichen Tian; Hongyu Liu

    2008-01-01

    Objectives To investigate the possible role of amino-terminal pro-brain natriuretic peptide (NT-proBNP) in the occurrence of atrial fibrillation (AF) after coronary artery bypass grafting (CABG).Methods This study group included 70 consecutive patients scheduled for elective off-pump CABG.The patients with ejection fraction (EF) less than 0.30,history of AF,use of class Ⅰor Ⅲ antiarrhythmic drug,implanted pacemaker,postoperative myocardial infarction or chest reopening for pericardial tamponade were excluded.Preoperative and postoperative serum NT-proBNP levels were measured by radioimmunoassay technique.Results Postoperative AF occurred in 15 patients (21.4%);these patients had significantly higher median NT-proBNP levels when compared with those without AF after the operation (P<0.01).Using multivariate logistic regression analyses,an increase in NT-proBNP level after CABG was found to be independently associated with AF (OR=3.78,95% IC=1.81~4.89,P<0.01 ).Increased age,diabetes mellitus,preoperative use of β-blocker,proximal right coronary artery involvement,and longer operation time were also associated with AF.Conclusions These results indicated that AF was associated with higher NT-proBNP concentrations after off pump CABG;the increase in NT-proBNP after CABG may play an important role in the occurrence of AF after the operation.The further studies are needed to define the reason that lead to higher NT-proBNP concentrations among the patients who present AF after off pump CABG.

  13. Serum procollagen 1 amino-terminal propeptide (P1NP) in prostate cancer; pitfalls of its use as an early surrogate marker for bone metastasis

    International Nuclear Information System (INIS)

    Procollagen 1 amino-terminal propeptide (P1NP) is a bone formation marker and has been shown to have a strong association with the extent of bone metastases (BM) in patients with advanced prostate cancer. More recently, its levels were found to be affected by androgen deprivation therapies and bisphosphonates. We investigated the role of P1NP as a surrogate marker of sub-radiological skeletal metastases in prostate cancer patients with biochemical failure (BF). BePrepared is a prospective longitudinal substudy of RADAR trial in which serial P1NPs were collected at regular intervals for 123 patients who had completed RADAR protocol treatment. There was no trend identified in P1NP levels prior to diagnosis of BM. We found that there was no difference in P1NP concentrations at the time of diagnosis of BM in the group that developed BM compared with P1NP levels in groups with only nodal metastases or BF. In the group of patients who did not experience BF, P1NP was affected by previous luteinizing hormone-releasing hormone-agonist and bisphosphonate therapy. Hence, patients who received an 18-month course of androgen deprivation without bisphosphonates had significantly higher P1NP values than patients with shorter androgen deprivation therapy (ADT) course combined with a course of bisphosphonates. P1NP is not a sensitive serum marker of early BM in high-risk prostate cancer patients with BF and low prostate-specific antigen levels as its levels are affected by prior history of bone remodelling therapies such as ADT and bisphosphonates.

  14. Structure of the TPR domain of AIP: lack of client protein interaction with the C-terminal α-7 helix of the TPR domain of AIP is sufficient for pituitary adenoma predisposition.

    Directory of Open Access Journals (Sweden)

    Rhodri M L Morgan

    Full Text Available Mutations of the aryl hydrocarbon receptor interacting protein (AIP have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h mutations, R304* (nonsense mutation, R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE of TOMM20.

  15. Structure of the TPR domain of AIP: lack of client protein interaction with the C-terminal α-7 helix of the TPR domain of AIP is sufficient for pituitary adenoma predisposition.

    Science.gov (United States)

    Morgan, Rhodri M L; Hernández-Ramírez, Laura C; Trivellin, Giampaolo; Zhou, Lihong; Roe, S Mark; Korbonits, Márta; Prodromou, Chrisostomos

    2012-01-01

    Mutations of the aryl hydrocarbon receptor interacting protein (AIP) have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h) mutations, R304* (nonsense mutation), R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE) of TOMM20. PMID:23300914

  16. Structure of the TPR domain of AIP: lack of client protein interaction with the C-terminal α-7 helix of the TPR domain of AIP is sufficient for pituitary adenoma predisposition.

    Science.gov (United States)

    Morgan, Rhodri M L; Hernández-Ramírez, Laura C; Trivellin, Giampaolo; Zhou, Lihong; Roe, S Mark; Korbonits, Márta; Prodromou, Chrisostomos

    2012-01-01

    Mutations of the aryl hydrocarbon receptor interacting protein (AIP) have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h) mutations, R304* (nonsense mutation), R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE) of TOMM20.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein

    NARCIS (Netherlands)

    Godeke, G J; de Haan, C A; Rossen, J W; Vennema, H; Rottier, P J

    2000-01-01

    The type I glycoprotein S of coronavirus, trimers of which constitute the typical viral spikes, is assembled into virions through noncovalent interactions with the M protein. Here we demonstrate that incorporation is mediated by the short carboxy-terminal segment comprising the transmembrane and end

  20. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

    Science.gov (United States)

    Wieczorek, Anna; McHenry, Charles S

    2006-05-01

    The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

  1. Structure-Function Analysis of the Human TFIIB-Related Factor II Protein Reveals an Essential Role for the C-Terminal Domain in RNA Polymerase III Transcription

    OpenAIRE

    Saxena, Ashish; Ma, Beicong; Schramm, Laura; Hernandez, Nouria

    2005-01-01

    The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2...

  2. Structure of the starch-debranching enzyme barley limit dextrinase reveals homology of the N-terminal domain to CBM21

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Abou Hachem, Maher; Svensson, Birte;

    2012-01-01

    molecule in the active site and is virtually identical to the structures of HvLD in complex with the competitive inhibitors α-cyclodextrin and β-cyclodextrin solved to 2.5 and 2.1 Å resolution, respectively. However, three loops in the N-terminal domain that are shown here to resemble carbohydrate......-binding module family 21 were traceable and were included in the present HvLD structure but were too flexible to be traced and included in the structures of the two HvLD-inhibitor complexes....

  3. A systematic study of nuclear interactome of C-terminal domain small phosphatase-like 2 using inducible expression system and shotgun proteomics.

    Science.gov (United States)

    Kang, NaNa; Koo, JaeHyung; Wang, Sen; Hur, Sun Jin; Bahk, Young Yil

    2016-06-01

    RNA polymerase II C-terminal domain phosphatases are newly emerging family of phosphatases that contain FCPH domain with Mg+2-binding DXDX(T/V) signature motif. Its subfamily includes small CTD phosphatases (SCPs). Recently, we identified several interacting partners of human SCP1 with appearance of dephosphorylation and O-GlcNAcylation. In this study, using an established cell line with inducible CTDSPL2 protein (a member of the new phosphatase family), proteomic screening was conducted to identify binding partners of CTDSPL2 in nuclear extract through immunoprecipitation of CTDSPL2 with its associated. This approach led to the identification of several interacting partners of CTDSPL2. This will provide a better understanding on CTDSPL2. [BMB Reports 2016; 49(6): 319-324]. PMID:26674342

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

    Science.gov (United States)

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

    2016-06-01

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

  5. Regions within the N-terminal domain of human topoisomerase I exert important functions during strand rotation and DNA binding

    DEFF Research Database (Denmark)

    Hougaard, Rikke Frølich; Andersen, Félicie Faucon; Westergaard, Ole;

    2004-01-01

    amino acid residues 1–202 or 191–206 or having tryptophane-205 substituted by glycine in a broad variety of in vitro activity assays. As a result of these investigations we find that mutants altered in the region 191–206 distinguished themselves from the wild-type enzyme by a faster strand rotation step......, insensitivity towards the anti-cancer drug camptothecin in relaxation and the inability to ligate blunt end DNA fragments. The mutant lacking amino acid residues 1–202 was impaired in blunt end DNA ligation and showed wild-type sensitivity towards camptothecin in relaxation. Taken together, the presented data...... support a model according to which tryptophane-205 and possibly other residues located between position 191–206 coordinates the restriction of free strand rotation during the topoisomerization step of catalysis. Moreover, tryptophane-205 appears important for the function of the bulk part of the N...

  6. A novel COL4A1 frameshift mutation in familial kidney disease: the importance of the C-terminal NC1 domain of type IV collagen

    Science.gov (United States)

    Gale, Daniel P.; Oygar, D. Deren; Lin, Fujun; Oygar, P. Derin; Khan, Nadia; Connor, Thomas M.F.; Lapsley, Marta; Maxwell, Patrick H.; Neild, Guy H.

    2016-01-01

    Background Hereditary microscopic haematuria often segregates with mutations of COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts. Methods We used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses. Results We identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested. Conclusions Missense mutations of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome (hereditary angiopathy, nephropathy, aneurysms and cramps). Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the α1 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans. PMID:27190376

  7. Identification of the C-Terminal GH5 Domain from CbCel9B/Man5A as the First Glycoside Hydrolase with Thermal Activation Property from a Multimodular Bifunctional Enzyme.

    Directory of Open Access Journals (Sweden)

    Rong Wang

    Full Text Available Caldicellulosiruptor bescii encodes at least six unique multimodular glycoside hydrolases crucial for plant cell wall polysaccharides degradation, with each having two catalytic domains separated by two to three carbohydrate binding modules. Among the six enzymes, three have one N- or C-terminal GH5 domain with identical amino acid sequences. Despite a few reports on some of these multimodular enzymes, little is known about how the conserved GH5 domains behave, which are believed to be important due to the gene duplication. We thus cloned a representative GH5 domain from the C-terminus of a multimodular protein, i.e. the bifunctional cellulase/mannanase CbCel9B/Man5A which has been reported, and expressed it in Escherichia coli. Without any appending CBMs, the recombinant CbMan5A was still able to hydrolyze a variety of mannan substrates with different backbone linkages or side-chain decorations. While CbMan5A displayed the same pH optimum as CbCel9B/Man5A, it had an increased optimal temperature (90°C and moreover, was activated by heating at 70°C and 80°C, a property not ever reported for the full-length protein. The turnover numbers of CbMan5A on mannan substrates were, however, lower than those of CbCel9B/Man5A. These data suggested that evolution of CbMan5A and the other domains into a single polypeptide is not a simple assembly; rather, the behavior of one module may be affected by the other ones in the full-length enzyme. The differential scanning calorimetry analysis further indicated that heating CbMan5A was not a simple transition state process. To the best knowledge of the authors, CbMan5A is the first glycoside hydrolase with thermal activation property identified from a multimodular bifunctional enzyme.

  8. The N-terminal extension domain of the C. elegans half-molecule ABC transporter, HMT-1, is required for protein-protein interactions and function.

    Directory of Open Access Journals (Sweden)

    Sungjin Kim

    Full Text Available BACKGROUND: Members of the HMT-1 (heavy metal tolerance factor 1 subfamily of the ATP-binding cassette (ABC transporter superfamily detoxify heavy metals and have unique topology: they are half-molecule ABC transporters that, in addition to a single transmembrane domain (TMD1 and a single nucleotide-binding domain (NBD1, possess a hydrophobic NH2-terminal extension (NTE. These structural features distinguish HMTs from other ABC transporters in different species including Drosophila and humans. Functional ABC transporters, however, are comprised of at least four-domains (two TMDs and two NDBs formed from either a single polypeptide or by the association of two or four separate subunits. Whether HMTs act as oligomers and what role the NTE domain plays in their function have not been determined. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined the oligomeric status of Caenorhabditis elegans HMT-1 and the functional significance of its NTE using gel-filtration chromatography in combination with the mating-based split-ubiquitin yeast two-hybrid system (mbSUS and functional in vivo assays. We found that HMT-1 exists in a protein complex in C. elegans. Studies in S. cerevisiae showed that HMT-1 at a minimum homodimerizes and that oligomerization is essential for HMT-1 to confer cadmium tolerance. We also established that the NTE domain plays an important structural and functional role: it is essential for HMT-1 oligomerization and Cd-detoxification function. However, the NTE itself was not sufficient for oligomerization suggesting that multiple structural features of HMT-1 must associate to form a functional transporter. CONCLUSIONS: The prominence of heavy metals as environmental toxins and the remarkable conservation of HMT-1 structural architecture and function in different species reinforce the value of continued studies of HMT-1 in model systems for identifying functional domains in HMT1 of humans.

  9. Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

    International Nuclear Information System (INIS)

    The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structure was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins

  10. Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

    Energy Technology Data Exchange (ETDEWEB)

    Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu [Pathology and Microbiology Department, 986495 Nebraska Medical Center, Omaha, NE 68198-6495 (United States)

    2011-05-01

    The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structure was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.

  11. Ligand-controlled interaction of histone acetyltransferase binding to ORC-1 (HBO1) with the N-terminal transactivating domain of progesterone receptor induces steroid receptor coactivator 1-dependent coactivation of transcription

    NARCIS (Netherlands)

    M. Georgiakaki (Maria); L.J. Blok (Leen); R. Milgrom (Roni); M. Lombès (Marc); A. Guiochon-Mantel (Anne); H. Loosfelt (Hugues); N. Chabbert-Buffet (Nathalie); B. Dasen (Boris); G. Meduri (Geri); S. Wenk (Sandra); L. Rajhi (Leila); L. Amazit (Larbi); A. Chauchereau (Anne); C.W. Burger (Curt)

    2006-01-01

    textabstractModulators of cofactor recruitment by nuclear receptors are expected to play an important role in the coordination of hormone-induced transactivation processes. To identify such factors interacting with the N-terminal domain (NTD) of the progesterone receptor (PR), we used this domain as

  12. Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

    International Nuclear Information System (INIS)

    We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly 13C, 15N labeled protein sample, sequential chemical-shift information for 74% of the N, Cα, Cβ triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.

  13. Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

    Energy Technology Data Exchange (ETDEWEB)

    Habenstein, Birgit [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France); Wasmer, Christian [Harvard Medical School (United States); Bousset, Luc; Sourigues, Yannick [UPR 3082 CNRS, Laboratoire d' Enzymologie et Biochimie Structurales (France); Schuetz, Anne [ETH Zurich, Physical Chemistry (Switzerland); Loquet, Antoine [Max Planck Institute for Biophysical Chemistry (Germany); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); Melki, Ronald, E-mail: melki@lebs.cnrs-gif.fr [UPR 3082 CNRS, Laboratoire d' Enzymologie et Biochimie Structurales (France); Boeckmann, Anja, E-mail: a.bockmann@ibcp.fr [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France)

    2011-11-15

    We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly {sup 13}C, {sup 15}N labeled protein sample, sequential chemical-shift information for 74% of the N, C{alpha}, C{beta} triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.

  14. Crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of the human spliceosomal DExD/H-box protein hPrp22

    International Nuclear Information System (INIS)

    The cloning, purification and crystallization of the C-terminal domain of human hPrp22 are reported. This communication also contains data for the preliminary X-ray diffraction analysis. The Homo sapiens DExD/H-box protein hPrp22 is a crucial component of the eukaryotic pre-mRNA splicing machinery. Within the splicing cycle, it is involved in the ligation of exons and generation of the lariat and it additionally catalyzes the release of mature mRNA from the spliceosomal U5 snRNP. The yeast homologue of this protein, yPrp22, shows ATP-dependent RNA-helicase activity and is capable of unwinding RNA/RNA duplex molecules. A truncated construct coding for residues 950–1183 of human Prp22, comprising the structurally and functionally uncharacterized C-terminal domain, was cloned into an Escherichia coli expression vector. The protein was subsequently overproduced, purified and crystallized. The crystals obtained diffracted to 2.1 Å resolution, belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = b = 78.2, c = 88.4 Å, and contained one molecule in the asymmetric unit

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

    Energy Technology Data Exchange (ETDEWEB)

    El Omari, Kamel; Iourin, Oleg; Kadlec, Jan [University of Oxford, Oxford OX3 7BN (United Kingdom); Fearn, Richard; Hall, David R. [Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Harlos, Karl [University of Oxford, Oxford OX3 7BN (United Kingdom); Grimes, Jonathan M.; Stuart, David I., E-mail: dave@strubi.ox.ac.uk [University of Oxford, Oxford OX3 7BN (United Kingdom); Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom)

    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.

  16. Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated Cysteines

    Science.gov (United States)

    Bischak, Connor G.; Longhi, Sonia; Snead, David M.; Costanzo, Stéphanie; Terrer, Elodie; Londergan, Casey H.

    2010-01-01

    Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) were cyanylated at cysteine and their infrared spectra in the C≡N stretching region were recorded both in the absence and in the presence of one of the physiological partners of NTAIL, namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within NTAIL, the C≡N stretching bands of the infrared probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and infrared spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level. PMID:20816082

  17. Prokaryotic expression and purification of fibronectin leucine rich transmembrane protein 3 C-terminal domain proteins in rats

    Institute of Scientific and Technical Information of China (English)

    Yan Cai; Jing Yang; He Huang; Fang Li; Ganqiu Wu; Jing Yang; Xuegang Luo

    2009-01-01

    BACKGROUND: Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood.OBJECTIVE: To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins.DESIGN, TIME AND SETTING: An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008.MATERIALS: Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coli (E. Coli) JM109 were purchased from Promega. E. Coil BL21 was provided by Novagen.METHODS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. Coli BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained.MAIN OUTCOME MEASURES: Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods.RESULTS: FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then cloned into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass

  18. Role of C-Terminal Domains in Surface Attachment of the Fructosyltransferase of Streptococcus salivarius ATCC 25975

    Science.gov (United States)

    Rathsam, Catherine; Jacques, Nicholas A.

    1998-01-01

    The cell-associated β-d-fructosyltransferase of Streptococcus salivarius, which is devoid of the cell wall anchoring motif, LPXTG, is released on exposure to its substrate, sucrose. Deletions within the C terminus of the enzyme implicated both the hydrophobic and the proline-glycine-serine-threonine-rich wall-associated domain in stabilizing the enzyme on the cell surface. PMID:9829954

  19. Disposable amperometric magnetoimmunosensor for the sensitive detection of the cardiac biomarker amino-terminal pro-B-type natriuretic peptide in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Esteban-Fernández de Ávila, Berta, E-mail: berta.efa@quim.ucm.es; Escamilla-Gómez, Vanessa, E-mail: vaneeg@quim.ucm.es; Campuzano, Susana, E-mail: susanacr@quim.ucm.es; Pedrero, María, E-mail: mpedrero@quim.ucm.es; Pingarrón, José M., E-mail: pingarro@quim.ucm.es

    2013-06-19

    Graphical abstract: -- Highlights: •Novel and sensitive amperometric magnetoimmunosensor for NT-proBNP detection. •Indirect competitive immunoassay onto HOOC-MBs and Au/SPEs as transducers. •Excellent analytical performance at levels clinically relevant in human serum. •Useful in clinical diagnosis and prognosis of cardiac diseases. -- Abstract: A novel amperometric magnetoimmunosensor using an indirect competitive format is developed for the sensitive detection of the amino-terminal pro-B-type natriuretic peptide (NT-proBNP). The immunosensor design involves the covalent immobilization of the antigen onto carboxylic-modified magnetic beads (HOOC-MBs) activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), and further incubation in a mixture solution containing variable concentrations of the antigen and a fixed concentration of an HRP-labeled detection antibody. Accordingly, the target NT-proBNP in the sample and that immobilized on the MBs compete for binding to a fixed amount of the specific HRP-labeled secondary antibody. The immunoconjugate-bearing MBs are captured by a magnet placed under the surface of a disposable gold screen-printed electrode (Au/SPE). The amperometric responses measured at –0.10 V (vs. a Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H{sub 2}O{sub 2} as the enzyme substrate, are used to monitor the affinity reaction. The developed magnetoimmunosensor provides attractive analytical characteristics in 10-times diluted human serum samples, exhibiting a linear range of clinical usefulness (0.12–42.9 ng mL{sup −1}) and a detection limit of 0.02 ng mL{sup −1}, which can be used in clinical diagnosis of chronic heart failure in the elderly and for classifying patients at risk of death after heart transplantation. The magnetoimmunosensor was successfully applied to the analysis of spiked human serum

  20. Amino Acid Activation of mTORC1 by a PB1-Domain-Driven Kinase Complex Cascade

    Directory of Open Access Journals (Sweden)

    Juan F. Linares

    2015-08-01

    Full Text Available The mTORC1 complex is central to the cellular response to changes in nutrient availability. The signaling adaptor p62 contributes to mTORC1 activation in response to amino acids and interacts with TRAF6, which is required for the translocation of mTORC1 to the lysosome and the subsequent K63 polyubiquitination and activation of mTOR. However, the signal initiating these p62-driven processes was previously unknown. Here, we show that p62 is phosphorylated via a cascade that includes MEK3/6 and p38δ and is driven by the PB1-containing kinase MEKK3. This phosphorylation results in the recruitment of TRAF6 to p62, the ubiquitination and activation of mTOR, and the regulation of autophagy and cell proliferation. Genetic inactivation of MEKK3 or p38δ mimics that of p62 in that it leads to inhibited growth of PTEN-deficient prostate organoids. Analysis of human prostate cancer samples showed upregulation of these three components of the pathway, which correlated with enhanced mTORC1 activation.

  1. Apical localization of ASIP/PAR-3:EGFP in zebrafish neuroepithelial cells involves the oligomerization domain CR1, the PDZ domains, and the C-terminal portion of the protein.

    Science.gov (United States)

    von Trotha, Jakob W; Campos-Ortega, José A; Reugels, Alexander M

    2006-04-01

    Neurulation in zebrafish (Danio rerio) embryos is characterized by oriented cell divisions and the progressive establishment of cellular polarity. Mitoses in the neural plate and neural tube are planar, but in the neural keel/rod stage, the mitotic spindle rotates by 90 degrees, causing cell divisions to occur perpendicular to the plane of the neuroepithelium. The mechanisms and molecules that establish cellular polarity and cause the stereotypic orientation of the mitotic spindle during neurulation are largely unknown. In Caenorhabditis elegans and Drosophila, the PAR/aPKC complex has been shown to be involved in both establishment of cellular polarity and spindle orientation. Here, we show that the conserved N-terminal oligomerization domain (CR1) and the PDZ domains of ASIP/PAR-3:EGFP are involved in its localization to the apical membrane in zebrafish neuroepithelial cells. We further show that the C-terminal part of ASIP/PAR-3 contributes to proper localization and that the apical localization signals in ASIP/PAR-3 prevent the basolateral localization of a Numb:PAR-3 fusion protein. The parallel orientation of the mitotic spindle in the neural tube, however, is only weakly impaired upon overexpression of various ASIP/PAR-3:EGFP constructs.

  2. Expression, purification and preliminary structural analysis of the head domain of Deinococcus radiodurans RecN

    OpenAIRE

    Pellegrino, Simone; Radzimanowski, Jens; McSweeney, Sean; Timmins, Joanna

    2011-01-01

    The head domain of the DNA-repair protein RecN from D. radiodurans, composed of the amino- and carboxy-terminal domains, was crystallized. X-ray diffraction data were collected to 3.0 Å resolution and the crystals belonged to space group P21.

  3. The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.

    Directory of Open Access Journals (Sweden)

    Luke J Alderwick

    2011-02-01

    Full Text Available The D-arabinan-containing polymers arabinogalactan (AG and lipoarabinomannan (LAM are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM. Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985 at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis.

  4. Crystal structures of histone and p53 methyltransferase SmyD2 reveal a conformational flexibility of the autoinhibitory C-terminal domain.

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    Yuanyuan Jiang

    Full Text Available SmyD2 belongs to a new class of chromatin regulators that control gene expression in heart development and tumorigenesis. Besides methylation of histone H3 K4, SmyD2 can methylate non-histone targets including p53 and the retinoblastoma tumor suppressor. The methyltransferase activity of SmyD proteins has been proposed to be regulated by autoinhibition via the intra- and interdomain bending of the conserved C-terminal domain (CTD. However, there has been no direct evidence of a conformational change in the CTD. Here, we report two crystal structures of SmyD2 bound either to the cofactor product S-adenosylhomocysteine or to the inhibitor sinefungin. SmyD2 has a two-lobed structure with the active site located at the bottom of a deep crevice formed between the CTD and the catalytic domain. By extensive engagement with the methyltransferase domain, the CTD stabilizes the autoinhibited conformation of SmyD2 and restricts access to the catalytic site. Unexpectedly, despite that the two SmyD2 structures are highly superimposable, significant differences are observed in the first two helices of the CTDs: the two helices bend outwards and move away from the catalytic domain to generate a less closed conformation in the sinefungin-bound structure. Although the overall fold of the individual domains is structurally conserved among SmyD proteins, SmyD2 appear to be a conformational "intermediate" between a close form of SmyD3 and an open form of SmyD1. In addition, the structures reveal that the CTD is structurally similar to tetratricopeptide repeats (TPR, a motif through which many cochaperones bind to the heat shock protein Hsp90. Our results thus provide the first evidence for the intradomain flexibility of the TPR-like CTD, which may be important for the activation of SmyD proteins by Hsp90.

  5. The TAF9 C-terminal conserved region domain is required for SAGA and TFIID promoter occupancy to promote transcriptional activation.

    Science.gov (United States)

    Saint, Malika; Sawhney, Sonal; Sinha, Ishani; Singh, Rana Pratap; Dahiya, Rashmi; Thakur, Anushikha; Siddharthan, Rahul; Natarajan, Krishnamurthy

    2014-05-01

    A common function of the TFIID and SAGA complexes, which are recruited by transcriptional activators, is to deliver TBP to promoters to stimulate transcription. Neither the relative contributions of the five shared TBP-associated factor (TAF) subunits in TFIID and SAGA nor the requirement for different domains in shared TAFs for transcriptional activation is well understood. In this study, we uncovered the essential requirement for the highly conserved C-terminal region (CRD) of Taf9, a shared TAF, for transcriptional activation in yeast. Transcriptome profiling performed under Gcn4-activating conditions showed that the Taf9 CRD is required for induced expression of ∼9% of the yeast genome. The CRD was not essential for the Taf9-Taf6 interaction, TFIID or SAGA integrity, or Gcn4 interaction with SAGA in cell extracts. Microarray profiling of a SAGA mutant (spt20Δ) yielded a common set of genes induced by Spt20 and the Taf9 CRD. Chromatin immunoprecipitation (ChIP) assays showed that, although the Taf9 CRD mutation did not impair Gcn4 occupancy, the occupancies of TFIID, SAGA, and the preinitiation complex were severely impaired at several promoters. These results suggest a crucial role for the Taf9 CRD in genome-wide transcription and highlight the importance of conserved domains, other than histone fold domains, as a common determinant for TFIID and SAGA functions.

  6. Interaction of the C-terminal acidic domain of the insulin receptor with histone modulates the receptor kinase activity.

    Science.gov (United States)

    Baron, V; Kaliman, P; Alengrin, F; Van Obberghen, E

    1995-04-01

    In this study, we investigated the role of the insulin receptor domain 1270-1280, an acid-rich sequence located in the receptor C-terminus. Antipeptide IgG raised against this sequence were obtained and used to analyze their effect on receptor function. Antipeptide IgG inhibited receptor autophosphorylation at Tyr1146, Tyr1150 and Tyr1151. These sites are known to be key modulators of the receptor activity. Autophosphorylation at other sites may also have been inhibited. The antipeptide antibody decreased the receptor kinase activity measured with poly(Glu80Tyr20) and a synthetic peptide corresponding to the proreceptor sequence 1142-1158. We provide evidence that the effect of the antibody on substrate phosphorylation may result from the control of the phosphorylation level of the receptor. Concerning the action of the antipeptide IgG on the receptor kinase activity, histone did not behave similarly to poly(Glu80Tyr20). The antibody recognizing sequence 1270-1280 competed with histone for an overlapping binding site. Histone also modulated insulin receptor autophosphorylation, supporting the idea that interference with domain 1270-1280 alters the receptor kinase. Our data suggest that the acidic region including residues 1270-1280 of the insulin receptor C-terminus is involved in the following events: (a) receptor binding with histone, an exogenous substrate of the receptor kinase, and (b) the regulation of receptor autophosphorylation and kinase activity. Based on these observations, we would like to propose that this insulin receptor domain could interact with cellular proteins modulating the receptor kinase. PMID:7744039

  7. A novel C-terminal domain of RecJ is critical for interaction with HerA in Deinococcus radiodurans

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    Kaiying eCheng

    2015-11-01

    Full Text Available Homologous recombination (HR generates error-free repair products, which plays an important role in double strand break repair and replication fork rescue processes. DNA end resection, the critical step in HR, is usually performed by a series of nuclease/helicase. RecJ was identified as a 5’-3’ exonuclease involved in bacterial DNA end resection. Typical RecJ possesses a conserved DHH domain, a DHHA1 domain, and an oligonucleotide/oligosaccharide-binding (OB fold. However, RecJs from Deinococcus-Thermus phylum, such as Deinococcus radiodurans RecJ (DrRecJ, possess an extra C-terminal domain (CTD, of which the function has not been characterized. Here, we showed that a CTD-deletion of DrRecJ (DrRecJΔC could not restore drrecJ mutant growth and mitomycin C (MMC-sensitive phenotypes, indicating that this domain is essential for DrRecJ in vivo. DrRecJΔC displayed reduced DNA nuclease activity and DNA binding ability. Direct interaction was identified between DrRecJ-CTD and DrHerA, which stimulates DrRecJ nuclease activity by enhancing its DNA binding affinity. Moreover, DrNurA nuclease, another partner of DrHerA, inhibited the stimulation of DrHerA on DrRecJ nuclease activity by interaction with DrHerA. Opposing growth and MMC-resistance phenotypes between the recJ and nurA mutants were observed. A novel modulation mechanism among DrRecJ, DrHerA, and DrNurA was also suggested.

  8. Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490963–1138 domain of TamB from Escherichia coli

    Science.gov (United States)

    Josts, Inokentijs; Grinter, Rhys; Kelly, Sharon M.; Mosbahi, Khedidja; Roszak, Aleksander; Cogdell, Richard; Smith, Brian O.; Byron, Olwyn; Walker, Daniel

    2014-01-01

    TamB is a recently described inner membrane protein that, together with its partner protein TamA, is required for the efficient secretion of a subset of autotransporter proteins in Gram-negative bacteria. In this study, the C-terminal DUF490963–1138 domain of TamB was overexpressed in Escherichia coli K-12, purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the primitive trigonal space group P3121, with unit-cell parameters a = b = 57.34, c = 220.74 Å, and diffracted to 2.1 Å resolution. Preliminary secondary-structure and X-ray diffraction analyses are reported. Two molecules are predicted to be present in the asymmetric unit. Experimental phasing using selenomethionine-labelled protein will be undertaken in the future. PMID:25195908

  9. Photosynthetic control of the plasma membrane H+-ATPase in Vallisneria leaves. II. Presence of putative isogenes and a protein equipped with a C-terminal autoinhibitory domain.

    Science.gov (United States)

    Harada, Akiko; Fukuhara, Toshiyuki; Takagi, Shingo

    2002-04-01

    In vitro treatment with trypsin of plasma membrane (PM) vesicles isolated from the leaves of Vallisneria gigantea Graebner, an aquatic monocot, produced a marked decrease in the Km for ATP and an increase in the Vmax of H+-transporting activity. Concomitantly, the removal of 8 kDa of the C-terminal domain from the 94-kDa PM H+-ATPase was confirmed by immunoblotting using different kinds of polyclonal antibody. Three partial clones of putative PM H+-ATPase genes (Vga1, 2, and 3) were isolated from leaves by reverse transcription polymerase chain reaction. Northern blotting analysis revealed that the expression level of Vga3 was high and that of the other two genes was much lower. The H+-transporting activity of PM vesicles was substantially suppressed in the presence of inorganic phosphate (Pi), which has been supposed to be a noncompetitive inhibitor of the PM H+-ATPase, coincident with an increase in the Km for ATP and a decrease in the Vmax. After treatment of the isolated PM vesicles with trypsin, the inhibitory effect of Pi was no longer evident. This result indicates that Pi inhibited the activity through the C-terminal autoinhibitory domain of the PM H+-ATPase. Furthermore, Pi increased the Km for ATP of the H+-transporting activity in the PM vesicles isolated from both dark-adapted and red-light-irradiated leaves. The results suggest that regulation of the Km for ATP through the operation of photosynthesis is independent of regulation through the cytoplasmic level of Pi. PMID:11941463

  10. Evolutionary genomics of plant genes encoding N-terminal-TM-C2 domain proteins and the similar FAM62 genes and synaptotagmin genes of metazoans

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    Craxton Molly

    2007-07-01

    Full Text Available Abstract Background Synaptotagmin genes are found in animal genomes and are known to function in the nervous system. Genes with a similar domain architecture as well as sequence similarity to synaptotagmin C2 domains have also been found in plant genomes. The plant genes share an additional region of sequence similarity with a group of animal genes named FAM62. FAM62 genes also have a similar domain architecture. Little is known about the functions of the plant genes and animal FAM62 genes. Indeed, many members of the large and diverse Syt gene family await functional characterization. Understanding the evolutionary relationships among these genes will help to realize the full implications of functional studies and lead to improved genome annotation. Results I collected and compared plant Syt-like sequences from the primary nucleotide sequence databases at NCBI. The collection comprises six groups of plant genes conserved in embryophytes: NTMC2Type1 to NTMC2Type6. I collected and compared metazoan FAM62 sequences and identified some similar sequences from other eukaryotic lineages. I found evidence of RNA editing and alternative splicing. I compared the intron patterns of Syt genes. I also compared Rabphilin and Doc2 genes. Conclusion Genes encoding proteins with N-terminal-transmembrane-C2 domain architectures resembling synaptotagmins, are widespread in eukaryotes. A collection of these genes is presented here. The collection provides a resource for studies of intron evolution. I have classified the collection into homologous gene families according to distinctive patterns of sequence conservation and intron position. The evolutionary histories of these gene families are traceable through the appearance of family members in different eukaryotic lineages. Assuming an intron-rich eukaryotic ancestor, the conserved intron patterns distinctive of individual gene families, indicate independent origins of Syt, FAM62 and NTMC2 genes. Resemblances

  11. A second disulfide bridge from the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39

    DEFF Research Database (Denmark)

    Storjohann, Laura; Holst, Birgitte; Schwartz, Thue W

    2008-01-01

    . Disruption of the nonconserved disulfide bridge by mutagenesis led to an increase in the Zn (2+) potency. This phenotype, with an approximate 10-fold increase in agonist potency and a slight increase in E max, was mimicked by treatment of the wild-type receptor with TCEP at low concentrations, which had no......A highly conserved feature across all families of 7TM receptors is a disulfide bridge between a Cys residue located at the extracellular end of transmembrane segment III (TM-III) and one in extracellular loop 2 (ECL-2). The zinc sensor GPR39 contains four Cys residues in the extracellular domains....... By using mutagenesis, treatment with the reducing agent TCEP, and a labeling procedure for free sulfhydryl groups, we identify the pairing of these Cys residues in two disulfide bridges: the prototypical bridge between Cys (108) in TM-III and Cys (210) in ECL-2 and a second disulfide bridge...

  12. BtcA, A class IA type III chaperone, interacts with the BteA N-terminal domain through a globular/non-globular mechanism.

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    Chen Guttman

    Full Text Available Bordetella pertussis, the etiological agent of "whooping cough" disease, utilizes the type III secretion system (T3SS to deliver a 69 kDa cytotoxic effector protein, BteA, directly into the host cells. As with other T3SS effectors, prior to its secretion BteA binds BtcA, a 13.9 kDa protein predicted to act as a T3SS class IA chaperone. While this interaction had been characterized for such effector-chaperone pairs in other pathogens, it has yet to be fully investigated in Bordetella. Here we provide the first biochemical proof that BtcA is indeed a class IA chaperone, responsible for the binding of BteA's N-terminal domain. We bring forth extensive evidence that BtcA binds its substrate effector through a dual-interface binding mechanism comprising of non-globular and bi-globular interactions at a moderate micromolar level binding affinity. We demonstrate that the non-globular interactions involve the first 31 N-terminal residues of BteA287 and their removal leads to destabilization of the effector-chaperone complex and lower binding affinities to BtcA. These findings represent an important first step towards a molecular understanding of BteA secretion and cell entry.

  13. A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation.

    Science.gov (United States)

    Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie; Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique; Revers, Frédéric; Ziegler-Graff, Véronique; Brault, Véronique

    2015-12-01

    Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RTCter) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RTCter. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells.

  14. CBF mediates adenovirus Ela trans-activation by interaction at the C-terminal promoter targeting domain of conserved region 3.

    Science.gov (United States)

    Agoff, S N; Wu, B

    1994-12-01

    Genetic and biochemical evidence suggest that conserved region 3 (CR3) of the adenovirus Ela polypeptide can provide two distinct and separable functions: an N-terminal transcriptional activation region and a C-terminal promoter targeting region. It is thought that the promoter targeting region of Ela CR3 interacts with promoter-specific transcription factors, thereby bringing the activation region of Ela CR3 in proximity of the promoter. Here we report that CBF, a CCAAT-box-binding factor that regulates hsp70 gene expression and mediates Ela trans-activation in vivo, interacts with the promoter targeting region of Ela CR3 in vitro. Point mutations in Ela CR3 that are defective in stimulating transcription from the hsp70 promoter are also defective in stimulating transcription directed by a synthetic activator, GAL-CBF, composed of the DNA-binding domain of yeast GAL4 fused to CBF. These mutations fall into two classes with respect to their abilities to interact with CBF in vitro. Mutations in the transcriptional activation region of Ela CR3 do not affect binding to CBF, but mutation of the promoter targeting region of Ela CR3 prevents association with CBF in vitro.

  15. RAD51AP2, a novel vertebrate- and meiotic-specific protein, sharesa conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    Energy Technology Data Exchange (ETDEWEB)

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-07-25

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1.

  16. A Novel Bmal1 Mutant Mouse Reveals Essential Roles of the C-Terminal Domain on Circadian Rhythms.

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    Noheon Park

    Full Text Available The mammalian circadian clock is an endogenous biological timer comprised of transcriptional/translational feedback loops of clock genes. Bmal1 encodes an indispensable transcription factor for the generation of circadian rhythms. Here, we report a new circadian mutant mouse from gene-trapped embryonic stem cells harboring a C-terminus truncated Bmal1 (Bmal1GTΔC allele. The homozygous mutant (Bmal1GTΔC/GTΔC mice immediately lost circadian behavioral rhythms under constant darkness. The heterozygous (Bmal1+/GTΔC mice displayed a gradual loss of rhythms, in contrast to Bmal1+/- mice where rhythms were sustained. Bmal1GTΔC/GTΔC mice also showed arrhythmic mRNA and protein expression in the SCN and liver. Lack of circadian reporter oscillation was also observed in cultured fibroblast cells, indicating that the arrhythmicity of Bmal1GTΔC/GTΔC mice resulted from impaired molecular clock machinery. Expression of clock genes exhibited distinct responses to the mutant allele in Bmal1+/GTΔC and Bmal1GTΔC/GTΔC mice. Despite normal cellular localization and heterodimerization with CLOCK, overexpressed BMAL1GTΔC was unable to activate transcription of Per1 promoter and BMAL1-dependent CLOCK degradation. These results indicate that the C-terminal region of Bmal1 has pivotal roles in the regulation of circadian rhythms and the Bmal1GTΔC mice constitute a novel model system to evaluate circadian functional mechanism of BMAL1.

  17. 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.

    Science.gov (United States)

    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

  18. Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation.

    Science.gov (United States)

    Ordaz, Benito; Tang, Jisen; Xiao, Rui; Salgado, Alfonso; Sampieri, Alicia; Zhu, Michael X; Vaca, Luis

    2005-09-01

    TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.

  19. Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A

    Institute of Scientific and Technical Information of China (English)

    YUAN; Chong; (

    2001-01-01

    [1]Price, N. C., Assembly of multi-subunit structures, in Mechanisms of Protein Folding (ed. Pain, R. H.), New York: Oxford University Press, 1994, 160-193.[2]Casal, J. I., Ahern, T. J., Davenport, R. C. et al., Subunit interface of triosephosphate isomerase: Site-directed mutagenesis and characterization of the altered enzyme, Biochemistry, 1987, 26: 1258-1264.[3]Chakerian, A. E., Matthews, K. S., Characterization of mutations in oligomerization domain of lac repressor protein, J. Biol. Chem., 1991, 266: 22206-22214.[4]Mandelman, D., Schwarz, F. P., Li, H. Y. et al., The role of quaternary interactions on the stability and activity of ascorbate peroxidase, Protein Sci., 1998, 7: 2089-2098.[5]Thomas, M. C., Ballantine, S. P., Bethell, S. S. et al., Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii, Biochemistry, 1998, 37: 11629-11636.[6]Holbrook, J. J., Liljas, A., Steindel, S. J. et al., Lactate dehydrogenase, in The Enzymes (ed. Boyer, P. D.), Vol. 11, 3rd ed., New York: Academic Press, 1975, 191-292.[7]Zettlmeissl, G., Rudolph, R., Jaenicke, R., Reconstitution of lactic dehydrogenase after acid dissociation, Eur. J. Biochem., 1981, 121: 169-175.[8]Zettlmeissl, G., Rudolph, R., Jaenicke, R., Rate-determining folding and association reactions on the reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride, Biochemistry, 1982, 21: 3946-3950.[9]Hermann, R., Jaenicke, R., Rudolph, R., Analysis of the reconstitution of oligomeric enzymes by cross-linking with glutaraldehyde: Kinetics of reassociation of lactic dehydrogenase, Biochemistry, 1981, 20: 5195-5201.[10]Jaenicke, R., Folding and association of protein, Prog. Biophys. Mol. Biol., 1987, 49: 117-237.[11]Opitz, U., Rudolph, R., Jaenicke, R. et al., Proteolytic dimeric of porcine muscle lactate dehydrogenase: Characterization, folding, and

  20. Structural Insights into the Calcium-Mediated Allosteric Transition in the C-Terminal Domain of Calmodulin from Nuclear Magnetic Resonance Measurements.

    Science.gov (United States)

    Kukic, Predrag; Lundström, Patrik; Camilloni, Carlo; Evenäs, Johan; Akke, Mikael; Vendruscolo, Michele

    2016-01-12

    Calmodulin is a two-domain signaling protein that becomes activated upon binding cooperatively two pairs of calcium ions, leading to large-scale conformational changes that expose its binding site. Despite significant advances in understanding the structural biology of calmodulin functions, the mechanistic details of the conformational transition between closed and open states have remained unclear. To investigate this transition, we used a combination of molecular dynamics simulations and nuclear magnetic resonance (NMR) experiments on the Ca(2+)-saturated E140Q C-terminal domain variant. Using chemical shift restraints in replica-averaged metadynamics simulations, we obtained a high-resolution structural ensemble consisting of two conformational states and validated such an ensemble against three independent experimental data sets, namely, interproton nuclear Overhauser enhancements, (15)N order parameters, and chemical shift differences between the exchanging states. Through a detailed analysis of this structural ensemble and of the corresponding statistical weights, we characterized a calcium-mediated conformational transition whereby the coordination of Ca(2+) by just one oxygen of the bidentate ligand E140 triggers a concerted movement of the two EF-hands that exposes the target binding site. This analysis provides atomistic insights into a possible Ca(2+)-mediated activation mechanism of calmodulin that cannot be achieved from static structures alone or from ensemble NMR measurements of the transition between conformations.

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

    Directory of Open Access Journals (Sweden)

    Benny Chain

    Full Text Available 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.

  2. Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization-Fragmentation Reaction Sequence.

    Science.gov (United States)

    Harris, Golda G; Lombardi, Patrick M; Pemberton, Travis A; Matsui, Tsutomu; Weiss, Thomas M; Cole, Kathryn E; Köksal, Mustafa; Murphy, Frank V; Vedula, L Sangeetha; Chou, Wayne K W; Cane, David E; Christianson, David W

    2015-12-01

    Geosmin synthase from Streptomyces coelicolor (ScGS) catalyzes an unusual, metal-dependent terpenoid cyclization and fragmentation reaction sequence. Two distinct active sites are required for catalysis: the N-terminal domain catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate (PPi), and the C-terminal domain catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone through a retro-Prins reaction. A unique αα domain architecture is predicted for ScGS based on amino acid sequence: each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg(2+) for catalysis. Here, we report the X-ray crystal structure of the unliganded N-terminal domain of ScGS and the structure of its complex with three Mg(2+) ions and alendronate. These structures highlight conformational changes required for active site closure and catalysis. Although neither full-length ScGS nor constructs of the C-terminal domain could be crystallized, homology models of the C-terminal domain were constructed on the basis of ∼36% sequence identity with the N-terminal domain. Small-angle X-ray scattering experiments yield low-resolution molecular envelopes into which the N-terminal domain crystal structure and the C-terminal domain homology model were fit, suggesting possible αα domain architectures as frameworks for bifunctional catalysis.

  3. Homophilic interactions mediated by receptor tyrosine phosphatases mu and kappa. A critical role for the novel extracellular MAM domain

    DEFF Research Database (Denmark)

    Zondag, G C; Koningstein, G M; Jiang, Y P;

    1995-01-01

    The receptor-like protein tyrosine phosphatases (RPTP) mu and RPTP kappa have a modular ectodomain consisting of four fibronectin type III-like repeats, a single Ig-like domain, and a newly identified N-terminal MAM domain. The function of the latter module, which comprises about 160 amino acids...

  4. Epigenetic blockade of neoplastic transformation by bromodomain and extra-terminal (BET) domain protein inhibitor JQ-1.

    Science.gov (United States)

    Zhang, Chengyue; Su, Zheng-Yuan; Wang, Ling; Shu, Limin; Yang, Yuqing; Guo, Yue; Pung, Douglas; Bountra, Chas; Kong, Ah-Ng

    2016-10-01

    The neoplastic transformation of cells and inflammation are processes that contribute to tumor initiation. Recently, emerging evidence has suggested that epigenetic alterations are also implicated in the early stages of carcinogenesis. Therefore, potent small molecules targeting epigenetic regulators have been developed as novel cancer therapeutic and preventive strategies. Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that play key roles at the interface between chromatin modification and transcriptional regulation. In this study, we investigated the effect of the BET inhibitor JQ-1 on malignant transformation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin epidermal JB6 P+ cells. Treatment with JQ-1 effectively impaired TPA-induced colony formation in vitro. At the molecular level, the expression of several key TPA-induced pro-survival and pro-proliferative genes (Bcl2, Cyclin D1, and c-Myc) decreased rapidly after BET inhibition. In addition, JQ-1 treatment attenuated the activation of inflammatory NF-κB signaling triggered by TPA. Luciferase reporter assays using plasmids carrying different elements from the COX2 or IL6 promoters demonstrated that JQ-1 does not directly inhibit interactions between NF-κB and its binding sequence; rather, it affects CRE-element-associated transcriptional enhancement. Through siRNA gene silencing, we found that JQ-1 inhibits the p300-dependent transcriptional activation of COX2, which correlates with the results of the luciferase assay. Chromatin immunoprecipitation assays showed that TPA elevated H3K27Ac enrichment in the COX2 promoter region, which is mediated by p300, and Brd4. JQ-1 treatment did not change H3K27Ac levels but decreased the recruitment of Brd4 and RNA Polymerase II. Collectively, our study reveals that the BET inhibitor JQ-1 exerts potent anti-cancer and anti-inflammatory effects by interfering with the core transcriptional program of neoplastic transformation

  5. Crystallization and preliminary X-ray analysis of a putative sensor histidine kinase domain: the C-terminal domain of HksP4 from Aquifex aeolicus VF5

    International Nuclear Information System (INIS)

    The putative sensor histidine kinase domain of the cytoplasmic protein HksP4 from the hyperthermophilic bacterium A. aeolicus VF5 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained in the presence of ATP and AMPPNP; they were found to belong to the same space group P212121 and diffracted X-rays to 3.1 and 2.9 Å resolution, respectively. The histidine kinase domain of the cytoplasmic protein HksP4 from the hyperthermophilic bacterium Aquifex aeolicus VF5, located in the C-terminal half of the protein, was expressed, purified and crystallized. Diffraction-quality crystals were obtained in the presence of adenosine triphosphate (ATP) or adenosine 5′-(β,γ-imido)triphosphate (AMPPNP) by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals obtained in the presence of ATP and AMPPNP diffracted X-rays to 3.1 and 2.9 Å resolution, respectively, on BL-5A at Photon Factory (Ibaraki, Japan) and were found to belong to the same space group P212121, with unit-cell parameters a = 80.2, b = 105.5, c = 122.0 Å and a = 81.5, b = 105.5, c = 130.9 Å, respectively. Their Matthews coefficients (VM = 2.74 and 2.51 Å3 Da−1, respectively) indicated that both crystals contained four protein molecules per asymmetric unit

  6. Amino acids

    Science.gov (United States)

    ... amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan , and valine. Nonessential amino acids "Nonessential" means that our bodies produce an amino ...

  7. Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.

    Directory of Open Access Journals (Sweden)

    Tobias Haase

    Full Text Available BACKGROUND: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC Förster resonance energy transfer (FRET was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC beta1 and alpha subunits. METHODOLOGY/PRINCIPAL FINDINGS: Cyan fluorescent protein (CFP was used as FRET donor and yellow fluorescent protein (YFP as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged alpha subunits with the carboxy-terminally tagged beta1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the alpha subunits is close to the carboxy-terminus of the beta1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. CONCLUSIONS/SIGNIFICANCE: Based on the ability of an amino-terminal fragment of the beta1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alphacatbetacat, Winger and Marletta (Biochemistry 2005, 44:4083-90 have proposed a direct interaction of the amino-terminal region of beta1 with the catalytic domains. In support of such a concept of "trans" regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed "fluorescent-conjoined" sGC's by fusion of the alpha amino-terminus to the beta1 carboxy-terminus leading to a

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

    Science.gov (United States)

    Brown, Laura E; Nicholson, Martin W; Arama, Jessica E; Mercer, Audrey; Thomson, Alex M; Jovanovic, Jasmina N

    2016-07-01

    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

  9. 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

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States); Whittaker, Gary R., E-mail: grw7@cornell.edu [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States)

    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.

  10. 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

    International Nuclear Information System (INIS)

    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.

  11. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells.

    Science.gov (United States)

    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

  12. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells.

    Science.gov (United States)

    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

  13. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells

    Science.gov (United States)

    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

  14. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells

    Science.gov (United States)

    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

  15. 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

    DEFF Research Database (Denmark)

    Frandsen, Kristian Erik Høpfner; Rasmussen, Kim K.; Jensen, Malene Ringkjøbing;

    2013-01-01

    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 ...

  16. Chaperone-like activities of different molecular forms of beta-casein. Importance of polarity of N-terminal hydrophilic domain.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  18. Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation.

    Science.gov (United States)

    Hao, Yu-Jun; Song, Qing-Xin; Chen, Hao-Wei; Zou, Hong-Feng; Wei, Wei; Kang, Xu-Sheng; Ma, Biao; Zhang, Wan-Ke; Zhang, Jin-Song; Chen, Shou-Yi

    2010-10-01

    Plant-specific transcription factor NAC proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. In the NAC family, some members function as transcription activators while others act as repressors. In the present study we found that though the full-length GmNAC20 from soybean did not have transcriptional activation activity, the carboxy-terminal activation domain of GmNAC20 had high transcriptional activation activity in the yeast assay system. Deletion experiments revealed an active repression domain with 35 amino acids, named NARD (NAC Repression Domain), in the d subdomain of NAC DNA-binding domain. NARD can reduce the transcriptional activation ability of diverse transcription factors when fused to either the amino-terminal or the carboxy-terminal of the transcription factors. NARD-like sequences are also present in other NAC family members and they are functional repression domain when fused to VP16 in plant protoplast assay system. Mutation analysis of conserved amino acid residues in NARD showed that the hydrophobic LVFY motif may partially contribute to the repression function. It is hypothesized that the interactions between the repression domain NARD and the carboxy-terminal activation domain may finally determine the ability of NAC family proteins to regulate downstream gene expressions.

  19. A form of mitofusin 2 (Mfn2) lacking the transmembrane domains and the COOH-terminal end stimulates metabolism in muscle and liver cells.

    Science.gov (United States)

    Segalés, Jessica; Paz, José C; Hernández-Alvarez, María Isabel; Sala, David; Muñoz, Juan Pablo; Noguera, Eduard; Pich, Sara; Palacín, Manuel; Enríquez, José Antonio; Zorzano, Antonio

    2013-11-15

    Mitofusin 2 (Mfn2), a protein that participates in mitochondrial fusion, is required to maintain normal mitochondrial metabolism in skeletal muscle and liver. Given that muscle Mfn2 is repressed in obese or type 2 diabetic subjects, this protein may have a potential pathophysiological role in these conditions. To evaluate whether the metabolic effects of Mfn2 can be dissociated from its function in mitochondrial dynamics, we studied a form of human Mfn2, lacking the two transmembrane domains and the COOH-terminal coiled coil (ΔMfn2). This form localized in mitochondria but did not alter mitochondrial morphology in cells or in skeletal muscle fibers. The expression of ΔMfn2 in mouse skeletal muscle stimulated glucose oxidation and enhanced respiratory control ratio, which occurred in the absence of changes in mitochondrial mass. ΔMfn2 did not stimulate mitochondrial respiration in Mfn2-deficient muscle cells. The expression of ΔMfn2 in mouse liver or in hepatoma cells stimulated gluconeogenesis. In addition, ΔMfn2 activated basal and maximal respiration both in muscle and liver cells. In all, we show that a form of Mfn2 lacking mitochondrial fusion activity stimulates mitochondrial function and enhances glucose metabolism in muscle and liver tissues. This study suggests that Mfn2 regulates metabolism independently of changes in mitochondrial morphology.

  20. Transient stability of the helical pattern of region F19-L22 of the N-terminal domain of p53: a molecular dynamics simulation study.

    Science.gov (United States)

    Espinoza-Fonseca, L Michel; Trujillo-Ferrara, José G

    2006-04-28

    Two molecular dynamics simulations of the region E17-N29 of p53 (p53(17-29)) at different temperatures were performed for a total time of 0.2 micros, to study the conformational landscape of this region. Previous studies have suggested that this region displays different structural motifs, such as helix of a double beta-turn, and that its secondary structure might be transiently stable. Interestingly, in this study it was found that the region F19-L25, and particularly its fragment F19-L22, display a stable, transient helical pattern at sub-microsecond periods. The region F19-L22, which contains one of the most important residues needed for the interaction of p53 with MDM2, seems to be formed and stabilized by the existence of one hydrophobic and one aromatic cluster. The main function of these clusters is to help their surrounding area to desolvate, to allow the hydrogen bond network, therefore favoring the formation of a stable helix. This preliminary study would be useful for a better understanding of the structure and function of the N-terminal domain of p53 and its implications for the control of different types of cancer. PMID:16530164

  1. Identification of a human ABCC10 orthologue in Catharanthus roseus reveals a U12-type intron determinant for the N-terminal domain feature

    Indian Academy of Sciences (India)

    Taissir El-Guizani; Clotilde Guibert; Saïda Triki; Benoit St-Pierre; Eric Ducos

    2014-04-01

    ABC (ATP-binding cassette) transporters are members of a large superfamily of proteins that utilize ATP hydrolysis to translocate a wide range of substrates across biological membranes. In general, members of C subfamily (ABCC) are structurally characterized by an additional (N-terminal) transmembrane domain (TMD0). Phylogenetic analysis of plant ABCCs separates their protein sequences into three distinct clusters: I and II are plant specific whereas cluster III contains both human and plant ABCCs. Screening of the Plant Medicinal Genomics Resource database allowed us to identify 16 ABCCs partial sequences in Catharanthus roseus; two of which belong to the unique CrABCC1 transcript that we identified in cluster III. Genomic organization of CrABCC1 TMD0 coding sequence displays an AT-AC U12-type intron that is conserved in higher plant orthologues. We showed that CrABCC1, like its human orthologue ABCC10, produces alternative transcripts that encode protein sequences with a truncated form of TMD0 without the first transmembrane span (TM1). Subcellular localization of CrABCC1 TMD0 variants using yellow fluorescent protein fusions reveals that the TM1 is required for a correct routing of the TMD0 to the tonoplast. Finally, the specific repartition of CrABCC1 orthologues in some species suggests that this gene was lost several times during evolution and that its physiological function may, rely on a common feature of multicellular eukaryotes.

  2. Cyclin-dependent kinase 2 phosphorylates s/t-p sites in the hepadnavirus core protein C-terminal domain and is incorporated into viral capsids.

    Science.gov (United States)

    Ludgate, Laurie; Ning, Xiaojun; Nguyen, David H; Adams, Christina; Mentzer, Laura; Hu, Jianming

    2012-11-01

    Phosphorylation of the hepadnavirus core protein C-terminal domain (CTD) is important for viral RNA packaging, reverse transcription, and subcellular localization. Hepadnavirus capsids also package a cellular kinase. The identity of the host kinase that phosphorylates the core CTD or gets packaged remains to be resolved. In particular, both the human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) core CTDs harbor several conserved serine/threonine-proline (S/T-P) sites whose phosphorylation state is known to regulate CTD functions. We report here that the endogenous kinase in the HBV capsids was blocked by chemical inhibitors of the cyclin-dependent kinases (CDKs), in particular, CDK2 inhibitors. The kinase phosphorylated the HBV CTD at the serine-proline (S-P) sites. Furthermore, we were able to detect CDK2 in purified HBV capsids by immunoblotting. Purified CDK2 phosphorylated the S/T-P sites of the HBV and DHBV CTD in vitro. Inhibitors of CDKs, of CDK2 in particular, decreased both HBV and DHBV CTD phosphorylation in vivo. Moreover, CDK2 inhibitors blocked DHBV CTD phosphorylation, specifically at the S/T-P sites, in a mammalian cell lysate. These results indicate that cellular CDK2 phosphorylates the functionally critical S/T-P sites of the hepadnavirus core CTD and is incorporated into viral capsids.

  3. Deficiency of syntrophin, dystroglycan, and merosin in a female infant with a congenital muscular dystrophy phenotype lacking cysteine-rich and C-terminal domains of dystrophin.

    Science.gov (United States)

    Tachi, N; Ohya, K; Chiba, S; Matsuo, M; Patria, S Y; Matsumura, K

    1997-08-01

    Primary deficiency of merosin is the cause of the classic form of congenital muscular dystrophy (CMD) accompanied by brain white matter abnormalities. We report a female infant with dystrophinopathy who was deficient in merosin in skeletal muscle. The patient had a phenotype of typical CMD and white matter abnormalities on brain MRI. Merosin was greatly reduced in the biopsied skeletal muscle. However, the expression of dystroglycan and syntrophin was also greatly reduced, and the immunoreactivity for the antibodies against the cysteine-rich/C-terminal domains of dystrophin was absent in the sarcolemma. Reverse transcriptase polymerase chain reaction analysis of the dystrophin gene revealed a complete lack of exons 71 through 74. In skeletal muscle, only the mutant gene was expressed. These results suggest that the patient is a symptomatic Duchenne muscular dystrophy carrier with skewed X-inactivation. This patient illustrates for the first time that a dystrophin abnormality can cause a secondary deficiency of merosin in dystrophinopathy. The reduction of merosin may account for the clinical phenotype of CMD and correlate with the white matter abnormalities in our patient.

  4. Characterization of the promoter and extended C-terminal domain of Arabidopsis WRKY33 and functional analysis of tomato WRKY33 homologues in plant stress responses.

    Science.gov (United States)

    Zhou, Jie; Wang, Jian; Zheng, Zuyu; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2015-08-01

    Arabidopsis AtWRKY33 plays a critical role in broad plant stress responses. Whether there are evolutionarily conserved homologues of AtWRKY33 in other plants and what make AtWRKY33 such an important protein in plant stress responses are largely unknown. We compared AtWRKY33 with its close homologues to identify AtWRKY33-specific regulatory and structural elements, which were then functionally analysed through complementation. We also performed phylogenetic analysis to identify structural AtWRKY33 homologues in other plants and functionally analysed two tomato homologues through complementation and gene silencing. AtWRKY33 has an extended C-terminal domain (CTD) absent in its close homologue AtWRKY25. Both its CTD and the strong pathogen/stress-responsive expression of AtWRKY33 are necessary to complement the critical phenotypes of atwrky33. Structural AtWRKY33 homologues were identified in both dicot and monocot plants including two (SlWRKY33A and SlWRKY33B) in tomato. Molecular complementation and gene silencing confirmed that the two tomato WRKY genes play a critical role similar to that of AtWRKY33 in plant stress responses. Thus, WRKY33 proteins are evolutionarily conserved with a critical role in broad plant stress responses. Both its CTD and promoter are critical for the uniquely important roles of WRKY33 in plant stress responses.

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

    Science.gov (United States)

    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

  6. Identification of a human ABCC10 orthologue in Catharanthus roseus reveals a U12-type intron determinant for the N-terminal domain feature.

    Science.gov (United States)

    El-Guizani, Taissir; Guibert, Clotilde; Triki, Saida; St-Pierre, Benoit; Ducos, Eric

    2014-04-01

    ABC (ATP-binding cassette) transporters are members of a large superfamily of proteins that utilize ATP hydrolysis to translocate a wide range of substrates across biological membranes. In general, members of C subfamily (ABCC) are structurally characterized by an additional (N-terminal) transmembrane domain (TMD0). Phylogenetic analysis of plant ABCCs separates their protein sequences into three distinct clusters: I and II are plant specific whereas cluster III contains both human and plant ABCCs. Screening of the Plant Medicinal Genomics Resource database allowed us to identify 16 ABCCs partial sequences in Catharanthus roseus; two of which belong to the unique CrABCC1 transcript that we identified in cluster III. Genomic organization of CrABCC1 TMD0 coding sequence displays an AT-AC U12-type intron that is conserved in higher plant orthologues. We showed that CrABCC1, like its human orthologue ABCC10, produces alternative transcripts that encode protein sequences with a truncated form of TMD0 without the first transmembrane span (TM1). Subcellular localization of CrABCC1 TMD0 variants using yellow fluorescent protein fusions reveals that the TM1 is required for a correct routing of the TMD0 to the tonoplast. Finally, the specific repartition of CrABCC1 orthologues in some species suggests that this gene was lost several times during evolution and that its physiological function may, rely on a common feature of multicellular eukaryotes. PMID:24840820

  7. Global gene expression analysis of fission yeast mutants impaired in Ser-2 phosphorylation of the RNA pol II carboxy terminal domain.

    Directory of Open Access Journals (Sweden)

    Reza Saberianfar

    Full Text Available In Schizosaccharomyces pombe the nuclear-localized Lsk1p-Lsc1p cyclin dependent kinase complex promotes Ser-2 phosphorylation of the heptad repeats found within the RNA pol II carboxy terminal domain (CTD. Here, we first provide evidence supporting the existence of a third previously uncharacterized Ser-2 CTD kinase subunit, Lsg1p. As expected for a component of the complex, Lsg1p localizes to the nucleus, promotes Ser-2 phosphorylation of the CTD, and physically interacts with both Lsk1p and Lsc1p in vivo. Interestingly, we also demonstrate that lsg1Δ mutants--just like lsk1Δ and lsc1Δ strains--are compromised in their ability to faithfully and reliably complete cytokinesis. Next, to address whether kinase mediated alterations in CTD phosphorylation might selectively alter the expression of genes with roles in cytokinesis and/or the cytoskeleton, global gene expression profiles were analyzed. Mutants impaired in Ser-2 phosphorylation display little change with respect to the level of transcription of most genes. However, genes affecting cytokinesis--including the actin interacting protein gene, aip1--as well as genes with roles in meiosis, are included in a small subset that are differentially regulated. Significantly, genetic analysis of lsk1Δ aip1Δ double mutants is consistent with Lsk1p and Aip1p acting in a linear pathway with respect to the regulation of cytokinesis.

  8. Loss of c-Kit and bone marrow failure upon conditional removal of the GATA-2 C-terminal zinc finger domain in adult mice.

    Science.gov (United States)

    Li, Haiyan S; Jin, Jin; Liang, Xiaoxuan; Matatall, Katie A; Ma, Ying; Zhang, Huiyuan; Ullrich, Stephen E; King, Katherine Y; Sun, Shao-Cong; Watowich, Stephanie S

    2016-09-01

    Heterozygous mutations in the transcriptional regulator GATA-2 associate with multilineage immunodeficiency, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The majority of these mutations localize in the zinc finger (ZnF) domains, which mediate GATA-2 DNA binding. Deregulated hematopoiesis with GATA-2 mutation frequently develops in adulthood, yet GATA-2 function in the bone marrow remains unresolved. To investigate this, we conditionally deleted the GATA-2 C-terminal ZnF (C-ZnF) coding sequences in adult mice. Upon Gata2 C-ZnF deletion, we observed rapid peripheral cytopenia, bone marrow failure, and decreased c-Kit expression on hematopoietic progenitors. Transplant studies indicated GATA-2 has a cell-autonomous role in bone marrow hematopoiesis. Moreover, myeloid lineage populations were particularly sensitive to Gata2 hemizygosity, while molecular assays indicated GATA-2 regulates c-Kit expression in multilineage progenitor cells. Enforced c-Kit expression in Gata2 C-ZnF-deficient hematopoietic progenitors enhanced myeloid colony activity, suggesting GATA-2 sustains myelopoiesis via a cell intrinsic role involving maintenance of c-Kit expression. Our results provide insight into mechanisms regulating hematopoiesis in bone marrow and may contribute to a better understanding of immunodeficiency and bone marrow failure associated with GATA-2 mutation.

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

    Science.gov (United States)

    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.

  10. 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.

    Science.gov (United States)

    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.

  11. 鸡大肠杆菌FimH基因C端结构域克隆及特性分析%Cloning and characterization of C terminal domain of avian E .coli ’Fim H gene

    Institute of Scientific and Technical Information of China (English)

    白宇琛; 谢金峰; 周广防; 冯秀丽

    2016-01-01

    Objective]Through further study of Fim H ,the the main structure gene of avian Escherichia coli type I , the research aimed to provide necessary theoretical foundation for pathogenic mechanism of avian E .coli and develop-ment of gene engineering vaccinE.[Methods]Based on the published C terminal domain nucleotide sequence of Fim H gene for reference,this research designed and synthesized primers,amplified three strains of the relevant Fim H genes in avian E .coli including JS1,JS2 and JS6,used Lasergene software for sequence alignment and protein structure pre-diction,and the evolutionary tree was drew,etc.[Results]The results showed that the homology of gene nucleotide sequences between Reference strain and JS1,JS2 or JS6 were 97.3%,97.7% and 97.3%,and their amino acid se-quences was 95.3%,97.6% and 95.3%,respectively.Although their Fim H antigenic nucleotide sequences between Reference strain and JS1,JS2 or JS6 were 97.3%,97.7% and 97.3%,and their amino acid sequences was 95.3%, 97.6% and 95.3%,respectively.Although their Fim H antigenic determinants are basically similar,amino acids changes at 78 th and 79 th caused the different antigenic determinant at C terminal domain of Fim H ,which indicated that amino acid variation of Fim H sequences might have the minor effect on the antigenic property of Fim H .Further-more,the evolutionary tree analysis showed the close evolutionary relationship among three isolated strains and the do-mestic avian E .coli strains.[Conclusion]The results indicated that the Fim H gene of avian E .coli had no significant variation,which makes important foundation for further research on molecular mechanism of avian E .coli and the con-trol strategies against pathogenic avian E .coli.%[目的]通过进一步研究禽大肠杆菌 I 型菌毛主要结构基因 FimH 的基因结构及其抗原特性,为深入探索鸡大肠杆菌致病机理及研制基因工程疫苗奠定必要的理论基础。[方法]本文以已发表的 Fim H

  12. Stability of the Human Papillomavirus Type 18 E2 Protein Is Regulated by a Proteasome Degradation Pathway through Its Amino-Terminal Transactivation Domain

    OpenAIRE

    Bellanger, Sophie; Demeret, Caroline; Goyat, Sylvain; Thierry, Françoise

    2001-01-01

    The E2 proteins of papillomaviruses regulate both viral transcription and DNA replication. The human papillomavirus type 18 (HPV18) E2 protein has been shown to repress transcription of the oncogenic E6 and E7 genes, inducing growth arrest in HeLa cells. Using HPV18 E2 fused to the green fluorescent protein (GFP), we showed that this protein was short-lived in transfected HeLa cells. Real-time microscopy experiments indicated that the E2-dependent signal increased for roughly 24 h after trans...

  13. The 5-amino acid N-terminal extension of non-sulfated drosulfakinin II is a unique target to generate novel agonists.

    Science.gov (United States)

    Leander, M; Heimonen, J; Brocke, T; Rasmussen, M; Bass, C; Palmer, G; Egle, J; Mispelon, M; Berry, K; Nichols, R

    2016-09-01

    The ability to design agonists that target peptide signaling is a strategy to delineate underlying mechanisms and influence biology. A sequence that uniquely characterizes a peptide provides a distinct site to generate novel agonists. Drosophila melanogaster sulfakinin encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2). Drosulfakinin is typical of sulfakinin precursors, which are conserved throughout invertebrates. Non-sulfated DSK II is structurally related to DSK I, however, it contains a unique 5-residue N-terminal extension; drosulfakinins signal through G-protein coupled receptors, DSK-R1 and DSK-R2. Drosulfakinin II distinctly influences adult and larval gut motility and larval locomotion; yet, its structure-activity relationship was unreported. We hypothesized substitution of an N-terminal extension residue may alter nsDSK II activity. By targeting the extension we identified, not unexpectedly, analogs mimicking nsDSK II, yet, surprisingly, we also discovered novel agonists with increased (super) and opposite (protean) effects. We determined [A3] nsDSK II increased larval gut contractility rather than, like nsDSK II, decrease it. [N4] nsDSK II impacted larval locomotion, although nsDSK II was inactive. In adult gut, [A1] nsDSK II, [A2] nsDSKII, and [A3] nsDSK II mimicked nsDSK II, and [A4] nsDSK II and [A5] nsDSK II were more potent; [N3] nsDSK II and [N4] nsDSK II mimicked nsDSK II. This study reports nsDSK II signals through DSK-R2 to influence gut motility and locomotion, identifying a novel role for the N-terminal extension in sulfakinin biology and receptor activation; it also led to the discovery of nsDSK II structural analogs that act as super and protean agonists. PMID:27397853

  14. Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating.

    Science.gov (United States)

    Murayama, Takashi; Kurebayashi, Nagomi; Oba, Toshiharu; Oyamada, Hideto; Oguchi, Katsuji; Sakurai, Takashi; Ogawa, Yasuo

    2011-10-14

    The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

  15. Engineering of halophilic enzymes: Two acidic amino acid residues at the carboxy-terminal region confer halophilic characteristics to Halomonas and Pseudomonas nucleoside diphosphate kinases

    OpenAIRE

    Tokunaga, Hiroko; Arakawa, Tsutomu; Tokunaga, Masao

    2008-01-01

    Nucleoside diphosphate kinase from Halomonas sp. 593 (HaNDK) exhibits halophilic characteristics. Residues 134 and 135 in the carboxy-terminal region of HaNDK are Glu–Glu, while those of its homologous counterpart of non-halophilic Pseudomonas NDK (PaNDK) are Ala–Ala. The double mutation, E134A-E135A, in HaNDK results in the loss of the halophilic characteristics, and, conversely, the double mutation of A134E-A135E in PaNDK confers halophilic characters to this enzyme, indicating that the cha...

  16. Cleavage of RseA by RseP requires a carboxyl-terminal hydrophobic amino acid following DegS cleavage

    OpenAIRE

    Li, Xiaochun; Wang, Boyuan; Feng, Lihui; Kang, Hui; Qi, Yang; Wang, Jiawei; Shi, Yigong

    2009-01-01

    Regulated intramembrane proteolysis (RIP) by the Site-2 protease (S2P) results in the release of a transmembrane signaling protein. Curiously, however, S2P cleavage must be preceded by the action of the Site-1 protease (S1P). To decipher the underlying mechanism, we reconstituted sequential, in vitro cleavages of the Escherichia coli transmembrane protein RseA by DegS (S1P) and RseP (S2P). After DegS cleavage, the newly exposed carboxyl-terminal residue Val-148 of RseA plays an essential role...

  17. The hemopexin and O-glycosylated domains tune gelatinase B/MMP-9 bioavailability via inhibition and binding to cargo receptors

    DEFF Research Database (Denmark)

    Van den Steen, Philippe E; Van Aelst, Ilse; Hvidberg, Vibeke;

    2006-01-01

    Gelatinase B/matrix metalloproteinase-9 (MMP-9), a key regulator and effector of immunity, contains a C-terminal hemopexin domain preceded by a unique linker sequence of approximately 64 amino acid residues. This linker sequence is demonstrated to be an extensively O-glycosylated (OG) domain with a...

  18. Viscocidad de la Dieta y su Efecto sobre la Pérdidad de Aminoácidos Endógenos Recuperados en Íleon Terminal de Cerdos

    OpenAIRE

    Edgar Chi-Moreno; Miguel Cervantes-Ramírez; J. Luis Figueroa-Velasco; Jorge Baeza-López; Manuel Cuca-García; Fernando Copado-Bueno; Jorge L. Yáñez-Hernández; Noemí Torrentera-Olivera

    2005-01-01

    La viscosidad de la dieta provocada por algunos compuestos antinutricionales en los cereales puede afectar la pérdida de aminoácidos (AA) endógenos. Por tanto, se realizó un estudio para evaluar el efecto de la viscosidad de la dieta, producida por la adición de goma Guar, en la pérdida de AA endógenos recuperados al final del intestino delgado. Seis cerdos (62.0±2.5 kg peso) con cánulas en íleon terminal se utilizaron en tres periodos experimentales en un diseño en Cuadro Latino 3×3 repetido...

  19. Influence of PAS Domain Flanking Regions on Oligomerisation and Redox Signalling By NifL

    OpenAIRE

    Richard Little; Peter Slavny; Ray Dixon

    2012-01-01

    Per-ARNT-Sim (PAS) domains constitute a typically dimeric, conserved α/β tertiary fold of approximately 110 amino acids that perform signalling roles in diverse proteins from all kingdoms of life. The amino terminal PAS1 domain of NifL from Azotobacter vinelandii accommodates a redox-active FAD group; elevation of cytosolic oxygen concentrations result in FAD oxidation and a concomitant conformational re-arrangement that is relayed via a short downstream linker to a second PAS domain, PAS2. A...

  20. TWEAK-independent Fn14 self-association and NF-κB activation is mediated by the C-terminal region of the Fn14 cytoplasmic domain.

    Directory of Open Access Journals (Sweden)

    Sharron A N Brown

    Full Text Available The tumor necrosis factor (TNF superfamily member TNF-like weak inducer of apoptosis (TWEAK is a pro-inflammatory and pro-angiogenic cytokine implicated in physiological tissue regeneration and wound repair. TWEAK binds to a 102-amino acid type I transmembrane cell surface receptor named fibroblast growth factor-inducible 14 (Fn14. TWEAK:Fn14 engagement activates several intracellular signaling cascades, including the NF-κB pathway, and sustained Fn14 signaling has been implicated in the pathogenesis of chronic inflammatory diseases and cancer. Although several groups are developing TWEAK- or Fn14-targeted agents for therapeutic use, much more basic science research is required before we fully understand the TWEAK/Fn14 signaling axis. For example, we and others have proposed that TWEAK-independent Fn14 signaling may occur in cells when Fn14 levels are highly elevated, but this idea has never been tested directly. In this report, we first demonstrate TWEAK-independent Fn14 signaling by showing that an Fn14 deletion mutant that is unable to bind TWEAK can activate the NF-κB pathway in transfected cells. We then show that ectopically-expressed, cell surface-localized Fn14 can self-associate into Fn14 dimers, and we show that Fn14 self-association is mediated by an 18-aa region within the Fn14 cytoplasmic domain. Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions. Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122. These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

  1. A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

    Directory of Open Access Journals (Sweden)

    Granier Thierry

    2011-08-01

    Full Text Available Abstract Background Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR. In grapevine (Vitis vinifera L., several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. Results In this work, we describe the effects of a single amino acid substitution (R69L located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5bL, the native protein being referred as VvMYB5bR was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5bR and VvMYB5bL genes in tobacco. Flowers from 35S::VvMYB5bL transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5bR and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. Conclusions Taken together, our findings indicate that VvMYB5bL is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s. In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment

  2. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part IV. Implication for the mechanism of folding of the parent protein

    OpenAIRE

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2010-01-01

    A 34-residue α/β peptide, [IG(28-61)], derived from the C-terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using CD and NMR spectroscopy at various temperatures, and by differential scanning calorimetry. It was found that the C-terminal part (a 16-residue-long fragment) of this peptide, which corresponds to the sequence of the β-hairpin in the native structure, forms structure similar to the β-hairpin only at T = 313 K, and the structure is...

  3. A prime-boost immunization with Tc52 N-terminal domain DNA and the recombinant protein expressed in Pichia pastoris protects against Trypanosoma cruzi infection.

    Science.gov (United States)

    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

  4. The C-terminal pentapeptide of Nanog tryptophan repeat domain interacts with Nac1 and regulates stem cell proliferation but not pluripotency.

    Science.gov (United States)

    Ma, Tianhua; Wang, Zhe; Guo, Yunqian; Pei, Duanqing

    2009-06-12

    Overexpression of Nanog in mouse embryonic stem (ES) cells has been shown to abrogate the requirement of leukemia inhibitory factor for self-renewal in culture. Little is known about the molecular mechanism of Nanog function. Here we describe the role of the tryptophan repeat (WR) domain, one of the two transactivators at its C terminus, in regulating stem cell proliferation as well as pluripotency. We first created a supertransactivator, W2W3x10, by duplicating repeats W2W3 10 times and discovered that it can functionally substitute for wild type WR at sustaining pluripotency, albeit with a significantly slower cell cycle, phenocopying Nanog(9W) with the C-terminal pentapeptide (WNAAP) of WR deleted. ES cells carrying both W2W3x10 and Nanog(9W) have a longer G1 phase, a shorter S phase in cell cycle distribution and progression analysis, and a lower level of pAkt(Ser473) compared with wild type Nanog, suggesting that both mutants impact the cell cycle machinery via the phosphatidylinositol 3-kinase/Akt pathway. Both mutants remain competent in dimerizing with Nanog but cannot form a complex with Nac1 efficiently, suggesting that WNAAP may be involved in Nac1 binding. By tagging Gal4DBD with WNAAP, we demonstrated that this pentapeptide is sufficient to confer Nac1 binding. Furthermore, we can rescue W2W3x10 by placing WNAAP at the corresponding locations. Finally, we found that Nanog and Nac1 synergistically up-regulate ERas expression and promote the proliferation of ES cells. These results suggest that Nanog interacts with Nac1 through WNAAP to regulate the cell cycle of ES cells via the ERas/phosphatidylinositol 3-kinase/Akt pathway, but not pluripotency, thus decoupling cell cycle control from pluripotency.

  5. Clostridium perfringens enterotoxin C-terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy-resistant ovarian cancer.

    Science.gov (United States)

    Cocco, Emiliano; Shapiro, Erik M; Gasparrini, Sara; Lopez, Salvatore; Schwab, Carlton L; Bellone, Stefania; Bortolomai, Ileana; Sumi, Natalia J; Bonazzoli, Elena; Nicoletti, Roberta; Deng, Yang; Saltzman, W Mark; Zeiss, Caroline J; Centritto, Floriana; Black, Jonathan D; Silasi, Dan-Arin; Ratner, Elena; Azodi, Masoud; Rutherford, Thomas J; Schwartz, Peter E; Pecorelli, Sergio; Santin, Alessandro D

    2015-12-01

    Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-naïve and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.

  6. RNA-Free and Ribonucleoprotein-Associated Influenza Virus Polymerases Directly Bind the Serine-5-Phosphorylated Carboxyl-Terminal Domain of Host RNA Polymerase II

    Science.gov (United States)

    Martínez-Alonso, Mónica; Hengrung, Narin

    2016-01-01

    ABSTRACT Influenza viruses subvert the transcriptional machinery of their hosts to synthesize their own viral mRNA. Ongoing transcription by cellular RNA polymerase II (Pol II) is required for viral mRNA synthesis. By a process known as cap snatching, the virus steals short 5′ capped RNA fragments from host capped RNAs and uses them to prime viral transcription. An interaction between the influenza A virus RNA polymerase and the C-terminal domain (CTD) of the large subunit of Pol II has been established, but the molecular details of this interaction remain unknown. We show here that the influenza virus ribonucleoprotein (vRNP) complex binds to the CTD of transcriptionally engaged Pol II. Furthermore, we provide evidence that the viral polymerase binds directly to the serine-5-phosphorylated form of the Pol II CTD, both in the presence and in the absence of viral RNA, and show that this interaction is conserved in evolutionarily distant influenza viruses. We propose a model in which direct binding of the viral RNA polymerase in the context of vRNPs to Pol II early in infection facilitates cap snatching, while we suggest that binding of free viral polymerase to Pol II late in infection may trigger Pol II degradation. IMPORTANCE Influenza viruses cause yearly epidemics and occasional pandemics that pose a threat to human health, as well as represent a large economic burden to health care systems globally. Existing vaccines are not always effective, as they may not exactly match the circulating viruses. Furthermore, there are a limited number of antivirals available, and development of resistance to these is a concern. New measures to combat influenza are needed, but before they can be developed, it is necessary to better understand the molecular interactions between influenza viruses and their host cells. By providing further insights into the molecular details of how influenza viruses hijack the host transcriptional machinery, we aim to uncover novel targets for

  7. Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin serotype A as a vaccine candidate using a bi-cistronic baculovirus system.

    Science.gov (United States)

    Villaflores, Oliver B; Hsei, Chein-Ming; Teng, Chao-Yi; Chen, Ying-Ju; Wey, Jiunn-Jye; Tsui, Pei-Yi; Shyu, Rong-Hwa; Tung, Kuo-Lun; Yeh, Jui-Ming; Chiao, Der-Jiang; Wu, Tzong-Yuan

    2013-04-01

    Clostridial botulinum neurotoxin (BoNT) is one of the most toxic proteins causing the food borne disease, botulism. In previous studies, recombinant BoNT production by Escherichia coli and yeast Pichia pastoris has been hampered by high AT content and codon bias in the gene encoding BoNT and required a synthetic gene to resolve this intrinsic bottleneck. This paper reports the simultaneous expression of the C-terminal heavy chain domain of BoNT (rBoNT/A-HC-6h) and enhanced green fluorescent protein (EGFP) using a bi-cistronic baculovirus-insect cell expression system. The expression of EGFP facilitated the monitoring of viral infection, virus titer determination, and isolation of the recombinant virus. Protein fusion with hexa-His-tag and one-step immobilized metal-ion affinity chromatography (IMAC) purification produced a homogenous, stable, and immunologically active 55-kDa rBoNT/A-HC-6h (about 3mg/L) with >90% purity. Furthermore, measured levels of serum titers were 8-folds for mice vaccinated with the purified rBoNT/A-HC-6h (2μg) than for mice administered with botulinum toxoid after initial immunization. Challenge experiment with botulinum A toxin demonstrated the immunoprotective activity of purified rBoNT/A-HC-6h providing the mice full protection against 10(2) LD50 botulinum A toxin with a dose as low as 0.2μg. This study provided supportive evidence for the use of a bi-cistronic baculovirus-Sf21 insect cell expression system in the facile expression of an immunogenically active rBoNT/A-HC. PMID:23313783

  8. The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks.

    Directory of Open Access Journals (Sweden)

    Audrey Costes

    Full Text Available We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB(Cter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB(Cter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.

  9. The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks.

    Science.gov (United States)

    Costes, Audrey; Lecointe, François; McGovern, Stephen; Quevillon-Cheruel, Sophie; Polard, Patrice

    2010-01-01

    We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter)) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter) interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB(Cter) deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB(Cter) acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome. PMID:21170359

  10. 重组蛋白药物C末端不同长度氨基酸序列的质谱分析%Mass spectrometry analysis of recombinant protein drugs with C-terminal amino acid sequence of different lengths

    Institute of Scientific and Technical Information of China (English)

    李萍; 赵永强; 薛燕; 刘锋; 刘炳玉; 何昆; 王红霞

    2011-01-01

    目的:基于本实验室已建立的溴化氰裂解蛋白质C末端方法结合优化后的质谱检测技术,对C末端长度分别为2~37个氨基酸,相对分子质量在200~5000的8个重组蛋白药物进行检测.方法:(1)针对重组蛋白药物的不同状态(SDS-PAGE、干粉或溶液)分别进行C末端胶内或溶液裂解;(2)质谱检测,正离子方式,雾化气为氮气,碰撞气体为氩气.源温80℃,锥孔电压50 V,MCP检测器电压为2.15 kV.结果:8个重组蛋白药物的C末端全部成功检测出,且基本为基峰.结论:建立的重组蛋白药物C末端测序联用方法应用于实际药物的检测具有很高的实用价值和学术意义.%Objective : Based on the method established by cyanogen bromide cleavage of proteins C - terminal combined with the optimized mass spectrometry in our laboratory, detection of C - terminal lengths of 2 to 37 amino acids, relative molecular mass 200 -5000 of the 8 recombinant protein drugs. Methods : ( 1 ) For different states of recombinant protein drugs ( SDS - PAGE,dry powder or solution) to C terminal cleavage in gel or solution, respectively. (2) Mass spectrometry detection, positive ion mode,atomization gas was nitrogen, collision gas was argon,source temperature 80 ℃ ,cone voltage 50 V ,MCP detector voltage of 2. 15 kV. Results : All of C - terminal of the 8 recombinant protein drugs successfully detected as the base peak. Conclusions: The established C - terminal sequencing method of the recombinant protein was applied to the actual drugs testing , have high practical value and academic significance.

  11. Structure of NS1A effector domain from the influenza A/Udorn/72 virus

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Shuangluo; Monzingo, Arthur F.; Robertus, Jon D., E-mail: jrobertus@mail.utexas.edu [Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas, 1 University Station A5300, Austin, TX 78712 (United States)

    2009-01-01

    The structure of the effector domain of the influenza protein NS1, a validated antiviral drug target, has been solved in two space groups. The nonstructural protein NS1A from influenza virus is a multifunctional virulence factor and a potent inhibitor of host immunity. It has two functional domains: an N-terminal 73-amino-acid RNA-binding domain and a C-terminal effector domain. Here, the crystallographic structure of the NS1A effector domain of influenza A/Udorn/72 virus is presented. Structure comparison with the NS1 effector domain from mouse-adapted influenza A/Puerto Rico/8/34 (PR8) virus strain reveals a similar monomer conformation but a different dimer interface. Further analysis and evaluation shows that the dimer interface observed in the structure of the PR8 NS1 effector domain is likely to be a crystallographic packing effect. A hypothetical model of the intact NS1 dimer is presented.

  12. Structure of NS1A effector domain from the influenza A/Udorn/72 virus

    International Nuclear Information System (INIS)

    The structure of the effector domain of the influenza protein NS1, a validated antiviral drug target, has been solved in two space groups. The nonstructural protein NS1A from influenza virus is a multifunctional virulence factor and a potent inhibitor of host immunity. It has two functional domains: an N-terminal 73-amino-acid RNA-binding domain and a C-terminal effector domain. Here, the crystallographic structure of the NS1A effector domain of influenza A/Udorn/72 virus is presented. Structure comparison with the NS1 effector domain from mouse-adapted influenza A/Puerto Rico/8/34 (PR8) virus strain reveals a similar monomer conformation but a different dimer interface. Further analysis and evaluation shows that the dimer interface observed in the structure of the PR8 NS1 effector domain is likely to be a crystallographic packing effect. A hypothetical model of the intact NS1 dimer is presented

  13. Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein

    International Nuclear Information System (INIS)

    A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO4-polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, the authors developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. The recombinant hTF/2N appeared to behave identically with the proteolytically derived half-molecule, but to show a higher degree of monodispersity than the latter protein. Addition of m-fluorotyrosine to the culture medium resulted in random incorporation of this amino acid into cellular protein in lieu of tyrosine. Purified recombinant 19F-Tyr hTF/2N gave four well-resolved 19F NMR resonances of 20-40 Hz line width, two with suggestions of shoulders

  14. Purification, characterization and N-terminal amino acid sequence of a new major allergen from European chestnut pollen--Cas s 1.

    Science.gov (United States)

    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.

  15. The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

    Directory of Open Access Journals (Sweden)

    Cheng Xu

    2014-08-01

    Full Text Available 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 TM (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.

  16. Exploring the interaction between the human copper transporter, CTR1, c-terminal domain and a methionine motif in the presence of Cu(I) and Ag(I) ions, using EPR spectroscopy

    Science.gov (United States)

    Shenberger, Yulia; Yarmiayev, Valeria; Ruthstein, Sharon

    2013-10-01

    The essentiality and toxicity of copper in human, yeast, and bacteria cells requires precise mechanisms for acquisition, intimately linked to controlled distribution, which have yet to be fully understood. Herein, we utilise continuous wave and pulsed electron paramagnetic resonance (EPR) spectroscopy to explore one aspect in the controlled copper transportation mechanism. This was achieved by probing structural changes that occur in the c-terminal domain of the human copper transporter, CTR1, upon interacting with a methionine segment. The copper transporter CTR1 transports Cu(I) and Ag(I) ions to various intracellular pathways. Methionine motifs are methionine-rich metal binding segments found in many proteins involved in the transportation of copper ions to other cellular pathways. They are also found to bind Ag(I) with an affinity comparable to Cu(I). This study indicates that the methionine motif experiences conformational changes in the presence of the CTR1 c-terminal domain. These structural changes are dependent on the nature of the metal ion, Cu(I) vs. Ag(I). In addition, the data collected in this study emphasise how important the cysteine residue of the CTR1 c-terminal domain is to a correct conformational state of the target metal binding site.

  17. Monoclonal Antibody 16D10 to the C-Terminal Domain of the Feto-Acinar Pancreatic Protein Binds to Membrane of Human Pancreatic Tumoral SOJ-6 Cells and Inhibits the Growth of Tumor Xenografts

    Directory of Open Access Journals (Sweden)

    Laurence Panicot-Dubois

    2004-11-01

    Full Text Available Feto-acinar pancreatic protein (FAPP characterized by mAbJ28 reactivity is a specific component associated with ontogenesis and behaves as an oncodevelopment-associated antigen. We attempted to determine whether pancreatic tumoral SOJ-6 cells are expressed at their surface FAPP antigens and to examine if specific antibodies directed against these FAPP epitopes could decrease the growth of pancreatic tumors in a mice model. For this purpose, we used specific antibodies against either the whole FAPP, the O-glycosylated C-terminal domain, or the N-terminal domain of the protein. Our results indicate that SOJ-6 cells expressed at their surface a 32-kDa peptide corresponding to the C-terminal domain of the FAPP. Furthermore, we show, by using endoproteinase Lys-C or geldanamycin, a drug able to impair the FAPP secretion, that this 32-kDa peptide expressed on the SOJ-6 cell surface comes from the degradation of the FAPP. Finally, an in vivo prospective study using a preventative tumor model in nude mice indicates that targeting this peptide by the use of mAb16D10 inhibits the growth of SOJ-6 xenografts. The specificity of mAb16D10 for pancreatic tumors and the possibility to obtain recombinant structures of mucin-like peptides recognized by mAb16D10 and mAbJ28 are promising tools in immunologic approaches to cure pancreatic cancers.

  18. The identification and structure of an N-terminal PR domain show that FOG1 is a member of the PRDM family of proteins.

    Directory of Open Access Journals (Sweden)

    Molly K Clifton

    Full Text Available 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.

  19. Structures of three members of Pfam PF02663 (FmdE) implicated in microbial methanogenesis reveal a conserved α+β core domain and an auxiliary C-terminal treble-clef zinc finger

    International Nuclear Information System (INIS)

    The first structures from the FmdE Pfam family (PF02663) reveal that some members of this family form tightly intertwined dimers consisting of two domains (N-terminal α+β core and C-terminal zinc-finger domains), whereas others contain only the core domain. The presence of the zinc-finger domain suggests that some members of this family may perform functions associated with transcriptional regulation, protein–protein interaction, RNA binding or metal-ion sensing. Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2-DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23-SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63-THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63-THEAC and Q2LQ23-SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2-DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63-THEAC and Q2LQ23-SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63-THEAC and Q2LQ23-SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63-THEAC, but is absent from the NTD of Q2LQ23-SYNAS. Second, whereas the structure of the CTD of Q2LQ23-SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure

  20. Analysis of Tb{sup 3+}- and melittin-binding with the C-terminal domain of centrin in Euplotes octocarinatus

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Yaqin; Diao Xiuling; Yan Jun; Feng Yanan [Key Laboratory of Chemical Biology and Microcular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006 (China); Wang Zhijun [Chemical Department, Changzhi University, Changzhi 046011 (China); Liang Aihua, E-mail: aliang@sxu.edu.cn [Key Laboratory of Chemical Biology and Microcular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006 (China); Yang Binsheng, E-mail: yangbs@sxu.edu.cn [Key Laboratory of Chemical Biology and Microcular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006 (China)

    2012-04-15

    Centrin is a low molecular mass (20 KDa) protein that belongs to the EF-hand superfamily. In this work, the interaction between the Tb{sup 3+}-saturated C-terminal domain of Euplotes octocarinatus centrin (Tb{sub 2}-C-EoCen) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was investigated using difference UV-vis spectra and the fluorescence spectra methods. In 100 mM N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid (Hepes) at pH 7.4, with the addition of Tb{sub 2}-C-EoCen, four new peaks were observed at 265 nm, 278 nm, 317 nm and 360 nm by absorptivity compared with blank solution of TNS. At the same time, the reaction could be measured by fluorescence spectra. The fluorescence emission of TNS was shifted from 480 nm to 445 nm in the presence of Tb{sub 2}-C-EoCen. Meanwhile, its fluorescence intensity was increased markedly. The 1:1 stoichiometric ratio of C-EoCen to TNS was confirmed by fluorescence titration curves. The conditional binding constants of TNS with C-EoCen and Tb{sub 2}-C-EoCen were calculated to be log K{sub (C-EoCen-TNS)}=5.32{+-}0.04 M{sup -1} and log K{sub (Tb2-C-EoCen-TNS)}=5.58{+-}0.12 M{sup -1}, respectively. In addition, the protein of Tb{sub 2}-C-EoCen binding with melittin was also studied. Based on the fluorescence titration curves, the 1:1 stoichiometric ratio of Tb{sub 2}-C-EoCen to melittin was confirmed. And the conditional binding constant of C-EoCen with melittin was calculated to be log Ka Prime =6.79{+-}0.17 M{sup -1}. - Highlights: Black-Right-Pointing-Pointer Tb{sup 3+} induced conformational changes of protein C-EoCen from closed state to open state. Black-Right-Pointing-Pointer Conformational changes resulted in the exposure of hydrophobic surfaces on C-EoCen. Black-Right-Pointing-Pointer Tb{sub 2}-C-EoCen may bind with target peptide melittin.

  1. Creation of a novel telomere-cutting endonuclease based on the EN domain of telomere-specific non-long terminal repeat retrotransposon, TRAS1

    Directory of Open Access Journals (Sweden)

    Yoshitake Kazutoshi

    2010-04-01

    Full Text Available Abstract Background The ends of chromosomes, termed telomeres consist of repetitive DNA. The telomeric sequences shorten with cell division and, when telomeres are critically abbreviated, cells stop proliferating. However, in cancer cells, by the expression of telomerase which elongates telomeres, the cells can continue proliferating. Many approaches for telomere shortening have been pursued in the past, but to our knowledge, cutting telomeres in vivo has not so far been demonstrated. In addition, there is lack of information on the cellular effects of telomere shortening in human cells. Results Here, we created novel chimeric endonucleases to cut telomeres by fusing the endonuclease domain (TRAS1EN of the silkworm's telomere specific non-long terminal repeat retrotransposon TRAS1 to the human telomere-binding protein, TRF1. An in vitro assay demonstrated that the TRAS1EN-TRF1 chimeric endonucleases (T-EN and EN-T cut the human (TTAGGGn repeats specifically. The concentration of TRAS1EN-TRF1 chimeric endonucleases necessary for the cleavage of (TTAGGGn repeats was about 40-fold lower than that of TRAS1EN alone. When TRAS1EN-TRF1 endonucleases were introduced into human U2OS cancer cells using adenovirus vectors, the enzymes localized at telomeres of nuclei, cleaved and shortened the telomeric DNA by double-strand breaks. When human U2OS and HFL-1 fibroblast cells were infected with EN-T recombinant adenovirus, their cellular proliferation was suppressed for about 2 weeks after infection. In contrast, the TRAS1EN mutant (H258A chimeric endonuclease fused with TRF1 (ENmut-T did not show the suppression effect. The EN-T recombinant adenovirus induced telomere shortening in U2OS cells, activated the p53-dependent pathway and caused the senescence associated cellular responses, while the ENmut-T construct did not show such effects. Conclusions A novel TRAS1EN-TRF1 chimeric endonuclease (EN-T cuts the human telomeric repeats (TTAGGGn specifically in

  2. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production

    Science.gov (United States)

    Ng, Ivan H. W.; Chan, Kitti W. K.; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Jans, David A.; Forwood, Jade K.

    2016-01-01

    Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization. PMID:27622521

  3. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production.

    Science.gov (United States)

    Tay, Moon Y F; Smith, Kate; Ng, Ivan H W; Chan, Kitti W K; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Luo, Dahai; Jans, David A; Forwood, Jade K; Vasudevan, Subhash G

    2016-09-01

    Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization. PMID:27622521

  4. 端胺基非异氰酸酯预聚体嵌段共聚聚醚型聚氨酯%Prepolymers of Amino-terminal Non-isocyanate Block Copolymerized with Polyether-based Polyurethane

    Institute of Scientific and Technical Information of China (English)

    宋赫; 邓新华; 孙元

    2012-01-01

    Modified polyether polyurethane and its film were prepared from prepolymer of amino- terminal non-isocyanate polyurethane and prepolymer of polyether-based polyurethane via block copolymerization. Synthesis condition of non-isocyanate prepolymer was analyzed; The impact of film-forming temperature and value of NCO and NH2 ratio on mechanical properties of membrane were investigated; Mechanical properties of different polyurethane materials were compared; Degree of phase separation was studied using differential scanning calorimetry(DSC). It showed that carbamate group was introduced into the amino-terminal non-isocyanate prepolymer; The best film-forming temperature was 140 ℃; When NCO/NH2 of prepolymer was 1/0. 9, the best performance was obtained, tensile strength of 25.1 MPa, modulus of 100% elongation of 5 MPa. Compared with regular polyurethane, polyurethane block with amino-terminal non-isocyanate polyurethane prepolymer had higher mechanical properties; DSC curves showed two different glass transition temperatures indicating phase separation.%用端胺基非异氰酸酯基聚氨酯预聚体与聚醚型聚氨酯预聚体嵌段共聚制备了改性聚醚型聚氨酯及其膜,分析了端胺基非异氰酸酯基聚氨酯预聚体,考查了聚醚型聚氨脂树脂成膜温度和预聚体的NCO/NH2配比对膜力学性能的影响,同时对比了聚氨酯材料的力学性能,采用差示扫描量热法(DSC)研究了相分离程度。结果表明,合成的端胺基非异氰酸酯基聚氨酯预聚体中成功地引入了氨基甲酸酯基团;最佳成膜温度为140℃;当预聚体的NCO/NH2=1/0.9(摩尔比,下同)时膜的性能最好,拉伸强度为25.1MPa,伸长100%模量为5MPa;与普通聚氨酯相比,端胺基非异氰酸酯基聚氨酯预聚体嵌段的聚氨酯力学性能更高;DSC曲线显示其有2个不同的玻璃化转变温度,相分离明昂。

  5. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes

    Energy Technology Data Exchange (ETDEWEB)

    Arino, J.; Woon, Chee Wai; Brautigan, D.L.; Miller, T.B. Jr.; Johnson, G.L. (Univ. of Massachusetts Medical School, Worcester (USA))

    1988-06-01

    Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity.

  6. Functional Analyses of Human and Zebrafish 18-Amino Acid In-Frame Deletion Pave the Way for Domain Mapping of the Cerebral Cavernous Malformation 3 Protein

    NARCIS (Netherlands)

    Voss, Katrin; Stahl, Sonja; Hogan, Benjamin M.; Reinders, Joerg; Schleider, Elisa; Schulte-Merker, Stefan; Felbor, Ute

    2009-01-01

    Cerebral cavernous malformations (CCMs) may cause recurrent headaches, seizures, and hemorrhagic stroke and have been associated with loss-of-function mutations in CCM1/KRIT1, CCM2, and CCM3/programmed cell death 10 (PDCD10). The CCM3/PDCD10 amino acid sequence does not reveal significant homologies

  7. Functional analyses of human and zebrafish 18-amino acid in-frame deletion pave the way for domain mapping of the cerebral cavernous malformation 3 protein

    NARCIS (Netherlands)

    Voss, K.; Stahl, S.; Hogan, B.M.; Reinders, J.; Schleider, E.; Schulte-Merker, S.; Felbor, U.

    2009-01-01

    Cerebral cavernous malformations (CCMs) may cause recurrent headaches, seizures, and hemorrhagic stroke and have been associated with loss-of-function mutations in CCM1/KRIT1, CCM2, and CCM3/programmed cell death 10 (PDCD10). The CCM3/PDCD10 amino acid sequence does not reveal significant homologies

  8. Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase.

    OpenAIRE

    Wong, H; Davis, R. C.; Nikazy, J; Seebart, K E; Schotz, M C

    1991-01-01

    Hepatic lipase and lipoprotein lipase hydrolyze fatty acids from triacylglycerols and are critical in the metabolism of circulating lipoproteins. The two lipases are similar in size and amino acid sequence but are distinguished by functional differences in substrate preference and cofactor requirement. Presumably, these distinctions result from structural differences in functional domains. To begin localization of these domains, a chimeric lipase was constructed composed of the N-terminal 329...

  9. Deorphanization of GPRC6A: a promiscuous L-alpha-amino acid receptor with preference for basic amino acids

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Hansen, Kasper B; Balsgaard, Anders;

    2005-01-01

    and was employed to rationally select potential ligands. Measurement of Ca2+-dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of h6A/5.24 and identification of L-alpha-amino acids as agonists. The most active agonists were basic L-alpha-amino acids, L-Arg, L-Lys, and L......One of the most important tasks of molecular pharmacology is the deorphanization of the large number of G-protein-coupled receptors with unidentified endogenous agonists. We recently reported the cloning and analysis of expression of a novel human family C G-protein-coupled receptor, termed hGPRC6A....... To identify agonists at this orphan receptor, we faced the challenges of achieving surface expression in mammalian cell lines and establishing an appropriate functional assay. Generating a chimeric receptor construct, h6A/5.24, containing the ligand binding amino-terminal domain (ATD) of hGPRC6A...

  10. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part IV. Implication for the mechanism of folding of the parent protein

    Science.gov (United States)

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2010-01-01

    A 34-residue α/β peptide, [IG(28-61)], derived from the C-terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using CD and NMR spectroscopy at various temperatures, and by differential scanning calorimetry. It was found that the C-terminal part (a 16-residue-long fragment) of this peptide, which corresponds to the sequence of the β-hairpin in the native structure, forms structure similar to the β-hairpin only at T = 313 K, and the structure is stabilized by non-native long-range hydrophobic interactions (Val47 – Val59). On the other hand, the N-terminal part of IG(28-61), which corresponds to the middle α-helix in the native structure, is unstructured at low temperature (283 K), and forms an α-helix-like structure at 305 K and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305 and 313 K), we do not observe any long-range connectivities which would have supported packing between the C-terminal (β-hairpin) and the N-terminal (α-helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Koliński [Kmiecik, S.; Kolinski, A. Biophys J 2008, 94, 726-736], based on Monte Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. PMID:20049918

  11. Mechanism of formation of the C-terminal beta-hairpin of the B3 domain of the immunoglobulin-binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein.

    Science.gov (United States)

    Lewandowska, Agnieszka; Ołdziej, Stanislaw; Liwo, Adam; Scheraga, Harold A

    2010-05-01

    A 34-residue alpha/beta peptide [IG(28-61)], derived from the C-terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C-terminal part (a 16-residue-long fragment) of this peptide, which corresponds to the sequence of the beta-hairpin in the native structure, forms structure similar to the beta-hairpin only at T = 313 K, and the structure is stabilized by non-native long-range hydrophobic interactions (Val47-Val59). On the other hand, the N-terminal part of IG(28-61), which corresponds to the middle alpha-helix in the native structure, is unstructured at low temperature (283 K) and forms an alpha-helix-like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long-range connectivities which would have supported packing between the C-terminal (beta-hairpin) and the N-terminal (alpha-helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726-736), based on Monte-Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. PMID:20049918

  12. The phosphoCTD-interacting domain of Topoisomerase I

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jianhong; Phatnani, Hemali P.; Hsieh, Tao-Shih [Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 (United States); Greenleaf, Arno L., E-mail: arno.greenleaf@duke.edu [Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 (United States)

    2010-06-18

    The N-terminal domain (NTD) of Drosophila melanogaster (Dm) Topoisomerase I has been shown to bind to RNA polymerase II, but the domain of RNAPII with which it interacts is not known. Using bacterially-expressed fusion proteins carrying all or half of the NTDs of Dm and human (Homo sapiens, Hs) Topo I, we demonstrate that the N-terminal half of each NTD binds directly to the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of the largest RNAPII subunit, Rpb1. Thus, the amino terminal segment of metazoan Topo I (1-157 for Dm and 1-114 for Hs) contains a novel phosphoCTD-interacting domain that we designate the Topo I-Rpb1 interacting (TRI) domain. The long-known in vivo association of Topo I with active genes presumably can be attributed, wholly or in part, to the TRI domain-mediated binding of Topo I to the phosphoCTD of transcribing RNAPII.

  13. The phosphoCTD-interacting domain of Topoisomerase I

    International Nuclear Information System (INIS)

    The N-terminal domain (NTD) of Drosophila melanogaster (Dm) Topoisomerase I has been shown to bind to RNA polymerase II, but the domain of RNAPII with which it interacts is not known. Using bacterially-expressed fusion proteins carrying all or half of the NTDs of Dm and human (Homo sapiens, Hs) Topo I, we demonstrate that the N-terminal half of each NTD binds directly to the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of the largest RNAPII subunit, Rpb1. Thus, the amino terminal segment of metazoan Topo I (1-157 for Dm and 1-114 for Hs) contains a novel phosphoCTD-interacting domain that we designate the Topo I-Rpb1 interacting (TRI) domain. The long-known in vivo association of Topo I with active genes presumably can be attributed, wholly or in part, to the TRI domain-mediated binding of Topo I to the phosphoCTD of transcribing RNAPII.

  14. Biosynthesis of the Aromatic Amino Acids.

    Science.gov (United States)

    Pittard, James; Yang, Ji

    2008-09-01

    This chapter describes in detail the genes and proteins of Escherichia coli involved in the biosynthesis and transport of the three aromatic amino acids tyrosine, phenylalanine, and tryptophan. It provides a historical perspective on the elaboration of the various reactions of the common pathway converting erythrose-4-phosphate and phosphoenolpyruvate to chorismate and those of the three terminal pathways converting chorismate to phenylalanine, tyrosine, and tryptophan. The regulation of key reactions by feedback inhibition, attenuation, repression, and activation are also discussed. Two regulatory proteins, TrpR (108 amino acids) and TyrR (513 amino acids), play a major role in transcriptional regulation. The TrpR protein functions only as a dimer which, in the presence of tryptophan, represses the expression of trp operon plus four other genes (the TrpR regulon). The TyrR protein, which can function both as a dimer and as a hexamer, regulates the expression of nine genes constituting the TyrR regulon. TyrR can bind each of the three aromatic amino acids and ATP and under their influence can act as a repressor or activator of gene expression. The various domains of this protein involved in binding the aromatic amino acids and ATP, recognizing DNA binding sites, interacting with the alpha subunit of RNA polymerase, and