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Sample records for c-terminal domain structure

  1. 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

  2. Structural and Functional Comparisons of Retroviral Envelope Protein C-Terminal Domains: Still Much to Learn

    Directory of Open Access Journals (Sweden)

    Jonathan D. Steckbeck

    2014-01-01

    Full Text Available Retroviruses are a family of viruses that cause a broad range of pathologies in animals and humans, from the apparently harmless, long-term genomic insertion of endogenous retroviruses, to tumors induced by the oncogenic retroviruses and acquired immunodeficiency syndrome (AIDS resulting from human immunodeficiency virus infection. Disease can be the result of diverse mechanisms, including tumorigenesis induced by viral oncogenes or immune destruction, leading to the gradual loss of CD4 T-cells. Of the virally encoded proteins common to all retroviruses, the envelope (Env displays perhaps the most diverse functionality. Env is primarily responsible for binding the cellular receptor and for effecting the fusion process, with these functions mediated by protein domains localized to the exterior of the virus. The remaining C-terminal domain may have the most variable functionality of all retroviral proteins. The C-terminal domains from three prototypical retroviruses are discussed, focusing on the different structures and functions, which include fusion activation, tumorigenesis and viral assembly and lifecycle influences. Despite these genetic and functional differences, however, the C-terminal domains of these viruses share a common feature in the modulation of Env ectodomain conformation. Despite their differences, perhaps each system still has information to share with the others.

  3. 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.

  4. 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...

  5. Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Ji-Hye; Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Park, Jung Eun [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Jung Sup, E-mail: jsplee@mail.chosun.ac.kr [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We have determined solution structures of vEP C-terminal regulatory domain. Black-Right-Pointing-Pointer vEP C-ter100 has a compact {beta}-barrel structure with eight anti-parallel {beta}-strands. Black-Right-Pointing-Pointer Solution structure of vEP C-ter100 shares its molecular topology with that of the collagen-binding domain of collagenase. Black-Right-Pointing-Pointer Residues in the {beta}3 region of vEP C-ter100 might be important in putative ligand/receptor binding. Black-Right-Pointing-Pointer vEP C-ter100 interacts strongly with iron ion. -- Abstract: An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel {beta}-strands with a unique fold that has a compact {beta}-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe{sup 3+}). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates

  6. Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

    International Nuclear Information System (INIS)

    Highlights: ► We have determined solution structures of vEP C-terminal regulatory domain. ► vEP C-ter100 has a compact β-barrel structure with eight anti-parallel β-strands. ► Solution structure of vEP C-ter100 shares its molecular topology with that of the collagen-binding domain of collagenase. ► Residues in the β3 region of vEP C-ter100 might be important in putative ligand/receptor binding. ► vEP C-ter100 interacts strongly with iron ion. -- Abstract: An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel β-strands with a unique fold that has a compact β-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe3+). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates in iron uptake from iron-withholding proteins of the host cell during infection.

  7. Crystal Structure and Mode of Helicase Binding of the C-Terminal Domain of Primase from Helicobacter pylori

    OpenAIRE

    Abdul Rehman, Syed Arif; Verma, Vijay; Mazumder, Mohit; Dhar, Suman K.; Gourinath, S.

    2013-01-01

    To better understand the poor conservation of the helicase binding domain of primases (DnaGs) among the eubacteria, we determined the crystal structure of the Helicobacter pylori DnaG C-terminal domain (HpDnaG-CTD) at 1.78 Å. The structure has a globular subdomain connected to a helical hairpin. Structural comparison has revealed that globular subdomains, despite the variation in number of helices, have broadly similar arrangements across the species, whereas helical hairpins show different o...

  8. 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

  9. Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain.

    Science.gov (United States)

    Alexandrovich, A; Czisch, M; Frenkiel, T A; Kelly, G P; Goosen, N; Moolenaar, G F; Chowdhry, B Z; Sanderson, M R; Lane, A N

    2001-10-01

    The solution structure, thermodynamic stability and hydrodynamic properties of the 55-residue C-terminal domain of UvrB that interacts with UvrC during excision repair in E. coli have been determined using a combination of high resolution NMR, ultracentrifugation, 15N NMR relaxation, gel permeation, NMR diffusion, circular dichroism and differential scanning calorimetry. The subunit molecular weight is 7,438 kDa., compared with 14.5+/-1.0 kDa. determined by equilibrium sedimentation, indicating a dimeric structure. The structure determined from NMR showed a stable dimer of anti-parallel helical hairpins that associate in an unusual manner, with a small and hydrophobic interface. The Stokes radius of the protein decreases from a high plateau value (ca. 22 A) at protein concentrations greater than 4 microM to about 18 A at concentrations less than 0.1 microM. The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (Tm ca. 71.5 degrees C at 36 microM). The simplest model consistent with these data was a dimer dissociating into folded monomers that then unfolds co-operatively. The van't Hoff enthalpy and dissociation constant for both transition was derived by fitting, with deltaH1=23 kJ mol(-1). K1(298)=0.4 microM and deltaH2= 184 kJ mol(-1). This is in good agreement with direct calorimetric analysis of the thermal unfolding of the protein, which gave a calorimetric enthalpy change of 181 kJ mol(-1) and a van't Hoff enthalpy change of 354 kJ mol(-1), confirming the dimer to monomer unfolding. The thermodynamic data can be reconciled with the observed mode of dimerisation. 15N NMR relaxation measurements at 14.1 T and 11.75 T confirmed that the protein behaves as an asymmetric dimer at mM concentrations, with a flexible N-terminal linker for attachment to the remainder of the UvrB protein. The role of dimerisation of this domain in the excision repair mechanism is discussed. PMID:11697728

  10. The solution structure of the C-terminal domain of the Mu B transposition protein

    OpenAIRE

    Hung, Ling-Hong; Chaconas, George; Shaw, Gary S.

    2000-01-01

    Mu B is one of four proteins required for the strand transfer step of bacteriophage Mu DNA transposition and the only one where no high resolution structural data is available. Structural work on Mu B has been hampered primarily by solubility problems and its tendency to aggregate. We have overcome this problem by determination of the three-dimensional structure of the C-terminal domain of Mu B (B223–312) in 1.5 M NaCl using NMR spectroscopic methods. The structure of Mu B223–312 comprises fo...

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Structure of the C-terminal head domain of the fowl adenovirus type 1 short fibre

    International Nuclear Information System (INIS)

    There are more than 100 known adenovirus serotypes, including 50 human serotypes. They can infect all 5 major vertebrate classes but only Aviadenovirus infecting birds and Mastadenovirus infecting mammals have been well studied. CELO (chicken embryo lethal orphan) adenovirus is responsible for mild respiratory pathologies in birds. Most studies on CELO virus have focussed on its genome sequence and organisation whereas the structural work on CELO proteins has only recently started. Contrary to most adenoviruses, the vertices of CELO virus reveal pentons with two fibres of different lengths. The distal parts (or head) of those fibres are involved in cellular receptor binding. Here we have determined the atomic structure of the short-fibre head of CELO (amino acids 201-410) at 2.0 A resolution. Despite low sequence identity, this structure is conserved compared to the other adenovirus fibre heads. We have used the existing CELO long-fibre head structure and the one we show here for a structure-based alignment of 11 known adenovirus fibre heads which was subsequently used for the construction of an evolutionary tree. Both the fibre head sequence and structural alignments suggest that enteric human group F adenovirus 41 (short fibre) is closer to the CELO fibre heads than the canine CAdV-2 fibre head, that lies closer to the human virus fibre heads

  19. 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...

  20. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase

    International Nuclear Information System (INIS)

    The crystal structure of a bacterial acetyltransferase with 27% sequence identity to the C-terminal domain of human O-GlcNAcase has been solved at 1.5 Å resolution. This S. sviceus protein is compared with known GCN5-related acetyltransferases, adding to the diversity observed in this superfamily. The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP-05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain

  1. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase

    Energy Technology Data Exchange (ETDEWEB)

    He, Yuan [Northwest University, Xi’an 710069 (China); The University of York, York YO10 5DD (United Kingdom); Roth, Christian; Turkenburg, Johan P.; Davies, Gideon J., E-mail: gideon.davies@york.ac.uk [The University of York, York YO10 5DD (United Kingdom); Northwest University, Xi’an 710069 (China)

    2014-01-01

    The crystal structure of a bacterial acetyltransferase with 27% sequence identity to the C-terminal domain of human O-GlcNAcase has been solved at 1.5 Å resolution. This S. sviceus protein is compared with known GCN5-related acetyltransferases, adding to the diversity observed in this superfamily. The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP-05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain.

  2. High-resolution crystal structure reveals a HEPN domain at the C-terminal region of S. cerevisiae RNA endonuclease Swt1

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Shuxia, E-mail: pengsx@ihep.ac.cn; Zhou, Ke; Wang, Wenjia; Gao, Zengqiang; Dong, Yuhui; Liu, Quansheng

    2014-10-31

    Highlights: • Crystal structure of the C-terminal (CT) domain of Swt1 was determined at 2.3 Å. • Structure of the CT domain was identified as HEPN domain superfamily member. • Low-resolution envelope of Swt1 full-length in solution was analyzed by SAXS. • The middle and CT domains gave good fit to SAXS structural model. - Abstract: Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.

  3. 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.

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

    International Nuclear Information System (INIS)

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

  5. 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

  6. 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

  7. Structures of the nucleoid occlusion protein SlmA bound to DNA and the C-terminal domain of the cytoskeletal protein FtsZ.

    Science.gov (United States)

    Schumacher, Maria A; Zeng, Wenjie

    2016-05-01

    Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called "nucleoid occlusion" (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator-CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA-DNA-FtsZ CTD regulatory interaction and provide insight into FtsZ-regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA-DNA-FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA-DNA-FtsZ interaction with implications for SlmA's NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins. PMID:27091999

  8. A structural constraint for functional interaction between N-terminal and C-terminal domains in simian immunodeficiency virus capsid proteins

    Directory of Open Access Journals (Sweden)

    Kawada Miki

    2010-10-01

    Full Text Available Abstract Background The Gag capsid (CA is one of the most conserved proteins in highly-diversified human and simian immunodeficiency viruses (HIV and SIV. Understanding the limitations imposed on amino acid sequences in CA could provide valuable information for vaccine immunogen design or anti-HIV drug development. Here, by comparing two pathogenic SIV strains, SIVmac239 and SIVsmE543-3, we found critical amino acid residues for functional interaction between the N-terminal and the C-terminal domains in CA. Results We first examined the impact of Gag residue 205, aspartate (Gag205D in SIVmac239 and glutamate (Gag205E in SIVsmE543-3, on viral replication; due to this difference, Gag206-216 (IINEEAADWDL epitope-specific cytotoxic T lymphocytes (CTLs were previously shown to respond to SIVmac239 but not SIVsmE543-3 infection. A mutant SIVmac239, SIVmac239Gag205E, whose Gag205D is replaced with Gag205E showed lower replicative ability. Interestingly, however, SIVmac239Gag205E passaged in macaque T cell culture often resulted in selection of an additional mutation at Gag residue 340, a change from SIVmac239 valine (Gag340V to SIVsmE543-3 methionine (Gag340M, with recovery of viral fitness. Structural modeling analysis suggested possible intermolecular interaction between the Gag205 residue in the N-terminal domain and Gag340 in the C-terminal in CA hexamers. The Gag205D-to-Gag205E substitution in SIVmac239 resulted in loss of in vitro core stability, which was recovered by additional Gag340V-to-Gag340M substitution. Finally, selection of Gag205E plus Gag340M mutations, but not Gag205E alone was observed in a chronically SIVmac239-infected rhesus macaque eliciting Gag206-216-specific CTL responses. Conclusions These results present in vitro and in vivo evidence implicating the interaction between Gag residues 205 in CA NTD and 340 in CA CTD in SIV replication. Thus, this study indicates a structural constraint for functional interaction between SIV CA

  9. The pH-sensitive structure of the C-terminal domain of voltage-gated proton channel and the thermodynamic characteristics of Zn2+ binding to this domain

    International Nuclear Information System (INIS)

    Highlights: • The α-helical content of the C-terminus is decreased with a pH increase. • The thermostability of the C-terminus is decreased with a pH increase. • Zn2+ binds to His244 and His266 residues within the C-terminal domain. • The binding of Zn2+ to His244 residue is an endothermic heat reaction. • The binding of Zn2+ to His266 residue is an exothermic heat reaction. - Abstract: The voltage-gated proton channel Hv1 is strongly sensitive to Zn2+. The H+ conduction is decreased at a high concentration of Zn2+ and Hv1 channel closing is slowed by the internal application of Zn2+. Although the recent studies demonstrated that Zn2+ interacts with the intracellular C-terminal domain, the binding sites and details of the interaction remain unknown. Here, we studied the pH-dependent structural stability of the intracellular C-terminal domain of human Hv1 and showed that Zn2+ binds to His244 and His266 residues. The thermodynamics signature of Zn2+ binding to the two sites was investigated by isothermal titration calorimetry. The binding of Zn2+ to His244 (mutant H266A) and His266 (mutant H244A) were an endothermic heat reaction and an exothermic heat reaction, respectively

  10. The pH-sensitive structure of the C-terminal domain of voltage-gated proton channel and the thermodynamic characteristics of Zn{sup 2+} binding to this domain

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Qing; Li, Chuanyong; Li, Shu Jie, E-mail: shujieli@nankai.edu.cn

    2015-01-02

    Highlights: • The α-helical content of the C-terminus is decreased with a pH increase. • The thermostability of the C-terminus is decreased with a pH increase. • Zn{sup 2+} binds to His{sup 244} and His{sup 266} residues within the C-terminal domain. • The binding of Zn{sup 2+} to His{sup 244} residue is an endothermic heat reaction. • The binding of Zn{sup 2+} to His{sup 266} residue is an exothermic heat reaction. - Abstract: The voltage-gated proton channel Hv1 is strongly sensitive to Zn{sup 2+}. The H{sup +} conduction is decreased at a high concentration of Zn{sup 2+} and Hv1 channel closing is slowed by the internal application of Zn{sup 2+}. Although the recent studies demonstrated that Zn{sup 2+} interacts with the intracellular C-terminal domain, the binding sites and details of the interaction remain unknown. Here, we studied the pH-dependent structural stability of the intracellular C-terminal domain of human Hv1 and showed that Zn{sup 2+} binds to His{sup 244} and His{sup 266} residues. The thermodynamics signature of Zn{sup 2+} binding to the two sites was investigated by isothermal titration calorimetry. The binding of Zn{sup 2+} to His{sup 244} (mutant H266A) and His{sup 266} (mutant H244A) were an endothermic heat reaction and an exothermic heat reaction, respectively.

  11. 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

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

    International Nuclear Information System (INIS)

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

  13. 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...

  14. 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

  15. 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.

  16. 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.

  17. 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...

  18. 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

  19. Forster Resonance Energy Transfer (FRET) Analysis of Dual CFP/YFP Labeled AMPA Receptors Reveals Structural Rearrangement within the C-Terminal Domain during Receptor Activation

    DEFF Research Database (Denmark)

    Zachariassen, Linda Grønborg; Katchan, Mila; Plested, Andrew;

    2014-01-01

    variants (CFP and YFP, respectively) of green fluorescent protein at various positions in the GluA2 AMPAR subunit to enable measurements of intra- receptor conformational changes using Fo¨ rster Resonance Energy Transfer (FRET) in live cells. We identify dual CFP/YFP-tagged GluA2 subunit con- structs that...... retain function and display intrareceptor FRET. This includes a construct (GluA2-6Y-10C) containing YFP in the intracellular loop between the M1 and M2 membrane-embedded segments and CFP inserted in the C-ter- minal domain (CTD). GluA2-6Y-10C displays FRET with an efficiency of 0.11 while retaining wild......-type receptor expression and kinetic properties. We have used GluA2-6Y-10C to study conformational changes in homomeric GluA2 receptors during receptor activation. Our results show that the FRET efficiency is dependent on functional state of GluA2-6Y-10C and hereby indi- cates that the intracellular CTD...

  20. Crystallization of the C-terminal globular domain of avian reovirus fibre

    Energy Technology Data Exchange (ETDEWEB)

    Raaij, Mark J. van, E-mail: vanraaij@usc.es [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Hermo Parrado, X. Lois; Guardado Calvo, Pablo [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Fox, Gavin C. [Spanish CRG Beamline BM16, European Synchrotron Radiation Facility (ESRF), 6 Rue Jules Horowitz, BP 220, F-38043 Grenoble (France); Llamas-Saiz, Antonio L. [Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Costas, Celina; Martínez-Costas, José; Benavente, Javier [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain)

    2005-07-01

    Partial proteolysis of the avian reovirus cell-attachment protein σC yields a major homotrimeric C-terminal fragment that presumably contains the receptor-binding domain. This fragment has been crystallized in the presence and absence of zinc sulfate and cadmium sulfate. One of the crystal forms diffracts synchrotron X-rays to 2.2–2.3 Å. Avian reovirus fibre, a homotrimer of the σC protein, is responsible for primary host-cell attachment. Using the protease trypsin, a C-terminal σC fragment containing amino acids 156–326 has been generated which was subsequently purified and crystallized. Two different crystal forms were obtained, one grown in the absence of divalent cations and belonging to space group P6{sub 3}22 (unit-cell parameters a = 75.6, c = 243.1 Å) and one grown in the presence of either zinc or cadmium sulfate and belonging to space group P321 (unit-cell parameters a = 74.7, c = 74.5 Å and a = 73.1, c = 69.9 Å for the Zn{sup II}- and Cd{sup II}-grown crystals, respectively). The first crystal form diffracted synchrotron radiation to 3.0 Å resolution and the second form to 2.2–2.3 Å. Its closest related structure, the C-terminal fragment of mammalian reovirus fibre, has only 18% sequence identity and molecular-replacement attempts were unsuccessful. Therefore, a search is under way for suitable heavy-atom derivatives and attempts are being made to grow protein crystals containing selenomethionine instead of methionine.

  1. Crystallization of the C-terminal globular domain of avian reovirus fibre

    International Nuclear Information System (INIS)

    Partial proteolysis of the avian reovirus cell-attachment protein σC yields a major homotrimeric C-terminal fragment that presumably contains the receptor-binding domain. This fragment has been crystallized in the presence and absence of zinc sulfate and cadmium sulfate. One of the crystal forms diffracts synchrotron X-rays to 2.2–2.3 Å. Avian reovirus fibre, a homotrimer of the σC protein, is responsible for primary host-cell attachment. Using the protease trypsin, a C-terminal σC fragment containing amino acids 156–326 has been generated which was subsequently purified and crystallized. Two different crystal forms were obtained, one grown in the absence of divalent cations and belonging to space group P6322 (unit-cell parameters a = 75.6, c = 243.1 Å) and one grown in the presence of either zinc or cadmium sulfate and belonging to space group P321 (unit-cell parameters a = 74.7, c = 74.5 Å and a = 73.1, c = 69.9 Å for the ZnII- and CdII-grown crystals, respectively). The first crystal form diffracted synchrotron radiation to 3.0 Å resolution and the second form to 2.2–2.3 Å. Its closest related structure, the C-terminal fragment of mammalian reovirus fibre, has only 18% sequence identity and molecular-replacement attempts were unsuccessful. Therefore, a search is under way for suitable heavy-atom derivatives and attempts are being made to grow protein crystals containing selenomethionine instead of methionine

  2. Interaction of the Tim44 C-terminal domain with negatively charged phospholipids.

    Science.gov (United States)

    Marom, Milit; Safonov, Roman; Amram, Shay; Avneon, Yoav; Nachliel, Esther; Gutman, Menachem; Zohary, Keren; Azem, Abdussalam; Tsfadia, Yossi

    2009-12-01

    The translocation of proteins from the cytosol into the mitochondrial matrix is mediated by the coordinated action of the TOM complex in the outer membrane, as well as the TIM23 complex and its associated protein import motor in the inner membrane. The focus of this work is the peripheral inner membrane protein Tim44. Tim44 is a vital component of the mitochondrial protein translocation motor that anchors components of the motor to the TIM23 complex. For this purpose, Tim44 associates with the import channel by direct interaction with the Tim23 protein. Additionally, it was shown in vitro that Tim44 associates with acidic model membranes, in particular those containing cardiolipin. The latter interaction was shown to be mediated by the carboxy-terminal domain of Tim44 [Weiss, C., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8890-8894]. The aim of this study was to determine the precise recognition site for negative lipids in the C-terminal domain of Tim44. In particular, we wanted to examine the recently suggested hypothesis that acidic phospholipids associate with Tim44 via a hydrophobic cavity that is observed in the high-resolution structure of the C-terminal domain of the protein [Josyula, R., et al. (2006) J. Mol. Biol. 359, 798-804]. Molecular dynamics simulations suggest that (i) the hydrophobic tail of lipids may interact with Tim44 via the latter's hydrophobic cavity and (ii) a region, located in the N-terminal alpha-helix of the C-terminal domain (helices A1 and A2), may serve as a membrane attachment site. To validate this assumption, N-terminal truncations of yeast Tim44 were examined for their ability to bind cardiolipin-containing phospholipid vesicles. The results indicate that removal of the N-terminal alpha-helix (helix A1) abolishes the capacity of Tim44 to associate with cardiolipin-containing liposomes. We suggest that helices A1 and A2, in Tim44, jointly promote the association of the protein with acidic phospholipids. PMID:19863062

  3. 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.

  4. MscCG from Corynebacterium glutamicum: functional significance of the C-terminal domain.

    Science.gov (United States)

    Becker, Michael; Krämer, Reinhard

    2015-10-01

    Corynebacterium glutamicum is used in microbial biotechnology for the production of amino acids, e.g., glutamate and lysine. Excretion of glutamate into the surrounding medium under production conditions is mediated by MscCG, an MscS-type mechanosensitive channel. In difference to most other MscS-type channel proteins, MscCG carries, in addition to the N-terminal pore domain, a long C-terminal domain that amounts to about half of the size of the protein and harbors an additional transmembrane segment. Here we study the impact of the C-terminal domain on both functions of MscCG as mechanosensitive channel and as glutamate exporter. Sequential truncations of the C-terminal domain were applied, as well as deletion of particular subdomains, replacement of these segments by other amino acid sequences, and sequence randomization. Several parameters of cell physiology and bioenergetics of the obtained mutants related to both glutamate excretion and response to osmotic stress were quantified. All three subdomains of the C-terminal domain, i.e., the periplasmic loop, the fourth transmembrane segment, and the cytoplasmic loop, proved to be of core significance for MscCG function, in particular for glutamate excretion. PMID:26033538

  5. 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,

  6. 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...

  7. 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.

  8. Determinants for Dephosphorylation of the RNA Polymerase II C-Terminal Domain by Scp1

    OpenAIRE

    Yan ZHANG; Kim, Youngjun; Genoud, Nicolas; Gao, Jianmin; Kelly, Jeffery W.; Pfaff, Samuel L.; Gill, Gordon N.; Dixon, Jack E.; Noel, Joseph P.

    2006-01-01

    Phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) represent a critical regulatory checkpoint for transcription. Transcription initiation requires Fcp1/Scp1-mediated dephosphorylation of phospho-CTD. Fcp1 and Scp1 belong to a family of Mg2+-dependent phosphoserine (P.Ser)/phosphothreonine (P.Thr)-specific phosphatases. We recently showed that Scp1 is an evolutionarily conserved regulator of neuronal gene silencing. Here, we present the X-ray cry...

  9. Conserved C-Terminal Domain of Spider Tubuliform Spidroin 1 Contributes to Extensibility in Synthetic Fibers

    Energy Technology Data Exchange (ETDEWEB)

    Gnesa, Eric; Hsia, Yang; Yarger, Jeffery L.; Weber, Warner; Lin-Cereghino, Joan; Lin-Cereghino, Geoff; Tang, Simon; Agari, Kimiko; Vierra, Craig (AZU); (Pacific)

    2012-05-24

    Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.

  10. Peptide library approach to uncover phosphomimetic inhibitors of the BRCA1 C-terminal domain.

    Science.gov (United States)

    White, E Railey; Sun, Luxin; Ma, Zhong; Beckta, Jason M; Danzig, Brittany A; Hacker, David E; Huie, Melissa; Williams, David C; Edwards, Ross A; Valerie, Kristoffer; Glover, J N Mark; Hartman, Matthew C T

    2015-05-15

    Many intracellular protein-protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein-protein interactions mediated by serine phosphorylation. PMID:25654734

  11. Role of C-terminal domain and transmembrane helices 5 and 6 in function and quaternary structure of major intrinsic proteins: analysis of aquaporin/glycerol facilitator chimeric proteins.

    Science.gov (United States)

    Duchesne, Laurence; Pellerin, Isabelle; Delamarche, Christian; Deschamps, Stephane; Lagree, Valerie; Froger, Alexandrine; Bonnec, Georgette; Thomas, Daniel; Hubert, Jean-Francois

    2002-06-01

    We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the C-terminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability. PMID:11927589

  12. 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

  13. 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

  14. Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II

    International Nuclear Information System (INIS)

    Eukaryotic RNA polymerase II consists of three subspecies, designated II0, II/sub A/, and II/sub B/, which differ in the apparent M/sub r/ of their largest subunit, IIo, IIa, and IIb, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. Subunit IIa (IIo) has been shown to contain an unusual C-terminal domain composed of 52 repeats of a seven amino acid block with the consensus sequence tyr-ser-pro-thr-ser-pro-ser. In an effort to purify the C-terminal domain, purified calf thymus RNA polymerase II was cleaved with CNBr. Following SDS polyacrylamide gel electrophoresis the CNBr digests were transferred and probed with IIa mono-specific antibody. A single immunoreactive peptide with an apparent M/sub r/ of 65,000 was detected. A peptide of similar M/sub r/ was found when purified subunit IIa was digested with CNBr while no immunoreactive peptide was detected in digests of subunit IIb. Purified RNA polymerase II was phosphorylated with γ[32P]-ATP in the presence of casein kinase I and 32P-labeled subunits IIa and IIo purified. CNBr clIIa revealed a major phosphopeptide with an apparent M/sub r/ of 65,000. Cleavage of 32P-labeled age of IIo revealed a broad phosphopeptide band of M/sub r/ 75,000-90,000. The C-terminal peptide from subunit IIa was purified by gel filtration on HPLC. Experiments are in progress to map in vivo phosphorylation sites within subunits IIo and IIa and to examine the effects of the purified C-terminal peptide on in vitro transcription

  15. Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II

    Energy Technology Data Exchange (ETDEWEB)

    Cadena, D.; Dahmus, M.E.

    1986-05-01

    Eukaryotic RNA polymerase II consists of three subspecies, designated II/sub 0/, II/sub A/, and II/sub B/, which differ in the apparent M/sub r/ of their largest subunit, IIo, IIa, and IIb, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. Subunit IIa (IIo) has been shown to contain an unusual C-terminal domain composed of 52 repeats of a seven amino acid block with the consensus sequence tyr-ser-pro-thr-ser-pro-ser. In an effort to purify the C-terminal domain, purified calf thymus RNA polymerase II was cleaved with CNBr. Following SDS polyacrylamide gel electrophoresis the CNBr digests were transferred and probed with IIa mono-specific antibody. A single immunoreactive peptide with an apparent M/sub r/ of 65,000 was detected. A peptide of similar M/sub r/ was found when purified subunit IIa was digested with CNBr while no immunoreactive peptide was detected in digests of subunit IIb. Purified RNA polymerase II was phosphorylated with ..gamma..(/sup 32/P)-ATP in the presence of casein kinase I and /sup 32/P-labeled subunits IIa and IIo purified. CNBr clIIa revealed a major phosphopeptide with an apparent M/sub r/ of 65,000. Cleavage of /sup 32/P-labeled age of IIo revealed a broad phosphopeptide band of M/sub r/ 75,000-90,000. The C-terminal peptide from subunit IIa was purified by gel filtration on HPLC. Experiments are in progress to map in vivo phosphorylation sites within subunits IIo and IIa and to examine the effects of the purified C-terminal peptide on in vitro transcription.

  16. Crystallization and preliminary crystallographic studies of the C-terminal domain of outer membrane protein A from enterohaemorrhagic Escherichia coli

    International Nuclear Information System (INIS)

    In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. Outer membrane protein A (OmpA) of enterohaemorrhagic Escherichia coli (EHEC) plays multiple roles in bacterial physiology and pathogenesis, such as mediation of bacterial conjunction, maintenance of cell shape, induction of adhesion of EHEC to host cells etc. Better understanding of the functions of OmpA will help in the control of EHEC infections. OmpA is composed of two domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is a β-barrel structure and embeds in the outer membrane of the bacterium. The structure and function of the C-terminal domain of OmpA (OmpAC) remain elusive. In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. The crystals belonged to space group I4132, with unit-cell parameter a = 158.99 Å. The Matthews coefficient and solvent content were calculated to be 2.55 Å3 Da−1 and 51.77%, respectively, for two molecules in the asymmetric unit

  17. 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.

  18. 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 ...

  19. Structure and function of C-terminal catalytic region of pasteurella multocida toxin

    International Nuclear Information System (INIS)

    Pasteurella multocida toxin (PMT) is one of virulence factors responsible for the pathogenesis in some Pasteurellosis. We determined the crystal structure of the C-terminal region of PMT (C-PMT), which carries an intracellularly active moiety. The overall structure of C-PMT displays three different domains designated C1, C2 and C3. We found in the C3 domain the Cys-His-Asp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying the Cys-His-Asp triad. Our results demonstrate that PMT is an enzymatic toxin carrying the cysteine-protease like catalytic triad, which is organized only under reducing conditions. (author)

  20. Expression, crystallization and preliminary crystallographic study of mouse hepatitis virus (MHV) nucleocapsid protein C-terminal domain

    International Nuclear Information System (INIS)

    The C-terminal domain of mouse hepatitis virus nucleocapsid protein has been overexpressed in E. coli, purified and crystallized. The crystal belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å, and diffracted to 2.20 Å resolution. Mouse hepatitis virus (MHV) belongs to the group II coronaviruses. The virus produces nine genes encoding 11 proteins that could be recognized as structural proteins and nonstructural proteins and are crucial for viral RNA synthesis. The nucleocapsid (N) protein, one of the structural proteins, interacts with the 30.4 kb virus genomic RNA to form the helical nucleocapsid and associates with the membrane glycoprotein via its C-terminus to stabilize virion assembly. Here, the expression and crystallization of the MHV nucleocapsid protein C-terminal domain are reported. The crystals diffracted to 2.20 Å resolution and belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content is 43.0% (VM = 2.16 Å3 Da−1)

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

    International Nuclear Information System (INIS)

    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

  2. C-terminal domain of SMYD3 serves as a unique HSP90-regulated motif in oncogenesis

    Science.gov (United States)

    Harriss, June; Das, Chhaya; Zhu, Li; Edwards, Melissa; Shaaban, Salam; Tucker, Haley

    2015-01-01

    The SMYD3 histone methyl transferase (HMTase) and the nuclear chaperone, HSP90, have been independently implicated as proto-oncogenes in several human malignancies. We show that a degenerate tetratricopeptide repeat (TPR)-like domain encoded in the SMYD3 C-terminal domain (CTD) mediates physical interaction with HSP90. We further demonstrate that the CTD of SMYD3 is essential for its basal HMTase activity and that the TPR-like structure is required for HSP90-enhanced enzyme activity. Loss of SMYD3-HSP90 interaction leads to SMYD3 mislocalization within the nucleus, thereby losing its chromatin association. This results in reduction of SMYD3-mediated cell proliferation and, potentially, impairment of SMYD3′s oncogenic activity. These results suggest a novel approach for blocking HSP90-driven malignancy in SMYD3-overexpressing cells with a reduced toxicity profile over current HSP90 inhibitors. PMID:25738358

  3. 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.

  4. Crystallization and preliminary crystallographic studies of CCM3 in complex with the C-terminal domain of MST4

    International Nuclear Information System (INIS)

    The complex of CCM3 and the C-terminal domain of MST4 has been successfully constructed, purified and crystallized. The crystal diffracted to a resolution of 2.4 Å. MST4 is a member of the GCKIII kinases. The interaction between cerebral cavernous malformation 3 (CCM3) and GCKIII kinases plays a critical role in cardiovascular development and in cerebral cavernous malformations. The complex of CCM3 and the C-terminal domain of MST4 has been constructed, purified and crystallized, and a diffraction data set has been collected to 2.4 Å resolution. The crystal of the CCM3–MST4 C-terminal domain complex belonged to space group P41212 or P43212, with unit-cell parameters a = 69.10, b = 69.10, c = 117.57 Å

  5. The C-terminal domain of Cernunnos/XLF is dispensable for DNA repair in vivo.

    Science.gov (United States)

    Malivert, Laurent; Callebaut, Isabelle; Rivera-Munoz, Paola; Fischer, Alain; Mornon, Jean-Paul; Revy, Patrick; de Villartay, Jean-Pierre

    2009-03-01

    The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair. PMID:19103754

  6. 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...

  7. 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

  8. The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum.

    Science.gov (United States)

    Nakayama, Yoshitaka; Becker, Michael; Ebrahimian, Haleh; Konishi, Tomoyuki; Kawasaki, Hisashi; Krämer, Reinhard; Martinac, Boris

    2016-01-01

    The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes. PMID:26494188

  9. Cloning, expression, purification, crystallization and preliminary X-ray studies of the C-terminal domain of Rv3262 (FbiB) from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    The C-terminal domain of FbiB, a bifunctional protein that is essential for the biosynthesis of cofactor F420 in M. tuberculosis, has been expressed, purified and crystallized. The crystals diffracted to 2.0 Å resolution and were suitable for structure determination. During cofactor F420 biosynthesis, the enzyme F420-γ-glutamyl ligase (FbiB) catalyzes the addition of γ-linked l-glutamate residues to form polyglutamylated F420 derivatives. In Mycobacterium tuberculosis, Rv3262 (FbiB) consists of two domains: an N-terminal domain from the F420 ligase superfamily and a C-terminal domain with sequence similarity to nitro-FMN reductase superfamily proteins. To characterize the role of the C-terminal domain of FbiB in polyglutamyl ligation, it has been purified and crystallized in an apo form. The crystals diffracted to 2.0 Å resolution using a synchrotron source and belonged to the tetragonal space group P41212 (or P43212), with unit-cell parameters a = b = 136.6, c = 101.7 Å, α = β = γ = 90°

  10. Functional and structural analysis of C-terminal BRCA1 missense variants.

    Directory of Open Access Journals (Sweden)

    Francisco Quiles

    Full Text Available Germline inactivating mutations in BRCA1 and BRCA2 genes are responsible for Hereditary Breast and Ovarian Cancer Syndrome (HBOCS. Genetic testing of these genes is available, although approximately 15% of tests identify variants of uncertain significance (VUS. Classification of these variants into pathogenic or non-pathogenic type is an important challenge in genetic diagnosis and counseling. The aim of the present study is to functionally assess a set of 7 missense VUS (Q1409L, S1473P, E1586G, R1589H, Y1703S, W1718L and G1770V located in the C-terminal region of BRCA1 by combining in silico prediction tools and structural analysis with a transcription activation (TA assay. The in silico prediction programs gave discrepant results making its interpretation difficult. Structural analysis of the three variants located in the BRCT domains (Y1703S, W1718L and G1770V reveals significant alterations of BRCT structure. The TA assay shows that variants Y1703S, W1718L and G1770V dramatically compromise the transcriptional activity of BRCA1, while variants Q1409L, S1473P, E1586G and R1589H behave like wild-type BRCA1. In conclusion, our results suggest that variants Y1703S, W1718L and G1770V can be classified as likely pathogenic BRCA1 mutations.

  11. Crystallization and preliminary X-ray analysis of the C-terminal RNase III domain of human Dicer

    International Nuclear Information System (INIS)

    The C-terminal RNase III domain (RNase IIIb) of human Dicer has been expressed, purified and crystallized by the sitting-drop vapour-diffusion method. Human Dicer protein contains two RNase III domains (RNase IIIa and RNase IIIb) which are involved in the production of short interfering RNAs (siRNAs). The C-terminal RNase III domain (RNase IIIb) of human Dicer was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group C2221, with unit-cell parameters a = 88.6, b = 199.7, c = 119.6 Å, and diffracted X-rays to 2.0 Å resolution. The asymmetric unit contained three molecules of the RNase IIIb and the solvent content was 67%

  12. 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

  13. 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.

  14. Both the RGS Domain and the Six C-Terminal Amino Acids of Mouse Axin Are Required for Normal Embryogenesis

    OpenAIRE

    Chia, Ian V.; Kim, Min Jung; Itoh, Keiji; Sokol, Sergei Y.; Costantini, Frank

    2009-01-01

    Axin is a negative regulator of canonical Wnt signaling, which promotes the degradation of β-catenin, the major effector in this signaling cascade. While many protein-binding domains of Axin have been identified, their significance has not been evaluated in vivo. Here, we report the generation and analysis of mice carrying modified Axin alleles in which either the RGS domain or the six C-terminal amino acids (C6 motif) were deleted. The RGS domain is required for APC-binding, while the C6 mot...

  15. 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 ...

  16. 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.

  17. 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

  18. 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...

  19. 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.

  20. 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

  1. 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...

  2. 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.

  3. Dandelion PPO-1/PPO-2 domain-swaps: the C-terminal domain modulates the pH optimum and the linker affects SDS-mediated activation and stability.

    Science.gov (United States)

    Leufken, Christine M; Moerschbacher, Bruno M; Dirks-Hofmeister, Mareike E

    2015-02-01

    Plant polyphenol oxidases (PPOs) have a conserved three-domain structure: (i) the N-terminal domain (containing the active site) is connected via (ii) a linker to (iii) the C-terminal domain. The latter covers the active site, thereby maintaining the enzyme in a latent state. Activation can be achieved with SDS but little is known about the mechanism. We prepared domain-swap variants of dandelion PPO-1 and PPO-2 to test the specific functions of individual domains and their impact on enzyme characteristics. Our experiments revealed that the C-terminal domain modulates the pH optimum curve and has a strong influence on the optimal pH value. The linker determines the SDS concentration required for full activation. It also influences the SDS concentration required for half maximal activation (kSDS) and the stability of the enzyme during prolonged incubation in buffers containing SDS, but the N-terminal domain has the strongest effect on these parameters. The N-terminal domain also determines the IC50 of SDS and the stability in buffers containing or lacking SDS. We propose that the linker and C-terminal domain fine-tune the activation of plant PPOs. The C-terminal domain adjusts the pH optimum and the linker probably contains an SDS-binding/interaction site that influences inactivation and determines the SDS concentration required for activation. For the first time, we have determined the influence of the three PPO domains on enzyme activation and stability providing insight into the regulation and activation mechanisms of type-3 copper proteins in general. PMID:25484281

  4. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain.

    Science.gov (United States)

    de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A; Enghild, Jan J; Thøgersen, Ida B; Gao, Jinlong; Kwan, Ann H; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F Xavier; Nguyen, Ky-Anh; Potempa, Jan

    2016-01-01

    In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

  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. 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...

  7. 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

  8. Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2

    International Nuclear Information System (INIS)

    The C-terminal protease domain of Venezuelan equine encephalitis virus (VEEV) nsP2 has been overexpressed in E. coli, purified and successfully crystallized. Native crystals diffract to beyond 2.5 Å resolution and isomorphous heavy-atom derivatives suitable for phase analysis have been identified. The C-terminal region of Venezuelan equine encephalitis virus (VEEV) nsP2 is responsible for proteolytic processing of the VEEV polyprotein replication complex. This action regulates the activity of the replication complex and is essential for viral replication, thus making nsP2 a very attractive target for development of VEEV therapeutics. The 338-amino-acid C-terminal region of VEEV nsP2 has been overexpressed in Escherichia coli, purified and crystallized. Crystals diffract to beyond 2.5 Å resolution and belong to the orthorhombic space group P212121. Isomorphous heavy-atom derivatives suitable for phase analysis have been obtained and work on building a complete structural model is under way

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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...

  14. Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha subunit.

    Directory of Open Access Journals (Sweden)

    Michiko M Nakano

    Full Text Available BACKGROUND: Spx, an ArsC (arsenate reductase family member, is a global transcriptional regulator of the microbial stress response and is highly conserved amongst Gram-positive bacteria. Bacillus subtilis Spx protein exerts positive and negative control of transcription through its interaction with the C-terminal domain of the RNA polymerase (RNAP alpha subunit (alphaCTD. Spx activates trxA (thioredoxin and trxB (thioredoxin reductase in response to thiol stress, and bears an N-terminal C10XXC13 redox disulfide center that is oxidized in active Spx. METHODOLOGY/PRINCIPAL FINDINGS: The structure of mutant Spx(C10S showed a change in the conformation of helix alpha4. Amino acid substitutions R60E and K62E within and adjacent to helix alpha4 conferred defects in Spx-activated transcription but not Spx-dependent repression. Electrophoretic mobility-shift assays showed alphaCTD interaction with trxB promoter DNA, but addition of Spx generated a supershifted complex that was disrupted in the presence of reductant (DTT. Interaction of alphaCTD/Spx complex with promoter DNA required the cis-acting elements -45AGCA-42 and -34AGCG-31 of the trxB promoter. The Spx(G52R mutant, defective in alphaCTD binding, did not interact with the alphaCTD-trxB complex. Spx(R60E not only failed to complex with alphaCTD-trxB, but also disrupted alphaCTD-trxB DNA interaction. CONCLUSIONS/SIGNIFICANCE: The results show that Spx and alphaCTD form a complex that recognizes the promoter DNA of an Spx-controlled gene. A conformational change during oxidation of Spx to the disulfide form likely alters the structure of Spx alpha helix alpha4, which contains residues that function in transcriptional activation and alphaCTD/Spx-promoter interaction. The results suggest that one of these residues, R60 of the alpha4 region of oxidized Spx, functions in alphaCTD/Spx-promoter contact but not in alphaCTD interaction.

  15. 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

  16. 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.

  17. Identification of Residues in the C-terminal Domain of HIV-1 Integrase That Mediate Binding to the Transportin-SR2 Protein*

    Science.gov (United States)

    De Houwer, Stephanie; Demeulemeester, Jonas; Thys, Wannes; Taltynov, Oliver; Zmajkovicova, Katarina; Christ, Frauke; Debyser, Zeger

    2012-01-01

    Transportin-SR2 (TRN-SR2 and TNPO3) is a cellular cofactor of HIV replication that has been implicated in the nuclear import of HIV. TRN-SR2 was originally identified in a yeast two-hybrid screen as an interaction partner of HIV integrase (IN) and in two independent siRNA screens as a cofactor of viral replication. We have now studied the interaction of TRN-SR2 and HIV IN in molecular detail and identified the TRN-SR2 interacting regions of IN. A weak interaction with the catalytic core domain (CCD) and a strong interaction with the C-terminal domain (CTD) of IN were detected. By dissecting the catalytic core domain (CCD) of IN into short structural fragments, we identified a peptide (INIP1, amino acids 170EHLKTAVQMAVFIHNFKRKGGI191) retaining the ability to interact with TRN-SR2. By dissecting the C-terminal domain (CTD) of IN, we could identify two interacting peptides (amino acids 214QKQITKIQNFRVYYR228 and 262RRKVKIIRDYGK273) that come together in the CTD tertiary structure to form an exposed antiparallel β-sheet. Through site-specific mutagenesis, we defined the following sets of amino acids in IN as important for the interaction with TRN-SR2: Phe-185/Lys-186/Arg-187/Lys-188 in the CCD and Arg-262/Arg-263/Lys-264 and Lys-266/Arg-269 in the CTD. An HIV-1 strain carrying K266A/R269A in IN was replication-defective due to a block in reverse transcription, confounding the study of nuclear import. Insight into the IN/TRN-SR2 interaction interface is necessary to guide drug discovery efforts targeting the nuclear entry step of replication. PMID:22872638

  18. The C-terminal domain controls the mobility of Crumbs 3 isoforms.

    Science.gov (United States)

    Djuric, Ivona; Siebrasse, Jan Peter; Schulze, Ulf; Granado, Daniel; Schlüter, Marc A; Kubitscheck, Ulrich; Pavenstädt, Hermann; Weide, Thomas

    2016-06-01

    The physiological function of epithelia depends on an asymmetric distribution of their membrane domains. Polarity proteins play a crucial role for distribution processes, however, little is known about their mobility in epithelial cells. In this study, we analyzed the intracellular and plasma-membrane-associated mobility of fluorescence-labeled Crb3A and Crb3B. Both variants belong to the Crumbs protein family, which control size and identity of apical membranes in epithelial cells. Fluorescence recovery after photo-bleaching measurements revealed different mobilities for the two Crb3 variants. They also differentially affected mobility and localization of the Pals1/Mpp5 protein, which binds to Crb3A but not to Crb3B. In addition, tracking of intracellular vesicles indicated that Crb3A containing vesicles are slightly more immobile than Crb3B ones. Taken together, our data revealed different intracellular mobility patterns for Crb3A and Crb3B. PMID:26975581

  19. Crystallization of the C-terminal head domain of the avian adenovirus CELO long fibre

    International Nuclear Information System (INIS)

    Avian adenovirus long-fibre head trimers were expressed, purified and crystallized. The crystals belong to space group C2 (unit-cell parameters a = 216.5, b = 59.2, c = 57.5 Å, β = 101.3°). A complete highly redundant data set was collected to 2.2 Å resolution at 100 K using a rotating-anode X-ray source. Avian adenovirus CELO contains two different fibres: fibre 1, the long fibre, and fibre 2, the short fibre. The short fibre is responsible for binding to an unknown avian receptor and is essential for infection of birds. The long fibre is not essential, but is known to bind the coxsackievirus and adenovirus receptor protein. Both trimeric fibres are attached to the same penton base, of which each icosahedral virus contains 12 copies. The short fibre extends straight outwards, while the long fibre emerges at an angle. The carboxy-terminal amino acids 579–793 of the avian adenovirus long fibre have been expressed with an amino-terminal hexahistidine tag and the expressed trimeric protein has been purified by nickel-affinity chromatography and crystallized. Crystals were grown at low pH using PEG 10 000 as precipitant and belonged to space group C2. The crystals diffracted rotating-anode Cu Kα radiation to at least 1.9 Å resolution and a complete data set was collected from a single crystal to 2.2 Å resolution. Unit-cell parameters were a = 216.5, b = 59.2, c = 57.5 Å, β = 101.3°, suggesting one trimer per asymmetric unit and a solvent content of 46%. The long fibre head does not have significant sequence homology to any other protein of known structure and molecular-replacement attempts with known fibre-head structures were unsuccessful. However, a map calculated using SIRAS phasing shows a clear trimer with a shape similar to known adenovirus fibre-head structures. Structure solution is in progress

  20. 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.

  1. Comparison of fast backbone dynamics at amide nitrogen and carbonyl sites in dematin headpiece C-terminal domain and its S74E mutant

    International Nuclear Information System (INIS)

    We perform a detailed comparison of fast backbone dynamics probed at amide nitrogen versus carbonyl carbon sites for dematin headpiece C-terminal domain (DHP) and its S74E mutant (DHPS74E). Carbonyl dynamics is probed via auto-correlated longitudinal rates and transverse C'/C'-Cα CSA/dipolar and C'/C'-N CSA/dipolar cross-correlated rates, while 15N data are taken from a previous study. Resulting values of effective order parameters and internal correlation times support the conclusion that C' relaxation reports on a different subset of fast motions compared to those probed at N-H bond vectors in the same peptide planes. 13C' order parameters are on the average 0.08 lower than 15N order parameters with the exception of the flexible loop region in DHP. The reduction of mobility in the loop region upon the S74E mutation can be seen from the 15N order parameters but not from the 13C order parameters. Internal correlation times at 13C' sites are on the average an order of magnitude longer than those at 15N sites for the well-structured C-terminal subdomains, while the more flexible N-terminal subdomains have more comparable average internal correlation times.

  2. Structure of the DNA-bound BRCA1 C-terminal Region from Human Replication Factor C p140 and Model of the Protein-DNA Complex

    OpenAIRE

    2010-01-01

    BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5′-phosphoryl...

  3. Human replication protein A: Global fold of the N-terminal RPA-70 domain reveals a basic cleft and flexible C-terminal linker+

    International Nuclear Information System (INIS)

    Human Replication Protein A (hsRPA) is required for multiple cellular processes in DNA metabolism including DNA repair, replication and recombination. It binds single-stranded DNA with high affinity and interacts specifically with multiple proteins. hsRPA forms a heterotrimeric complex composed of 70-, 32- and 14-kDa subunits (henceforth RPA70, RPA32, and RPA14). The N-terminal 168 residues of RPA70 form a structurally distinct domain that stimulates DNA polymerase α activity, interacts with several transcriptional activators including tumor suppressor p53, and during the cell cycle it signals escape from the DNA damage induced G2/M checkpoint. We have solved the global fold of the fragment corresponding to this domain (RPA70Δ169) and we find residues 8-108 of the N-terminal domain are structured. The remaining C-terminal residues are unstructured and may form a flexible linker to the DNA-binding domain of RPA70. The globular region forms a five-stranded anti-parallel β-barrel. The ends of the barrel are capped by short helices. Two loops on one side of the barrel form a large basic cleft which is a likely site for binding the acidic motifs of transcriptional activators. Many lethal or conditional lethal yeast point mutants map to this cleft, whereas no mutations with severe phenotype have been found in the linker region

  4. 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.

  5. Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I: OUTER MEMBRANE INSERTION, TRIMERIZATION, AND PORIN FUNCTION REQUIRE A C-TERMINAL β-BARREL DOMAIN.

    Science.gov (United States)

    Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D

    2015-05-01

    We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP. PMID:25805501

  6. The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation. Role of disulfide bonds

    DEFF Research Database (Denmark)

    Roher, N; Miró, F; Boldyreff, B; Llorens, F; Plana, M; Issinger, O G; Itarte, E

    2001-01-01

    The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment with...

  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. Guiding strand passage: DNA-induced movement of the gyrase C-terminal domains defines an early step in the supercoiling cycle

    OpenAIRE

    Lanz, Martin A.; Klostermeier, Dagmar

    2011-01-01

    DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA in a strand passage mechanism. A double-stranded segment of DNA, the T-segment, is passed through the gap in a transiently cleaved G-segment by coordinated closing and opening of three protein interfaces in gyrase. T-segment capture is thought to be guided by the C-terminal domains of the GyrA subunit of gyrase that wrap DNA around their perimeter and cause a DNA-crossing with a positive handedness. We show here that the C-termin...

  9. 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.

  10. 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.

  11. 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

  12. Heteronuclear multidimensional NMR and homology modelling studies of the C-terminal nucleotide-binding domain of the human mitochondrial ABC transporter ABCB6

    International Nuclear Information System (INIS)

    Human ATP-binding cassette, sub-family B, member 6 (ABCB6) is a mitochondrial ABC transporter, and presumably contributes to iron homeostasis. Aimed at understanding the structural basis for the conformational changes accompanying the substrate-transportation cycle, we have studied the C-terminal nucleotide-binding domain of ABCB6 (ABCB6-C) in both the nucleotide-free and ADP-bound states by heteronuclear multidimensional NMR and homology modelling. A non-linear sampling scheme was utilised for indirectly acquired 13C and 15N dimensions of all 3D triple-resonance NMR experiments, in order to overcome the instability and the low solubility of ABCB6-C. The backbone resonances for approximately 25% of non-proline residues, which are mostly distributed around the functionally important loops and in the Helical domain, were not observed for nucleotide-free form of ABCB6-C. From the pH, temperature and magnetic field strength dependencies of the resonance intensities, we concluded that this incompleteness in the assignments is mainly due to the exchange between multiple conformations at an intermediate rate on the NMR timescale. These localised conformational dynamics remained in ADP-bound ABCB6-C except for the loops responsible for adenine base and α/β-phosphate binding. These results revealed that the localised dynamic cooperativity, which was recently proposed for a prokaryotic ABC MJ1267, also exists in a higher eukaryotic ABC, and is presumably shared by all members of the ABC family. Since the Helical domain is the putative interface to the transmembrane domain, this cooperativity may explain the coupled functions between domains in the substrate-transportation cycle

  13. Crystallization and preliminary crystallographic studies of Mycobacterium tuberculosis DNA gyrase B C-terminal domain, part of the enzyme reaction core

    International Nuclear Information System (INIS)

    The crystallization of DNA gyrase B C-terminal domain is described. DNA gyrase subunit B C-terminal domain (GyrB-CTD) is a functional module of DNA gyrase which participates in forming the core of DNA gyrase and plays critical roles in G-segment binding and T-segment loading and passage. Here, the purification, crystallization and preliminary X-ray crystallographic studies of GyrB-CTD from Mycobacterium tuberculosis H37Rv are reported. Diffraction data were collected from crystals of native GyrB-CTD and its selenomethionine derivative to resolutions of 2.8 and 3.0 Å, respectively. These crystals belonged to space group P212121 with similar unit-cell parameters. The native protein crystals had unit-cell parameters a = 52.831, b = 52.763, c = 192.579 Å

  14. 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, ...

  15. 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

  16. 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

  17. The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains

    Directory of Open Access Journals (Sweden)

    Solbak Sara MØ

    2011-12-01

    Full Text Available Abstract Background Cyclophilin A (CypA represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1 replication. CypA interacts with the virus proteins Capsid (CA and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear. Results Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues 75GCRHSRIGVTRQRRAR90, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr75-90 R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA. Conclusions For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are

  18. Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity

    Science.gov (United States)

    Faye, Audrey; Borthwick, Karen; Esnous, Catherine; Price, Nigel T.; Gobin, Stéphanie; Jackson, Vicky N.; Zammit, Victor A.; Girard, Jean; Prip-Buus, Carina

    2004-01-01

    We have previously proposed that changes in malonyl-CoA sensitivity of rat L-CPT1 (liver carnitine palmitoyltransferase 1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains. By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we have now shown the existence of such N–C (N- and C-terminal domain) intramolecular interactions both in wild-type L-CPT1 expressed in Saccharomyces cerevisiae and in the native L-CPT1 in fed rat liver mitochondria. These N–C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity. Moreover, increasing the outer membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitization, attenuated the N–C interactions. This indicates that the changes in malonyl-CoA sens-itivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N–C interactions. Finally, we show that mutations in the regulatory regions of the N-terminal domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased [S24A (Ser24→Ala)/Q30A] or abrogated (E3A) malonyl-CoA sensitivity. Moreover, we have identified the region immediately N-terminal to transmembrane domain 1 (residues 40–47) as being involved in the chemical N–C cross-linking. These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains, and that this determines its degree of malonyl-CoA sensitivity depending on the physiological state

  19. 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

  20. 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°

  1. 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.

  2. 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.

  3. The Shapes of Z-α1-Antitrypsin Polymers in Solution Support the C-Terminal Domain-Swap Mechanism of Polymerization

    DEFF Research Database (Denmark)

    Behrens, Manja Annette; Sendall, Timothy J.; Pedersen, Jan Skov; Kjeldgaard, Morten; Huntington, James A.; Jensen, Jan Kristian

    2014-01-01

    Emphysema and liver cirrhosis can be caused by the Z mutation (Glu342Lys) in the serine protease inhibitor α1-antitrypsin (α1AT), which is found in more than 4% of the Northern European population. Homozygotes experience deficiency in the lung concomitantly with a massive accumulation of polymers...... within hepatocytes, causing their destruction. Recently, it was proposed that Z-α1AT polymerizes by a C-terminal domain swap. In this study, small-angle x-ray scattering (SAXS) was used to characterize Z-α1AT polymers in solution. The data show that the Z-α1AT trimer, tetramer, and pentamer all form ring...

  4. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

    Energy Technology Data Exchange (ETDEWEB)

    Fernández-Fueyo, Elena [CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Acebes, Sandra [Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona (Spain); Ruiz-Dueñas, Francisco J.; Martínez, María Jesús; Romero, Antonio; Medrano, Francisco Javier, E-mail: fjmedrano@cib.csic.es [CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Guallar, Victor, E-mail: fjmedrano@cib.csic.es [Barcelona Supercomputing Center, Jordi Girona 29, 08034 Barcelona (Spain); ICREA, Passeig Lluís Companys 23, 08010 Barcelona (Spain); Martínez, Angel T., E-mail: fjmedrano@cib.csic.es [CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

    2014-12-01

    The variable C-terminal tail of manganese peroxidases, a group of enzymes involved in lignin degradation, is implicated in their catalytic and stability properties, as shown by new crystal structures, molecular-simulation and directed-mutagenesis data. Based on this structural–functional evaluation, short and long/extralong manganese peroxidase subfamilies have been accepted; the latter are characterized by exceptional stability, while it is shown for the first time that the former are able to oxidize other substrates at the same site where manganese(II) is oxidized. The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn{sup 2+}-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn{sup 2+} oxidation by the internal propionate, but prevents the oxidation of 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd{sup 2+} binds at the Mn{sup 2+}-oxidation site and competitively inhibits oxidation of both Mn{sup 2+} and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their

  5. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

    International Nuclear Information System (INIS)

    The variable C-terminal tail of manganese peroxidases, a group of enzymes involved in lignin degradation, is implicated in their catalytic and stability properties, as shown by new crystal structures, molecular-simulation and directed-mutagenesis data. Based on this structural–functional evaluation, short and long/extralong manganese peroxidase subfamilies have been accepted; the latter are characterized by exceptional stability, while it is shown for the first time that the former are able to oxidize other substrates at the same site where manganese(II) is oxidized. The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+ oxidation by the internal propionate, but prevents the oxidation of 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+ binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+ and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two

  6. 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

  7. 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

  8. 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.

  9. 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

  10. A Novel Bmal1 Mutant Mouse Reveals Essential Roles of the C-Terminal Domain on Circadian Rhythms.

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. 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

  13. The solution structure of the C-terminal modular pair from Clostridium perfringens mu-toxin reveals a noncellulosomal dockerin module.

    Science.gov (United States)

    Chitayat, Seth; Adams, Jarrett J; Furness, Heather S T; Bayer, Edward A; Smith, Steven P

    2008-09-19

    The genome of the opportunistic pathogen Clostridium perfringens encodes a large number of secreted glycoside hydrolases. Their predicted activities indicate that they are involved in the breakdown of complex carbohydrates and other glycans found in the mucosal layer of the human gastrointestinal tract, within the extracellular matrix, and on the surface of host cells. One such group of these enzymes is the family 84 glycoside hydrolases, which has predicted hyaluronidase activity and comprises five members [C. perfringens glycoside hydrolase family 84 (CpGH84) A-E]. The first identified member, CpGH84A, corresponds to the mu-toxin whose modular architecture includes an N-terminal catalytic domain, four family 32 carbohydrate-binding modules, three FIVAR modules of unknown function, and a C-terminal putative calcium-binding module. Here, we report the solution NMR structure of the C-terminal modular pair from the mu-toxin. The three-helix bundle FIVAR module displays structural homology to a heparin-binding module within the N-terminal of the a C protein from group B Streptoccocus. The C-terminal module has a typical calcium-binding dockerin fold comprising two anti-parallel helices that form a planar face with EF-hand calcium-binding loops at opposite ends of the module. The size of the helical face of the mu-toxin dockerin module is approximately equal to the planar region recently identified on the surface of a cohesin-like X82 module of CpGH84C. Size-exclusion chromatography and heteronuclear NMR-based chemical shift mapping studies indicate that the helical face of the dockerin module recognizes the CpGH84C X82 module. These studies represent the structural characterization of a noncellulolytic dockerin module and its interaction with a cohesin-like X82 module. Dockerin/X82-mediated enzyme complexes may have important implications in the pathogenic properties of C. perfringens. PMID:18602403

  14. 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...

  15. 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.

  16. Possible paraneoplastic syndrome case of bullous pemphigoid with immunoglobulin G anti-BP180 C-terminal domain antibodies associated with psoriasis and primary macroglobulinemia.

    Science.gov (United States)

    Maki, Nobuki; Demitsu, Toshio; Umemoto, Naoka; Nagashima, Kazutaka; Nakamura, Toshinobu; Kakurai, Maki; Nakamura, Satoshi; Yamada, Tomoko; Ishii, Norito; Hashimoto, Takashi

    2016-05-01

    A 61-year-old Japanese man developed bullous skin lesions during topical therapy for psoriasis vulgaris. Physical examination demonstrated numerous tense bullae and scaly erythemas on the trunk and extremities. Histopathology of the skin biopsy demonstrated subepidermal bullae and lymphocytic infiltration with eosinophils in the dermis. Direct immunofluorescence revealed linear deposits of immunoglobulin (Ig)G, IgA and C3 along the basement membrane zone. Indirect immunofluorescence of 1 mol/L NaCl-split skin showed IgG reactivity with both epidermal and the dermal sides. IgM reactivity with both the epidermal and dermal sides was also detected. Enzyme-linked immunosorbent assays showed negative results for both BP180 and BP230. Immunoelectrophoresis of serum and bone marrow aspiration revealed underlying primary macroglobulinemia with M-proteinemia of IgM-κ type. Immunoblot analysis revealed IgG, but not IgM, antibodies to recombinant protein of BP180 C-terminal domain. We diagnosed the present case as bullous pemphigoid with IgG anti-BP180 C-terminal domain autoantibodies associated with primary macroglobulinemia and psoriasis vulgaris. Systemic administration of prednisolone 30 mg/day resulted in dramatic improvement of both bullous and psoriatic skin lesions. When the bullous and psoriatic lesions relapsed, DRC chemotherapy (dexamethasone, rituximab and cyclophosphamide) for macroglobulinemia was performed. Then, the psoriatic lesions improved and the bullous lesions disappeared. We suggested that the present case may be paraneoplastic syndrome of bullous pemphigoid associated with primary macroglobulinemia and psoriasis vulgaris. PMID:26507447

  17. 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

  18. Acidic Residues C-Terminal to the A2 Domain Facilitate Thrombin-Catalyzed Activation of Factor VIII

    OpenAIRE

    Newell, Jennifer L.; Fay, Philip J.

    2008-01-01

    Factor VIII is activated by thrombin through proteolysis at Arg740, Arg372, and Arg1689. One region implicated in this exosite-dependent interaction is the factor VIII a2 segment (residues 711-740) separating the A2 and B domains. Residues 717-725 (DYYEDSYED) within this region consist of five acidic residues and three sulfo-Tyr residues, thus representing a high density of negative charge potential. The contributions of these residues to thrombin-catalyzed activation of factor VIII were asse...

  19. The X-ray structures of two mutant crystallin domains shed light on the evolution of multi-domain proteins.

    Science.gov (United States)

    Norledge, B V; Mayr, E M; Glockshuber, R; Bateman, O A; Slingsby, C; Jaenicke, R; Driessen, H P

    1996-03-01

    We use protein engineering and crystallography to simulate aspects of the early evolution of beta gamma-crystallins by observing how a single domain oligomerizes in response to changes in a sequence extension. The crystal structure of the C-terminal domain of gamma beta-crystallin with its four-residue C-terminal extension shows that the domain does not form a symmetric homodimer analogous to the two-domain pairing in beta gamma-crystallins. Instead the C-terminal extension now forms heterologous interactions with other domains leading to the solvent exposure of the natural hydrophobic interface with a consequent loss in protein solubility. However, this domain truncated by just the C-terminal tyrosine forms a symmetric homodimer of domains in the crystal lattice. PMID:8605629

  20. A C-terminal segment of the V1R vasopressin receptor is unstructured in the crystal structure of its chimera with the maltose-binding protein

    International Nuclear Information System (INIS)

    The 1.8 Å crystal structure of an MBP-fusion protein with the C-terminal cytoplasmic segment of the V1 vasopressin receptor reveals that the receptor segment is unstructured. The V1 vascular vasopressin receptor (V1R) is a G-protein-coupled receptor (GPCR) involved in the regulation of body-fluid osmolality, blood volume and blood pressure. Signal transduction is mediated by the third intracellular loop of this seven-transmembrane protein as well as by the C-terminal cytoplasmic segment. A chimera of the maltose-binding protein (MBP) and the C-terminal segment of V1R has been cloned, expressed, purified and crystallized. The crystals belong to space group P21, with unit-cell parameters a = 51.10, b = 66.56, c = 115.72 Å, β = 95.99°. The 1.8 Å crystal structure reveals the conformation of MBP and part of the linker region of this chimera, with the C-terminal segment being unstructured. This may reflect a conformational plasticity in the C-terminal segment that may be necessary for proper function of V1R

  1. Evidence against extracellular exposure of a highly immunogenic region in the C-terminal domain of the simian immunodeficiency virus gp41 transmembrane protein.

    Science.gov (United States)

    Postler, Thomas S; Martinez-Navio, José M; Yuste, Eloísa; Desrosiers, Ronald C

    2012-01-01

    The generally accepted model for human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein topology includes a single membrane-spanning domain. An alternate model has been proposed which features multiple membrane-spanning domains. Consistent with the alternate model, a high percentage of HIV-1-infected individuals produce unusually robust antibody responses to a region of envelope, the so-called "Kennedy epitope," that in the conventional model should be in the cytoplasm. Here we show analogous, robust antibody responses in simian immunodeficiency virus SIVmac239-infected rhesus macaques to a region of SIVmac239 envelope located in the C-terminal domain, which in the conventional model should be inside the cell. Sera from SIV-infected rhesus macaques consistently reacted with overlapping oligopeptides corresponding to a region located within the cytoplasmic domain of gp41 by the generally accepted model, at intensities comparable to those observed for immunodominant areas of the surface component gp120. Rabbit serum raised against this highly immunogenic region (HIR) reacted with SIV envelope in cell surface-staining experiments, as did monoclonal anti-HIR antibodies isolated from an SIVmac239-infected rhesus macaque. However, control experiments demonstrated that this surface staining could be explained in whole or in part by the release of envelope protein from expressing cells into the supernatant and the subsequent attachment to the surfaces of cells in the culture. Serum and monoclonal antibodies directed against the HIR failed to neutralize even the highly neutralization-sensitive strain SIVmac316. Furthermore, a potential N-linked glycosylation site located close to the HIR and postulated to be outside the cell in the alternate model was not glycosylated. An artificially introduced glycosylation site within the HIR was also not utilized for glycosylation. Together, these data support the conventional model of SIV envelope as a type Ia transmembrane

  2. 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

  3. 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.

  4. Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM located in the C-terminal domain of fission yeast DNA polymerase δ subunit Cdc27

    Directory of Open Access Journals (Sweden)

    Warbrick Emma

    2004-12-01

    Full Text Available Abstract Background DNA polymerases α and δ play essential roles in the replication of chromosomal DNA in eukaryotic cells. DNA polymerase α (Pol α-primase is required to prime synthesis of the leading strand and each Okazaki fragment on the lagging strand, whereas DNA polymerase δ (Pol δ is required for the elongation stages of replication, a function it appears capable of performing on both leading and lagging strands, at least in the absence of DNA polymerase ε (Pol ε. Results Here it is shown that the catalytic subunit of Pol α, Pol1, interacts with Cdc27, one of three non-catalytic subunits of fission yeast Pol δ, both in vivo and in vitro. Pol1 interacts with the C-terminal domain of Cdc27, at a site distinct from the previously identified binding sites for Cdc1 and PCNA. Comparative protein sequence analysis identifies a protein sequence motif, called the DNA polymerase interaction motif (DPIM, in Cdc27 orthologues from a wide variety of eukaryotic species, including mammals. Mutational analysis shows that the DPIM in fission yeast Cdc27 is not required for effective DNA replication, repair or checkpoint function. Conclusions The absence of any detectable phenotypic consequences arising from mutation of the DPIM suggests that despite its evolutionary conservation, the interaction between the two polymerases mediated by this motif is a non-essential one.

  5. Wolbachia transcription elongation factor "Wol GreA" interacts with α2ββ'σ subunits of RNA polymerase through its dimeric C-terminal domain.

    Directory of Open Access Journals (Sweden)

    Jeetendra Kumar Nag

    2014-06-01

    Full Text Available OBJECTIVES: Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for therapy against lymphatic filariasis. Transcription elongation factor GreA is an essential factor that mediates transcriptional transition from abortive initiation to productive elongation by stimulating the escape of RNA polymerase (RNAP from native prokaryotic promoters. Upon screening of 6257 essential bacterial genes, 57 were suggested as potential future drug targets, and GreA is among these. The current study emphasized the characterization of Wol GreA with its domains. METHODOLOGY/PRINCIPAL FINDINGS: Biophysical characterization of Wol GreA with its N-terminal domain (NTD and C-terminal domain (CTD was performed with fluorimetry, size exclusion chromatography, and chemical cross-linking. Filter trap and far western blotting were used to determine the domain responsible for the interaction with α2ββ'σ subunits of RNAP. Protein-protein docking studies were done to explore residual interaction of RNAP with Wol GreA. The factor and its domains were found to be biochemically active. Size exclusion and chemical cross-linking studies revealed that Wol GreA and CTD exist in a dimeric conformation while NTD subsists in monomeric conformation. Asp120, Val121, Ser122, Lys123, and Ser134 are the residues of CTD through which monomers of Wol GreA interact and shape into a dimeric conformation. Filter trap, far western blotting, and protein-protein docking studies revealed that dimeric CTD of Wol GreA through Lys82, Ser98, Asp104, Ser105, Glu106, Tyr109, Glu116, Asp120, Val121, Ser122, Ser127, Ser129, Lys140, Glu143, Val147, Ser151, Glu153, and Phe163 residues exclusively participates in binding with α2ββ'σ subunits of polymerase. CONCLUSIONS/SIGNIFICANCE: To the best of our knowledge, this research is the first documentation of the residual mode of action in wolbachial mutualist. Therefore, findings may be crucial to understanding the

  6. N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells

    International Nuclear Information System (INIS)

    Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved. The MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively. Both of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity. Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents

  7. Nuclear Trafficking of the Rabies Virus Interferon Antagonist P-Protein Is Regulated by an Importin-Binding Nuclear Localization Sequence in the C-Terminal Domain

    Science.gov (United States)

    Rowe, Caitlin L.; Wagstaff, Kylie M.; Oksayan, Sibil; Glover, Dominic J.

    2016-01-01

    Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P

  8. Nuclear Trafficking of the Rabies Virus Interferon Antagonist P-Protein Is Regulated by an Importin-Binding Nuclear Localization Sequence in the C-Terminal Domain.

    Science.gov (United States)

    Rowe, Caitlin L; Wagstaff, Kylie M; Oksayan, Sibil; Glover, Dominic J; Jans, David A; Moseley, Gregory W

    2016-01-01

    Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P

  9. 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

  10. Mutational analysis of Mdm2 C-terminal tail suggests an evolutionarily conserved role of its length in Mdm2 activity toward p53 and indicates structural differences between Mdm2 homodimers and Mdm2/MdmX heterodimers

    OpenAIRE

    Dolezelova, Pavlina; Cetkovska, Katerina; Vousden, Karen H.; Uldrijan, Stjepan

    2012-01-01

    Mdm2 can mediate p53 ubiquitylation and degradation either in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer. The ubiquitin ligase activity of these complexes resides mainly in their respective RING finger domains and also requires adjacent C-terminal tails. So far, structural studies have failed to show significant differences between Mdm2 RING homodimers and Mdm2/MdmX RING heterodimers. Here, we report that not only the primary amino acid sequence, but also the length of the C-term...

  11. 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 ...

  12. Structural basis of phosphodiesterase 6 inhibition by the C-terminal region of the γ-subunit

    OpenAIRE

    Barren, Brandy; Gakhar, Lokesh; Muradov, Hakim; Boyd, Kimberly K.; Ramaswamy, S; Artemyev, Nikolai O.

    2009-01-01

    The inhibitory interaction of phosphodiesterase-6 (PDE6) with its γ-subunit (Pγ) is pivotal in vertebrate phototransduction. Here, crystal structures of a chimaeric PDE5/PDE6 catalytic domain (PDE5/6cd) complexed with sildenafil or 3-isobutyl-1-methylxanthine and the Pγ-inhibitory peptide Pγ70−87 have been determined at 2.9 and 3.0 Å, respectively. These structures show the determinants and the mechanism of the PDE6 inhibition by Pγ and suggest the conformational change of Pγ on transducin ac...

  13. 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.

  14. Structure and potential C-terminal dimerization of a recombinant mutant of surfactant-associated protein C in chloroform/methanol.

    Science.gov (United States)

    Luy, Burkhard; Diener, Alexander; Hummel, Rolf-Peter; Sturm, Ernst; Ulrich, Wolf-Rüdiger; Griesinger, Christian

    2004-06-01

    The solution structure of a recombinant mutant [rSP-C (FFI)] of the human surfactant-associated protein C (hSP-C) in a mixture of chloroform and methanol was determined by high-resolution NMR spectroscopy. rSP-C (FFI) contains a helix from Phe5 to the C-terminal Leu34 and is thus longer by two residues than the helix of porcine SP-C (pSP-C), which is reported to start at Val7 in the same solvent. Two sets of resonances at the C-terminus of the peptide were observed, which are explained by low-order oligomerization, probably dimerization of rSP-C (FFI) in its alpha-helical form. The dimerization may be induced by hydrogen bonding of the C-terminal carboxylic groups or by the strictly conserved C-terminal heptapeptide segment with a motif similar to the GxxxG dimerization motif of glycophorin A. Dimerization at the heptapeptide segment would be consistent with findings based on electrospray ionization MS data, chemical cross-linking studies, and CNBr cleavage data. PMID:15153097

  15. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part III. Dynamics of long-range hydrophobic interactions

    Science.gov (United States)

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2010-01-01

    A 20-residue peptide, IG(42–61), derived from the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using CD, NMR spectroscopy at various temperatures and by differential scanning calorimetry. Unlike other related peptides studied so far, this peptide displays two heat capacity peaks in DSC measurements (at a scanning rate of 1.5 deg/min at a peptide concentration of 0.07mM) which suggests a three-state folding/unfolding process. The results from DSC and NMR measurements suggest the formation of a dynamic network of hydrophobic interactions stabilizing the structure, which resembles a β-hairpin shape over a wide range of temperatures (283 – 313 K). Our results show that IG(42–61) possesses a well-organized three-dimensional structure stabilized by long-range hydrophobic interactions (Tyr50 ··· Phe57 and Trp48 ··· Val59) at T = 283 K and (Trp48 ··· Val59) at 305 and 313 K. The mechanism of β-hairpin folding and unfolding, as well as the influence of peptide length on its conformational properties, are also discussed. PMID:19847914

  16. Mechanism of formation of the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. III. Dynamics of long-range hydrophobic interactions.

    Science.gov (United States)

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A

    2010-02-15

    A 20-residue peptide, IG(42-61), derived from the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using circular dichroism, nuclear magnetic resonance (NMR) spectroscopy at various temperatures and by differential scanning calorimetry (DSC). Unlike other related peptides studied so far, this peptide displays two heat capacity peaks in DSC measurements (at a scanning rate of 1.5 deg/min at a peptide concentration of 0.07 mM), which suggests a three-state folding/unfolding process. The results from DSC and NMR measurements suggest the formation of a dynamic network of hydrophobic interactions stabilizing the structure, which resembles a beta-hairpin shape over a wide range of temperatures (283-313 K). Our results show that IG (42-61) possesses a well-organized three-dimensional structure stabilized by long-range hydrophobic interactions (Tyr50 ... Phe57 and Trp48 ... Val59) at T = 283 K and (Trp48 ... Val59) at 305 and 313 K. The mechanism of beta-hairpin folding and unfolding, as well as the influence of peptide length on its conformational properties, are also discussed. PMID:19847914

  17. Identification of a novel pentatricopeptide repeat subfamily with a C-terminal domain of bacterial origin acquired via ancient horizontal gene transfer

    OpenAIRE

    Manna, Sam; Barth, Christian

    2013-01-01

    Background Pentatricopeptide repeat (PPR) proteins are a large family of sequence-specific RNA binding proteins involved in organelle RNA metabolism. Very little is known about the origin and evolution of these proteins, particularly outside of plants. Here, we report the identification of a novel subfamily of PPR proteins not found in plants and explore their evolution. Results We identified a novel subfamily of PPR proteins, which all contain a C-terminal tRNA guanine methyltransferase (TGM...

  18. Interaction of Cytosolic Glutamine Synthetase of Soybean Root Nodules with the C-terminal Domain of the Symbiosome Membrane Nodulin 26 Aquaglyceroporin*♦

    OpenAIRE

    Masalkar, Pintu; Wallace, Ian S.; Hwang, Jin Ha; Roberts, Daniel M.

    2010-01-01

    Nodulin 26 (nod26) is a major intrinsic protein that constitutes the major protein component on the symbiosome membrane (SM) of N2-fixing soybean nodules. Functionally, nod26 forms a low energy transport pathway for water, osmolytes, and NH3 across the SM. Besides their transport functions, emerging evidence suggests that high concentrations of major intrinsic proteins on membranes provide interaction and docking targets for various cytosolic proteins. Here it is shown that the C-terminal dom...

  19. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part II. Interplay of local backbone conformational dynamics and long-range hydrophobic interactions in hairpin formation

    OpenAIRE

    Skwierawska, Agnieszka; Żmudzińska, Wioletta; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2009-01-01

    Two peptides, corresponding to the turn region of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, consisting of residues 51–56 [IG(51–56)] and 50–57 [IG(50–57)], respectively, were studied by CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. Our results show that the part of the sequence corresponding to the β-turn in the native structure (DDATKT) of the B3 domain forms bent conformations similar to ...

  20. Structural basis of phosphodiesterase 6 inhibition by the C-terminal region of the [gamma]-subunit

    Energy Technology Data Exchange (ETDEWEB)

    Barren, Brandy; Gakhar, Lokesh; Muradov, Hakim; Boyd, Kimberly K.; Ramaswamy, S.; Artemyev, Nikolai O.; (Iowa)

    2010-03-16

    The inhibitory interaction of phosphodiesterase-6 (PDE6) with its {gamma}-subunit (P{gamma}) is pivotal in vertebrate phototransduction. Here, crystal structures of a chimaeric PDE5/PDE6 catalytic domain (PDE5/6cd) complexed with sildenafil or 3-isobutyl-1-methylxanthine and the P{gamma}-inhibitory peptide P{gamma}{sub 70-87} have been determined at 2.9 and 3.0 {angstrom}, respectively. These structures show the determinants and the mechanism of the PDE6 inhibition by P{gamma} and suggest the conformational change of P{gamma} on transducin activation. Two variable H- and M-loops of PDE5/6cd form a distinct interface that contributes to the P{gamma}-binding site. This allows the P{gamma} C-terminus to fit into the opening of the catalytic pocket, blocking cGMP access to the active site. Our analysis suggests that disruption of the H-M loop interface and P{gamma}-binding site is a molecular cause of retinal degeneration in atrd3 mice. Comparison of the two PDE5/6cd structures shows an overlap between the sildenafil and P{gamma}{sub 70-87}-binding sites, thereby providing critical insights into the side effects of PDE5 inhibitors on vision.

  1. Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure

    Directory of Open Access Journals (Sweden)

    Hayashi Nobuhiro

    2008-02-01

    Full Text Available Abstract Background The myelin sheath is a multilamellar membrane structure wrapped around the axon, enabling the saltatory conduction of nerve impulses in vertebrates. Myelin basic protein, one of the most abundant myelin-specific proteins, is an intrinsically disordered protein that has been shown to bind calmodulin. In this study, we focus on a 19-mer synthetic peptide from the predicted calmodulin-binding segment near the C-terminus of human myelin basic protein. Results The interaction of native human myelin basic protein with calmodulin was confirmed by affinity chromatography. The binding of the myelin basic protein peptide to calmodulin was tested with isothermal titration calorimetry (ITC in different temperatures, and Kd was observed to be in the low μM range, as previously observed for full-length myelin basic protein. Surface plasmon resonance showed that the peptide bound to calmodulin, and binding was accompanied by a conformational change; furthermore, gel filtration chromatography indicated a decrease in the hydrodynamic radius of calmodulin in the presence of the peptide. NMR spectroscopy was used to map the binding area to reside mainly within the hydrophobic pocket of the C-terminal lobe of calmodulin. The solution structure obtained by small-angle X-ray scattering indicates binding of the myelin basic protein peptide into the interlobal groove of calmodulin, while calmodulin remains in an extended conformation. Conclusion Taken together, our results give a detailed structural insight into the interaction of calmodulin with a C-terminal segment of a major myelin protein, the myelin basic protein. The used 19-mer peptide interacts mainly with the C-terminal lobe of calmodulin, and a conformational change accompanies binding, suggesting a novel mode of calmodulin-target protein interaction. Calmodulin does not collapse and wrap around the peptide tightly; instead, it remains in an extended conformation in the solution structure

  2. Structure of a C-terminal AHNAK peptide in a 1:2:2 complex with S100A10 and an acetylated N-terminal peptide of annexin A2

    Energy Technology Data Exchange (ETDEWEB)

    Ozorowski, Gabriel [University of California, Irvine, Irvine, CA 92697-3900 (United States); University of California, Irvine, Irvine, CA 92697-3900 (United States); Milton, Saskia [University of California, Irvine, Irvine, CA 92697-3900 (United States); Luecke, Hartmut, E-mail: hudel@uci.edu [University of California, Irvine, Irvine, CA 92697-3900 (United States); University of California, Irvine, Irvine, CA 92697-3900 (United States); University of California, Irvine, Irvine, CA 92697 (United States); University of California, Irvine, Irvine, CA 92697 (United States)

    2013-01-01

    Structure of a 20-amino-acid peptide of AHNAK bound asymmetrically to the AnxA2–S100A10A heterotetramer (1:2:2 symmetry) provides insights into the atomic level interactions that govern this membrane-repair scaffolding complex. AHNAK, a large 629 kDa protein, has been implicated in membrane repair, and the annexin A2–S100A10 heterotetramer [(p11){sub 2}(AnxA2){sub 2})] has high affinity for several regions of its 1002-amino-acid C-terminal domain. (p11){sub 2}(AnxA2){sub 2} is often localized near the plasma membrane, and this C2-symmetric platform is proposed to be involved in the bridging of membrane vesicles and trafficking of proteins to the plasma membrane. All three proteins co-localize at the intracellular face of the plasma membrane in a Ca{sup 2+}-dependent manner. The binding of AHNAK to (p11){sub 2}(AnxA2){sub 2} has been studied previously, and a minimal binding motif has been mapped to a 20-amino-acid peptide corresponding to residues 5654–5673 of the AHNAK C-terminal domain. Here, the 2.5 Å resolution crystal structure of this 20-amino-acid peptide of AHNAK bound to the AnxA2–S100A10 heterotetramer (1:2:2 symmetry) is presented, which confirms the asymmetric arrangement first described by Rezvanpour and coworkers and explains why the binding motif has high affinity for (p11){sub 2}(AnxA2){sub 2}. Binding of AHNAK to the surface of (p11){sub 2}(AnxA2){sub 2} is governed by several hydrophobic interactions between side chains of AHNAK and pockets on S100A10. The pockets are large enough to accommodate a variety of hydrophobic side chains, allowing the consensus sequence to be more general. Additionally, the various hydrogen bonds formed between the AHNAK peptide and (p11){sub 2}(AnxA2){sub 2} most often involve backbone atoms of AHNAK; as a result, the side chains, particularly those that point away from S100A10/AnxA2 towards the solvent, are largely interchangeable. While the structure-based consensus sequence allows interactions with various

  3. Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

    Energy Technology Data Exchange (ETDEWEB)

    González-Páez, Gonzalo E.; Wolan, Dennis W. (Scripps)

    2012-09-05

    Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 {angstrom} resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC{sub 50} values for trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.

  4. 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

  5. Crystal structure of the tetrameric fibrinogen-like recognition domain of Fibrinogen C domain containing 1 (FIBCD1)

    DEFF Research Database (Denmark)

    Shrive, Annette K; Moeller, Jesper B; Burns, Ian;

    2014-01-01

    The high-resolution crystal structures of a recombinant fragment of the C-terminal fibrinogen-like recognition domain of FIBCD1, a vertebrate receptor that binds chitin, have been determined. The overall tetrameric structure shows similarity in structure and aggregation to the horseshoe crab innate...

  6. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part III. Dynamics of long-range hydrophobic interactions

    OpenAIRE

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2010-01-01

    A 20-residue peptide, IG(42–61), derived from the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using CD, NMR spectroscopy at various temperatures and by differential scanning calorimetry. Unlike other related peptides studied so far, this peptide displays two heat capacity peaks in DSC measurements (at a scanning rate of 1.5 deg/min at a peptide concentration of 0.07mM) which suggests a three-state folding/unfolding process. The ...

  7. The MIK region rather than the C-terminal domain of AP3-like class B floral homeotic proteins determines functional specificity in the development and evolution of petals.

    Science.gov (United States)

    Su, Kunmei; Zhao, Suzhen; Shan, Hongyan; Kong, Hongzhi; Lu, Wenliang; Theissen, Günter; Chen, Zhiduan; Meng, Zheng

    2008-01-01

    In core eudicots, euAP3-type MADS-box genes encode a PISTILLATA (PI)-derived motif, as well as a C-terminal euAP3 motif that originated from a paleoAP3 motif of an ancestral APETALA3 (AP3)-like protein through a translational frameshift mutation. To determine the functional and evolutionary relevance of these motifs, a series of point mutation and domain-swap constructs were generated, involving CsAP3, a paleoAP3-type gene from the basal angiosperm Chloranthus spicatus encoding a truncated paleoAP3 motif, and AtAP3, a euAP3-type gene from the core eudicot Arabidopsis thaliana. The chimeric constructs were expressed in A. thaliana under the control of the AP3 promoter or the CaMV 35S promoter in an ap3 mutant or wild-type background, respectively. Significant recovery of AP3 function was obtained in both complementation and ectopic expression experiments whenever the region upstream of the C-terminal motifs (MIK region) from A. thaliana was taken, even when the PI-derived motif and the truncated paleoAP3 motif of CsAP3 substituted for the corresponding sequences from AtAP3. However, no or very weak complementation or gain-of-function was seen when the MIK region was from CsAP3. Our data suggest that changes in the MIK region rather than mutations in the C-terminal domain were of crucial importance for the evolution of the functional specificity of euAP3-type proteins in stamen and petal development. PMID:18298432

  8. Recombinant expression of two bacteriophage proteins that lyse clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal active domains.

    Science.gov (United States)

    Simmons, Mustafa; Donovan, David M; Siragusa, Gregory R; Seal, Bruce S

    2010-10-13

    Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium capable of producing four major toxins that are responsible for disease symptoms and pathogenesis in a variety of animals, humans, and poultry. The organism is the third leading cause of human foodborne bacterial disease, and C. perfringens is the presumptive etiologic agent of necrotic enteritis among chickens, which in the acute form can cause increased mortality among broiler flocks. Countries that have complied with the ban on antimicrobial growth promoters (AGP) in feeds have had increased incidences of C. perfringens-associated necrotic enteritis in poultry. To address this issue, new antimicrobial agents, putative lysins from the genomes of bacteriophages, are identified. Two putative phage lysin genes (ply) from the clostridial phages phiCP39O and phiCP26F were cloned and expressed in Escherichia coli , and the resultant proteins were purified to near homogeneity. Gene and protein sequencing revealed that the predicted and chemically determined amino acid sequences of the two recombinant proteins were homologous to N-acetylmuramoyl-l-alanine amidases. The proteins were identical in the C-terminal putative cell-wall binding domain, but only 55% identical to each other in the presumptive N-terminal catalytic domain. Both recombinant lysins were capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays. The observed reduction in turbidity was correlated with up to a 3 log cfu/mL reduction in viable C. perfringens on brain-heart infusion agar plates. However, other member species of the clostridia were resistant to the lytic activity by both assays. PMID:20825156

  9. Cyclic AMP-dependent protein kinase phosphorylates residues in the C-terminal domain of the cardiac L-type calcium channel alpha1 subunit.

    Science.gov (United States)

    Leach, R N; Brickley, K; Norman, R I

    1996-06-11

    The molecular basis of the regulation of cardiac L-type calcium channel activity by cAMP-dependent protein kinase (cA-PK) remains unclear. Direct cA-PK-dependent phosphorylation of the bovine ventricular alpha1 subunit in vitro has been demonstrated in microsomal membranes, detergent extracts and partially purified (+)-[3H]PN 200-110 receptor preparations. Two 32P-labeled phosphopeptides, derived from cyanogen bromide cleavage, of 4.7 and 9.5 kDa were immunoprecipitated specifically by site-directed antibodies against the rabbit cardiac alpha1 subunit amino acid sequences 1602-1616 and 1681-1694, respectively, consistent with phosphorylation at the cA-PK consensus sites at Ser(1627) and Ser(1700). No phosphopeptide products consistent with phosphorylation at three other C-terminal cA-PK consensus phosphorylation sites (Ser(1575), Ser(1848) and Ser(1928)) were identified using similar procedures suggesting that these sites are poor substrates for this kinase. Ser(1627) and Ser(1700) may represent sites of cA-PK phosphorylation involved in the physiological regulation of cardiac L-type calcium channel function. PMID:8664319

  10. The C-terminal domain of the nuclear factor I-B2 isoform is glycosylated and transactivates the WAP gene in the JEG-3 cells

    International Nuclear Information System (INIS)

    The transcription factor nuclear factor I (NFI) has been shown previously both in vivo and in vitro to be involved in the cooperative regulation of whey acidic protein (WAP) gene transcription along with the glucocorticoid receptor and STAT5. In addition, one of the specific NFI isoforms, NFI-B2, was demonstrated in transient co-transfection experiments in JEG cells, which lack endogenous NFI, to be preferentially involved in the cooperative regulation of WAP gene expression. A comparison of the DNA-binding specificities of the different NFI isoforms only partially explained their differential ability to activate the WAP gene transcription. Here, we analyzed the transactivation regions of two NFI isoforms by making chimeric proteins between the NFI-A and B isoforms. Though, their DNA-binding specificities were not altered as compared to the corresponding wild-type transcription factors, the C-terminal region of the NFI-B isoform was shown to preferentially activate WAP gene transcription in cooperation with GR and STAT5 in transient co-transfection assays in JEG-3 cells. Furthermore, determination of serine and threonine-specific glycosylation (O-linked N-acetylglucosamine) of the C-terminus of the NFI-B isoform suggested that the secondary modification by O-GlcNAc might play a role in the cooperative regulation of WAP gene transcription by NFI-B2 and STAT5

  11. Co-expression of the C-terminal domain of Yersinia enterocolitica invasin enhances the efficacy of classical swine-fever-vectored vaccine based on human adenovirus

    Indian Academy of Sciences (India)

    Helin Li; Pengbo Ning; Zhi Lin; Wulong Liang; Kai Kang; Lei He; Yanming Zhang

    2015-03-01

    The use of adenovirus vector-based vaccines is a promising approach for generating antigen-specific immune responses. Improving vaccine potency is necessary in other approaches to address their inadequate protection for the majority of infectious diseases. This study is the first to reconstruct a recombinant replication-defective human adenovirus co-expressing E2 and invasin C-terminal (InvC) glycoproteins (rAd-E2-InvC). rAd-E2-InvC with 2×106 TCID50 was intramuscularly administered two times to CSFV-free pigs at 14 day intervals. No adverse clinical reactions were observed in any of the pigs after the vaccination. The CSFV E2-specific antibody titer was significantly higher in the rAd-E2-InvC group than that in the rAdV-E2 group as measured by NPLA and blocking ELISA. Pigs immunized with rAd-E2-InvC were completely protected against lethal challenge. Neither CSFV RNA nor pathological changes were detected in the tissues after CSFV challenge. These results demonstrate that rAd-E2-InvC could be an alternative to the existing CSF vaccine. Moreover, InvC that acts as an adjuvant could enhance the immunogenicity of rAdV-E2 and induce high CSFV E2-specific antibody titer and protection level.

  12. Structural and binding studies of C-terminal half (C-lobe) of lactoferrin protein with COX-2-specific non-steroidal anti-inflammatory drugs (NSAIDs).

    Science.gov (United States)

    Mir, Rafia; Singh, Nagendra; Vikram, Gopalakrishnapillai; Sinha, Mau; Bhushan, Asha; Kaur, Punit; Srinivasan, Alagiri; Sharma, Sujata; Singh, Tej P

    2010-08-15

    Three COX-2-specific non-steroidal anti-inflammatory drugs (NSAIDs), etoricoxib, parecoxib, and nimesulide are widely prescribed against inflammatory conditions. However, their long term administration leads to severe conditions of cardiovascular complications and gastric ulceration. In order to minimize these side effects, C-terminal half (C-lobe) of colostrum protein lactoferrin has been indicated to be useful if co-administered with NSAIDs. Lactoferrin is an 80kDa glycoprotein with two similar halves designated as N- and C-lobes. Since NSAID-binding site is located in the C-terminal half of lactoferrin, C-lobe was prepared from lactoferrin by limited proteolysis using proteinase K. The incubation of lactoferrin with serine proteases for extended periods showed that N-lobe was completely digested but C-lobe was resistant for more than 72h indicating its long half life in the animal gut. The solution studies have shown that COX-2-specific NSAIDs bind to C-lobe with binding constants ranging from 10(-4) to 10(-5)M showing significant affinities for sequestering these compounds. In order to understand the mode of binding and sequestering properties, the complexes of C-lobe with all these three compounds, etoricoxib, parecoxib, and nimesulide were prepared and the structures of their complexes with C-lobe were determined at 2.2, 2.9, and 2.7A resolutions, respectively. The analysis of the structures of complexes of C-lobe with NSAIDs clearly show that all the three compounds bind firmly at the same ligand-binding site in the C-lobe revealing the details of the interactions between C-lobe and NSAIDs. The mode of binding of COX-2-specific NSAIDs to C-lobe is similar to that of the binding of COX-2 non-specific NSAIDs to C-lobe. PMID:20515646

  13. 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...

  14. Structural analysis of the C-terminal region (modules 18-20 of complement regulator factor H (FH.

    Directory of Open Access Journals (Sweden)

    Hugh P Morgan

    Full Text Available Factor H (FH is a soluble regulator of the human complement system affording protection to host tissues. It selectively inhibits amplification of C3b, the activation-specific fragment of the abundant complement component C3, in fluid phase and on self-surfaces and accelerates the decay of the alternative pathway C3 convertase, C3bBb. We have determined the crystal structure of the three carboxyl-terminal complement control protein (CCP modules of FH (FH18-20 that bind to C3b, and which additionally recognize polyanionic markers specific to self-surfaces. These CCPs harbour nearly 30 disease-linked missense mutations. We have also deployed small-angle X-ray scattering (SAXS to investigate FH18-20 flexibility in solution using FH18-20 and FH19-20 constructs. In the crystal lattice FH18-20 adopts a "J"-shape: A ~122-degree tilt between the structurally highly similar modules 18 and 19 precedes an extended, linear arrangement of modules 19 and 20 as observed in previously determined structures of these two modules alone. However, under solution conditions FH18-20 adopts multiple conformations mediated by flexibility between CCPs 18 and 19. We also pinpoint the locations of disease-associated missense mutations on the module 18 surface and discuss our data in the context of the C3b:FH interaction.

  15. Evidence against Extracellular Exposure of a Highly Immunogenic Region in the C-Terminal Domain of the Simian Immunodeficiency Virus gp41 Transmembrane Protein

    OpenAIRE

    Postler, Thomas S.; Martinez-Navio, José M.; Yuste, Eloísa; Desrosiers, Ronald C

    2012-01-01

    The generally accepted model for human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein topology includes a single membrane-spanning domain. An alternate model has been proposed which features multiple membrane-spanning domains. Consistent with the alternate model, a high percentage of HIV-1-infected individuals produce unusually robust antibody responses to a region of envelope, the so-called “Kennedy epitope,” that in the conventional model should be in the cytoplasm. Here we sho...

  16. The catalytic subunit of Dictyostelium cAMP-dependent protein kinase -- role of the N-terminal domain and of the C-terminal residues in catalytic activity and stability.

    Science.gov (United States)

    Etchebehere, L C; Van Bemmelen, M X; Anjard, C; Traincard, F; Assemat, K; Reymond, C; Véron, M

    1997-09-15

    The C subunit of Dictyostelium cAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI. Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequence-comparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe- motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases. PMID:9342234

  17. pHluorin-assisted expression, purification, crystallization and X-ray diffraction data analysis of the C-terminal domain of the HsdR subunit of the Escherichia coli type I restriction-modification system EcoR124I.

    Science.gov (United States)

    Grinkevich, Pavel; Iermak, Iuliia; Luedtke, Nicholas A; Mesters, Jeroen R; Ettrich, Rüdiger; Ludwig, Jost

    2016-09-01

    The HsdR subunit of the type I restriction-modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C-terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C-terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887-1038 and yielded crystals that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 83.42, b = 176.58, c = 126.03 Å, α = β = γ = 90.00° and two molecules in the asymmetric unit (VM = 2.55 Å(3) Da(-1), solvent content 50.47%). X-ray diffraction data were collected to a resolution of 2.45 Å. PMID:27599856

  18. A C-terminal segment of the V{sub 1}R vasopressin receptor is unstructured in the crystal structure of its chimera with the maltose-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Adikesavan, Nallini Vijayarangan; Mahmood, Syed Saad; Stanley, Nithianantham; Xu, Zhen; Wu, Nan [Department of Biochemistry, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935 (United States); Thibonnier, Marc [Department of Medicine, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935 (United States); Shoham, Menachem, E-mail: mxs10@case.edu [Department of Biochemistry, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935 (United States)

    2005-04-01

    The 1.8 Å crystal structure of an MBP-fusion protein with the C-terminal cytoplasmic segment of the V1 vasopressin receptor reveals that the receptor segment is unstructured. The V{sub 1} vascular vasopressin receptor (V{sub 1}R) is a G-protein-coupled receptor (GPCR) involved in the regulation of body-fluid osmolality, blood volume and blood pressure. Signal transduction is mediated by the third intracellular loop of this seven-transmembrane protein as well as by the C-terminal cytoplasmic segment. A chimera of the maltose-binding protein (MBP) and the C-terminal segment of V{sub 1}R has been cloned, expressed, purified and crystallized. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 51.10, b = 66.56, c = 115.72 Å, β = 95.99°. The 1.8 Å crystal structure reveals the conformation of MBP and part of the linker region of this chimera, with the C-terminal segment being unstructured. This may reflect a conformational plasticity in the C-terminal segment that may be necessary for proper function of V{sub 1}R.

  19. 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...

  20. A conserved BURP domain defines a novel group of plant proteins with unusual primary structures.

    Science.gov (United States)

    Hattori, J; Boutilier, K A; van Lookeren Campagne, M M; Miki, B L

    1998-09-01

    We have identified a new class of plant proteins containing a common C-terminal region, which we have termed the BURP domain. These proteins are defined not only by the BURP domain, but also by the overall similarity in their modular construction. The BURP domain proteins consist of either three or four modules: (i) an N-terminal hydrophobic domain -- a presumptive transit peptide, joined to (ii) a short conserved segment or other short segment, (iii) an optional segment consisting of repeated units which is unique to each member, and (iv) the C-terminal BURP domain. These individual modules appear to be combined to form two main classes of BURP domain proteins. The BURP domain proteins, despite the similarities in their primary structural features, show no obvious similarities in the tissues or conditions under which they are expressed. The presence of the conserved BURP domain in diverse plant proteins suggests an important and fundamental functional role for this domain. PMID:9790599

  1. Crystal structure of the fibrinogen-like recognition domain of FIBCD1

    DEFF Research Database (Denmark)

    Møller, Jesper Bonnet; Schlosser, Anders; Sørensen, Grith Lykke;

    The high resolution crystal structures of a recombinant fragment of the C-terminal fibrinogen-related domain of FIBCD1, a vertebrate chitin receptor, have been determined. The overall structure shows similarity in structure and aggregation to the homologous innate immune protein tachylectin 5A...

  2. The effect of pH on recombinant C-terminal domain of Botulinum Neurotoxin type E (rBoNT/E-HCC

    Directory of Open Access Journals (Sweden)

    Seyed Jafar Mousavy

    2013-12-01

    Full Text Available Recombinant proteins are tending to be the most favorable vaccine-candidates against botulism. Recombinant Carboxy-terminal of botulinum neurotoxin serotype E (rBoNT/E-HCC has been introduced as an efficient vaccine against botulism type E. In this report, we made an effort to investigate the effect of different pH on protein structure to assess if rBoNT/E-HCC could be used as a vaccine for oral administration. Initially, rBoNT/E-HCC was expressed and purified. Structural changes of rBoNT/E-HCC at several pH conditions were studied by various techniques including circular dichroism (CD, fluorescence, aggregation and UV-Vis spectroscopy. The results showed the more compact and more stable structure for rBoNT/E-HCC at acidic pH, and loosely folded structure at alkaline pH. Our finding as the first step of rBoNT/E-HCC evaluation, hopefully introduce it as a suitable vaccine candidate for oral administration.

  3. 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.

  4. Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

    OpenAIRE

    Lidell, Martin E.; Moncada, Darcy M.; Chadee, Kris; Hansson, Gunnar C.

    2006-01-01

    In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycos...

  5. Electrochemical Analysis of p53 Protein. Determination of GST Traces in Preparations of C-terminal Domain (aa320-393)

    Czech Academy of Sciences Publication Activity Database

    Kizek, René; Brázdová, Marie; Havran, Luděk; Fojta, Miroslav; Paleček, Emil

    2000-01-01

    Roč. 17, - (2000), s. 1144-1145. ISSN 0739-1102. [Mendel - Brno 2000. DNA Structure and Interactions. Their Biological Roles and Implications in Biomedicine and Biotechnologies. 19.07.2000-23.07.2000, Brno] R&D Projects: GA ČR GV204/97/K084 Institutional research plan: CEZ:A17/98:Z5-004-9-ii Subject RIV: BO - Biophysics

  6. The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation.

    Science.gov (United States)

    Chen, Yu-Tsung Shane; Wu, Jianhong; Modrich, Paul; Hsieh, Tao-Shih

    2016-06-17

    Eukaryotic topoisomerase 2 (Top2) and one of its interacting partners, topoisomerase IIβ binding protein 1 (TopBP1) are two proteins performing essential cellular functions. We mapped the interacting domains of these two proteins using co-immunoprecipitation and pulldown experiments with truncated or mutant Drosophila Top2 with various Ser-to-Ala substitutions. We discovered that the last 20 amino acids of Top2 represent the key region for binding with Mus101 (the Drosophila homolog of TopBP1) and that phosphorylation of Ser-1428 and Ser-1443 is important for Top2 to interact with the N terminus of Mus101, which contains the BRCT1/2 domains. The interaction between Mus101 and the Top2 C-terminal regulatory domain is phosphorylation-dependent because treatment with phosphatase abolishes their association in pulldown assays. The binding affinity of N-terminal Mus101 with a synthetic phosphorylated peptide spanning the last 25 amino acids of Top2 (with Ser(P)-1428 and Ser(P)-1443) was determined by surface plasmon resonance with a Kd of 0.57 μm In an in vitro decatenation assay, Mus101 can specifically reduce the decatenation activity of Top2, and dephosphorylation of Top2 attenuates this response. Next, we endeavored to establish a cellular system for testing the biological function of Top2-Mus101 interaction. Top2-silenced S2 cells rescued by Top2Δ20, Top2 with 20 amino acids truncated from the C terminus, developed abnormally high chromosome numbers, which implies that Top2-Mus101 interaction is important for maintaining the fidelity of chromosome segregation during mitosis. PMID:27129233

  7. Structure and biochemical function of a prototypical Arabidopsis U-box domain

    DEFF Research Database (Denmark)

    Andersen, Pernille; Kragelund, Birthe B; Olsen, Addie N;

    2004-01-01

    corresponding to zinc ion-coordinating residues in RING domains. Residues from a C-terminal alpha-helix interact with the core domain and contribute to stabilization. The Prp19p U-box lacks a corresponding C-terminal alpha-helix. Chemical shift analysis suggested that aromatic residues exposed at the N terminus...... and the C-terminal alpha-helix of the AtPUB14 U-box participate in dimerization. Thus, AtPUB14 may form a biologically relevant dimer. This is the first plant U-box structure to be determined, and it provides a model for studies of the many plant U-box proteins and their interactions. Structural insight...... into these interactions is important, because ubiquitin-dependent protein degradation is a prevalent regulatory mechanism in plants....

  8. Highly Conserved Structural Properties of the C-terminal Tail of HIV-1 gp41 Protein Despite Substantial Sequence Variation among Diverse Clades: IMPLICATIONS FOR FUNCTIONS IN VIRAL REPLICATION*

    OpenAIRE

    Steckbeck, Jonathan D.; Craigo, Jodi K.; Barnes, Christopher O.; Ronald C Montelaro

    2011-01-01

    Although the HIV-1 Env gp120 and gp41 ectodomain have been extensively characterized in terms of structure and function, similar characterizations of the C-terminal tail (CTT) of HIV gp41 remain relatively limited and contradictory. The current study was designed to examine in detail CTT sequence conservation relative to gp120 and the gp41 ectodomain and to examine the conservation of predicted physicochemical and structural properties across a number of divergent HIV clades and groups. Resul...

  9. 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.

  10. Structural and Histone Binding Ability Characterizations of Human PWWP Domains

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong (Toronto); (Penn)

    2013-09-25

    The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

  11. Roles of the N- and C-terminal domains of carnitine palmitoyltransferase I isoforms in malonyl-CoA sensitivity of the enzymes: insights from expression of chimaeric proteins and mutation of conserved histidine residues.

    Science.gov (United States)

    Swanson, S T; Foster, D W; McGarry, J D; Brown, N F

    1998-01-01

    The mitochondrial outer membrane enzyme carnitine palmitoyltransferase I (CPT I) plays a major role in the regulation of fatty acid entry into the mitochondrial matrix for beta-oxidation by virtue of its inhibition by malonyl-CoA. Two isoforms of CPT I, the liver type (L) and muscle type (M), have been identified, the latter being 100 times more sensitive to malonyl-CoA and having a much higher Km for the substrate carnitine. Here we have examined the roles of different regions of the CPT I molecules in their response to malonyl-CoA, etomoxir (an irreversible inhibitor) and carnitine. To this end, we analysed the properties of engineered rat CPT I constructs in which (a) the N-terminal domain of L-CPT I was deleted, (b) the N-terminal domains of L- and M-CPT I were switched, or (c) each of three conserved histidine residues located towards the N-terminus in L-CPT I was mutated. Several novel points emerged: (1) whereas the N-terminal domain is critical for a normal malonyl-CoA response, it does not itself account for the widely disparate sensitivities of the liver and muscle enzymes to the inhibitor; (2) His-5 and/or His-140 probably play a direct role in the malonyl-CoA response, but His-133 does not; (3) the truncated, chimaeric and point- mutant variants of the enzyme all bound the covalent, active-site- directed ligand, etomoxir; and (4) only the most radical alteration of L-CPT I, i.e. deletion of the N-terminal 82 residues, affected the response to carnitine. We conclude that the N-terminal domain of CPT I plays an essential, but permissive, role in the inhibition of the enzyme by malonyl-CoA. By contrast, the larger C-terminal region dictates the degree of sensitivity to malonyl-CoA, as well as the response to carnitine; it is also sufficient for etomoxir binding. Additionally, further weight is added to the notion that one or more histidine residues may be involved in the CPT I-malonyl-CoA interaction. PMID:9794789

  12. The crystal structure of the human polo-like kinase-1 polo box domain and its phospho-peptide complex

    OpenAIRE

    Cheng, Kin-Yip; Lowe, Edward D; Sinclair, John; Nigg, Erich A.; Johnson, Louise N

    2003-01-01

    Human polo-like kinase Plk1 localizes to the centrosomes, kinetochores and central spindle structures during mitosis. It plays an essential role in promoting mitosis and cytokinesis through phosphorylation of a number of different substrates. Kinase activity is regulated by a conserved C-terminal domain, termed the polo box domain (PBD), which acts both as an autoinhibitory domain and as a subcellular localization domain. We have determined the crystal structure of Plk1 PBD (residues 367–603)...

  13. Structure of the yeast Bre1 RING domain

    Science.gov (United States)

    Kumar, Pankaj; Wolberger, Cynthia

    2015-01-01

    Monoubiquitination of histone H2B at Lys123 in yeast plays a critical role in regulating transcription, mRNA export, DNA replication and the DNA damage response. The RING E3 ligase, Bre1, catalyzes monoubiquitination of H2B in concert with the E2 ubiquitin conjugating enzyme, Rad6. The crystal structure of a C-terminal fragment of Bre1 shows that the catalytic RING domain is preceded by an N-terminal helix that mediates coiled-coil interactions with a crystallographically related monomer. Homology modeling suggests that the human homologue of Bre1, RNF20/RNF40, heterodimerizes through similar coiled coil interactions. PMID:25864391

  14. Formation of C-terminally truncated version of the Taz1 protein employs cleavage-box structure in mRNA

    International Nuclear Information System (INIS)

    When expressed in various hosts the taz1+ gene encoding the fission yeast telomere-binding protein produces two forms of polypeptides: full-length (Taz1p) and truncated (Taz1pΔC) version lacking almost entire Myb-domain. Whereas Taz1p binds telomeric DNA in vitro, Taz1pΔC forms long filaments unable of DNA binding. The formation of Taz1pΔC is a result of neither site-specific proteolysis, nor premature termination of transcription. In silico analysis of the taz1+ RNA transcript revealed a stem-loop structure at the site of cleavage (cleavage box; CB). In order to explore whether it possesses inherent destabilizing effects, we cloned CB sequence into the open reading frame (ORF) of glutathione-S-transferase (GST) and observed that when expressed in Escherichia coli the engineered gene produced two forms of the reporter protein. The formation of the truncated version of GST was abolished, when CB was replaced with recoded sequence containing synonymous codons thus indicating that the truncation is based on structural properties of taz1+ mRNA.

  15. The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

    Science.gov (United States)

    Shukla, Ashutosh; Dey, Debajit; Banerjee, Kamalika; Nain, Anshu; Banerjee, Manidipa

    2015-10-01

    Viroporins are virally encoded, membrane-active proteins, which enhance viral replication and assist in egress of viruses from host cells. The 2B proteins in the picornaviridae family are known to have viroporin-like properties, and play critical roles during virus replication. The 2B protein of Hepatitis A Virus (2B), an unusual picornavirus, is somewhat dissimilar from its analogues in several respects. HAV 2B is approximately 2.5 times the length of other 2B proteins, and does not disrupt calcium homeostasis or glycoprotein trafficking. Additionally, its membrane penetrating properties are not yet clearly established. Here we show that the membrane interacting activity of HAV 2B is localized in its C-terminal region, which contains an alpha-helical hairpin motif. We show that this region is capable of forming small pores in membranes and demonstrates lipid specific activity, which partially rationalizes the intracellular localization of full-length 2B. Using a combination of biochemical assays and molecular dynamics simulation studies, we also show that HAV 2B demonstrates a marked propensity to dimerize in a crowded environment, and probably interacts with membranes in a multimeric form, a hallmark of other picornavirus viroporins. In sum, our study clearly establishes HAV 2B as a bona fide viroporin in the picornaviridae family.

  16. High-resolution NMR structures of the domains of Saccharomyces cerevisiae Tho1

    Energy Technology Data Exchange (ETDEWEB)

    Jacobsen, Julian O. B.; Allen, Mark D.; Freund, Stefan M. V.; Bycroft, Mark, E-mail: mxb@mrc-lmb.cam.ac.uk [MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH (United Kingdom)

    2016-05-23

    In this study, high-resolution structures of both the N-terminal DNA-binding SAP domain and the C-terminal RNA-binding domain of S. cerevisiae Tho1 have been determined. THO is a multi-protein complex involved in the formation of messenger ribonuclear particles (mRNPs) by coupling transcription with mRNA processing and export. THO is thought to be formed from five subunits, Tho2p, Hpr1p, Tex1p, Mft1p and Thp2p, and recent work has determined a low-resolution structure of the complex [Poulsen et al. (2014 ▸), PLoS One, 9, e103470]. A number of additional proteins are thought to be involved in the formation of mRNP in yeast, including Tho1, which has been shown to bind RNA in vitro and is recruited to actively transcribed chromatin in vivo in a THO-complex and RNA-dependent manner. Tho1 is known to contain a SAP domain at the N-terminus, but the ability to suppress the expression defects of the hpr1Δ mutant of THO was shown to reside in the RNA-binding C-terminal region. In this study, high-resolution structures of both the N-terminal DNA-binding SAP domain and C-terminal RNA-binding domain have been determined.

  17. 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

  18. 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.

  19. The C-terminal heavy-chain domain of botulinum neurotoxin a is not the only site that binds neurons, as the N-terminal heavy-chain domain also plays a very active role in toxin-cell binding and interactions.

    Science.gov (United States)

    Ayyar, B Vijayalakshmi; Aoki, K Roger; Atassi, M Zouhair

    2015-04-01

    Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729-845). HN729-845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729-845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729-845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin. PMID:25624352

  20. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  1. Structure of the thioredoxin-fold domain of human phosducin-like protein 2

    International Nuclear Information System (INIS)

    The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 Å resolution and resembled the Trx-fold domain of rat phosducin. Human phosducin-like protein 2 (hPDCL2) has been identified as belonging to subgroup II of the phosducin (Pdc) family. The members of this family share an N-terminal helix domain and a C-terminal thioredoxin-fold (Trx-fold) domain. The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 Å resolution and resembled the Trx-fold domain of rat phosducin. Comparative structural analysis revealed the structural basis of their putative functional divergence

  2. Crystal Structures of the Outer Membrane Domain of Intimin and Invasin from Enterohemorrhagic E. coli and Enteropathogenic Y. pseudotuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Fairman, James W.; Dautin, Nathalie; Wojtowicz, Damian; Liu, Wei; Noinaj, Nicholas; Barnard, Travis J.; Udho, Eshwar; Przytycka, Teresa M.; Cherezov, Vadim; Buchanan, Susan K. (CUA); (Einstein); (NIH); (Scripps)

    2012-12-10

    Intimins and invasins are virulence factors produced by pathogenic Gram-negative bacteria. They contain C-terminal extracellular passenger domains that are involved in adhesion to host cells and N-terminal {beta} domains that are embedded in the outer membrane. Here, we identify the domain boundaries of an E. coli intimin {beta} domain and use this information to solve its structure and the {beta} domain structure of a Y. pseudotuberculosis invasin. Both {beta} domain structures crystallized as monomers and reveal that the previous range of residues assigned to the {beta} domain also includes a protease-resistant domain that is part of the passenger. Additionally, we identify 146 nonredundant representative members of the intimin/invasin family based on the boundaries of the highly conserved intimin and invasin {beta} domains. We then use this set of sequences along with our structural data to find and map the evolutionarily constrained residues within the {beta} domain.

  3. Tyrosine kinase activity of a chimeric insulin-like-growth-factor-1 receptor containing the insulin receptor C-terminal domain. Comparison with the tyrosine kinase activities of the insulin and insulin-like-growth-factor-1 receptors using a cell-free system.

    Science.gov (United States)

    Mothe, I; Tartare, S; Kowalski-Chauvel, A; Kaliman, P; Van Obberghen, E; Ballotti, R

    1995-03-15

    In a previous study, we showed that a chimeric insulin-like-growth-factor-1 (IGF-1) receptor, with the beta subunit C-terminal part of the insulin receptor was more efficient in stimulating glycogen synthesis and p44mapk activity compared to the wild-type IFG-1 receptor [Tartare, S., Mothe, I., Kowalski-Chauvel, A., Breittmayer, J.-P., Ballotti, R. & Van Obberghen, E. (1994) J. Biol. Chem. 269, 11449-11455]. These data indicate that the receptor C-terminal domain plays an important role in the transmission of biological effects. To understand the molecular basis of the differences in receptor specificity, we studied the characteristics of insulin, IGF-1 and chimeric receptor tyrosine kinase activities in a cell-free system. We found that, compared to wild-type insulin and IGF-1 receptors, the chimeric receptor showed a decrease in (a) autophosphorylation, (b) tyrosine kinase activity towards insulin receptor substrate-1 and the insulin receptor-(1142-1158)-peptide, and (c) the ability to activate phosphatidylinositol 3-kinase. However, for all the effects measured in a cell-free system, the chimeric receptor displayed an increased response to IGF-1 compared to the native IGF-1 receptor. Concerning the cation dependence of the tyrosine kinase activity, we showed that, at 10 mM Mg2+, the ligand-stimulated phosphorylation of poly(Glu80Tyr20) by both insulin receptor and chimeric receptor was increased by Mn2+. Conversely at 50 mM Mg2+, the chimeric receptor behaved like the IGF-1 receptor, since the presence of Mn2+ decreased the stimulatory effect of IGF-1 on their kinase activity. Furthermore, the Km of the chimeric receptor for ATP was increased compared to the wild-type receptors. These data demonstrate that the replacement of the C-terminal tail of the IGF-1 receptor by that of the insulin receptor has changed the receptor characteristics studied in a cell-free system. Our findings indicate that the C-terminal domain of the insulin receptor beta subunit plays a

  4. A C-terminal truncated hepatitis C virus core protein variant assembles in vitro into virus-like particles in the absence of structured nucleic acids

    International Nuclear Information System (INIS)

    Little is known about the assembly pathway or structure of the hepatitis C virus (HCV). In this work a truncated HCcAg variant covering the first 120 aa (HCcAg.120) with a 32 aa N-terminal fusion peptide (6x Histag-Xpress epitope) was purified as a monomer under strong denaturing conditions. In addition, minor HCcAg.120 peaks exhibiting little different molecular mass by SDS-PAGE which possibly represents alternative forms harboring the N-termini of HCcAg.120 were detected. Analysis using gel filtration chromatography showed that HCcAg.120 assembled into high molecular weight structures in vitro in the absence of structured nucleic acids. The negative-stain electron microscopy analysis revealed that these structures correspond with spherical VLPs of uniform morphology and size distribution. The diameters of these particles ranged from 20 to 43 nm with an average diameter of approximately 30 nm and were specifically immunolabelled with a mouse monoclonal antibody against the residues 5-35 of HCcAg. Results presented in this work showed that HCcAg.120 assembled in vitro into VLPs in the absence of structured nucleic acids with similar morphology and size distribution to those found in sera and hepatocytes from HCV-infected patients. Therefore, these VLPs would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure

  5. Structuring very large domain models

    DEFF Research Database (Denmark)

    Störrle, Harald

    2010-01-01

    a lower level of detail have not been dealt with. This paper aims at filling this gap by reporting personal experiences from a very large scale industrial domain modeling project. There, structuring the logical view turned out to be a critical success factor. We explain the project and its setting......, analyze the role and repercussions of model structuring, and examine the implications model structuring decisions have on other parts of the project. We then explain the model structure abstracted from a very large scale industrial modeling project. Finally, we discuss lessons learned....

  6. Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro-β-1,3-glucanase

    OpenAIRE

    Shinshi, H.; Wenzler, H; Neuhaus, J.-M.; Felix, G.; Hofsteenge, J; Meins, F

    1988-01-01

    Tobacco glucan endo-1,3-β-glucosidase (β-1,3-glucanase; 1,3-β-D-glucan glucanohydrolase; EC 3.2.1.39) exhibits complex hormonal and developmental regulation and is induced when plants are infected with pathogens. We determined the primary structure of this enzyme from the nucleotide sequence of five partial cDNA clones and the amino acid sequence of five peptides covering a total of 70 residues. β-1,3-Glucanase is produced as a 359-residue preproenzyme with an N-terminal hydrophobic signal pe...

  7. Biochemical properties and catalytic domain structure of the CcmH protein from Escherichia coli.

    Science.gov (United States)

    Zheng, Xue-Ming; Hong, Jing; Li, Hai-Yin; Lin, Dong-Hai; Hu, Hong-Yu

    2012-12-01

    In the Gram-negative bacterium of Escherichia coli, eight genes organized as a ccm operon (ccmABCDEFGH) are involved in the maturation of c-type cytochromes. The proteins encoded by the last three genes ccmFGH are believed to form a lyase complex functioning in the reduction of apocytochrome c and haem attachment. Among them, CcmH is a membrane-associated protein; its N-terminus is a catalytic domain with the active CXXC motif and the C-terminus is predicted as a TPR-like domain with unknown function. By using SCAM (scanning cysteine accessibility mutagenesis) and Gaussia luciferase fusion assays, we provide experimental evidence for the entire topological structure of E. coli CcmH. The mature CcmH is a periplasm-resident oxidoreductase anchored to the inner membrane by two transmembrane segments. Both N- and C-terminal domains are located and function in the periplasmic compartment. Moreover, the N-terminal domain forms a monomer in solution, while the C-terminal domain is a compact fold with helical structures. The NMR solution structure of the catalytic domain in reduced form exhibits mainly a three-helix bundle, providing further information for the redox mechanism. The redox potential suggests that CcmH exhibits a strong reductase that may function in the last step of reduction of apocytochrome c for haem attachment. PMID:22789558

  8. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Chingakham R. [Kansas State University, 338 Ackert Hall, Manhattan, KS 66506 (United States); Lovell, Scott; Mehzabeen, Nurjahan [University of Kansas, Del Shankel Structural Biology Center, Lawrence, KS 66047 (United States); Chowdhury, Wasimul Q.; Geanes, Eric S. [Kansas State University, 338 Ackert Hall, Manhattan, KS 66506 (United States); Battaile, Kevin P. [IMCA-CAT Hauptman–Woodward Medical Research Institute, 9700 South Cass Avenue, Building 435A, Argonne, IL 60439 (United States); Roelofs, Jeroen, E-mail: jroelofs@ksu.edu [Kansas State University, 338 Ackert Hall, Manhattan, KS 66506 (United States)

    2014-03-25

    The proteasome-assembly chaperone Nas2 binds to the proteasome subunit Rpt5 using its PDZ domain. The structure of the Nas2 PDZ domain has been determined. The 26S proteasome is a 2.5 MDa protease dedicated to the degradation of ubiquitinated proteins in eukaryotes. The assembly of this complex containing 66 polypeptides is assisted by at least nine proteasome-specific chaperones. One of these, Nas2, binds to the proteasomal AAA-ATPase subunit Rpt5. The PDZ domain of Nas2 binds to the C-terminal tail of Rpt5; however, it does not require the C-terminus of Rpt5 for binding. Here, the 1.15 Å resolution structure of the PDZ domain of Nas2 is reported. This structure will provide a basis for further insights regarding the structure and function of Nas2 in proteasome assembly.

  9. The modular xylanase Xyn10A from Rhodothermus marinus is cell-attached, and its C-terminal domain has several putative homologues among cell-attached proteins within the phylum Bacteroidetes

    DEFF Research Database (Denmark)

    Karlsson, Eva Nordberg; Hachem, Maher Abou; Ramchuran, Santosh;

    2004-01-01

    the light of this, a revision of experimental data present on both Xyn10A and Man26A was performed, and the results all indicate a cell-anchoring role of the domain, suggesting that this domain represents a novel type of module that mediates cell attachment in proteins originating from members of the...

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

    Science.gov (United States)

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

    2009-01-27

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

  11. System using tandem repeats of the cA peptidoglycan-binding domain from Lactococcus lactis for display of both N- and C-terminal fusions on cell surfaces of lactic acid bacteria.

    Science.gov (United States)

    Okano, Kenji; Zhang, Qiao; Kimura, Sakurako; Narita, Junya; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2008-02-01

    Here, we established a system for displaying heterologous protein to the C terminus of the peptidoglycan-binding domain (cA domain) of AcmA (a major autolysin from Lactococcus lactis). Western blot and flow cytometric analyses revealed that the fusion proteins (cA-AmyA) of the cA domain and alpha-amylase from Streptococcus bovis 148 (AmyA) are efficiently expressed and successfully displayed on the surfaces of L. lactis cells. AmyA was also displayed on the cell surface while retaining its activity. Moreover, with an increase in the number of cA domains, the quantity of cA-AmyA fusion proteins displayed on the cell surface increased. When three repeats of the cA domain were used as an anchor protein, 82% of alpha-amylase activity was detected on the cells. The raw starch-degrading activity of AmyA was significantly higher when AmyA was fused to the C terminus of the cA domain than when it was fused to the N terminus. In addition, cA-AmyA fusion proteins were successfully displayed on the cell surfaces of Lactobacillus plantarum and Lactobacillus casei. PMID:18156338

  12. The Crystal Structures of EAP Domains from Staphylococcus aureus Reveal an Unexpected Homology to Bacterial Superantigens

    Energy Technology Data Exchange (ETDEWEB)

    Geisbrecht, B V; Hamaoka, B Y; Perman, B; Zemla, A; Leahy, D J

    2005-10-14

    The Eap (extracellular adherence protein) of Staphylococcus aureus functions as a secreted virulence factor by mediating interactions between the bacterial cell surface and several extracellular host proteins. Eap proteins from different Staphylococcal strains consist of four to six tandem repeats of a structurally uncharacterized domain (EAP domain). We have determined the three-dimensional structures of three different EAP domains to 1.8, 2.2, and 1.35 {angstrom} resolution, respectively. These structures reveal a core fold that is comprised of an {alpha}-helix lying diagonally across a five-stranded, mixed {beta}-sheet. Comparison of EAP domains with known structures reveals an unexpected homology with the C-terminal domain of bacterial superantigens. Examination of the structure of the superantigen SEC2 bound to the {beta}-chain of a T-cell receptor suggests a possible ligand-binding site within the EAP domain (Fields, B. A., Malchiodi, E. L., Li, H., Ysern, X., Stauffacher, C. V., Schlievert, P. M., Karjalainen, K., and Mariuzza, R. (1996) Nature 384, 188-192). These results provide the first structural characterization of EAP domains, relate EAP domains to a large class of bacterial toxins, and will guide the design of future experiments to analyze EAP domain structure/function relationships.

  13. The crystal structures of dystrophin and utrophin spectrin repeats: implications for domain boundaries.

    Directory of Open Access Journals (Sweden)

    Muralidharan Muthu

    Full Text Available Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1 from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.

  14. Protein structural domains: definition and prediction.

    Science.gov (United States)

    Ezkurdia, Iakes; Tress, Michael L

    2011-11-01

    Recognition and prediction of structural domains in proteins is an important part of structure and function prediction. This unit lists the range of tools available for domain prediction, and describes sequence and structural analysis tools that complement domain prediction methods. Also detailed are the basic domain prediction steps, along with suggested strategies for different protein sequences and potential pitfalls in domain boundary prediction. The difficult problem of domain orientation prediction is also discussed. All the resources necessary for domain boundary prediction are accessible via publicly available Web servers and databases and do not require computational expertise. PMID:22045561

  15. Domain Modeling: NP_055672.3 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_055672.3 chr1 Solution structure of the chimera of the C-terminal PID domain of ...Fe65L and the C-terminal tail peptide of APP p2ysza_ chr1/NP_055672.3/NP_055672.3_apo_116-314.pdb swppa 0 ...

  16. The first crystal structure of the peptidase domain of the U32 peptidase family.

    Science.gov (United States)

    Schacherl, Magdalena; Montada, Angelika A M; Brunstein, Elena; Baumann, Ulrich

    2015-12-01

    The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins from Helicobacter and Salmonella. The first crystal structure analysis of a U32 catalytic domain from Methanopyrus kandleri (gene mk0906) reveals a modified (βα)8 TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to a Strep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands. PMID:26627657

  17. The host-binding domain of the P2 phage tail spike reveals a trimeric iron-binding structure

    International Nuclear Information System (INIS)

    The C-terminal domain of a bacteriophage P2 tail-spike protein, gpV, was crystallized and its structure was solved at 1.27 Å resolution. The refined model showed a triple β-helix structure and the presence of iron, calcium and chloride ions. The adsorption and infection of bacteriophage P2 is mediated by tail fibres and tail spikes. The tail spikes on the tail baseplate are used to irreversibly adsorb to the host cells. Recently, a P2 phage tail-spike protein, gpV, was purified and it was shown that a C-terminal domain, Ser87–Leu211, is sufficient for the binding of gpV to host Escherichia coli membranes [Kageyama et al. (2009 ▶), Biochemistry, 48, 10129–10135]. In this paper, the crystal structure of the C-terminal domain of P2 gpV is reported. The structure is a triangular pyramid and looks like a spearhead composed of an intertwined β-sheet, a triple β-helix and a metal-binding region containing iron, calcium and chloride ions

  18. Crystallographic structure of porcine adenovirus type 4 fiber head and galectin domains.

    Science.gov (United States)

    Guardado-Calvo, Pablo; Muñoz, Eva M; Llamas-Saiz, Antonio L; Fox, Gavin C; Kahn, Richard; Curiel, David T; Glasgow, Joel N; van Raaij, Mark J

    2010-10-01

    Adenovirus isolate NADC-1, a strain of porcine adenovirus type 4, has a fiber containing an N-terminal virus attachment region, shaft and head domains, and a C-terminal galectin domain connected to the head by an RGD-containing sequence. The crystal structure of the head domain is similar to previously solved adenovirus fiber head domains, but specific residues for binding the coxsackievirus and adenovirus receptor (CAR), CD46, or sialic acid are not conserved. The structure of the galectin domain reveals an interaction interface between its two carbohydrate recognition domains, locating both sugar binding sites face to face. Sequence evidence suggests other tandem-repeat galectins have the same arrangement. We show that the galectin domain binds carbohydrates containing lactose and N-acetyl-lactosamine units, and we present structures of the galectin domain with lactose, N-acetyl-lactosamine, 3-aminopropyl-lacto-N-neotetraose, and 2-aminoethyl-tri(N-acetyl-lactosamine), confirming the domain as a bona fide galectin domain. PMID:20686025

  19. Amino acid residue Y196E substitution and C-terminal peptide synergistically alleviate the toxicity of Clostridium perfringens epsilon toxin.

    Science.gov (United States)

    Yao, Wenwu; Kang, Lin; Gao, Shan; Zhuang, Xiangjin; Zhang, Tao; Yang, Hao; Ji, Bin; Xin, Wenwen; Wang, Jinglin

    2015-06-15

    Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains, and is the causative agent of a lethal enterotoxemia in livestock animals and possibly in humans. However, many details of ETX structure and activity are not known. Therefore, it is important to clarify the relationship between ETX structure and activity. To explore the effect and mechanism of ETX amino acid residue Y196E substitution and C-terminal peptide on toxicity, four recombinant proteins, rETX (without 13 N-terminal peptides and 23 C-terminal peptides), rETX-C (rETX with 23 C-terminal peptides), rETX(Y196E) (rETX with an amino acid residue substitution at Y196) and rETX(Y196E)-C (rETX-C with a Y196E mutation), were constructed in this study. Both the amino acid residue Y196E substitution and the C-terminal peptide reduce ETX toxicity to a similar extent, and the two factors synergistically alleviate ETX toxicity. In addition, we demonstrated that the C-terminal peptides and Y196E amino acid mutation reduce the toxin toxicity in two different pathways: the C-terminal peptides inhibit the binding activity of toxins to target cells, and the Y196E amino acid mutation slightly inhibits the pore-forming or heptamer-forming process. Interaction between the two factors was not observed in pore-forming or binding assays but toxicity assays, which demonstrated that the relationship between domains of the toxin is more complicated than previously appreciated. However, the exact mechanism of synergistic action is not yet clarified. PMID:25912943

  20. Two Distinct Binding Modes Define the Interaction of Brox with the C-Terminal Tails of CHMP5 and CHMP4B

    Energy Technology Data Exchange (ETDEWEB)

    Mu, Ruiling; Dussupt, Vincent; Jiang, Jiansheng; Sette, Paola; Rudd, Victoria; Chuenchor, Watchalee; Bello, Nana F.; Bouamr, Fadila; Xiao, Tsan Sam (NIH)

    2012-05-21

    Interactions of the CHMP protein carboxyl terminal tails with effector proteins play important roles in retroviral budding, cytokinesis, and multivesicular body biogenesis. Here we demonstrate that hydrophobic residues at the CHMP4B C-terminal amphipathic {alpha} helix bind a concave surface of Brox, a mammalian paralog of Alix. Unexpectedly, CHMP5 was also found to bind Brox and specifically recruit endogenous Brox to detergent-resistant membrane fractions through its C-terminal 20 residues. Instead of an {alpha} helix, the CHMP5 C-terminal tail adopts a tandem {beta}-hairpin structure that binds Brox at the same site as CHMP4B. Additional Brox:CHMP5 interface is furnished by a unique CHMP5 hydrophobic pocket engaging the Brox residue Y348 that is not conserved among the Bro1 domains. Our studies thus unveil a {beta}-hairpin conformation of the CHMP5 protein C-terminal tail, and provide insights into the overlapping but distinct binding profiles of ESCRT-III and the Bro1 domain proteins.

  1. Analysis of DCC domain structure

    International Nuclear Information System (INIS)

    Wavelet-type methods are employed for the analysis of pion field configurations that have been obtained by dynamical simulations in idealized scenarios relevant to the formation of disoriented chiral condensates. It is illustrated how the measurement of the isospin domain structure depends on the ability to zoom in on limited parts of the phase space, due to the interplay between the pion correlation length and the effective source geometry. The need for advanced analysis methods is underscored by the fact that the extracted neutral-fraction distribution would differ significantly from the ideal form, even under perfect experimental conditions, and, moreover, by the circumstance that thermal sources with suitably adjusted temperatures can lead to distributions that may be practically indistinguishable from those arising from DCC-type nonequilibrium evolutions. copyright 1997 The American Physical Society

  2. Identification, structure, and differential expression of members of a BURP domain containing protein family in soybean.

    Science.gov (United States)

    Granger, Cheryl; Coryell, Virginia; Khanna, Anupama; Keim, Paul; Vodkin, Lila; Shoemaker, Randy C

    2002-08-01

    Expressed sequence tags (ESTs) exhibiting homology to a BURP domain containing gene family were identified from the Glycine max (L.) Merr. EST database. These ESTs were assembled into 16 contigs of variable sizes and lengths. Consistent with the structure of known BURP domain containing proteins, the translation products exhibit a modular structure consisting of a C-terminal BURP domain, an N-terminal signal sequence, and a variable internal region. The soybean family members exhibit 35-98% similarity in a -100-amino-acid C-terminal region, and a phylogenetic tree constructed using this region shows that some soybean family members group together in closely related pairs, triplets, and quartets, whereas others remain as singletons. The structure of these groups suggests that multiple gene duplication events occurred during the evolutionary history of this family. The depth and diversity of G. max EST libraries allowed tissue-specific expression patterns of the putative soybean BURPs to be examined. Consistent with known BURP proteins, the newly identified soybean BURPs have diverse expression patterns. Furthermore, putative paralogs can have both spatially and quantitatively distinct expression patterns. We discuss the functional and evolutionary implications of these findings, as well as the utility of EST-based analyses for identifying and characterizing gene families. PMID:12175072

  3. SARS Coronavirus-unique Domain (SUD): Three-domain Molecular Architecture in Solution and RNA Binding

    OpenAIRE

    Johnson, Margaret A.; Chatterjee, Amarnath; Neuman, Benjamin W.; Wüthrich, Kurt

    2010-01-01

    The nonstructural protein 3 (nsp3) of the severe acute respiratory syndrome coronavirus (SARS-CoV) includes a “SARS-unique region” (SUD) consisting of three globular domains separated by short linker peptide segments. This paper reports NMR structure determinations of the C-terminal domain (SUD-C) and of a two-domain construct (SUD-MC) containing the middle domain (SUD-M) and the C-terminal domain, and NMR data on the conformational states of the N-terminal domain (SUD-N) and the SUD-NM two-d...

  4. The C-Terminal Domain of Eukaryotic Initiation Factor 5 Promotes Start Codon Recognition by Its Dynamic Interplay with eIF1 and eIF2β

    Directory of Open Access Journals (Sweden)

    Rafael E. Luna

    2012-06-01

    Full Text Available Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD of eIF5 stimulates 43S preinitiation complex (PIC assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2β. This study provides mechanistic insight into the role of eIF5-CTD's dynamic interplay with eIF1 and eIF2β in switching PICs from an open to a closed state at start codons.

  5. Mutations in the C-Terminal Loop of the Nucleocapsid Protein Affect Vesicular Stomatitis Virus RNA Replication and Transcription Differentially▿

    OpenAIRE

    Harouaka, Djamila; Wertz, Gail W.

    2009-01-01

    The 2.9-Å structure of the vesicular stomatitis virus nucleocapsid (N) protein bound to RNA shows the RNA to be tightly sequestered between the two lobes of the N protein. Domain movement of the lobes of the N protein has been postulated to facilitate polymerase access to the RNA template. We investigated the roles of individual amino acid residues in the C-terminal loop, involved in long-range interactions between N protein monomers, in forming functional ribonucleoprotein (RNP) templates. T...

  6. Contribution of intracellular calcium and pH in ischemic uncoupling of cardiac gap junction channels formed of connexins 43, 40, and 45: a critical function of C-terminal domain.

    Directory of Open Access Journals (Sweden)

    Giriraj Sahu

    Full Text Available Ischemia is known to inhibit gap junction (GJ mediated intercellular communication. However the detail mechanisms of this inhibition are largely unknown. In the present study, we determined the vulnerability of different cardiac GJ channels formed of connexins (Cxs 43, 40, and 45 to simulated ischemia, by creating oxygen glucose deprived (OGD condition. 5 minutes of OGD decreased the junctional conductance (Gj of Cx43, Cx40 and Cx45 by 53±3%, 64±1% and 85±2% respectively. Reduction of Gj was prevented completely by restricting the change of both intracellular calcium ([Ca(2+]i and pH (pHi with potassium phosphate buffer. Clamping of either [Ca(2+]i or pHi, through BAPTA (2 mM or HEPES (80 mM respectively, offered partial resistance to ischemic uncoupling. Anti-calmodulin antibody attenuated the uncoupling of Cx43 and Cx45 significantly but not of Cx40. Furthermore, OGD could reduce only 26±2% of Gj in C-terminus (CT truncated Cx43 (Cx43-Δ257. Tethering CT of Cx43 to the CT-truncated Cx40 (Cx40-Δ249, and Cx45 (Cx45-Δ272 helped to resist OGD mediated uncoupling. Moreover, CT domain played a significant role in determining the junction current density and plaque diameter. Our results suggest; OGD mediated uncoupling of GJ channels is primarily due to elevated [Ca(2+]i and acidic pHi, though the latter contributes more. Among Cx43, Cx40 and Cx45, Cx43 is the most resistant to OGD while Cx45 is the most sensitive one. CT of Cx43 has major necessary elements for OGD induced uncoupling and it can complement CT of Cx40 and Cx45.

  7. Data on structural transitions in domains of hordeivirus TGB1 protein forming ribonucleoprotein complex.

    Science.gov (United States)

    Makarov, Valentin V; Makarova, Svetlana S; Kalinina, Natalia O

    2016-09-01

    This data article is related to the research article entitled "in vitro properties of hordeivirus TGB1 protein forming ribonucleoprotein complexes" (Makarov et al., 2015 [1]), demonstrating that upon incubation with viral RNA the poa semilatent hordeivirus (PSLV) TGB1 protein (the movement 63 K protein encoded by the first gene of the triple gene block) in vitro forms RNP structures resembling filamentous virus-like particles and its internal domain (ID) performs a major structural role in this process. This article reports the additional results on the structural lability of ID and the structural transitions in the C-terminal NTPase/helicase domain (HELD) induced by interaction with tRNA and phosphorylation. PMID:27331098

  8. Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

    International Nuclear Information System (INIS)

    Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering

  9. Crystal Structure of the Passenger Domain of the Escherichia coli Autotransporter EspP

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Shekeb; Mian, Hira S.; Sandercock, Linda E.; Chirgadze, Nickolay Y.; Pai, Emil F. (Toronto); (OCI)

    2013-03-07

    Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal 'passenger domain' responsible for the specific effector functions of the molecule and a C-terminal '{beta}-domain' responsible for translocation of the passenger across the bacterial outer membrane. Here, we present the 2.5-{angstrom} crystal structure of the passenger domain of the extracellular serine protease EspP, produced by the pathogen Escherichia coli O157:H7 and a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs). Like the previously structurally characterized SPATE passenger domains, the EspP passenger domain contains an extended right-handed parallel {beta}-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this {beta}-helix. We describe the structure of the EspP passenger domain in the context of previous results and provide an alternative hypothesis for the function of the {beta}-helix within SPATEs.

  10. Structure and RNA binding of the mouse Pumilio-2 Puf domain.

    Science.gov (United States)

    Jenkins, Huw T; Baker-Wilding, Rosanna; Edwards, Thomas A

    2009-09-01

    Puf proteins control translation through the interaction of a C-terminal Puf domain with specific sequences present in the 3' untranslated region of messenger RNAs. In Drosophila, binding of the protein Pumilio to mRNA leads to translational repression which is required for anterior/posterior patterning during embryogenesis. The vertebrate Pumilio homologue 2 (Pum2) has been implicated in controlling germ cell development through interactions with the RNA binding proteins deleted in azoospermia (DAZ), DAZ-like (DAZL) and BOULE. We present the 1.6A resolution X-ray crystal structure of the Puf domain from murine Pum2 and demonstrate that this domain is capable of binding with nanomolar affinity to RNA sequences from the hunchback Nanos response element (NRE) and a previously identified Pum2 binding element (PBE). PMID:19540345

  11. The Cold Denatured State of the C-terminal Domain of Protein L9 Is Compact and Contains Both Native and Non-native Structure

    OpenAIRE

    Shan, Bing; McClendon, Sebastian; Rospigliosi, Carla; Eliezer, David; Raleigh, Daniel P.

    2010-01-01

    Cold denaturation is a general property of globular proteins, and the process provides insight into the origins of the cooperativity of protein folding and the nature of partially folded states. Unfortunately, studies of protein cold denaturation have been hindered by the fact that the cold denatured state is normally difficult to access experimentally. Special conditions such as addition of high concentrations of denaturant, encapsulation into reverse micelles, the formation of emulsified so...

  12. Structure of the F-spondin Domain of Mindin an Integrin Ligand and Pattern Recognition Molecule

    Energy Technology Data Exchange (ETDEWEB)

    Y Li; C Cao; W Jia; L Yu; M Mo; Q Wang; Y Huang; J Lim; M Ishihara; et. al.

    2011-12-31

    Mindin (spondin-2) is an extracellular matrix protein of unknown structure that is required for efficient T-cell priming by dendritic cells. Additionally, mindin functions as a pattern recognition molecule for initiating innate immune responses. These dual functions are mediated by interactions with integrins and microbial pathogens, respectively. Mindin comprises an N-terminal F-spondin (FS) domain and C-terminal thrombospondin type 1 repeat (TSR). We determined the structure of the FS domain at 1.8-A resolution. The structure revealed an eight-stranded antiparallel beta-sandwich motif resembling that of membrane-targeting C2 domains, including a bound calcium ion. We demonstrated that the FS domain mediates integrin binding and identified the binding site by mutagenesis. The mindin FS domain therefore represents a new integrin ligand. We further showed that mindin recognizes lipopolysaccharide (LPS) through its TSR domain, and obtained evidence that C-mannosylation of the TSR influences LPS binding. Through these dual interactions, the FS and TSR domains of mindin promote activation of both adaptive and innate immune responses.

  13. Structure of the F-Spondin Domain of Mindin, an Integrin Ligand and Pattern Recognition Molecule

    Energy Technology Data Exchange (ETDEWEB)

    Li, Y.; Cao, C; Jia, W; Yu, L; Mo, M; Wang, Q; Huang, Y; Lim, J; Ishihara, M; et. al.

    2009-01-01

    Mindin (spondin-2) is an extracellular matrix protein of unknown structure that is required for efficient T-cell priming by dendritic cells. Additionally, mindin functions as a pattern recognition molecule for initiating innate immune responses. These dual functions are mediated by interactions with integrins and microbial pathogens, respectively. Mindin comprises an N-terminal F-spondin (FS) domain and C-terminal thrombospondin type 1 repeat (TSR). We determined the structure of the FS domain at 1.8-A resolution. The structure revealed an eight-stranded antiparallel ?-sandwich motif resembling that of membrane-targeting C2 domains, including a bound calcium ion. We demonstrated that the FS domain mediates integrin binding and identified the binding site by mutagenesis. The mindin FS domain therefore represents a new integrin ligand. We further showed that mindin recognizes lipopolysaccharide (LPS) through its TSR domain, and obtained evidence that C-mannosylation of the TSR influences LPS binding. Through these dual interactions, the FS and TSR domains of mindin promote activation of both adaptive and innate immune responses.

  14. The C-terminal polyproline-containing region of ELMO contributes to an increase in the life-time of the ELMO-DOCK complex.

    Science.gov (United States)

    Sévajol, Marion; Reiser, Jean-Baptiste; Chouquet, Anne; Pérard, Julien; Ayala, Isabel; Gans, Pierre; Kleman, Jean-Philippe; Housset, Dominique

    2012-03-01

    The eukaryotic Engulfment and CellMotility (ELMO) proteins form an evolutionary conserved family of key regulators which play a central role in Rho-dependent biological processes such as engulfment and cell motility/migration. ELMO proteins interact with a subset of Downstream of Crk (DOCK) family members, a new type of guanine exchange factors (GEF) for Rac and cdc42 GTPases. The physiological function of DOCK is to facilitate actin remodeling, a process which occurs only in presence of ELMO. Several studies have determined that the last 200 C-terminal residues of ELMO1 and the first 180 N-terminal residues of DOCK180 are responsible for the ELMO-DOCK interaction. However, the precise role of the different domains and motifs identified in these regions has remained elusive. Divergent functional, biochemical and structural data have been reported regarding the contribution of the C-terminal end of ELMO, comprising its polyproline motif, and of the DOCK SH3 domain. In the present study, we have investigated the contribution of the C-terminal end of ELMO1 to the interaction between ELMO1 and the SH3 domain of DOCK180 using nuclear magnetic resonance spectroscopy and surface plasmon resonance. Our data presented here demonstrate the ability of the SH3 domain of DOCK180 to interact with ELMO1, regardless of the presence of the polyproline-containing C-terminal end. However, the presence of the polyproline region leads to a significant increase in the half-life of the ELMO1-DOCK180 complex, along with a moderate increase on the affinity. PMID:22177965

  15. Investigation of peptidyl synthase activity of the Y Carboxypeptidase: influence of the primary structure of the substrate C-terminal fragment and application to radio-marking (3H or 14C) of three neuro hypophysis hormones

    International Nuclear Information System (INIS)

    As radio-marking of peptides of biological interest induced significant advances in the metabolism study, radio-immunology and molecular endocrinology, this research thesis reports investigation performed on hormonal peptides released by the post-hypophysis. The ultimate objective is to substitute in these hormones the C-terminal Gly-NH2 by its tritiated radioactive homologue to provide these peptides with a R.A.S equal to that of the tritiated Gly-NH2 in order to study reaction mechanisms and to generalize this method to a larger number of peptides

  16. Heparan sulfate regulates fibrillin-1 N- and C-terminal interactions

    DEFF Research Database (Denmark)

    Cain, Stuart A; Baldwin, Andrew K; Mahalingam, Yashithra; Raynal, Bertrand; Jowitt, Thomas A; Shuttleworth, C Adrian; Couchman, John R; Kielty, Cay M

    2008-01-01

    Fibrillin-1 N- and C-terminal heparin binding sites have been characterized. An unprocessed monomeric N-terminal fragment (PF1) induced a very high heparin binding response, indicating heparin-mediated multimerization. Using PF1 deletion and short fragments, a heparin binding site was localized...... response to soluble PF1. Within domains encoded by exons 59-62 near the fibrillin-1 C terminus are novel conformation-dependent high affinity heparin and tropoelastin binding sites. Heparin disrupted tropoelastin binding but did not disrupt N- and C-terminal fibrillin-1 interactions. Thus, fibrillin-1 N-terminal...... interactions with heparin/heparan sulfate directly influence cell behavior, whereas C-terminal interactions with heparin/heparan sulfate regulate elastin deposition. These data highlight how heparin/heparan sulfate controls fibrillin-1 interactions....

  17. Structure and Function of KH Domains

    Energy Technology Data Exchange (ETDEWEB)

    Valverde, R.; Regan, E

    2008-01-01

    The hnRNP K homology (KH) domain was first identified in the protein human heterogeneous nuclear ribonucleoprotein K (hnRNP K) 14 years ago. Since then, KH domains have been identified as nucleic acid recognition motifs in proteins that perform a wide range of cellular functions. KH domains bind RNA or ssDNA, and are found in proteins associated with transcriptional and translational regulation, along with other cellular processes. Several diseases, e.g. fragile X mental retardation syndrome and paraneoplastic disease, are associated with the loss of function of a particular KH domain. Here we discuss the progress made towards understanding both general and specific features of the molecular recognition of nucleic acids by KH domains. The typical binding surface of KH domains is a cleft that is versatile but that can typically accommodate only four unpaired bases. Van der Waals forces and hydrophobic interactions and, to a lesser extent, electrostatic interactions, contribute to the nucleic acid binding affinity. 'Augmented' KH domains or multiple copies of KH domains within a protein are two strategies that are used to achieve greater affinity and specificity of nucleic acid binding. Isolated KH domains have been seen to crystallize as monomers, dimers and tetramers, but no published data support the formation of noncovalent higher-order oligomers by KH domains in solution. Much attention has been given in the literature to a conserved hydrophobic residue (typically Ile or Leu) that is present in most KH domains. The interest derives from the observation that an individual with this Ile mutated to Asn, in the KH2 domain of fragile X mental retardation protein, exhibits a particularly severe form of the syndrome. The structural effects of this mutation in the fragile X mental retardation protein KH2 domain have recently been reported. We discuss the use of analogous point mutations at this position in other KH domains to dissect both structure and

  18. Domain structure of circular and ring magnets

    International Nuclear Information System (INIS)

    We present the results of a study of the domain structure and the magnetization reversal processes in arrays of circular and ring magnets, obtained by magnetic force microscopy and magnetization measurements. The particles, with outer diameter 0.55 and 2.2 μm, were made of Permalloy films of thickness 24 and 66 nm. In the circular elements two domain configurations were observed: a vortex structure with perpendicular magnetization at the core and a 'bud' state. In the ring elements the domain structure was found to depend on the ring width: a flux closed state was formed in wide rings and the 'onion' state was observed in narrow rings

  19. PDZ Binding Domains, Structural Disorder and Phosphorylation: A Menage-a-trois Tailing Dcp2 mRNA Decapping Enzymes.

    Science.gov (United States)

    Gunawardana, Dilantha

    2016-01-01

    Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein-protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer's disease, Parkinson's disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes. PMID:27151193

  20. Crystal structure and dynamics of Spt16N-domain of FACT complex from Cicer arietinum.

    Science.gov (United States)

    Are, Venkat N; Ghosh, Biplab; Kumar, Ashwani; Gadre, Rekha; Makde, Ravindra D

    2016-07-01

    The facilitates chromatin transcription (FACT) complex, a heterodimer of SSRP1 and Spt16 proteins, is an essential histone chaperone that transiently reorganizes nucleosomes during transcription, replication and repair. N-terminal domain of Spt16 subunit (Spt16N) is strictly conserved in all the known Spt16 orthologs. Genetic studies in yeast have revealed a partially redundant role of Spt16N for the FACT functionality. Here, we report the crystal structure of Spt16N from a plant origin (Spt16Nca, Cicer arietinum) and its comparisons with the known Spt16N structures from yeasts and human. The inter-domain angle in Spt16Nca is significantly different from that of the yeast and human Spt16N structures. Normal mode analysis and classical molecular dynamics simulations reveal inter-domain movement in Spt16Nca and later also shows conformational flexibility of the critical loops. Spt16Nca binds to histone H3/H4 complex, similar to its orthologs from yeast and human origins. Further, conservation of electrostatic surface potentials in Spt16N structures from evolutionary distinct domains of eukaryotes (plant, human and fungi) have provided the potential sites on Spt16N for histone interactions. The structural comparisons with M24 peptidases show that the hydrophobic pocket shielded by a flexible loop of C-terminal domain of Spt16N that may be functionally important. PMID:26995613

  1. Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA

    OpenAIRE

    Deretic, Dusanka; Schmerl, Sonia; Hargrave, Paul A.; Arendt, Anatol; McDowell, J. Hugh

    1998-01-01

    Several mutations that cause severe forms of the human disease autosomal dominant retinitis pigmentosa cluster in the C-terminal region of rhodopsin. Recent studies have implicated the C-terminal domain of rhodopsin in its trafficking on specialized post-Golgi membranes to the rod outer segment of the photoreceptor cell. Here we used synthetic peptides as competitive inhibitors of rhodopsin trafficking in the frog retinal cell-free system to delineate the potential regulatory sequence within ...

  2. Multilevel domain decomposition for electronic structure calculations

    CERN Document Server

    Barrault, M; Hager, W W; Le Bris, C

    2005-01-01

    We introduce a new multilevel domain decomposition method (MDD) for electronic structure calculations within semi-empirical and Density Functional Theory (DFT) frameworks. This method iterates between local fine solvers and global coarse solvers, in the spirit of domain decomposition methods.

  3. Four distinct structural domains in Clostridium difficile toxin B visualized using SAXS.

    Science.gov (United States)

    Albesa-Jové, David; Bertrand, Thomas; Carpenter, Elisabeth P; Swain, Gemma V; Lim, Jenson; Zhang, Jiancheng; Haire, Lesley F; Vasisht, Nishi; Braun, Veit; Lange, Anton; von Eichel-Streiber, Christoph; Svergun, Dmitri I; Fairweather, Neil F; Brown, Katherine A

    2010-03-12

    Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of approximately 275 A. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes. PMID:20070948

  4. Two-domain structure of the td intron-encoded endonuclease I-TevI correlates with the two-domain configuration of the homing site.

    Science.gov (United States)

    Derbyshire, V; Kowalski, J C; Dansereau, J T; Hauer, C R; Belfort, M

    1997-02-01

    I-TevI, the T4 td intron-encoded endonuclease, catalyzes the first step in intron homing by making a double-strand break in the intronless allele within a sequence designated the homing site. The 28 kDa enzyme, which interacts with the homing site over a span of 37 bp, binds as a monomer, contacting two domains of the substrate. In this study, limited proteolysis experiments indicate that I-TevI consists of two domains that behave as discrete physical entities as judged by a number of functional and structural criteria. Overexpression clones for each domain were constructed and the proteins were purified. The carboxy-terminal domain has DNA-binding activity coincident with the primary binding region of the homing site and binds with the same affinity as the full-length enzyme. The isolated amino-terminal domain, contains the conserved GIY-YIG motif, consistent with its being the catalytic domain. Furthermore, site-directed mutagenesis of a conserved arginine residue within the extended motif rendered the full-length protein catalytically inactive, although DNA-binding was maintained. This is the first evidence that the GIY-YIG motif is important for catalytic activity. An enzyme with an N-terminal catalytic domain and a C-terminal DNA-binding domain connected by a flexible linker is in accord with the bipartite structure of the homing site. PMID:9048944

  5. Structure of the stand-alone RAM-domain protein from Thermus thermophilus HB8

    International Nuclear Information System (INIS)

    The crystal structure of the stand-alone RAM domain from T. thermophilus HB8 has been determined at 2.4 Å resolution. The structure revealed that five dimers are arranged to form a ring. The stand-alone RAM (regulation of amino-acid metabolism) domain protein SraA from Thermus thermophilus HB8 (TTHA0845) was crystallized in the presence of zinc ions. The X-ray crystal structure was determined using a multiple-wavelength anomalous dispersion technique and was refined at 2.4 Å resolution to a final R factor of 25.0%. The monomeric structure is a βαββαβ fold and it dimerizes mainly through interactions between the antiparallel β-sheets. Furthermore, five SraA dimers form a ring with external and internal diameters of 70 and 20 Å, respectively. This decameric structure is unique compared with the octameric and dodecameric structures found for other stand-alone RAM-domain proteins and the C-terminal RAM domains of Lrp/AsnC-family proteins

  6. Role of the C-terminal YG repeats of the primer-dependent streptococcal glucosyltransferase, GtfJ, in binding to dextran and mutan.

    Science.gov (United States)

    Kingston, Kim B; Allen, Donna M; Jacques, Nicholas A

    2002-02-01

    The recombinant primer-dependent glucosyltransferase GtfJ of Streptococcus salivarius possesses a C-terminal glucan-binding domain composed of eighteen 21 aa YG repeats. By engineering a series of C-terminal truncated proteins, the position at which truncation prevented further mutan synthesis was defined to a region of 43 aa, confirming that not all of the YG motifs were required for the formation of mutan by GtfJ. The role of the YG repeats in glucan binding was investigated in detail. Three proteins consisting of 3.8, 7.2 or 11.0 C-terminal YG repeats were expressed in Escherichia coli. Each of the three purified proteins bound to both the 1,6-alpha-linked glucose residues of dextran and the 1,3-alpha-linked glucose residues of mutan, indicating that a protein consisting of nothing but 3.8 YG repeats could attach to either substrate. Secondary structure predictions of the primary amino acid sequence suggested that 37% of the amino acids were capable of forming a structure such that five regions of beta-sheet were separated by regions capable of forming beta-turns and random coils. CD spectral analysis showed that the purified 3.8 YG protein possessed an unordered secondary structure with some evidence of possible beta-sheet formation and that the protein maintained this relatively unordered structure on binding to dextran. PMID:11832518

  7. Role of N-terminal extension of Bacillus stearothermophilus RNase H2 and C-terminal extension of Thermotoga maritima RNase H2.

    Science.gov (United States)

    Permanasari, Etin-Diah; Angkawidjaja, Clement; Koga, Yuichi; Kanaya, Shigenori

    2013-10-01

    Bacillus stearothermophilus RNase H2 (BstRNH2) and Thermotoga maritima RNase H2 (TmaRNH2) have N-terminal and C-terminal extensions, respectively, as compared with Aquifex aeolicus RNase H2 (AaeRNH2). To analyze the role of these extensions, BstRNH2 and TmaRNH2 without these extensions were constructed, and their biochemical properties were compared with those of their intact partners and AaeRNH2. The far-UV CD spectra of all proteins were similar, suggesting that the protein structure is not significantly altered by removal of these extensions. However, both the junction ribonuclease and RNase H activities of BstRNH2 and TmaRNH2, as well as their substrate-binding affinities, were considerably decreased by removal of these extensions. The stability of BstRNH2 and TmaRNH2 was also decreased by removal of these extensions. The activity, substrate binding affinity and stability of TmaRNH2 without the C-terminal 46 residues were partly restored by the attachment of the N-terminal extension of BstRNH2. These results suggest that the N-terminal extension of BstRNH2 functions as a substrate-binding domain and stabilizes the RNase H domain. Because the C-terminal extension of TmaRNH2 assumes a helix hairpin structure and does not make direct contact with the substrate, this extension is probably required to make the conformation of the substrate-binding site functional. AaeRNH2 showed comparable junction ribonuclease activity to those of BstRNH2 and TmaRNH2, and was more stable than these proteins, indicating that bacterial RNases H2 do not always require an N-terminal or C-terminal extension to increase activity, substrate-binding affinity, and/or stability. PMID:23937561

  8. Structural investigations of recombinant urokinase growth factor-like domain.

    Science.gov (United States)

    Beloglazova, I B; Beabealashvilli, R Sh; Gursky, Ya G; Bocharov, E V; Mineev, K S; Parfenova, E V; Tkachuk, V A

    2013-05-01

    Urokinase-type plasminogen activator (uPA) is a serine protease that converts the plasminogen zymogen into the enzymatically active plasmin. uPA is synthesized and secreted as the single-chain molecule (scuPA) composed of an N-terminal domain (GFD) and kringle (KD) and C-terminal proteolytic (PD) domains. Earlier, the structure of ATF (which consists of GFD and KD) was solved by NMR (A. P. Hansen et al. (1994) Biochemistry, 33, 4847-4864) and by X-ray crystallography alone and in a complex with the soluble form of the urokinase receptor (uPAR, CD87) lacking GPI (C. Barinka et al. (2006) J. Mol. Biol., 363, 482-495). According to these data, GFD contains two β-sheet regions oriented perpendicularly to each other. The area in the GFD responsible for binding to uPAR is localized in the flexible Ω-loop, which consists of seven amino acid residues connecting two strings of antiparallel β-sheet. It was shown by site-directed mutagenesis that shortening of the Ω-loop length by one amino acid residue leads to the inability of GFD to bind to uPAR (V. Magdolen et al. (1996) Eur. J. Biochem., 237, 743-751). Here we show that, in contrast to the above-mentioned studies, we found no sign of the β-sheet regions in GFD in our uPA preparations either free or in a complex with uPAR. The GFD seems to be a rather flexible and unstructured domain, demonstrating in spite of its apparent flexibility highly specific interaction with uPAR both in vitro and in cell culture experiments. Circular dichroism, tryptophan fluorescence during thermal denaturation of the protein, and heteronuclear NMR spectroscopy of ¹⁵N/¹³C-labeled ATF both free and in complex with urokinase receptor were used to judge the secondary structure of GFD of uPA. PMID:23848154

  9. Automation of C-terminal sequence analysis of 2D-PAGE separated proteins

    Directory of Open Access Journals (Sweden)

    P.P. Moerman

    2014-06-01

    Full Text Available Experimental assignment of the protein termini remains essential to define the functional protein structure. Here, we report on the improvement of a proteomic C-terminal sequence analysis method. The approach aims to discriminate the C-terminal peptide in a CNBr-digest where Met-Xxx peptide bonds are cleaved in internal peptides ending at a homoserine lactone (hsl-derivative. pH-dependent partial opening of the lactone ring results in the formation of doublets for all internal peptides. C-terminal peptides are distinguished as singlet peaks by MALDI-TOF MS and MS/MS is then used for their identification. We present a fully automated protocol established on a robotic liquid-handling station.

  10. C-Terminally Truncated Derivatives of Escherichia coli Hfq Are Proficient in Riboregulation

    DEFF Research Database (Denmark)

    Olsen, Anders Steno; Møller-Jensen, Jakob; Brennan, Richard G; Valentin-Hansen, Poul

    2010-01-01

    Hfq derivatives, consisting of the conserved core and short C-terminal extensions, to support the regulation of rpoS expression and riboregulation by various well-characterized small regulatory RNAs. Our data show that, in all cases tested, the truncated proteins are fully capable of promoting...... interaction promotes annealing. So far, mutational and structural studies have established that Escherichia coli Hfq contains two separate RNA binding sites that are part of the conserved N-terminal portion of the protein. Moreover, it has been suggested that the nonconserved C-terminal extension of E. coli...... posttranscriptional control, indicating that the C-terminal tail of E. coli Hfq plays a small role or no role in riboregulation....

  11. Structure and function of the interacting domains of Spire and Fmn-family formins

    Energy Technology Data Exchange (ETDEWEB)

    Vizcarra, Christina L.; Kreutz, Barry; Rodal, Avital A.; Toms, Angela V.; Lu, Jun; Zheng, Wei; Quinlan, Margot E.; Eck, Michael J. (UCLA); (Brandeis); (DFCI)

    2012-07-11

    Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.

  12. Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation.

    Science.gov (United States)

    Schmitz, M L; dos Santos Silva, M A; Altmann, H; Czisch, M; Holak, T A; Baeuerle, P A

    1994-10-14

    The p65 subunit of the NF-kappa B transcription factor contains in its C-terminal 120 amino acids at least two transcription activation domains. One domain (TA1) is contained within only the 30 C-terminal amino acids. Structural studies employing CD and NMR spectroscopy revealed that the TA1 domain is unstructured. NMR analysis of a protein corresponding to the C-terminal 123 amino acids also showed a random coil conformation. However, a portion of TA1 was found to adopt an alpha-helical conformation in the presence of hydrophobic solvents. Transcriptional analysis of point mutants revealed the functional importance of two evolutionary conserved sequence repeats, which are located in the conditionally alpha-helical region of TA1. These repeats acted synergistically in transcription activation. The inhibitory effect of some mutants indicated secondary structure constraints on TA1 in intact cells. Inverting the sequence of two acidic activation domains significantly reduced their transactivating potential, suggesting that amino acid composition is not solely essential for activity; a defined primary structure is necessary as well. Acidic sequence motifs related in primary structure and squelching activity to those of TA1 are present in the activation domains of VP16, c-Rel, and several other transcription factors. We propose a model suggesting that primarily unstructured acidic activation domains can adopt a secondary structure upon contacting their target molecules by an "induced fit" mechanism. PMID:7929265

  13. Crystal Structure of the Dynein Motor Domain

    OpenAIRE

    Carter, Andrew P.; Cho, Carol; Jin, Lan; Vale, Ronald D.

    2011-01-01

    Dyneins are microtubule-based motor proteins that power ciliary beating, transport intracellular cargos, and help to construct the mitotic spindle. Evolved from ring-shaped hexameric AAA-family adenosine triphosphatases (ATPases), dynein’s large size and complexity have posed challenges for understanding its structure and mechanism. Here, we present a 6 angstrom crystal structure of a functional dimer of two ~300-kilodalton motor domains of yeast cytoplasmic dynein. The structure reveals an u...

  14. Structure of the caspase-recruitment domain from a zebrafish guanylate-binding protein

    International Nuclear Information System (INIS)

    The crystal structure of the first zebrafish caspase-recruitment domain at 1.47 Å resolution illustrates a six-helix bundle fold similar to that of the human NLRP1 CARD. The caspase-recruitment domain (CARD) mediates homotypic protein–protein interactions that assemble large oligomeric signaling complexes such as the inflammasomes during innate immune responses. Structural studies of the mammalian CARDs demonstrate that their six-helix bundle folds belong to the death-domain superfamily, whereas such studies have not been reported for other organisms. Here, the zebrafish interferon-induced guanylate-binding protein 1 (zIGBP1) was identified that contains an N-terminal GTPase domain and a helical domain typical of the mammalian guanylate-binding proteins, followed by a FIIND domain and a C-terminal CARD similar to the mammalian inflammasome proteins NLRP1 and CARD8. The structure of the zIGBP1 CARD as a fusion with maltose-binding protein was determined at 1.47 Å resolution. This revealed a six-helix bundle fold similar to the NLRP1 CARD structure with the bent α1 helix typical of all known CARD structures. The zIGBP1 CARD surface contains a positively charged patch near its α1 and α4 helices and a negatively charged patch near its α2, α3 and α5 helices, which may mediate its interaction with partner domains. Further studies using binding assays and other analyses will be required in order to address the physiological function(s) of this zebrafish protein

  15. Evolutionary diversification of an ancient gene family (rhs through C-terminal displacement

    Directory of Open Access Journals (Sweden)

    Parkhill Julian

    2009-12-01

    Full Text Available Abstract Background Rhs genes are prominent features of bacterial genomes that have previously been implicated in genomic rearrangements in E. coli. By comparing rhs repertoires across the Enterobacteriaceae, this study provides a robust explanation of rhs diversification and evolution, and a mechanistic model of how rhs diversity is gained and lost. Results Rhs genes are ubiquitous and comprise six structurally distinct lineages within the Enterobacteriaceae. There is considerable intergenomic variation in rhs repertoire; for instance, in Salmonella enterica, rhs are restricted to mobile elements, while in Escherichia coli one rhs lineage has diversified through transposition as older lineages have been deleted. Overall, comparative genomics reveals frequent, independent gene gains and losses, as well as occasional lateral gene transfer, in different genera. Furthermore, we demonstrate that Rhs 'core' domains and variable C-termini are evolutionarily decoupled, and propose that rhs diversity is driven by homologous recombination with circular intermediates. Existing C-termini are displaced by laterally acquired alternatives, creating long arrays of dissociated 'tips' that characterize the appearance of rhs loci. Conclusion Rhs repertoires are highly dynamic among Enterobacterial genomes, due to repeated gene gains and losses. In contrast, the primary structures of Rhs genes are evolutionarily conserved, indicating that rhs sequence diversity is driven, not by rapid mutation, but by the relatively slow evolution of novel core/tip combinations. Hence, we predict that a large pool of dissociated rhs C-terminal tips exists episomally and these are potentially transmitted across taxonomic boundaries.

  16. Structured hints : extracting and abstracting domain expertise.

    Energy Technology Data Exchange (ETDEWEB)

    Hereld, M.; Stevens, R.; Sterling, T.; Gao, G. R.; Mathematics and Computer Science; California Inst. of Tech.; Louisiana State Univ.; Univ. of Delaware

    2009-03-16

    We propose a new framework for providing information to help optimize domain-specific application codes. Its design addresses problems that derive from the widening gap between the domain problem statement by domain experts and the architectural details of new and future high-end computing systems. The design is particularly well suited to program execution models that incorporate dynamic adaptive methodologies for live tuning of program performance and resource utilization. This new framework, which we call 'structured hints', couples a vocabulary of annotations to a suite of performance metrics. The immediate target is development of a process by which a domain expert describes characteristics of objects and methods in the application code that would not be readily apparent to the compiler; the domain expert provides further information about what quantities might provide the best indications of desirable effect; and the interactive preprocessor identifies potential opportunities for the domain expert to evaluate. Our development of these ideas is progressing in stages from case study, through manual implementation, to automatic or semi-automatic implementation. In this paper we discuss results from our case study, an examination of a large simulation of a neural network modeled after the neocortex.

  17. Structural Basis for Phosphotyrosine Recognition by the Src Homology-2 Domains of the Adapter Proteins SH2-B and APS

    Energy Technology Data Exchange (ETDEWEB)

    Hu,J.; Hubbard, S.

    2006-01-01

    SH2-B, APS, and Lnk constitute a family of adapter proteins that modulate signaling by protein tyrosine kinases. These adapters contain an N-terminal dimerization region, a pleckstrin homology domain, and a C-terminal Src homology-2 (SH2) domain. SH2-B is recruited via its SH2 domain to various protein tyrosine kinases, including Janus kinase-2 (Jak2) and the insulin receptor. Here, we present the crystal structure at 2.35 Angstroms resolution of the SH2 domain of SH2-B in complex with a phosphopeptide representing the SH2-B recruitment site in Jak2 (pTyr813). The structure reveals a canonical SH2 domain-phosphopeptide binding mode, but with specific recognition of a glutamate at the +1 position relative to phosphotyrosine, in addition to recognition of a hydrophobic residue at the +3 position. Biochemical studies of SH2-B and APS demonstrate that, although the SH2 domains of these two adapter proteins share 79% sequence identity, the SH2-B SH2 domain binds preferentially to Jak2, whereas the APS SH2 domain has higher affinity for the insulin receptor. This differential specificity is attributable to the difference in the oligomeric states of the two SH2 domains: monomeric for SH2-B and dimeric for APS.

  18. Structural characterization of the regulatory domain of brain carnitine palmitoyltransferase 1.

    Science.gov (United States)

    Samanta, Soma; Situ, Alan J; Ulmer, Tobias S

    2014-04-01

    Neurons contain a mammalian-specific isoform of the enzyme carnitine palmitoyltransferase 1 (CPT1C) that couples malonyl-CoA to ceramide levels thereby contributing to systemic energy homeostasis and feeding behavior. In contrast to CPT1A, which controls the rate-limiting step of long-chain fatty acid β-oxidation in all tissues, the biochemical context and regulatory mechanism of CPT1C are unknown. CPT1 enzymes are comprised of an N-terminal regulatory domain and a C-terminal catalytic domain (CD) that are separated by two transmembrane helices. In CPT1A, the regulatory domain, termed N, adopts an inhibitory and non-inhibitory state, Nα and Nβ, respectively, which differ in their association with the CD. To provide insight into the regulatory mechanism of CPT1C, we have determined the structure of its regulatory domain (residues Met1-Phe50) by NMR spectroscopy. In relation to CPT1A, the inhibitory Nα state was found to be structurally homologues whereas the non-inhibitory Nβ state was severely destabilized, suggesting a change in overall regulation. The destabilization of Nβ may contribute to the low catalytic activity of CPT1C relative to CPT1A and makes its association with the CD unlikely. In analogy to the stabilization of Nβ by the CPT1A CD, non-inhibitory interactions of N of CPT1C with another protein may exist. PMID:24037959

  19. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

    OpenAIRE

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; Glover, J. N. Mark

    2009-01-01

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC282–202, which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DN...

  20. Fasciola hepatica calcium-binding protein FhCaBP2: structure of the dynein light chain-like domain.

    Science.gov (United States)

    Nguyen, Thanh H; Thomas, Charlotte M; Timson, David J; van Raaij, Mark J

    2016-07-01

    The common liver fluke Fasciola hepatica causes an increasing burden on human and animal health, partly because of the spread of drug-resistant isolates. As a consequence, there is considerable interest in developing new drugs to combat liver fluke infections. A group of potential targets is a family of calcium-binding proteins which combine an N-terminal domain with two EF-hand motifs and a C-terminal domain with predicted similarity to dynein light chains (DLC-like domain). The function of these proteins is unknown, although in several species, they have been localised to the tegument, an important structure at the host-parasite interface. Here, we report the X-ray crystal structure of the DLC-like domain of F. hepatica calcium-binding protein 2 (FhCaBP2), solved using single-wavelength anomalous diffraction and refined at 2.3 Å resolution in two different crystal forms. The FhCaBP2 DLC-like domain has a structure similar to other DLC domains, with an anti-parallel β-sheet packed against an α-helical hairpin. Like other DLC domains, it dimerises through its β2-strand, which extends in an arch and forms the fifth strand in an extended β-sheet of the other monomer. The structure provides molecular details of the dimerisation of FhCaBP2, the first example from this family of parasite proteins. PMID:27083189

  1. Multilevel domain decomposition for electronic structure calculations

    International Nuclear Information System (INIS)

    We introduce a new multilevel domain decomposition method (MDD) for electronic structure calculations within semi-empirical and density functional theory (DFT) frameworks. This method iterates between local fine solvers and global coarse solvers, in the spirit of domain decomposition methods. Using this approach, calculations have been successfully performed on several linear polymer chains containing up to 40,000 atoms and 200,000 atomic orbitals. Both the computational cost and the memory requirement scale linearly with the number of atoms. Additional speed-up can easily be obtained by parallelization. We show that this domain decomposition method outperforms the density matrix minimization (DMM) method for poor initial guesses. Our method provides an efficient preconditioner for DMM and other linear scaling methods, variational in nature, such as the orbital minimization (OM) procedure

  2. Physical association of GPR54 C-terminal with protein phosphatase 2A

    International Nuclear Information System (INIS)

    KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.

  3. Structural Analyses of the Ankyrin Repeat Domain of TRPV6 and Related TRPV Ion Channels

    Energy Technology Data Exchange (ETDEWEB)

    Phelps, C.B.; Huang, R.J.; Lishko, P.V.; Wang, R.R.; Gaudet, R. (Harvard)

    2008-06-03

    Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.

  4. Crystal structure and functional analysis of Dcp2p from Schizosaccharomyces pombe

    OpenAIRE

    She, Meipei; Decker, Carolyn J.; Chen, Nan; Tumati, Suneeta; Parker, Roy; Song, Haiwei

    2005-01-01

    Decapping is a key step in both general and nonsense-mediated 5′ → ′ mRNA-decay pathways. Removal of the cap structure is catalyzed by the Dcp1–Dcp2 complex. The crystal structure of a C-terminally truncated Schizosaccharomyces pombe Dcp2p reveals two distinct domains: an all-helical N-terminal domain and a C-terminal domain that is a classic Nudix fold. The C-terminal domain of both Saccharomyces cerevisiae and S. pombe Dcp2p proteins is sufficient for decapping activity, although the N-term...

  5. Structural and Functional Studies of the Ras-Associating and Pleckstrin Homology Domains of Grb10 and Grb14

    Energy Technology Data Exchange (ETDEWEB)

    Depetris, R.; Wu, J; Hubbard, S

    2009-01-01

    Growth factor receptor-binding proteins Grb7, Grb10 and Grb14 are adaptor proteins containing a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region and a C-terminal Src-homology-2 domain. Previous structural studies showed that the Grb14 BPS region binds as a pseudosubstrate inhibitor in the tyrosine kinase domain of the insulin receptor to suppress insulin signaling. Here we report the crystal structure of the RA and PH domains of Grb10 at 2.6-A resolution. The structure reveals that these two domains, along with the intervening linker, form an integrated, dimeric structural unit. Biochemical studies demonstrated that Grb14 binds to activated Ras, which may serve as a timing mechanism for downregulation of insulin signaling. Our results illuminate the membrane-recruitment mechanisms not only of Grb7, Grb10 and Grb14 but also of MIG-10, Rap1-interacting adaptor molecule, lamellipodin and Pico, proteins involved in actin-cytoskeleton rearrangement that share a structurally related RA-PH tandem unit.

  6. Synaptic Vesicle Tethering and the CaV2.2 Distal C-terminal

    Directory of Open Access Journals (Sweden)

    Fiona K Wong

    2014-03-01

    Full Text Available . Evidence that synaptic vesicles (SVs can be gated by a single voltage sensitive calcium channel (CaV2.2 predict a molecular linking mechanism or ‘tether’[Stanley 1993]. Recent studies have proposed that the SV binds to the distal C-terminal on the CaV2.2 calcium channel [Kaeser et al. 2011;Wong, Li, and Stanley 2013] while genetic analysis proposed a double tether mechanism via RIM: directly to the C terminus PDZ ligand domain or indirectly via a more proximal proline rich site [Kaeser et al. 2011]. Using a novel in vitro SV-PD binding assay, we reported that SVs bind to a fusion protein comprising the C-terminal distal third (C3, aa 2137-2357 [Wong, Li, and Stanley 2013]. Here we limit the binding site further to the last 58 aa, beyond the proline rich site, by the absence of SV capture by a truncated C3 fusion protein (aa 2137-2299. To test PDZ-dependent binding we generated two C terminus-mutant C3 fusion proteins and a mimetic blocking peptide (H-WC, aa 2349-2357 and validated these by elimination of MINT-1 or RIM binding. Persistence of SV capture with all three fusion proteins or with the full length C3 protein but in the presence of the blocking peptide, demonstrated that SVs can bind to the distal C-terminal via a PDZ-independent mechanism. These results were supported in situ by normal SV turnover in H-WC-loaded synaptosomes, as assayed by a novel peptide cryoloading method. Thus, SVs tether to the CaV2.2 C-terminal within a 49 aa region immediately prior to the terminus PDZ ligand domain. Long tethers that could reflect extended C termini were imaged by electron microscopy of synaptosome ghosts. To fully account for SV tethering we propose a model where SVs are initially captured, or ‘grabbed’, from the cytoplasm by a binding site on the distal region of the channel C-terminal and are then retracted to be ‘locked’ close to the channel by a second attachment mechanism in preparation for single channel domain gating.

  7. Structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor, P

    Energy Technology Data Exchange (ETDEWEB)

    Green, Todd J.; Luo, Ming; (UAB-MED)

    2009-10-05

    The negative-strand RNA viruses (NSRVs) are unique because their nucleocapsid, not the naked RNA, is the active template for transcription and replication. The viral polymerase of nonsegmented NSRVs contains a large polymerase catalytic subunit (L) and a nonenzymatic cofactor, the phosphoprotein (P). Insight into how P delivers the polymerase complex to the nucleocapsid has long been pursued by reverse genetics and biochemical approaches. Here, we present the X-ray crystal structure of the C-terminal domain of P of vesicular stomatitis virus, a prototypic nonsegmented NSRV, bound to nucleocapsid-like particles. P binds primarily to the C-terminal lobe of 2 adjacent N proteins within the nucleocapsid. This binding mode is exclusive to the nucleocapsid, not the nucleocapsid (N) protein in other existing forms. Localization of phosphorylation sites within P and their proximity to the RNA cavity give insight into how the L protein might be oriented to access the RNA template.

  8. Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria

    Directory of Open Access Journals (Sweden)

    Lindblad Peter

    2003-05-01

    Full Text Available Abstract Background Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102, only the bidirectional (Synechocystis PCC 6803 or both NiFe-hydrogenases (Anabaena PCC 7120 prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above. Results We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41% to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions. Conclusion Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known

  9. Domain structure of black hole space-times

    International Nuclear Information System (INIS)

    We introduce the domain structure for stationary black hole space-times. The domain structure lives on the submanifold of fixed points of the Killing vector fields. Depending on which Killing vector field has fixed points the submanifold is naturally divided into domains. The domain structure provides invariants of the space-time, both topological and continuous. It is defined for any space-time dimension and any number of Killing vector fields. We examine the domain structure for asymptotically flat space-times and find a canonical form for the metric of such space-times. The domain structure generalizes the rod structure introduced for space-times with D-2 commuting Killing vector fields. We analyze in detail the domain structure for Minkowski space, the Schwarzschild-Tangherlini black hole and the Myers-Perry black hole in six and seven dimensions. Finally, we consider the possible domain structures for asymptotically flat black holes in six and seven dimensions.

  10. [Construction and expression of six deletion mutants of human astrovirus C-terminal nsP1a/4 protein].

    Science.gov (United States)

    Zhao, Wei; Niu, Ke; Zhao, Jian; Jin, Yi-ming; Sui, Ting-ting; Wang, Wen

    2013-09-01

    Human astrovirus (HAstV) is one of the leading causes of actue virual diarrhea in infants. HAstV-induced epithdlial cell apoptosis plays an important role in the pathogenesis of HAstV infection. Our previous study indicated that HAstV non-structural protein nsPla C-terminal protein nsPla/4 was the major apoptosis functional protein and probably contained the main apoptosis domains. In order to screen for astrovirus encoded apoptotic protien, nsPla/4 and six turncated proteins, which possessed nsPla/4 protein different function domain ,were cloned into green fluorescent protein (GFP) vector pEG-FP-N3. After 24-72 h transfection, the fusion protein expression in BHK21 cells, was analysis by fluorescence microscope and Western blot. The results indicated seven fusion proteins were observed successfully in BHK21 cell after transfected for 24 h. Western blot analysis showed that the level of fusion protein expressed in BHK21 cells was increased significantly at 72h compared to 48h in transfected cells. The successful expression of deletion mutants of nsPla/4 protein was an important foundation to gain further insights into the function of apoptosis domains of nsPla/4 protein and it would also provide research platform to further confirm the molecule pathogenic mechanism of human astrovirus. PMID:24386845

  11. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

    Science.gov (United States)

    Williams, Alison A; Mehler, Vera J; Mueller, Christina; Vonhoff, Fernando; White, Robin; Duch, Carsten

    2016-01-01

    Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2. PMID:27442528

  12. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

    Directory of Open Access Journals (Sweden)

    Alison A Williams

    Full Text Available Methyl-CpG binding protein 2 (MeCP2 is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X, a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80 and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-27

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

  14. The structure of SSO2064, the first representative of Pfam family PF01796, reveals a novel two-domain zinc-ribbon OB-fold architecture with a potential acyl-CoA-binding role

    International Nuclear Information System (INIS)

    The crystal structure of SSO2064, the first structural representative of Pfam family PF01796 (DUF35), reveals a two-domain architecture comprising an N-terminal zinc-ribbon domain and a C-terminal OB-fold domain. Analysis of the domain architecture, operon organization and bacterial orthologs combined with the structural features of SSO2064 suggests a role involving acyl-CoA binding for this family of proteins. SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein–protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0)

  15. Insights into function of PSI domains from structure of the Met receptor PSI domain

    International Nuclear Information System (INIS)

    PSI domains are cysteine-rich modules found in extracellular fragments of hundreds of signaling proteins, including plexins, semaphorins, integrins, and attractins. Here, we report the solution structure of the PSI domain from the human Met receptor, a receptor tyrosine kinase critical for proliferation, motility, and differentiation. The structure represents a cysteine knot with short regions of secondary structure including a three-stranded antiparallel β-sheet and two α-helices. All eight cysteines are involved in disulfide bonds with the pattern consistent with that for the PSI domain from Sema4D. Comparison with the Sema4D structure identifies a structurally conserved core comprising the N-terminal half of the PSI domain. Interestingly, this part links adjacent SEMA and immunoglobulin domains in the Sema4D structure, suggesting that the PSI domain serves as a wedge between propeller and immunoglobulin domains and is responsible for the correct positioning of the ligand-binding site of the receptor

  16. The structure of the FnI-EGF-like tandem domain of coagulation factor XII solved using SIRAS

    International Nuclear Information System (INIS)

    The structure of the FnI-EGF-like tandem domain was solved to 1.6 Å using data sets of native and holmium chloride bound protein. Putative biological roles of this tandem domain are discussed based on the structure. Coagulation factor XII (FXII) is a key protein in the intrinsic coagulation and kallikrein–kinin pathways. It has been found that negative surfaces and amyloids, such as Aβ fibrils, can activate FXII. Additionally, it has been suggested that FXII simulates cells and that it plays an important role in thrombosis. To date, no structural data on FXII have been deposited, which makes it difficult to support any hypothesis on the mechanism of FXII function. The crystal structure of the FnI-EGF-like tandem domain of FXII presented here was solved using experimental phases. To determine the phases, a SIRAS approach was used with a native and a holmium chloride-soaked data set. The holmium cluster was coordinated by the C-terminal tails of two symmetry-related molecules. Another observation was that the FnI domain was much more ordered than the EGF-like domain owing to crystal packing. Furthermore, the structure shows the same domain orientation as the homologous FnI-EGF-like tandem domain of tPA. The plausibility of several proposed interactions of these domains of FXII is discussed. Based on this FXII FnI-EGF-like structure, it could be possible that FXII binding to amyloid and negatively charged surfaces is mediated via this part of FXII

  17. Modulation of voltage-gated potassium Kv2.1 via the cytoplasmic C terminal

    Institute of Scientific and Technical Information of China (English)

    Man Jin; Peiyuan Lu

    2011-01-01

    Voltage-gated potassium channels comprise 12 subtypes (Kv1-Kv12). Kv2.1, which is expressed in most mammalian central neurons, provides the majority of delayed-rectifier K current in cortical and hippocampal pyramidal neurons, and plays an especially prominent role in repolarizing membrane potential, as well as in facilitation of exocytosis. Kv2.1-encoded K efflux is essential for neuronal apoptosis programming. The human form of the Kv2.1 potassium channel contains large intracellular regions. The cytoplasmic C-terminal plays a key role in modulating Kv2.1 gating. The present manuscript summarized Kv2.1 structure and modulation in neurons and analyzed the roles of the cytoplasmic C-terminal.

  18. Reuse of structural domain–domain interactions in protein networks

    OpenAIRE

    Bateman Alex; Schuster-Böckler Benjamin

    2007-01-01

    Abstract Background Protein interactions are thought to be largely mediated by interactions between structural domains. Databases such as iPfam relate interactions in protein structures to known domain families. Here, we investigate how the domain interactions from the iPfam database are distributed in protein interactions taken from the HPRD, MPact, BioGRID, DIP and IntAct databases. Results We find that known structural domain interactions can only explain a subset of 4–19% of the available...

  19. Relationship of Structure and Function of DNA-Binding Domain in Vitamin D Receptor

    Directory of Open Access Journals (Sweden)

    Lin-Yan Wan

    2015-07-01

    Full Text Available While the structure of the DNA-binding domain (DBD of the vitamin D receptor (VDR has been determined in great detail, the roles of its domains and how to bind the motif of its target genes are still under debate. The VDR DBD consists of two zinc finger modules and a C-terminal extension (CTE, at the end of the C-terminal of each structure presenting α-helix. For the first zinc finger structure, N37 and S-box take part in forming a dimer with 9-cis retinoid X receptor (RXR, while V26, R50, P-box and S-box participate in binding with VDR response elements (VDRE. For the second zinc finger structure, P61, F62 and H75 are essential in the structure of the VDR homodimer with the residues N37, E92 and F93 of the downstream of partner VDR, which form the inter-DBD interface. T-box of the CTE, especially the F93 and I94, plays a critical role in heterodimerization and heterodimers–VDRE binding. Six essential residues (R102, K103, M106, I107, K109, and R110 of the CTE α-helix of VDR construct one interaction face, which packs against the DBD core of the adjacent symmetry mate. In 1,25(OH2D3-activated signaling, the VDR-RXR heterodimer may bind to DR3-type VDRE and ER9-type VDREs of its target gene directly resulting in transactivation and also bind to DR3-liked nVDRE of its target gene directly resulting in transrepression. Except for this, 1α,25(OH2D3 ligand VDR-RXR may bind to 1αnVDRE indirectly through VDIR, resulting in transrepression of the target gene. Upon binding of 1α,25(OH2D3, VDR can transactivate and transrepress its target genes depending on the DNA motif that DBD binds.

  20. 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.

  1. 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.

  2. Crystal Structure of the Mycoplasma arthritidis-Derived Mitogen in Apo Form Reveals a 3D Domain-Swapped Dimer

    Energy Technology Data Exchange (ETDEWEB)

    Liu, L.; Li, Z; Guo, Y; VanVranken, S; Mourad, W; Li, H

    2010-01-01

    Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular V{beta} elements of T cell receptor. Here, we report the crystal structure of a MAM mutant K201A in apo form (unliganded) at 2.8-{angstrom} resolutions. We also partially refined the crystal structures of the MAM wild type and another MAM mutant L50A in apo forms at low resolutions. Unexpectedly, the structures of these apo MAM molecules display a three-dimensional domain-swapped dimer. The entire C-terminal domains of these MAM molecules are involved in the domain swapping. Functional analyses demonstrated that the K201A and L50A mutants do not show altered ability to bind to their host receptors and that they stimulate the activation of T cells as efficiently as does the wild type. Structural comparisons indicated that the 'reconstituted' MAM monomer from the domain-swapped dimer displays large differences at the hinge regions from the MAM{sub wt} molecule in the receptor-bound form. Further comparison indicated that MAM has a flexible N-terminal loop, implying that conformational changes could occur upon receptor binding.

  3. Structural biology of the LRRK2 GTPase and Kinase domains: Implications for regulation

    Directory of Open Access Journals (Sweden)

    Arjan Kortholt

    2014-05-01

    Full Text Available Human leucine-rich-repeat kinase 2 (LRRK2 belongs to the Roco family of proteins, which are characterized by the presence of a Ras-like G-domain (Roc, a C-terminal of Roc domain (COR, and a kinase domain. Mutations in LRRK2 have been found to be thus far the most frequent cause of late-onset Parkinson’s disease (PD. Several of the pathogenic mutations in LRRK2 result in decreased GTPase activity and enhanced kinase activity, suggesting a possible PD-related gain of abnormal function. Important progress in the structural understanding of LRRK2 has come from our work with related Roco proteins from lower organisms. Atomic structures of Roco proteins from prokaryotes revealed that Roco proteins belong to the GAD class of molecular switches (G proteins activated by nucleotide dependent dimerization. As in LRRK2, PD-analogous mutations in Roco proteins from bacteria decrease the GTPase reaction. Studies with Roco proteins from the model organism Dictyostelium discoideum revealed that PD mutants have different effects and most importantly they explained the G2019S-related increased LRRK2 kinase activity. Furthermore, the structure of Dictyostelium Roco4 kinase in complex with the LRRK2 inhibitor H1152 showed that Roco4 and other Roco family proteins can be important for the optimization of the current, and identification of new, LRRK2 kinase inhibitors. In this review we highlight the recent progress in structural and biochemical characterization of Roco proteins and discuss its implication for the understanding of the complex regulatory mechanism of LRRK2.

  4. Crystal Structure of the APOBEC3G Catalytic Domain Reveals Potential Oligomerization Interfaces

    Energy Technology Data Exchange (ETDEWEB)

    Shandilya, Shivender M.D.; Nalam, Madhavi N.L.; Nalivaika, Ellen A.; Gross, Phillip J.; Valesano, Johnathan C.; Shindo, Keisuke; Li, Ming; Munson, Mary; Royer, William E.; Harjes, Elena; Kono, Takahide; Matsuo, Hiroshi; Harris, Reuben S.; Somasundaran, Mohan; Schiffer, Celia A. (UMASS, MED); (UMM)

    2010-02-11

    APOBEC3G is a DNA cytidine deaminase that has antiviral activity against HIV-1 and other pathogenic viruses. In this study the crystal structure of the catalytically active C-terminal domain was determined to 2.25 {angstrom}. This structure corroborates features previously observed in nuclear magnetic resonance (NMR) studies, a bulge in the second {beta} strand and a lengthening of the second {alpha} helix. Oligomerization is postulated to be critical for the function of APOBEC3G. In this structure, four extensive intermolecular interfaces are observed, suggesting potential models for APOBEC3G oligomerization. The structural and functional significance of these interfaces was probed by solution NMR and disruptive variants were designed and tested for DNA deaminase and anti-HIV activities. The variant designed to disrupt the most extensive interface lost both activities. NMR solution data provides evidence that another interface, which coordinates a novel zinc site, also exists. Thus, the observed crystallographic interfaces of APOBEC3G may be important for both oligomerization and function.

  5. Structure and Specificity of a Binary Tandem Domain F-Lectin from Striped Bass (Morone saxatilis)

    Energy Technology Data Exchange (ETDEWEB)

    Bianchet, M.; Odom, E; Vasta, J; Amzel, M

    2010-01-01

    The plasma of the striped bass Morone saxatilis contains a fucose-specific lectin (MsaFBP32) that consists of two F-type carbohydrate recognition domains (CRDs) in tandem. The crystal structure of the complex of MsaFBP32 with l-fucose reported here shows a cylindrical 81-A-long and 60-A-wide trimer divided into two globular halves: one containing N-terminal CRDs (N-CRDs) and the other containing C-terminal CRDs (C-CRDs). The resulting binding surfaces at the opposite ends of the cylindrical trimer have the potential to cross-link cell surface or humoral carbohydrate ligands. The N-CRDs and C-CRDs of MsaFBP32 exhibit significant structural differences, suggesting that they recognize different glycans. Analysis of the carbohydrate binding sites provides the structural basis for the observed specificity of MsaFBP32 for simple carbohydrates and suggests that the N-CRD recognizes more complex fucosylated oligosaccharides and with a relatively higher avidity than the C-CRD. Modeling of MsaFBP32 complexed with fucosylated glycans that are widely distributed in prokaryotes and eukaryotes rationalizes the observation that binary tandem CRD F-type lectins function as opsonins by cross-linking 'non-self' carbohydrate ligands and 'self' carbohydrate ligands, such as sugar structures displayed by microbial pathogens and glycans on the surface of phagocytic cells from the host.

  6. Low resolution solution structure of HAMLET and the importance of its alpha-domains in tumoricidal activity.

    Directory of Open Access Journals (Sweden)

    C S James Ho

    Full Text Available HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells.

  7. Identification of antigenic domains in the non-structural protein of Muscovy duck parvovirus.

    Science.gov (United States)

    Yu, Tian-Fei; Li, Ming; Yan, Bing; Shao, Shu-Li; Fan, Xing-Dong; Wang, Jia; Wang, Dan-Na

    2016-08-01

    Muscovy duck parvovirus (MDPV) infection is widespread in many Muscovy-duck-farming countries, leading to a huge economic loss. By means of overlapping peptides expressed in Escherichia coli in combination with Western blot, antigenic domains on the non-structural protein (NSP) of MDPV were identified for the first time. On the Western blot, the fragments NS(481-510), NS (501-530), NS (521-550), NS (541-570), NS (561-590), NS (581-610) and NS (601-627) were positive (the numbers in parentheses indicate the location of amino acids), and other fragments were negative. These seven fragments were also reactive in an indirect enzyme-linked immunosorbent assay (i-ELISA). We therefore conclude that a linear antigenic domain of the NSP is located at its C-terminal end (amino acid residues 481-627). These results may facilitate future investigations into the function of NSP of MDPV and the development of immunoassays for the diagnosis of MDPV infection. PMID:27154558

  8. Structure of a Thyroid Hormone Receptor DNA-Binding Domain Homodimer Bound to an Inverted Palindrome DNA Response Element

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yi; Young, Matthew A. (Michigan)

    2010-10-22

    Thyroid hormone receptor (TR), as a member of the nuclear hormone receptor family, can recognize and bind different classes of DNA response element targets as either a monomer, a homooligomer, or a heterooligomer. We report here the first crystal structure of a homodimer TR DNA-binding domain (DBD) in complex with an inverted repeat class of thyroid response element (TRE). The structure shows a nearly symmetric structure of the TR DBD assembled on the F2 TRE where the base recognition contacts in the homodimer DNA complex are conserved relative to the previously published structure of a TR-9-cis-retinoic acid receptor heterodimer DNA complex. The new structure also reveals that the T-box region of the DBD can function as a structural hinge that enables a large degree of flexibility in the position of the C-terminal extension helix that connects the DBD to the ligand-binding domain. Although the isolated TR DBDs exist as monomers in solution, we have measured highly cooperative binding of the two TR DBD subunits onto the inverted repeat DNA sequence. This suggests that elements of the DBD can influence the specific TR oligomerization at target genes, and it is not just interactions between the ligand-binding domains that are responsible for TR oligomerization at target genes. Mutational analysis shows that intersubunit contacts at the DBD C terminus account for some, but not all, of the cooperative homodimer TR binding to the inverted repeat class TRE.

  9. Phase transitions and domain structures in multiferroics

    Science.gov (United States)

    Vlahos, Eftihia

    2011-12-01

    Thin film ferroelectrics and multiferroics are two important classes of materials interesting both from a scientific and a technological prospective. The volatility of lead and bismuth as well as environmental issues regarding the toxicity of lead are two disadvantages of the most commonly used ferroelectric random access memory (FeRAM) materials such as Pb(Zr,Ti)O3 and SrBi2Ta2O9. Therefore lead-free thin film ferroelectrics are promising substitutes as long as (a) they can be grown on technologically important substrates such as silicon, and (b) their T c and Pr become comparable to that of well established ferroelectrics. On the other hand, the development of functional room temperature ferroelectric ferromagnetic multiferroics could lead to very interesting phenomena such as control of magnetism with electric fields and control of electrical polarization with magnetic fields. This thesis focuses on the understanding of material structure-property relations using nonlinear optical spectroscopy. Nonlinear spectroscopy is an excellent tool for probing the onset of ferroelectricity, and domain dynamics in strained ferroelectrics and multiferroics. Second harmonic generation was used to detect ferroelectricity and the antiferrodistortive phase transition in thin film SrTiO3. Incipient ferroelectric CaTiO3 has been shown to become ferroelectric when strained with a combination of SHG and dielectric measurements. The tensorial nature of the induced nonlinear polarization allows for probing of the BaTiO3 and SrTiO3 polarization contributions in nanoscale BaTiO3/SrTiO3 superlattices. In addition, nonlinear optics was used to demonstrate ferroelectricity in multiferroic EuTiO3. Finally, confocal SHG and Raman microscopy were utilized to visualize polar domains in incipient ferroelectric and ferroelastic CaTiO3.

  10. All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors

    Science.gov (United States)

    Mohammadiarani, Hossein; Vashisth, Harish

    2016-01-01

    The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane–solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane–solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor. PMID

  11. Crystal structure of the sensory domain of Escherichia coli CadC, a member of the ToxR-like protein family

    OpenAIRE

    Eichinger, Andreas; Haneburger, Ina; Koller, Christiane; Jung, Kirsten; Skerra, Arne

    2011-01-01

    The membrane-integral transcriptional activator CadC comprises sensory and transcriptional regulatory functions within one polypeptide chain. Its C-terminal periplasmic domain, CadCpd, is responsible for sensing of environmental pH as well as for binding of the feedback inhibitor cadaverine. Here we describe the crystal structure of CadCpd (residues 188–512) solved at a resolution of 1.8 Å via multiple wavelength anomalous dispersion (MAD) using a ReCl62− derivative. CadCpd reveals a novel fo...

  12. A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain.

    Science.gov (United States)

    González-Rivera, Christian; Campbell, Anne M; Rutherford, Stacey A; Pyburn, Tasia M; Foegeding, Nora J; Barke, Theresa L; Spiller, Benjamin W; McClain, Mark S; Ohi, Melanie D; Lacy, D Borden; Cover, Timothy L

    2016-09-01

    Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly β-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the β-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the β-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly. PMID:27382020

  13. Sequential 1H NMR assignments and secondary structure of the B domain of staphylococcal protein A: Structural changes between the free B domain in solution and the Fc-bound B domain in crystal

    International Nuclear Information System (INIS)

    The recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G (IgG), has been investigated with the use of two-dimensional proton nuclear magnetic resonance spectroscopy. All backbone and side-chain proton resonances of FB (60 amino acid residues), except the amide proton resonance of Ala2, were assigned by the sequential assignment procedures by using double-quantum-filtered correlated spectroscopy (DQF-COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), and nuclear Overhauser enhancement spectroscopy (NOESY). On the basis of the NOESY data, three helical regions, Glu9-His19, Glu25-Asp37, and Ser42-Ala55, were identified in the free FB in solution. Existence of two of the three helical regions, Glu9-His19 and Glu25-Asp37, is consistent with the X-ray crystallographic structure of the Fc-bound FB. By contrast, in the Fc-bound FB as revealed by the X-ray analysis, the Ser42-Glu48 segment and no structural information has been available in the Ala49-Ala55 segment. The authors suggest that a significant conformation change is induced in the C-terminal region of FB when it is bound to the Fc portion of IgG

  14. Computational Methods for Domain Partitioning of Protein Structures

    Science.gov (United States)

    Veretnik, Stella; Shindyalov, Ilya

    Analysis of protein structures typically begins with decomposition of structure into more basic units, called "structural domains". The underlying goal is to reduce a complex protein structure to a set of simpler yet structurally meaningful units, each of which can be analyzed independently. Structural semi-independence of domains is their hallmark: domains often have compact structure and can fold or function independently. Domains can undergo so-called "domain shuffling"when they reappear in different combinations in different proteins thus implementing different biological functions (Doolittle, 1995). Proteins can then be conceived as being built of such basic blocks: some, especially small proteins, consist usually of just one domain, while other proteins possess a more complex architecture containing multiple domains. Therefore, the methods for partitioning a structure into domains are of critical importance: their outcome defines the set of basic units upon which structural classifications are built and evolutionary analysis is performed. This is especially true nowadays in the era of structural genomics. Today there are many methods that decompose the structure into domains: some of them are manual (i.e., based on human judgment), others are semiautomatic, and still others are completely automatic (based on algorithms implemented as software). Overall there is a high level of consistency and robustness in the process of partitioning a structure into domains (for ˜80% of proteins); at least for structures where domain location is obvious. The picture is less bright when we consider proteins with more complex architectures—neither human experts nor computational methods can reach consistent partitioning in many such cases. This is a rather accurate reflection of biological phenomena in general since domains are formed by different mechanisms, hence it is nearly impossible to come up with a set of well-defined rules that captures all of the observed cases.

  15. The Crystal Structure of Escherichia coli Group 4 Capsule Protein GfcC Reveals a Domain Organization Resembling That of Wza

    Energy Technology Data Exchange (ETDEWEB)

    Sathiyamoorthy, Karthik; Mills, Erez; Franzmann, Titus M.; Rosenshine, Ilan; Saper, Mark A. (Michigan); (Hebrew)

    2012-03-15

    We report the 1.9 {angstrom} resolution crystal structure of enteropathogenic Escherichia coli GfcC, a periplasmic protein encoded by the gfc operon, which is essential for assembly of group 4 polysaccharide capsule (O-antigen capsule). Presumed gene orthologs of gfcC are present in capsule-encoding regions of at least 29 genera of Gram-negative bacteria. GfcC, a member of the DUF1017 family, is comprised of tandem {beta}-grasp (ubiquitin-like) domains (D2 and D3) and a carboxyl-terminal amphipathic helix, a domain arrangement reminiscent of that of Wza that forms an exit pore for group 1 capsule export. Unlike the membrane-spanning C-terminal helix from Wza, the GfcC C-terminal helix packs against D3. Previously unobserved in a {beta}-grasp domain structure is a 48-residue helical hairpin insert in D2 that binds to D3, constraining its position and sequestering the carboxyl-terminal amphipathic helix. A centrally located and invariant Arg115 not only is essential for proper localization but also forms one of two mostly conserved pockets. Finally, we draw analogies between a GfcC protein fused to an outer membrane {beta}-barrel pore in some species and fusion proteins necessary for secreting biofilm-forming exopolysaccharides.

  16. The crystal structure of Escherichia coli group 4 capsule protein GfcC reveals a domain organization resembling that of Wza.

    Science.gov (United States)

    Sathiyamoorthy, Karthik; Mills, Erez; Franzmann, Titus M; Rosenshine, Ilan; Saper, Mark A

    2011-06-21

    We report the 1.9 Å resolution crystal structure of enteropathogenic Escherichia coli GfcC, a periplasmic protein encoded by the gfc operon, which is essential for assembly of group 4 polysaccharide capsule (O-antigen capsule). Presumed gene orthologs of gfcC are present in capsule-encoding regions of at least 29 genera of Gram-negative bacteria. GfcC, a member of the DUF1017 family, is comprised of tandem β-grasp (ubiquitin-like) domains (D2 and D3) and a carboxyl-terminal amphipathic helix, a domain arrangement reminiscent of that of Wza that forms an exit pore for group 1 capsule export. Unlike the membrane-spanning C-terminal helix from Wza, the GfcC C-terminal helix packs against D3. Previously unobserved in a β-grasp domain structure is a 48-residue helical hairpin insert in D2 that binds to D3, constraining its position and sequestering the carboxyl-terminal amphipathic helix. A centrally located and invariant Arg115 not only is essential for proper localization but also forms one of two mostly conserved pockets. Finally, we draw analogies between a GfcC protein fused to an outer membrane β-barrel pore in some species and fusion proteins necessary for secreting biofilm-forming exopolysaccharides. PMID:21449614

  17. One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

    Directory of Open Access Journals (Sweden)

    Merkx Maarten

    2008-10-01

    Full Text Available Abstract Background Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed. Results A novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation. Conclusion An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.

  18. Domain Structures in Nematic Liquid Crystals on a Polycarbonate Surface

    OpenAIRE

    Vasily F. Shabanov; Parshin, Alexander M.; Victor Y. Zyryanov; Gunyakov, Vladimir A.

    2013-01-01

    Alignment of nematic liquid crystals on polycarbonate films obtained with the use of solvents with different solvations is studied. Domain structures occurring during the growth on the polymer surface against the background of the initial thread-like or schlieren texture are demonstrated. It is established by optical methods that the domains are stable formations visualizing the polymer surface structures. In nematic droplets, the temperature-induced transition from the domain structure with ...

  19. Tunable conductance of magnetic nanowires with structured domain walls.

    Science.gov (United States)

    Dugaev, V K; Berakdar, J; Barnaś, J

    2006-02-01

    We show that in a magnetic nanowire with double magnetic domain walls, quantum interference results in spin-split quasistationary states localized mainly between the domain walls. Spin-flip-assisted transmission through the domain structure increases strongly when these size-quantized states are tuned on resonance with the Fermi energy, e.g., upon varying the distance between the domain walls which results in resonance-type peaks of the wire conductance. This novel phenomenon is shown to be utilizable to manipulate the spin density in the domain vicinity. The domain wall parameters are readily controllable, and the predicted effect is hence exploitable in spintronic devices. PMID:16486888

  20. Nonlinear dynamics of C-terminal tails in cellular microtubules.

    Science.gov (United States)

    Sekulic, Dalibor L; Sataric, Bogdan M; Zdravkovic, Slobodan; Bugay, Aleksandr N; Sataric, Miljko V

    2016-07-01

    The mechanical and electrical properties, and information processing capabilities of microtubules are the permanent subject of interest for carrying out experiments in vitro and in silico, as well as for theoretical attempts to elucidate the underlying processes. In this paper, we developed a new model of the mechano-electrical waves elicited in the rows of very flexible C-terminal tails which decorate the outer surface of each microtubule. The fact that C-terminal tails play very diverse roles in many cellular functions, such as recruitment of motor proteins and microtubule-associated proteins, motivated us to consider their collective dynamics as the source of localized waves aimed for communication between microtubule and associated proteins. Our approach is based on the ferroelectric liquid crystal model and it leads to the effective asymmetric double-well potential which brings about the conditions for the appearance of kink-waves conducted by intrinsic electric fields embedded in microtubules. These kinks can serve as the signals for control and regulation of intracellular traffic along microtubules performed by processive motions of motor proteins, primarly from kinesin and dynein families. On the other hand, they can be precursors for initiation of dynamical instability of microtubules by recruiting the proper proteins responsible for the depolymerization process. PMID:27475079

  1. Structure and Functional Studies of the CS Domain of the Essential H/ACA Ribonucleoparticle Assembly Protein SHQ1

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Mahavir; Gonzales, Fernando A.; Cascio, Duilio; Heckmann, Nathanael; Chanfreau, Guillaume; Feigon, Juli; (UCLA)

    2009-03-16

    H/ACA ribonucleoprotein particles are essential for ribosomal RNA and telomerase RNA processing and metabolism. Shq1p has been identified as an essential eukaryotic H/ACA small nucleolar (sno) ribonucleoparticle (snoRNP) biogenesis and assembly factor. Shq1p is postulated to be involved in the early biogenesis steps of H/ACA snoRNP complexes, and Shq1p depletion leads to a specific decrease in H/ACA small nucleolar RNA levels and to defects in ribosomal RNA processing. Shq1p contains two predicted domains as follows: an N-terminal CS (named after CHORD-containing proteins and SGT1) or HSP20-like domain, and a C-terminal region of high sequence homology called the Shq1 domain. Here we report the crystal structure and functional studies of the Saccharomyces cerevisiae Shq1p CS domain. The structure consists of a compact anti-parallel {beta}-sandwich fold that is composed of two {beta}-sheets containing four and three {beta}-strands, respectively, and a short {alpha}-helix. Deletion studies showed that the CS domain is required for the essential functions of Shq1p. Point mutations in residues Phe-6, Gln-10, and Lys-80 destabilize Shq1p in vivo and induce a temperature-sensitive phenotype with depletion of H/ACA small nucleolar RNAs and defects in rRNA processing. Although CS domains are frequently found in co-chaperones of the Hsp90 molecular chaperone, no interaction was detected between the Shq1p CS domain and yeast Hsp90 in vitro. These results show that the CS domain is essential for Shq1p function in H/ACA snoRNP biogenesis in vivo, possibly in an Hsp90-independent manner.

  2. Tunable conductance of magnetic nanowires with structured domain walls

    OpenAIRE

    Dugaev, V. K.; Berakdar, J.; Barnas, J.

    2006-01-01

    We show that in a magnetic nanowire with double magnetic domain walls, quantum interference results in spin-split quasistationary states localized mainly between the domain walls. Spin-flip-assisted transmission through the domain structure increases strongly when these size-quantized states are tuned on resonance with the Fermi energy, e.g. upon varying the distance between the domain walls which results in resonance-type peaks of the wire conductance. This novel phenomena is shown to be uti...

  3. C1 inhibitor serpin domain structure reveals the likely mechanism of heparin potentiation and conformational disease.

    Science.gov (United States)

    Beinrohr, László; Harmat, Veronika; Dobó, József; Lörincz, Zsolt; Gál, Péter; Závodszky, Péter

    2007-07-20

    C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded beta-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack. PMID:17488724

  4. Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1.

    Science.gov (United States)

    Drmota Prebil, Sara; Slapšak, Urška; Pavšič, Miha; Ilc, Gregor; Puž, Vid; de Almeida Ribeiro, Euripedes; Anrather, Dorothea; Hartl, Markus; Backman, Lars; Plavec, Janez; Lenarčič, Brigita; Djinović-Carugo, Kristina

    2016-01-01

    The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin. PMID:27272015

  5. Structure-dependent electrical conductivity of protein: its differences between alpha-domain and beta-domain structures.

    Science.gov (United States)

    Zhang, X Y; Shao, Jian; Jiang, S X; Wang, Biao; Zheng, Yue

    2015-03-27

    Electron transports in the α-domain and β-domain of proteins have been comprehensively investigated. The structure-dependent electron transport of proteins has been experimentally measured and theoretically simulated, and both the theoretical and experimental results demonstrate significant differences in electrical conductivity between the α-domain and β-domain. By controlling the feedback system of the scanning tunneling microscope (STM), the conductance of a single α-domain protein hemoglobin (Hgb) and a β-domain protein superoxide dismutase enzyme (SOD) were measured, respectively. The current signal of Hgb is obviously stronger, indicating that the α-domain is more conductive. To confirm our finding, molecular orbitals of both the β-domain in SOD and α-domain in Hgb have been analyzed based on first-principles calculations. As expected, tunneling transport and hopping in the α-domain are both more efficient, indicating that it is easier for electrons to transport through the α-domain, which are in great agreement with our experimental data. In order to explain our results, molecular structures of α- and β-domains have been carefully analyzed and show that the explanation should lie in the differences in packing mode between the α-domain and β-domain. This research should be very important to application prospects in molecular electronics. PMID:25736549

  6. Structure-dependent electrical conductivity of protein: its differences between alpha-domain and beta-domain structures

    International Nuclear Information System (INIS)

    Electron transports in the α-domain and β-domain of proteins have been comprehensively investigated. The structure-dependent electron transport of proteins has been experimentally measured and theoretically simulated, and both the theoretical and experimental results demonstrate significant differences in electrical conductivity between the α-domain and β-domain. By controlling the feedback system of the scanning tunneling microscope (STM), the conductance of a single α-domain protein hemoglobin (Hgb) and a β-domain protein superoxide dismutase enzyme (SOD) were measured, respectively. The current signal of Hgb is obviously stronger, indicating that the α-domain is more conductive. To confirm our finding, molecular orbitals of both the β-domain in SOD and α-domain in Hgb have been analyzed based on first-principles calculations. As expected, tunneling transport and hopping in the α-domain are both more efficient, indicating that it is easier for electrons to transport through the α-domain, which are in great agreement with our experimental data. In order to explain our results, molecular structures of α- and β-domains have been carefully analyzed and show that the explanation should lie in the differences in packing mode between the α-domain and β-domain. This research should be very important to application prospects in molecular electronics. (paper)

  7. Reuse of structural domain–domain interactions in protein networks

    Directory of Open Access Journals (Sweden)

    Bateman Alex

    2007-07-01

    Full Text Available Abstract Background Protein interactions are thought to be largely mediated by interactions between structural domains. Databases such as iPfam relate interactions in protein structures to known domain families. Here, we investigate how the domain interactions from the iPfam database are distributed in protein interactions taken from the HPRD, MPact, BioGRID, DIP and IntAct databases. Results We find that known structural domain interactions can only explain a subset of 4–19% of the available protein interactions, nevertheless this fraction is still significantly bigger than expected by chance. There is a correlation between the frequency of a domain interaction and the connectivity of the proteins it occurs in. Furthermore, a large proportion of protein interactions can be attributed to a small number of domain interactions. We conclude that many, but not all, domain interactions constitute reusable modules of molecular recognition. A substantial proportion of domain interactions are conserved between E. coli, S. cerevisiae and H. sapiens. These domains are related to essential cellular functions, suggesting that many domain interactions were already present in the last universal common ancestor. Conclusion Our results support the concept of domain interactions as reusable, conserved building blocks of protein interactions, but also highlight the limitations currently imposed by the small number of available protein structures.

  8. A hybrid method for identification of structural domains

    Science.gov (United States)

    Hua, Yongpan; Zhu, Min; Wang, Yuelong; Xie, Zhaoyang; Li, Menglong

    2014-12-01

    Structural domains in proteins are the basic units to form various proteins. In the protein's evolution and functioning, domains play important roles. But the definition of domain is not yet precisely given, and the update cycle of structural domain databases is long. The automatic algorithms identify domains slowly, while protein entities with great structural complexity are on the rise. Here, we present a method which recognizes the compact and modular segments of polypeptide chains to identify structural domains, and contrast some data sets to illuminate their effect. The method combines support vector machine (SVM) with K-means algorithm. It is faster and more stable than most current algorithms and performs better. It also indicates that when proteins are presented as some Alpha-carbon atoms in 3D space, it is feasible to identify structural domains by the spatially structural properties. We have developed a web-server, which would be helpful in identification of structural domains (http://vis.sculab.org/~huayongpan/cgi-bin/domainAssignment.cgi).

  9. X-ray crystal structure of the passenger domain of plasmid encoded toxin(Pet), an autotransporter enterotoxin from enteroaggregative Escherichia coli (EAEC)

    Energy Technology Data Exchange (ETDEWEB)

    Domingo Meza-Aguilar, J. [Departamento de Salud Pública Facultad de Medicina UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato Oncología e Investigación, Hospital Infantil de México Federico Gómez 06720, D.F. (Mexico); Fromme, Petra [Department of Chemistry and Biochemistry, Arizona State University, Physical Sciences BLDG D-102, Tempe, AZ 85287 (United States); Torres-Larios, Alfredo [Instituto de Fisiología Celular UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Mendoza-Hernández, Guillermo [Instituto de Química UNAM, Ciudad Universitaria Coyoacán 04510, D.F (Mexico); Hernandez-Chiñas, Ulises [Departamento de Salud Pública Facultad de Medicina UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato Oncología e Investigación, Hospital Infantil de México Federico Gómez 06720, D.F. (Mexico); Arreguin-Espinosa de los Monteros, Roberto A. [Instituto de Química UNAM, Ciudad Universitaria Coyoacán 04510, D.F (Mexico); and others

    2014-03-07

    Highlights: • X-ray crystal structure of the passenger domain of Plasmid encoded toxin at 2.3 Å. • Structural differences between Pet passenger domain and EspP protein are described. • High flexibility of the C-terminal beta helix is structurally assigned. - Abstract: Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a β-pleaded sheet at the sequence region of residues 181–190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP.

  10. X-ray crystal structure of the passenger domain of plasmid encoded toxin(Pet), an autotransporter enterotoxin from enteroaggregative Escherichia coli (EAEC)

    International Nuclear Information System (INIS)

    Highlights: • X-ray crystal structure of the passenger domain of Plasmid encoded toxin at 2.3 Å. • Structural differences between Pet passenger domain and EspP protein are described. • High flexibility of the C-terminal beta helix is structurally assigned. - Abstract: Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a β-pleaded sheet at the sequence region of residues 181–190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Domain Modeling: NP_061971.3 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_061971.3 chr8 CRYSTAL STRUCTURE OF THE C-TERMINAL DOMAIN OF BCLA, THE MAJOR ANTI...GEN OF THE EXOSPORIUM OF THE BACILLUS ANTHRACIS SPORE. p1wcka_ chr8/NP_061971.3/NP_061971.3_holo_674-808.pdb swppa 677K,729E,731E,805E CAC 0 ...

  13. The Lectin Domain of the Polypeptide GalNAc Transferase Family of Glycosyltransferases (ppGalNAc Ts) Acts as a Switch Directing Glycopeptide Substrate Glycosylation in an N- or C-terminal Direction, Further Controlling Mucin Type O-Glycosylation

    DEFF Research Database (Denmark)

    Gerken, Thomas A; Revoredo, Leslie; Thome, Joseph J C; Tabak, Lawrence A; Vester-Christensen, Malene Bech; Clausen, Henrik; Gahlay, Gagandeep K; Jarvis, Donald L; Johnson, Roy W; Moniz, Heather A; Moremen, Kelley

    2013-01-01

    Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain an...

  14. Catalytic domain structure and hypothesis for function of GIY-YIG intron endonuclease I-TevI.

    Science.gov (United States)

    Van Roey, Patrick; Meehan, Lisa; Kowalski, Joseph C; Belfort, Marlene; Derbyshire, Victoria

    2002-11-01

    I-TevI, a member of the GIY-YIG family of homing endonucleases, consists of an N-terminal catalytic domain and a C-terminal DNA-binding domain joined by a flexible linker. The GIY-YIG motif is in the N-terminal domain of I-TevI, which corresponds to a phylogenetically widespread catalytic cartridge that is often associated with mobile genetic elements. The crystal structure of the catalytic domain of I-TevI, the first of any GIY-YIG endonuclease, reveals a novel alpha/beta-fold with a central three-stranded antiparallel beta-sheet flanked by three helices. The most conserved and putative catalytic residues are located on a shallow, concave surface and include a metal coordination site. Similarities in the three-dimensional arrangement of the catalytically important residues and the cation-binding site with those of the His-Cys box endonuclease I-PpoI suggest the possibility of mechanistic relationships among these different families of homing endonucleases despite completely different folds. PMID:12379841

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

    International Nuclear Information System (INIS)

    During autophagy a crescent shaped like membrane is formed, which engulfs the material that is to be degraded. This membrane grows further until its edges fuse to form the double membrane covered autophagosome. Atg8 is a protein, which is required for this initial step of autophagy. Therefore, a multistage conjugation process of newly synthesized Atg8 to phosphatidylethanolamine is of critical importance. Here we present the high resolution structure of unprocessed Atg8 determined by nuclear magnetic resonance spectroscopy. Its C-terminal subdomain shows a well-defined ubiquitin-like fold with slightly elevated mobility in the pico- to nanosecond timescale as determined by heteronuclear NOE data. In comparison to unprocessed Atg8, cleaved Atg8G116 shows a decreased mobility behaviour. The N-terminal domain adopts different conformations within the micro- to millisecond timescale. The possible biological relevance of the differences in dynamic behaviours between both subdomains as well as between the cleaved and uncleaved forms is discussed.

  16. Structural and functional characterization of the recombinant death domain from death-associated protein kinase.

    Directory of Open Access Journals (Sweden)

    Evangelos Dioletis

    Full Text Available Death-associated protein kinase (DAPk is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD, NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2 and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent 'D-motif' located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural

  17. Structural Insights into the Functional Role of the Hcn Sub-domain of the Receptor-Binding Domain of the Botulinum Neurotoxin Mosaic Serotype C/D

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Gardberg, Anna; Edwards, Tom E.; Sankaran, Banumathi; Robinson, Howard; Varnum, Susan M.; Buchko, Garry W.

    2013-07-01

    Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. Produced by different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. Endocytosis, the first step of cellular intoxication, is triggered by the ~50 kDa, heavy-chain receptor-binding module (HCR) that is specific for a ganglioside and a protein receptor on neuronal cell surfaces. This dual receptor recognition mechanism between BoNT and the host cell’s membrane is well documented and occurs via specific intermolecular interactions with the C-terminal sub-domain, Hcc, of BoNT-HCR. The N-terminal sub-domain of BoNT-HCR, Hcn, comprises ~50% of BoNT-HCR and adopts a B-sheet jelly roll fold. While suspected in assisting cell surface recognition, no unambiguous function for the Hcn sub-domain in BoNT has been indentified. To obtain insights into the potential function of the Hcn sub-domain in BoNT, the first crystal structure of a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. Here, we describe the crystal structure of BoNT/CD-HCR determined at 1.70 Å resolution with a tetraethylene glycol (PG4) molecule bound in an hydrophobic cleft between B-strands in the B-sheet jelly fold roll of the Hcn sub-domain. The molecule is completely engulfed in the cleft, making numerous hydrophobic (Y932, S959, W966, and D1042) and hydrophilic (S935, W977, L979, N1013, and I1066) contacts with the protein’s side chain and backbone that may mimic in vivo interactions with the phospholipid membranes on neuronal cell surfaces. A sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 in the Hcc sub-domain of BoNT/CD-HCR. In the crystal structure of a similar protein, BoNT/D-HCR, a sialic acid

  18. Structural Characterization of the Regulatory Domain of Brain Carnitine Palmitoyltransferase 1

    OpenAIRE

    Samanta, Soma; Situ, Alan J.; Ulmer, Tobias S.

    2014-01-01

    Neurons contain a mammalian-specific isoform of the enzyme carnitine palmitoyltransferase 1 (CPT1C) that couples malonyl-CoA to ceramide levels thereby contributing to systemic energy homeostasis and feeding behavior. In contrast to CPT1A, which controls the rate-limiting step of long-chain fatty acid β-oxidation in all tissues, the biochemical context and regulatory mechanism of CPT1C are unknown. CPT1 enzymes are comprised of an N-terminal regulatory domain and a C-terminal catalytic domain...

  19. Domain swapping in the low-similarity isomerase/hydratase superfamily: the crystal structure of rat mitochondrial Delta3, Delta2-enoyl-CoA isomerase.

    Science.gov (United States)

    Hubbard, Paul A; Yu, Wenfeng; Schulz, Horst; Kim, Jung-Ja P

    2005-06-01

    Two monofunctional Delta(3), Delta(2)-enoyl-CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low-similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl-CoA substrate for re-entry into the beta-oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI. To elucidate whether Glu165 of mECI is correctly positioned for catalysis, the crystal structure of rat mECI has been solved. Crystal packing suggests the enzyme is trimeric, in contrast to other members of the superfamily, which appear crystallographically to be dimers of trimers. The polypeptide fold of mECI, like pECI, belongs to a subset of this superfamily in which the C-terminal domain of a given monomer interacts with its own N-terminal domain. This differs from that of crotonase and 1,4-dihydroxy-2-naphtoyl-CoA synthase, whose C-terminal domains are involved in domain swapping with an adjacent monomer. The structure confirms Glu165 as the putative catalytic acid/base, positioned to abstract the pro-R proton from C2 and reprotonate at C4 of the acyl chain. The large tunnel-shaped active site cavity observed in the mECI structure explains the relative substrate promiscuity in acyl-chain length and stereochemistry. Comparison with the crystal structure of pECI suggests the catalytic residues from both enzymes are spatially conserved but not in their primary structures, providing a powerful reminder of how catalytic residues cannot be determined solely by sequence alignments. PMID:15883186

  20. Axial-symmetrical domain structures in ferrite-garnet films

    International Nuclear Information System (INIS)

    Applying the improved technique of double ultrahigh-speed photography one studied occurrence and progress of domain structure upon magnetization of a small section of (BiLaTm)3(FeGa)5O12 ferrite-garnet film prior to saturation. The radial deformation affecting the specimen on the magnetized coil side was determined to break single-axis anisotropy resulting in occurrence of axial-oriented band domain structure inside the magnetized range. That structure period reduced with growth of the pulse field amplitude. Band axial-oriented domain structure is formed under the effect of the magnetostatic axisymmetric field

  1. Extra-domain B in Oncofetal Fibronectin Structurally Promotes Fibrillar Head-to-tail Dimerization of Extracellular Matrix Protein*

    Science.gov (United States)

    Schiefner, André; Gebauer, Michaela; Skerra, Arne

    2012-01-01

    The type III extra-domain B (ED-B) is specifically spliced into fibronectin (Fn) during embryogenesis and neoangiogenesis, including many cancers. The x-ray structure of the recombinant four-domain fragment FnIII7B89 reveals a tightly associated, extended head-to-tail dimer, which is stabilized via pair-wise shape and charge complementarity. A tendency toward ED-B-dependent dimer formation in solution was supported by size exclusion chromatography and analytical ultracentrifugation. When amending the model with the known three-dimensional structure of the FnIII10 domain, its RGD loop as well as the adhesion synergy region in FnIII9–10 become displayed on the same face of the dimer; this should allow simultaneous binding of at least two integrins and, thus, receptor clustering on the cell surface and intracellular signaling. Insertion of ED-B appears to stabilize overall head-to-tail dimerization of two separate Fn chains, which, together with alternating homodimer formation via disulfide bridges at the C-terminal Fn tail, should lead to the known macromolecular fibril formation. PMID:22442152

  2. Structure of an isolated unglycosylated antibody CH2 domain

    International Nuclear Information System (INIS)

    The crystal structure of an isolated unglycosylated antibody CH2 domain has been determined at 1.7 Å resolution. The CH2 (CH3 for IgM and IgE) domain of an antibody plays an important role in mediating effector functions and preserving antibody stability. It is the only domain in human immunoglobulins (Igs) which is involved in weak interchain protein–protein interactions with another CH2 domain solely through sugar moieties. The N-linked glycosylation at Asn297 is conserved in mammalian IgGs as well as in homologous regions of other antibody isotypes. To examine the structural details of the CH2 domain in the absence of glycosylation and other antibody domains, the crystal structure of an isolated unglycosylated antibody γ1 CH2 domain was determined at 1.7 Å resolution and compared with corresponding CH2 structures from intact Fc, IgG and Fc receptor complexes. Furthermore, the oligomeric state of the protein in solution was studied using size-exclusion chromatography. The results suggested that the unglycosylated human antibody CH2 domain is a monomer and that its structure is similar to that found in the intact Fc, IgG and Fc receptor complex structures. However, certain structural variations were observed in the Fc receptor-binding sites. Owing to its small size, stability and non-immunogenic Ig template, the CH2-domain structure could be useful for the development by protein design of antibody domains exerting effector functions and/or antigen specificity and as a robust scaffold in protein-engineering applications

  3. Mirror Domain Structures Induced by Interlayer Magnetic Wall Coupling

    Science.gov (United States)

    Lew, W. S.; Li, S. P.; Lopez-Diaz, L.; Hatton, D. C.; Bland, J. A.

    2003-05-01

    We have found that during giant magnetoresistance measurements in ˜10×10 mm2 NiFe/Cu/Co continuous film spin-valve structures, the resistance value suddenly drops to its absolute minimum during the NiFe reversal. The results reveal that the alignment of all magnetic domains in the NiFe film follow exactly that of corresponding domains in the Co film for an appropriate applied field strength. This phenomenon is caused by trapping of the NiFe domain walls through the magnetostatic interaction with the Co domain-wall stray fields. Consequently, the interlayer domain-wall coupling induces a mirror domain structure in the magnetic trilayer.

  4. 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.

  5. The history of the CATH structural classification of protein domains.

    Science.gov (United States)

    Sillitoe, Ian; Dawson, Natalie; Thornton, Janet; Orengo, Christine

    2015-12-01

    This article presents a historical review of the protein structure classification database CATH. Together with the SCOP database, CATH remains comprehensive and reasonably up-to-date with the now more than 100,000 protein structures in the PDB. We review the expansion of the CATH and SCOP resources to capture predicted domain structures in the genome sequence data and to provide information on the likely functions of proteins mediated by their constituent domains. The establishment of comprehensive function annotation resources has also meant that domain families can be functionally annotated allowing insights into functional divergence and evolution within protein families. PMID:26253692

  6. Quantifying information transfer by protein domains: Analysis of the Fyn SH2 domain structure

    Directory of Open Access Journals (Sweden)

    Serrano Luis

    2008-10-01

    Full Text Available Abstract Background Efficient communication between distant sites within a protein is essential for cooperative biological response. Although often associated with large allosteric movements, more subtle changes in protein dynamics can also induce long-range correlations. However, an appropriate formalism that directly relates protein structural dynamics to information exchange between functional sites is still lacking. Results Here we introduce a method to analyze protein dynamics within the framework of information theory and show that signal transduction within proteins can be considered as a particular instance of communication over a noisy channel. In particular, we analyze the conformational correlations between protein residues and apply the concept of mutual information to quantify information exchange. Mapping out changes of mutual information on the protein structure then allows visualizing how distal communication is achieved. We illustrate the approach by analyzing information transfer by the SH2 domain of Fyn tyrosine kinase, obtained from Monte Carlo dynamics simulations. Our analysis reveals that the Fyn SH2 domain forms a noisy communication channel that couples residues located in the phosphopeptide and specificity binding sites and a number of residues at the other side of the domain near the linkers that connect the SH2 domain to the SH3 and kinase domains. We find that for this particular domain, communication is affected by a series of contiguous residues that connect distal sites by crossing the core of the SH2 domain. Conclusion As a result, our method provides a means to directly map the exchange of biological information on the structure of protein domains, making it clear how binding triggers conformational changes in the protein structure. As such it provides a structural road, next to the existing attempts at sequence level, to predict long-range interactions within protein structures.

  7. Magnetic domain structures in high purity single crystal terbium

    International Nuclear Information System (INIS)

    Domain structures in the ferromagnetic phase have been observed down to 950K in a single crystal of terbium grown by electron beam float zone melting in UHV. The demagnetized configuration is of slab domains, some ten microns thick, separated by 1800 walls lying in the basal plane

  8. Structural Disorder within Paramyxoviral Nucleoproteins and Phosphoproteins in Their Free and Bound Forms: From Predictions to Experimental Assessment

    OpenAIRE

    Johnny Habchi; Sonia Longhi

    2015-01-01

    We herein review available computational and experimental data pointing to the abundance of structural disorder within the nucleoprotein (N) and phosphoprotein (P) from three paramyxoviruses, namely the measles (MeV), Nipah (NiV) and Hendra (HeV) viruses. We provide a detailed molecular description of the mechanisms governing the disorder-to-order transition that the intrinsically disordered C-terminal domain (NTAIL) of their N proteins undergoes upon binding to the C-terminal X domain (PXD)...

  9. The capping domain in RalF regulates effector functions.

    Directory of Open Access Journals (Sweden)

    Eric Alix

    Full Text Available The Legionella pneumophila effector protein RalF functions as a guanine nucleotide exchange factor (GEF that activates the host small GTPase protein ADP-ribosylation factor (Arf, and recruits this host protein to the vacuoles in which this pathogen resides. GEF activity is conferred by the Sec7 domain located in the N-terminal region of RalF. Structural studies indicate that the C-terminal region of RalF makes contacts with residues in the Sec7 domain important for Arf interactions. Theoretically, the C-terminal region of RalF could prevent nucleotide exchange activity by blocking the ability of Arf to interact with the Sec7 domain. For this reason, the C-terminal region of RalF has been termed a capping domain. Here, the role of the RalF capping domain was investigated by comparing biochemical and effector activities mediated by this domain in both the Legionella RalF protein (LpRalF and in a RalF ortholog isolated from the unrelated intracellular pathogen Rickettsia prowazekii (RpRalF. These data indicate that both RalF proteins contain a functional Sec7 domain and that the capping domain regulates RalF GEF activity. The capping domain has intrinsic determinants that mediate localization of the RalF protein inside of host cells and confer distinct effector activities. Localization mediated by the capping domain of LpRalF enables the GEF to modulate membrane transport in the secretory pathway, whereas, the capping domain of RpRalF enables this bacterial GEF to modulate actin dynamics occurring near the plasma membrane. Thus, these data reveal that divergence in the function of the C-terminal capping domain alters the in vivo functions of the RalF proteins.

  10. Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Ping; Paterson, Reay G.; Leser, George P.; Lamb, Robert A.; Jardetzky, Theodore S. (Stanford-MED); (NWU)

    2012-09-06

    Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN0) form has to be cleaved to render HN biologically active. Here we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase {beta}-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN{sub 0} and associated reduced virulence.

  11. Solution structure of the human BTK SH3 domain complexed with a proline-rich peptide from p120cbl

    International Nuclear Information System (INIS)

    X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B cell differentiation which incapacitates antibody production in XLA patients leading to, sometimes lethal, bacterial infections. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in one patient family. To understand the role of BTK in B cell development, we have determined the solution structure of BTK SH3 domain complexed with a proline-rich peptide from the protein product of c-cbl protooncogene (p120cbl). Like other SH3 domains, BTK SH3 domain consists of five β-strands packed in two β-sheets forming a β-barrel-like structure. The rmsd calculated from the averaged coordinates for the BTK SH3 domain residues 218-271 and the p120cbl peptide residues 6-12 of the complex was 0.87 A (±0.16 A) for the backbone heavy atoms (N, C, and Cα) and 1.64 A (±0.16 A) for all heavy atoms. Based on chemical shift changes and inter-molecular NOEs, we have found that the residues located in the RT loop, n-Src loop and helix-like loop between β4 and β5 of BTK SH3 domain are involved in ligand binding. We have also determined that the proline-rich peptide from p120cbl binds to BTK SH3 domain in a class I orientation. These results correlate well with our earlier observation that the truncated BTK SH3 domain (deletion of β4, β5 and the helix-like loop) exhibits weaker affinity for the p120cbl peptide. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context and hence the weaker binding. These results delineate the importance of the C-terminus in the binding of SH3 domains and also indicate that improper folding and the altered binding behavior of mutant BTK SH3 domain likely lead to XLA

  12. Solution structure of the human BTK SH3 domain complexed with a proline-rich peptide from p120cbl

    Energy Technology Data Exchange (ETDEWEB)

    Tzeng, S.-R.; Lou, Y.-C.; Pai, M.-T.; Jain, Moti L.; Cheng, J.-W. [National Tsing Hua University, Division of Structural Biology and Biomedical Science, Department of Life Science (China)

    2000-04-15

    X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B cell differentiation which incapacitates antibody production in XLA patients leading to, sometimes lethal, bacterial infections. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in one patient family. To understand the role of BTK in B cell development, we have determined the solution structure of BTK SH3 domain complexed with a proline-rich peptide from the protein product of c-cbl protooncogene (p120{sup cbl}). Like other SH3 domains, BTK SH3 domain consists of five {beta}-strands packed in two {beta}-sheets forming a {beta}-barrel-like structure. The rmsd calculated from the averaged coordinates for the BTK SH3 domain residues 218-271 and the p120{sup cbl} peptide residues 6-12 of the complex was 0.87 A ({+-}0.16 A) for the backbone heavy atoms (N, C, and C{sub {alpha}}) and 1.64 A ({+-}0.16 A) for all heavy atoms. Based on chemical shift changes and inter-molecular NOEs, we have found that the residues located in the RT loop, n-Src loop and helix-like loop between {beta}4 and {beta}5 of BTK SH3 domain are involved in ligand binding. We have also determined that the proline-rich peptide from p120{sup cbl} binds to BTK SH3 domain in a class I orientation. These results correlate well with our earlier observation that the truncated BTK SH3 domain (deletion of {beta}4, {beta}5 and the helix-like loop) exhibits weaker affinity for the p120{sup cbl} peptide. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context and hence the weaker binding. These results delineate the importance of the C-terminus in the binding of SH3 domains and also indicate that improper folding and the altered binding behavior of mutant BTK SH3 domain likely lead to XLA.

  13. Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

    Science.gov (United States)

    Dupré, Mathieu; Cantel, Sonia; Martinez, Jean; Enjalbal, Christine

    2012-02-01

    By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1 + H2O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2O and NH3) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in

  14. Structured and Unstructured Cache Models for SMT Domain Adaptation

    OpenAIRE

    Louis, Annie; Webber, Bonnie L.

    2014-01-01

    We present a French to English translation system for Wikipedia biography articles. We use training data from out-of-domain corpora and adapt the system for biographies. We propose two forms of domain adaptation. The first biases the system towards words likely in biographies and encourages repetition of words across the document. Since biographies in Wikipedia follow a regular structure, our second model exploits this structure as a sequence of topic segments, where each segment discusses a ...

  15. Structural Time Domain Identification (STDI) Toolbox for Use with MATLAB

    OpenAIRE

    KIRKEGAARD, Poul Henning; Andersen, P.; Brincker, Rune

    1996-01-01

    The Structural Time Domain Identification (STDI) toolbox for use with MATLABTM is developed at Aalborg University, Denmark, based on the system identification research performed during recent years. By now, a reliable set of functions offers a wide spectrum of services for all the important steps of multivariate time domain system identification of time-variant as well as time-invariant civil engineering structures from ambient testing data. A graphical user interface (GUI) is also developed ...

  16. Domain structures of ferroelectric films under different electrical boundary conditions

    OpenAIRE

    Z. D. Zhou; Wu, D Y

    2015-01-01

    A two-dimensional phase field simulation of ferroelectric films is used that incorporates Landau-Devonshire energy, gradient energy and depolarization electrical energy. A new intermediate electrical boundary condition is firstly presented to study the effects on domain structures of ferroelectric films. Two-dimensional simulations of domain structures are carried out under the open circuit (OC), short circuit (SC) and intermediate (IM) electrical boundary conditions. The simulation results s...

  17. Structurally distinct Arabidopsis thaliana NLR immune receptors recognize tandem WY domains of an oomycete effector.

    Science.gov (United States)

    Goritschnig, Sandra; Steinbrenner, Adam D; Grunwald, Derrick J; Staskawicz, Brian J

    2016-05-01

    Nucleotide-binding leucine-rich repeat (NB-LRR, or NLR) receptors mediate pathogen recognition. The Arabidopsis thaliana NLR RPP1 recognizes the tandem WY-domain effector ATR1 from the oomycete Hyaloperonospora arabidopsidis through direct association with C-terminal LRRs. We isolated and characterized homologous NLR genes RPP1-EstA and RPP1-ZdrA from two Arabidopsis ecotypes, Estland (Est-1) and Zdarec (Zdr-1), responsible for recognizing a novel spectrum of ATR1 alleles. RPP1-EstA and -ZdrA encode nearly identical NLRs that are phylogenetically distinct from known immunity-activating RPP1 homologs and possess greatly expanded LRR domains. Site-directed mutagenesis and truncation analysis of ATR1 suggests that these homologs recognize a novel surface of the 2(nd) WY domain of ATR1, partially specified by a C-terminal region of the LRR domain. Synteny comparison with RPP1 loci involved in hybrid incompatibility suggests that these functions evolved independently. Closely related RPP1 homologs have diversified their recognition spectra through LRR expansion and sequence variation, allowing them to detect multiple surfaces of the same pathogen effector. Engineering NLR receptor specificity may require a similar combination of repeat expansion and tailored amino acid variation. PMID:26725254

  18. C-terminal Src kinase-mediated EPIYA phosphorylation of Pragmin creates a feed-forward C-terminal Src kinase activation loop that promotes cell motility.

    Science.gov (United States)

    Senda, Yoshie; Murata-Kamiya, Naoko; Hatakeyama, Masanori

    2016-07-01

    Pragmin is one of the few mammalian proteins containing the Glu-Pro-Ile-Tyr-Ala (EPIYA) tyrosine-phosphorylation motif that was originally discovered in the Helicobacter pylori CagA oncoprotein. Following delivery into gastric epithelial cells by type IV secretion and subsequent tyrosine phosphorylation at the EPIYA motifs, CagA serves as an oncogenic scaffold/adaptor that promiscuously interacts with SH2 domain-containing mammalian proteins such as the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) and the C-terminal Src kinase (Csk), a negative regulator of Src family kinases. Like CagA, Pragmin also forms a physical complex with Csk. In the present study, we found that Pragmin directly binds to Csk by the tyrosine-phosphorylated EPIYA motif. The complex formation potentiates kinase activity of Csk, which in turn phosphorylates Pragmin on tyrosine-238 (Y238), Y343, and Y391. As Y391 of Pragmin comprises the EPIYA motif, Pragmin-Csk interaction creates a feed-forward regulatory loop of Csk activation. Together with the finding that Pragmin and Csk are colocalized to focal adhesions, these observations indicate that the Pragmin-Csk interaction, triggered by Pragmin EPIYA phosphorylation, robustly stimulates the kinase activity of Csk at focal adhesions, which direct cell-matrix adhesion that regulates cell morphology and cell motility. As a consequence, expression of Pragmin and/or Csk in epithelial cells induces an elongated cell shape with elevated cell scattering in a manner that is mutually dependent on Pragmin and Csk. Deregulation of the Pragmin-Csk axis may therefore induce aberrant cell migration that contributes to tumor invasion and metastasis. PMID:27116701

  19. Comparative structural analysis of lipid binding START domains.

    Directory of Open Access Journals (Sweden)

    Ann-Gerd Thorsell

    Full Text Available BACKGROUND: Steroidogenic acute regulatory (StAR protein related lipid transfer (START domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease. PRINCIPAL FINDINGS: We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains. SIGNIFICANCE: Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  20. Wnts grasp the WIF domain of Wnt Inhibitory Factor 1 at two distinct binding sites.

    Science.gov (United States)

    Kerekes, Krisztina; Bányai, László; Patthy, László

    2015-10-01

    Wnts have a structure resembling a hand with "thumb" and "index" fingers that grasp the cysteine rich domains of Frizzled receptors at two distinct binding sites. In the present work we show that the WIF domain of Wnt Inhibitory Factor 1 is also bound by Wnts at two sites. Using C-terminal domains of Wnt5a and Wnt7a and arginine-scanning mutagenesis of the WIF domain we demonstrate that, whereas the N-terminal, lipid-modified "thumb" of Wnts interacts with the alkyl-binding site of the WIF domain, the C-terminal domain of Wnts (Wnt-CTD) binds to a surface on the opposite side of the WIF domain. PMID:26342861

  1. Function of C-terminal hydrophobic region in fructose dehydrogenase

    International Nuclear Information System (INIS)

    Fructose dehydrogenase (FDH) catalyzes oxidation of D-fructose into 2-keto-D-fructose and is one of the enzymes allowing a direct electron transfer (DET)-type bioelectrocatalysis. FDH is a heterotrimeric membrane-bound enzyme (subunit I, II, and III) and subunit II has a C terminal hydrophobic region (CHR), which was expected to play a role in anchoring to membranes from the amino acid sequence. We have constructed a mutated FDH lacking of CHR (ΔchrFDH). Contrary to the expected function of CHR, ΔchrFDH is expressed in the membrane fraction, and subunit I/III subcomplex (ΔcFDH) is also expressed in a similar activity level but in the soluble fraction. In addition, the enzyme activity of the purified ΔchrFDH is about one twentieth of the native FDH. These results indicate that CHR is concerned with the binding between subunit I(/III) and subunit II and then with the enzyme activity. ΔchrFDH has clear DET activity that is larger than that expected from the solution activity, and the characteristics of the catalytic wave of ΔchrFDH are very similar to those of FDH. The deletion of CHR seems to increase the amounts of the enzyme with the proper orientation for the DET reaction at electrode surfaces. Gel filtration chromatography coupled with urea treatment shows that the binding in ΔchrFDH is stronger than that in FDH. It can be considered that the rigid binding between subunit I(/III) and II without CHR results in a conformation different from the native one, which leads to the decrease in the enzyme activity in solution

  2. Domain and wall structures in films with helical magnetization profile

    Energy Technology Data Exchange (ETDEWEB)

    Dubuget, Vincent [Laboratoire d' Electrodynamique des Materiaux Avances, Universite Francois Rabelais, CNRS UMR 6157, Parc de Grandmont, F-37200 Tours (France); CEA, DAM, Le Ripault, F-37260 Monts (France); Thiaville, Andre [Laboratoire de Physique des Solides, Universite Paris-Sud, CNRS UMR 8502, Bat. 510, F-91405 Orsay (France); Adenot-Engelvin, Anne-Lise, E-mail: anne-lise.adenot-engelvin@cea.f [CEA, DAM, Le Ripault, F-37260 Monts (France); Duverger, Francois; Dubourg, Sebastien [CEA, DAM, Le Ripault, F-37260 Monts (France)

    2011-06-15

    We study soft magnetic bilayers having orthogonal, in-plane easy axes. The layers are thicker than the Bloch wall width linked to the anisotropy, so that a helical magnetization with a large angle exists across the sample thickness. The magnetic domains structure has been investigated at both sample surfaces, using magneto-optical microscopy. The domain structure is found to be similar to that of double films with biquadratic coupling. Two kinds of domain walls are identified, namely with a 90{sup o} and 180{sup o} rotation of the average magnetization. The detailed structure and energy of these walls are studied by micromagnetic calculations. - Research highlights: This paper is devoted to the peculiar domain structure resulting from an anisotropy distribution in the thickness of the sample, realized through specific elaboration conditions. The helical magnetization profile obtained leads to a complex dynamic behaviour described and modelled in Phys.Rev. B 80, 134412 (published in October 2009) which has been already cited three times. This paper sheds light on of the demagnetized state of such samples: a variety of domains structure has been observed by Kerr microscopy, under various saturation fields. The most striking conclusion is driven by the analysis of the magnetization process which implies the co-existence of two types of domain walls in the sample, with four possible directions for the mean magnetization. The magnetization profile of the two walls has been confirmed by numerical simulation.

  3. Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains

    DEFF Research Database (Denmark)

    Dellgren, Christoffer; Nehlin, Jan O; Barington, Torben

    2015-01-01

    . Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly......Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses...... expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of...

  4. C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar [Research Laboratory for Nuclear Reactors and Department of Nuclear Engineering, Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo 152-8550 (Japan); Adachi, Noritaka [Graduate School of Nanobioscience, Yokohama City University, Yokohama 236-0027 (Japan); Matsumoto, Yoshihisa, E-mail: yoshim@nr.titech.ac.jp [Research Laboratory for Nuclear Reactors and Department of Nuclear Engineering, Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo 152-8550 (Japan)

    2013-09-20

    Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  5. Magnetic domain structure in thin CoPt perpendicular magnetic anisotropy films

    OpenAIRE

    Komine T.; Tojo R.; Onose M.; Kawada Y.; Sugita R.

    2013-01-01

    The relation between thickness and domain structure of Co80Pt20 perpendicular magnetic anisotropy films was investigated through experiments and micromagnetic simulation. The films with thickness over 10 nm exhibited clear maze domain structure, while for the films thinner than 10 nm the domain structure abruptly changed from maze domain to irregular and large domain as the thickness became thinner. The irregular domain had narrower domain wall width than maze domain.

  6. Magnetic domain structure in thin CoPt perpendicular magnetic anisotropy films

    Directory of Open Access Journals (Sweden)

    Komine T.

    2013-01-01

    Full Text Available The relation between thickness and domain structure of Co80Pt20 perpendicular magnetic anisotropy films was investigated through experiments and micromagnetic simulation. The films with thickness over 10 nm exhibited clear maze domain structure, while for the films thinner than 10 nm the domain structure abruptly changed from maze domain to irregular and large domain as the thickness became thinner. The irregular domain had narrower domain wall width than maze domain.

  7. Conservation of structural and functional domains in complement component C3 of Xenopus and mammals.

    OpenAIRE

    Grossberger, D; Marcuz, A; Du Pasquier, L; LAMBRIS, J. D.

    1989-01-01

    The cDNA sequence and the deduced amino acid sequence of the Mr 34,000 C-terminal fragment of Xenopus laevis complement component C3 are presented. The sequence of Xenopus C3 has 57% nucleotide identity to the corresponding sequence of human C3 and approximately 49% amino acid identity to C3 from human, mouse, and rabbit. The Xenopus C3 sequence shows clusters of high and of low similarity to the mammalian C3 sequences. One of these regions of high similarity represents the domain of mammalia...

  8. Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response.

    Science.gov (United States)

    Dinesh, Dhurvas Chandrasekaran; Kovermann, Michael; Gopalswamy, Mohanraj; Hellmuth, Antje; Calderón Villalobos, Luz Irina A; Lilie, Hauke; Balbach, Jochen; Abel, Steffen

    2015-05-12

    The plant hormone auxin activates primary response genes by facilitating proteolytic removal of auxin/indole-3-acetic acid (AUX/IAA)-inducible repressors, which directly bind to transcriptional auxin response factors (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like β-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (KD ∼ 6.4 μM) were determined by isothermal titration calorimetry. In silico protein-protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein-protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression. PMID:25918389

  9. The role of C-terminal amidation in the membrane interactions of the anionic antimicrobial peptide, maximin H5.

    Science.gov (United States)

    Dennison, Sarah R; Mura, Manuela; Harris, Frederick; Morton, Leslie H G; Zvelindovsky, Andrei; Phoenix, David A

    2015-05-01

    Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m(-1)) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 m Nm(-1)) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptide's N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives. PMID:25640709

  10. Domain Structures in Nematic Liquid Crystals on a Polycarbonate Surface

    Directory of Open Access Journals (Sweden)

    Vasily F. Shabanov

    2013-08-01

    Full Text Available Alignment of nematic liquid crystals on polycarbonate films obtained with the use of solvents with different solvations is studied. Domain structures occurring during the growth on the polymer surface against the background of the initial thread-like or schlieren texture are demonstrated. It is established by optical methods that the domains are stable formations visualizing the polymer surface structures. In nematic droplets, the temperature-induced transition from the domain structure with two extinction bands to the structure with four bands is observed. This transition is shown to be caused by reorientation of the nematic director in the liquid crystal volume from the planar alignment to the homeotropic state with the pronounced radial configuration of nematic molecules on the surface. The observed textures are compared with different combinations of the volume LC orientations and the radial distribution of the director field and the disclination lines at the polycarbonate surface.

  11. Optical and thermal control of domain structures in ferroelectric crystals

    OpenAIRE

    Brown, Paul Thomas

    1999-01-01

    This thesis presents the results of investigations into the thermal and optical control of ferroelectric domains within lithium tantalate and strontium barium niobate crystals. The aim of the work was to develop techniques for optically pattering domain inverted structures within ferroelectric crystals. Initial studies involving strontium barium niobate revealed an enhanced temperature sensitivity for transient repoling occurring at room temperatures for this material. This has important...

  12. Solution Structure of Proinsulin: CONNECTING DOMAIN FLEXIBILITY AND PROHORMONE PROCESSING*

    OpenAIRE

    Yang, Yanwu; Hua, Qing-Xin; Liu, Jin; Shimizu, Eri H.; Choquette, Meredith H.; Mackin, Robert B.; Weiss, Michael A.

    2010-01-01

    The folding of proinsulin, the single-chain precursor of insulin, ensures native disulfide pairing in pancreatic β-cells. Mutations that impair folding cause neonatal diabetes mellitus. Although the classical structure of insulin is well established, proinsulin is refractory to crystallization. Here, we employ heteronuclear NMR spectroscopy to characterize a monomeric analogue. Proinsulin contains a native-like insulin moiety (A- and B-domains); the tethered connecting (C) domain (as probed b...

  13. Structural and biophysical investigation of the interaction of a mutant Grb2 SH2 domain (W121G) with its cognate phosphopeptide.

    Science.gov (United States)

    Papaioannou, Danai; Geibel, Sebastian; Kunze, Micha B A; Kay, Christopher W M; Waksman, Gabriel

    2016-03-01

    The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C-terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β-turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X-ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre-organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild-type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide. PMID:26645482

  14. DOMAIN TIPS : STRUCTURE AND MOBILITY IN UNIAXIAL AMORPHOUS THIN FILMS

    OpenAIRE

    Labrune, M.; Hamzaoui, S.; Puchalska, I.

    1988-01-01

    Statics and dynamics of domain tips have been investigated by high resolution Kerr effect using digital contrast enhancement. The magnetic structure is affected by demagnetizing effect. The internal wall structure is visible and a transition between asymmetric Bloch wall to Néel one is shown. Tip propagation along the easy axis is measured.

  15. Structural and functional characterization of DUF1471 domains of Salmonella proteins SrfN, YdgH/SssB, and YahO.

    Directory of Open Access Journals (Sweden)

    Alexander Eletsky

    Full Text Available Bacterial species in the Enterobacteriaceae typically contain multiple paralogues of a small domain of unknown function (DUF1471 from a family of conserved proteins also known as YhcN or BhsA/McbA. Proteins containing DUF1471 may have a single or three copies of this domain. Representatives of this family have been demonstrated to play roles in several cellular processes including stress response, biofilm formation, and pathogenesis. We have conducted NMR and X-ray crystallographic studies of four DUF1471 domains from Salmonella representing three different paralogous DUF1471 subfamilies: SrfN, YahO, and SssB/YdgH (two of its three DUF1471 domains: the N-terminal domain I (residues 21-91, and the C-terminal domain III (residues 244-314. Notably, SrfN has been shown to have a role in intracellular infection by Salmonella Typhimurium. These domains share less than 35% pairwise sequence identity. Structures of all four domains show a mixed α+β fold that is most similar to that of bacterial lipoprotein RcsF. However, all four DUF1471 sequences lack the redox sensitive cysteine residues essential for RcsF activity in a phospho-relay pathway, suggesting that DUF1471 domains perform a different function(s. SrfN forms a dimer in contrast to YahO and SssB domains I and III, which are monomers in solution. A putative binding site for oxyanions such as phosphate and sulfate was identified in SrfN, and an interaction between the SrfN dimer and sulfated polysaccharides was demonstrated, suggesting a direct role for this DUF1471 domain at the host-pathogen interface.

  16. Structural basis of Smoothened regulation by its extracellular domains

    Science.gov (United States)

    Byrne, Eamon F. X.; Sircar, Ria; Miller, Paul S.; Hedger, George; Luchetti, Giovanni; Nachtergaele, Sigrid; Tully, Mark D.; Mydock-McGrane, Laurel; Covey, Douglas F.; Rambo, Robert P.; Sansom, Mark S. P.; Newstead, Simon; Rohatgi, Rajat; Siebold, Christian

    2016-07-01

    Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD–linker domain–TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

  17. Selective enzymatic hydrolysis of C-terminal tert-butyl esters of peptides

    OpenAIRE

    Eggen, I.F.; Boeriu, C.G.

    2007-01-01

    The present invention relates to a process for the selective enzymatic hydrolysis of C-terminal esters of peptide substrates in the synthesis of peptides, comprising hydrolysing C-terminal tert-butyl esters using the protease subtilisin. This process is useful in the production of protected or unprotected peptides.

  18. Selective enzymatic hydrolysis of C-terminal tert-butyl esters of peptides

    NARCIS (Netherlands)

    Eggen, I.F.; Boeriu, C.G.

    2007-01-01

    The present invention relates to a process for the selective enzymatic hydrolysis of C-terminal esters of peptide substrates in the synthesis of peptides, comprising hydrolysing C-terminal tert-butyl esters using the protease subtilisin. This process is useful in the production of protected or unpro

  19. Structural Evidence for Direct Interactions Between the BRCT Domains of Human BRCA1 and a Phospho-Peptide from Human ACC1

    Energy Technology Data Exchange (ETDEWEB)

    Shen,Y.; Tong, L.

    2008-01-01

    The tandem BRCA1 C-terminal (BRCT) domains are phospho-serine/threonine recognition modules essential for the function of BRCA1. Recent studies suggest that acetyl-CoA carboxylase 1 (ACC1), an enzyme with crucial roles in de novo fatty acid biosynthesis and lipogenesis and essential for cancer cell survival, may be a novel binding partner for BRCA1, through interactions with its BRCT domains. We report here the crystal structure at 3.2 Angstroms resolution of human BRCA1 BRCT domains in complex with a phospho-peptide from human ACC1 (p-ACC1 peptide, with the sequence 1258-DSPPQ-pS-PTFPEAGH-1271), which provides molecular evidence for direct interactions between BRCA1 and ACC1. The p-ACC1 peptide is bound in an extended conformation, located in a groove between the tandem BRCT domains. There are recognizable and significant structural differences to the binding modes of two other phospho-peptides to these domains, from BACH1 and CtIP, even though they share a conserved pSer-Pro-(Thr/Val)-Phe motif. Our studies establish a framework for understanding the regulation of lipid biosynthesis by BRCA1 through its inhibition of ACC1 activity, which could be a novel tumor suppressor function of BRCA1.

  20. Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import

    Directory of Open Access Journals (Sweden)

    Fowke Keith R

    2005-10-01

    Full Text Available Abstract Background In addition to mediating the integration process, HIV-1 integrase (IN has also been implicated in different steps during viral life cycle including reverse transcription and viral DNA nuclear import. Although the karyophilic property of HIV-1 IN has been well demonstrated using a variety of experimental approaches, the definition of domain(s and/or motif(s within the protein that mediate viral DNA nuclear import and its mechanism are still disputed and controversial. In this study, we performed mutagenic analyses to investigate the contribution of different regions in the C-terminal domain of HIV-1 IN to protein nuclear localization as well as their effects on virus infection. Results Our analysis showed that replacing lysine residues in two highly conserved tri-lysine regions, which are located within previously described Region C (235WKGPAKLLWKGEGAVV and sequence Q (211KELQKQITK in the C-terminal domain of HIV-1 IN, impaired protein nuclear accumulation, while mutations for RK263,4 had no significant effect. Analysis of their effects on viral infection in a VSV-G pseudotyped RT/IN trans-complemented HIV-1 single cycle replication system revealed that all three C-terminal mutant viruses (KK215,9AA, KK240,4AE and RK263,4AA exhibited more severe defect of induction of β-Gal positive cells and luciferase activity than an IN class 1 mutant D64E in HeLa-CD4-CCR5-β-Gal cells, and in dividing as well as non-dividing C8166 T cells, suggesting that some viral defects are occurring prior to viral integration. Furthermore, by analyzing viral DNA synthesis and the nucleus-associated viral DNA level, the results clearly showed that, although all three C-terminal mutants inhibited viral reverse transcription to different extents, the KK240,4AE mutant exhibited most profound effect on this step, whereas KK215,9AA significantly impaired viral DNA nuclear import. In addition, our analysis could not detect viral DNA integration in each C-terminal

  1. Structural Time Domain Identification (STDI) Toolbox for Use with MATLAB

    DEFF Research Database (Denmark)

    Kirkegaard, Poul Henning; Andersen, P.; Brincker, Rune

    1997-01-01

    The Structural Time Domain Identification (STDI) toolbox for use with MATLABTM is developed at Aalborg University, Denmark, based on the system identification research performed during recent years. By now, a reliable set of functions offers a wide spectrum of services for all the important steps...... of multivariate time domain system identification of time-variant as well as time-invariant civil engineering structures from ambient testing data. A graphical user interface (GUI) is also developed to make the toolbox more user friendly.......The Structural Time Domain Identification (STDI) toolbox for use with MATLABTM is developed at Aalborg University, Denmark, based on the system identification research performed during recent years. By now, a reliable set of functions offers a wide spectrum of services for all the important steps...

  2. Structural Time Domain Identification (STDI) Toolbox for Use with MATLAB

    DEFF Research Database (Denmark)

    Kirkegaard, Poul Henning; Andersen, P.; Brincker, Rune

    The Structural Time Domain Identification (STDI) toolbox for use with MATLABTM is developed at Aalborg University, Denmark, based on the system identification research performed during recent years. By now, a reliable set of functions offers a wide spectrum of services for all the important steps...... of multivariate time domain system identification of time-variant as well as time-invariant civil engineering structures from ambient testing data. A graphical user interface (GUI) is also developed to make the toolbox more user friendly.......The Structural Time Domain Identification (STDI) toolbox for use with MATLABTM is developed at Aalborg University, Denmark, based on the system identification research performed during recent years. By now, a reliable set of functions offers a wide spectrum of services for all the important steps...

  3. Structures of the spectrin-ankyrin interaction binding domains

    Energy Technology Data Exchange (ETDEWEB)

    Ipsaro, Jonathan J.; Huang, Lei; Mondragón, Alfonso; (NWU)

    2010-01-07

    As key components of the erythrocyte membrane skeleton, spectrin and ankyrin specifically interact to tether the spectrin cytoskeleton to the cell membrane. The structure of the spectrin binding domain of ankyrin and the ankyrin binding domain of spectrin have been solved to elucidate the structural basis for ankyrin-spectrin recognition. The structure of repeats 14 and 15 of spectrin shows that these repeats are similar to all other spectrin repeats. One feature that could account for the preference of ankyrin for these repeats is the presence of a conserved, negatively charged patch on one side of repeat 14. The structure of the ankyrin ZU5 domain shows a novel structure containing a {beta} core. The structure reveals that the canonical ZU5 consensus sequence is likely to be missing an important region that codes for a {beta} strand that forms part of the core of the domain. In addition, a positively charged region is suggestive of a binding surface for the negatively charged spectrin repeat 14. Previously reported mutants of ankyrin that map to this region lie mostly on the surface of the protein, although at least one is likely to be part of the core.

  4. Modeling Microwave Structures in Time Domain Using Laguerre Polynomials

    Directory of Open Access Journals (Sweden)

    Z. Raida

    2006-09-01

    Full Text Available The paper is focused on time domain modeling of microwave structures by the method of moments. Two alternative schemes with weighted Laguerre polynomials are presented. Thanks to their properties, these schemes are free of late time oscillations. Further, the paper is aimed to effective and accurate evaluation of Green's functions integrals within these schemes. For this evaluation, a first- and second-order polynomial approximation is developed. The last part of the paper deals with modeling microstrip structures in the time domain. Conditions of impedance matching are derived, and the proposed approach is verified by modeling a microstrip filter.

  5. EFFICIENT TIME DOMAIN ANALYSIS of ARBITRARILY SHAPED CONDUCTING WIRE STRUCTURES

    Institute of Scientific and Technical Information of China (English)

    AI-Majdi Kadhum; Ming Qi

    2007-01-01

    We developed an efficient analysis the current induced in the wire structure. The analysis based on the time-Domain Integral Equation, in which a thin wire approximation is used. The time-domain electric field integral equation is used with the moment method to develop a numerical procedure for treating problems of scattering by arbitrary shaped bodies. We present an efficient numerical method for calculating the electromagnetic scattering from arbitrary shaped conducting bodies in the time domain with a comprehensive treatment of a single, straight thin wire.A time domain electric field integral equation is formulated for the problem of an arbitrary shape. The solution method is based on the moment method to solve the straight thin-wire problem.

  6. Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

    International Nuclear Information System (INIS)

    Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-α-helical regulatory domains classified into two related Pfam families: FadR-C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni2+ ions but that it is able to bind Zn2+ with Kd < 70 nM. It is concluded that Zn2+ is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors

  7. Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn{sup 2+}-binding FCD domains

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Meiying; Cooper, David R. [Integrated Center for Structure-Function Innovation, Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908-0736 (United States); Grossoehme, Nickolas E. [Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102 (United States); Yu, Minmin [Physical Biosciences Division, Lawrence Berkeley National Laboratory, MS4R0230, Berkeley, CA 94720 (United States); Hung, Li-Wei [Physical Biosciences Division, Lawrence Berkeley National Laboratory, MS4R0230, Berkeley, CA 94720 (United States); Physics Division, MS D454, Los Alamos National Laboratory, Los Alamos, NM 87545 (United States); Cieslik, Marcin; Derewenda, Urszula [Integrated Center for Structure-Function Innovation, Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908-0736 (United States); Lesley, Scott A. [The Scripps Research Institute, North Torrey Pines Road, La Jolla, CA 92037 (United States); Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121 (United States); Wilson, Ian A. [The Scripps Research Institute, North Torrey Pines Road, La Jolla, CA 92037 (United States); Giedroc, David P. [Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102 (United States); Derewenda, Zygmunt S., E-mail: zsd4n@virginia.edu [Integrated Center for Structure-Function Innovation, Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908-0736 (United States)

    2009-04-01

    Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-α-helical regulatory domains classified into two related Pfam families: FadR-C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni{sup 2+} ions but that it is able to bind Zn{sup 2+} with K{sub d} < 70 nM. It is concluded that Zn{sup 2+} is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.

  8. Structural Basis of the Interaction between Tuberous Sclerosis Complex 1 (TSC1) and Tre2-Bub2-Cdc16 Domain Family Member 7 (TBC1D7).

    Science.gov (United States)

    Qin, Jiayue; Wang, Zhizhi; Hoogeveen-Westerveld, Marianne; Shen, Guobo; Gong, Weimin; Nellist, Mark; Xu, Wenqing

    2016-04-15

    Mutations in TSC1 or TSC2 cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterized by the occurrence of benign tumors in various vital organs and tissues. TSC1 and TSC2, the TSC1 and TSC2 gene products, form the TSC protein complex that senses specific cellular growth conditions to control mTORC1 signaling. TBC1D7 is the third subunit of the TSC complex, and helps to stabilize the TSC1-TSC2 complex through its direct interaction with TSC1. Homozygous inactivation of TBC1D7 causes intellectual disability and megaencephaly. Here we report the crystal structure of a TSC1-TBC1D7 complex and biochemical characterization of the TSC1-TBC1D7 interaction. TBC1D7 interacts with the C-terminal region of the predicted coiled-coil domain of TSC1. The TSC1-TBC1D7 interface is largely hydrophobic, involving the α4 helix of TBC1D7. Each TBC1D7 molecule interacts simultaneously with two parallel TSC1 helices from two TSC1 molecules, suggesting that TBC1D7 may stabilize the TSC complex by tethering the C-terminal ends of two TSC1 coiled-coils. PMID:26893383

  9. Structure-function relations in the NTPase domain of the antiviral tRNA ribotoxin Escherichia coli PrrC

    Energy Technology Data Exchange (ETDEWEB)

    Meineke, Birthe; Shuman, Stewart, E-mail: s-shuman@ski.mskcc.org

    2012-06-05

    Breakage of tRNA by Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection. Expression of EcoPrrC is cytocidal in yeast, signifying that PrrC ribotoxicity crosses phylogenetic domain boundaries. EcoPrrC consists of an N-terminal NTPase module that resembles ABC transporters and a C-terminal nuclease module that is sui generis. PrrC homologs are prevalent in many other bacteria. Here we report that Haemophilus influenzae PrrC is toxic in E. coli and yeast. To illuminate structure-activity relations, we conducted a new round of mutational analysis of EcoPrrC guided by primary structure conservation among toxic PrrC homologs. We indentify 17 candidate active site residues in the NTPase module that are essential for toxicity in yeast when EcoPrrC is expressed at high gene dosage. Their functions could be educed by integrating mutational data with the atomic structure of the transition-state complex of a homologous ABC protein.

  10. Soil-structure-interaction analysis in time domain

    International Nuclear Information System (INIS)

    The procedures to perform nonlinear soil-structure-interaction analysis in the time domain are summarized. The nonlinearity is restricted to the structure and possibly an adjacent irregular soil region. The unbounded soil (far field) must remain linear in this formulation. Besides the direct method where local frequency-independent boundary conditions are enforced on the artificial boundary, various formulations based on the substructure method are addressed, ranging from a discrete model with springs, dashpots and masses to boundary-element methods with convolution integrals involving either the dynamic-stiffness coefficients or the Green's functions in the time domain via the iterative hybrid-frequency-time-domain analysis procedure with the nonlinearities affecting only the right-hand side of the equations of motion. (orig.)

  11. Identification and characterization of the role of c-terminal Src kinase in dengue virus replication.

    Science.gov (United States)

    Kumar, Rinki; Agrawal, Tanvi; Khan, Naseem Ahmed; Nakayama, Yuji; Medigeshi, Guruprasad R

    2016-01-01

    We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication. PMID:27457684

  12. DOG 1.0: illustrator of protein domain structures

    Institute of Scientific and Technical Information of China (English)

    Jian Ren; Longping Wen; Xinjiao Gao; Changjiang Jin; Yu Xue; Xuebiao Yao

    2009-01-01

    @@ Dear Editor, A good picture is worth a thousand words. Schematic diagram of protein domain structures with functional motifs/sites in a concise and illustrative drawing is greatly helpful for a broad readership to grasp the old and novel functions of proteins rapidly.

  13. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

    Energy Technology Data Exchange (ETDEWEB)

    Saikatendu, Kumar Singh; Zhang, Xuejun; Kinch, Lisa; Leybourne, Matthew; Grishin, Nick V.; Zhang, Hong (Texas-D); (U. of Texas-SMED)

    2009-01-26

    The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. The structure of SA1388 has been solved to 2.0{angstrom} resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric 'lids' formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric

  14. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

    International Nuclear Information System (INIS)

    The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. The structure of SA1388 has been solved to 2.0(angstrom) resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric 'lids' formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric 'lids' that

  15. Structural studies of the PARP-1 BRCT domain

    Directory of Open Access Journals (Sweden)

    Gabel Scott A

    2011-10-01

    Full Text Available Abstract Background Poly(ADP-ribose polymerase-1 (PARP-1 is one of the first proteins localized to foci of DNA damage. Upon activation by encountering nicked DNA, the PARP-1 mediated trans-poly(ADP-ribosylation of DNA binding proteins occurs, facilitating access and accumulation of DNA repair factors. PARP-1 also auto-(ADP-ribosylates its central BRCT-containing domain forming part of an interaction site for the DNA repair scaffolding protein X-ray cross complementing group 1 protein (XRCC1. The co-localization of XRCC1, as well as bound DNA repair factors, to sites of DNA damage is important for cell survival and genomic integrity. Results Here we present the solution structure and biophysical characterization of the BRCT domain of rat PARP-1. The PARP-1 BRCT domain has the globular α/β fold characteristic of BRCT domains and has a thermal melting transition of 43.0°C. In contrast to a previous characterization of this domain, we demonstrate that it is monomeric in solution using both gel-filtration chromatography and small-angle X-ray scattering. Additionally, we report that the first BRCT domain of XRCC1 does not interact significantly with the PARP-1 BRCT domain in the absence of ADP-ribosylation. Moreover, none of the interactions with other longer PARP-1 constructs which previously had been demonstrated in a pull-down assay of mammalian cell extracts were detected. Conclusions The PARP-1 BRCT domain has the conserved BRCT fold that is known to be an important protein:protein interaction module in DNA repair and cell signalling pathways. Data indicating no significant protein:protein interactions between PARP-1 and XRCC1 likely results from the absence of poly(ADP-ribose in one or both binding partners, and further implicates a poly(ADP-ribose-dependent mechanism for localization of XRCC1 to sites of DNA damage.

  16. Relevance of amyloid precursor-like protein 2 C-terminal fragments in pancreatic cancer cells

    OpenAIRE

    PETERS, HALEY L.; Tuli, Amit; Wang, Xiaojian; Liu, Cuiling; Pan, Zenggang; Ouellette, Michel M.; Hollingsworth, Michael A.; MacDonald, Richard G.; Solheim, Joyce C.

    2012-01-01

    In some cellular systems, particularly neurons, amyloid precursor-like protein 2 (APLP2), and its highly homologous family member amyloid precursor protein (APP), have been linked to cellular growth. APLP2 and APP undergo regulated intramembrane proteolysis to produce C-terminal fragments. In this study, we found comprehensive expression of APLP2 C-terminal fragments in a panel of pancreatic cancer cell lines; however, APP C-terminal fragments were notably limited to the BxPC3 cell line. Exte...

  17. ASH structure alignment package: Sensitivity and selectivity in domain classification

    Directory of Open Access Journals (Sweden)

    Toh Hiroyuki

    2007-04-01

    Full Text Available Abstract Background Structure alignment methods offer the possibility of measuring distant evolutionary relationships between proteins that are not visible by sequence-based analysis. However, the question of how structural differences and similarities ought to be quantified in this regard remains open. In this study we construct a training set of sequence-unique CATH and SCOP domains, from which we develop a scoring function that can reliably identify domains with the same CATH topology and SCOP fold classification. The score is implemented in the ASH structure alignment package, for which the source code and a web service are freely available from the PDBj website http://www.pdbj.org/ASH/. Results The new ASH score shows increased selectivity and sensitivity compared with values reported for several popular programs using the same test set of 4,298,905 structure pairs, yielding an area of .96 under the receiver operating characteristic (ROC curve. In addition, weak sequence homologies between similar domains are revealed that could not be detected by BLAST sequence alignment. Also, a subset of domain pairs is identified that exhibit high similarity, even though their CATH and SCOP classification differs. Finally, we show that the ranking of alignment programs based solely on geometric measures depends on the choice of the quality measure. Conclusion ASH shows high selectivity and sensitivity with regard to domain classification, an important step in defining distantly related protein sequence families. Moreover, the CPU cost per alignment is competitive with the fastest programs, making ASH a practical option for large-scale structure classification studies.

  18. Structural basis for the recognition of two consecutive mutually interacting DPF motifs by the SGIP1 μ homology domain

    Science.gov (United States)

    Shimada, Atsushi; Yamaguchi, Atsuko; Kohda, Daisuke

    2016-01-01

    FCHo1, FCHo2, and SGIP1 are key regulators of clathrin-mediated endocytosis. Their μ homology domains (μHDs) interact with the C-terminal region of an endocytic scaffold protein, Eps15, containing fifteen Asp-Pro-Phe (DPF) motifs. Here, we show that the high-affinity μHD-binding site in Eps15 is a region encompassing six consecutive DPF motifs, while the minimal μHD-binding unit is two consecutive DPF motifs. We present the crystal structures of the SGIP1 μHD in complex with peptides containing two DPF motifs. The peptides bind to a novel ligand-binding site of the μHD, which is distinct from those of other distantly related μHD-containing proteins. The two DPF motifs, which adopt three-dimensional structures stabilized by sequence-specific intramotif and intermotif interactions, are extensively recognized by the μHD and are both required for binding. Thus, consecutive and singly scattered DPF motifs play distinct roles in μHD binding.

  19. Polar and chemical domain structures of lead scandium tantalate (PST)

    International Nuclear Information System (INIS)

    The local structure of chemical and polar domains and domain walls is determined directly by atomic resolution high-resolution electron microscopy. Thus the Pb, Ta and Sc atomic positions may be located in the images of very thin crystals. Furthermore the Pb cation displacements away from the ideal perovskite A-site have been measured directly for the first time. Local variations in polarization direction may be mapped directly off the images, provided certain electron optical conditions are met. The results are relevant to recent theories of polar-glass behaviour in relaxor-type complex oxide functional ceramics. 17 refs., 9 figs

  20. Structure of the first PDZ domain of human PSD-93

    DEFF Research Database (Denmark)

    Fiorentini, Monica; Nielsen, Ann Kallehauge; Kristensen, Ole;

    2009-01-01

    The crystal structure of the PDZ1 domain of human PSD-93 has been determined to 2.0 A resolution. The PDZ1 domain forms a crystallographic trimer that is also predicted to be stable in solution. The main contributions to the stabilization of the trimer seem to arise from interactions involving the...... well as of the closely related human PSD-95 PDZ1 shows that they are very similar in terms of amino-acid composition. However, the cleft is significantly narrower in PSD-95. This could be part of the basis of peptide selectivity between PSD-93 PDZ1 and PSD-95 PDZ1....

  1. Magnetoresistance due to domain walls in semiconducting magnetic nano structures

    International Nuclear Information System (INIS)

    Magnetoresistance of a semiconducting ferromagnetic nano structure with a laterally constrained domain wall is analyzed theoretically in the limit of sharp domain walls and fully polarized electron gas is considered. The spin-orbit interaction of Rash ba type is included into considerations. It is shown that the magnetoresistance in such a case can be relatively large, which is in a qualitative agreement with recent experimental observations. It is also shown that spin-orbit interaction can enhance the magnetoresistance. The role of localization corrections is also briefly discussed

  2. Magnetoresistance due to domain walls in semiconducting magnetic nano structures

    Energy Technology Data Exchange (ETDEWEB)

    Dugaev, V.K. [Max-Planck-Institut fuer Mikrostrukturphysik, Weinberg 2, 06120 Halle (Germany) and Institute for Problems of Materials Science, Vilde 5, 58001 Chernovtsy (Ukraine)]. E-mail: vdugaev@mpi-halle.de; Berakdar, J. [Max-Planck-Institut fuer Mikrostrukturphysik, Weinberg 2, 06120 Halle (Germany); Barnas, J. [Department of Physics, Adam Mickiewicz University, ul. Umultowska 85, 61-614 Poznan, and Institute of Molecular Physics PAN, ul. M. Smoluchowskiego 17, 60-179 Poznan (Poland); Dobrowolski, W. [Institute of Physics PAN, Al. Lotnikow 32/46, 02-668 Warsaw (Poland); Mitin, V.F. [Institute of Semiconductor Physics, NANU, pr. Nauki, 03108 Kiev (Ukraine); Vieira, M. [ISEL-DEETC, Rua Cons. Emidio Navarro, 1950-062 Lisbon (Portugal)

    2005-12-15

    Magnetoresistance of a semiconducting ferromagnetic nano structure with a laterally constrained domain wall is analyzed theoretically in the limit of sharp domain walls and fully polarized electron gas is considered. The spin-orbit interaction of Rash ba type is included into considerations. It is shown that the magnetoresistance in such a case can be relatively large, which is in a qualitative agreement with recent experimental observations. It is also shown that spin-orbit interaction can enhance the magnetoresistance. The role of localization corrections is also briefly discussed.

  3. Domain structures in PbTiO3 thin films

    OpenAIRE

    Fløystad, Jostein Bø

    2010-01-01

    We have studied thin films (27 and 50 unit cells) of ferroelectric PbTiO3 by acquiring reciprocal space maps using wide-angle x-ray scattering. Diffuse satelites were found around the PbTiO3 Bragg peaks, consistent with a ferroelectric domain structure within the film. The satelites showed a shift towards higher wavevector transfers normal to the film plane.We have developed a Markov chain based model to account for a random distribution of domain sizes in the film. The model is in good agree...

  4. Sequence and modified group analysis on C-terminal modified analogs of endomorphin-2 using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometer

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In this paper, a series of C-terminal modified analogs of endomorphin-2 is investigated using ESI-FT-ICR-MS. Some b, y″, a, and internal ions are found in the CID spectra and slight mass differ- ences between the calculated and observed results are obtained. Moreover, if the C-terminal modified group is t-butyloxy, it can lose butene through McLafferty rearrangement. FT-ICR MS shows its power in peptide sequencing successfully helping us obtain the structure of peptide analogs.

  5. Nanoscaled domain structures and magnetization processes in thin films

    International Nuclear Information System (INIS)

    Magnetic thin films and multilayers display a fascinating variety of domain structures and magnetization processes due to competing interactions. For single layers one has to consider intra- and intergrain exchange interactions and magnetostatic interactions versus complex anisotropies. In multilayer systems, additional interlayer interactions (e.g. exchange bias, antiferromagnetic or ferromagnetic coupling) broaden the range of possible coupling scenarios and thus the diversity of the magnetic behavior. For a microscopic understanding of the magnetization processes we perform magnetic force microscopy (MFM), which offers a direct observation of the domain structure on a length scale down to 10 nm, and its evolution in a magnetic field. We present multilayers with perpendicular anisotropy and antiferromagnetic interlayer coupling of the type[(Co/Pt)XCo/Ru]N, which possess complex antiferromagnetically and ferromagnetically coupled domain structures, depending on temperature and magnetic history. As an example of a highly anisotropic single layer film with in-plane easy axis orientation, the magnetization process of a nanocrystalline epitaxial SmCo5 film is investigated. Whereas the magnetizing process proceeds on a small length scale of 100 nm, in the demagnetizing process the switching occurs via a fast propagation of few interaction domain walls

  6. Differential contributions of porcine bocavirus NP1 protein N- and C-terminal regions to its nuclear localization and immune regulation.

    Science.gov (United States)

    Zhang, Ruoxi; Fang, Liurong; Cai, Kaimei; Zeng, Songlin; Wu, Wei; An, Kang; Chen, Huanchun; Xiao, Shaobo

    2016-05-01

    Porcine bocavirus (PBoV), a newly identified parvovirus in the family Parvoviridae, has been reported worldwide in swine with post-weaning multisystemic wasting syndrome, respiratory disease or diarrhoea and in asymptomatic swine. NP1 is a protein unique to the genus Bocavirus and its function is not fully understood. In this study, we show that the N-terminal region of PBoV NP1 contains two classical nuclear localization signals (cNLSs) and a non-classical NLS. The N-terminal region also inhibits the promoter activity of IFN-β and IFN-stimulated response element activity the same as full-length NP1 protein, but the PBoV NP1 C-terminal region does not. PBoV NP1 also induces NFκB activation by increasing the phosphorylation of p65, and we demonstrate that the C-terminal region (aa 168-218) is responsible for the induction of NFκB, although the cNLS region of NP1 enhances this activation. The data suggest that PBoV NP1 contains two functionally independent domains in its N- and C-terminal regions. Thus, the N-terminal region of PBoV NP1 is critical for its nuclear localization and IFN-related promoter inhibition, and the C-terminal region is critical for its induction of NFκB. PMID:26813332

  7. Domain structure effects in the relaxor ferroelectric PZN-PT

    International Nuclear Information System (INIS)

    Full text: PZN-PT (Pb(Zn1/3Nb2/3)O33-x%PbTiO33) exhibits a maximum strain versus electric field exceeding 1% if it is poled along Two suggestions exist for the origin of this exceptional strain the domain structure created during poling, 'polarisation rotation' due to the existence of a monoclinic phase reported for PZN 8%PT Neutron diffraction data will be presented that shows the monoclmic phase is in fact a distortion of the ambient phase resulting from intersecting domain structures This interpretation also reconciles PZN-4 5%PT which shows similar properties to PZN 8%PT yet does not exist in the monoclmic structure. Copyright (2005) Australian Institute of Physics

  8. Structural analysis of the DNA-binding domain of the Erwinia amylovora RcsB protein and its interaction with the RcsAB box.

    Science.gov (United States)

    Pristovsek, Primoz; Sengupta, Kaushik; Löhr, Frank; Schäfer, Birgit; von Trebra, Markus Wehland; Rüterjans, Heinz; Bernhard, Frank

    2003-05-16

    The transcriptional regulator RcsB interacts with other coactivators to control the expression of biosynthetic operons in enterobacteria. While in a heterodimer complex with the regulator RcsA the RcsAB box consensus is recognized, DNA binding sites for RcsB without RcsA have also been identified. The conformation of RcsB might therefore be modulated upon interaction with various coactivators, resulting in the recognition of different DNA targets. We report the solution structure of the C-terminal DNA-binding domain of the RcsB protein from Erwinia amylovora spanning amino acid residues 129-215 solved by heteronuclear magnetic resonance (NMR) spectroscopy. The C-terminal domain is composed of four alpha-helices where two central helices form a helix-turn-helix motif similar to the structures of the regulatory proteins GerE, NarL, and TraR. Amino acid residues involved in the RcsA independent DNA binding of RcsB were identified by titration studies with a RcsAB box consensus fragment. Data obtained from NMR spectroscopy together with surface plasmon resonance measurements demonstrate that the RcsAB box is specifically recognized by the RcsAB heterodimer as well as by RcsB alone. However, the binding constant of RcsB alone at target promoters from Escherichia coli, E. amylovora, and Pantoea stewartii was approximately 1 order of magnitude higher compared with that of the RcsAB heterodimer. We present evidence that the obvious role of RcsA is not to alter the DNA binding specificity of RcsB but to stabilize RcsB-DNA complexes. PMID:12740396

  9. Structure and evolution of the magnetochrome domains: no longer alone

    Directory of Open Access Journals (Sweden)

    Pascal eArnoux

    2014-03-01

    Full Text Available Magnetotactic bacteria (MTB can swim along Earth’s magnetic field lines, thanks to the alignment of dedicated cytoplasmic organelles. These organelles, termed magnetosomes, are proteolipidic vesicles filled by a 35-120 nm crystal of either magnetite or greigite. The formation and alignment of magnetosomes are mediated by a group of specific genes, the mam genes, encoding the magnetosome-associated proteins. The whole process of magnetosome biogenesis can be divided into four sequential steps; (i cytoplasmic membrane invagination, (ii magnetosomes alignment, (iii iron crystal nucleation and (iv species-dependent mineral size and shape control. Since both magnetite and greigite are a mix of iron(III and iron(II, iron redox state management within the magnetosome vesicle is a key issue. Recently, studies have started pointing out the importance of a MTB-specific c-type cytochrome domain found in several magnetosome-associated proteins (MamE, P, T and X. This magnetochrome (MCR domain is almost always found in tandem, and this tandem is either found alone (MamT, in combination with a PDZ domain (MamP, a domain of unknown function (MamX or with a trypsin combined to one or two PDZ domains (MamE. By taking advantage of new genomic data available on MTB and a recent structural study of MamP, which helped define the MCR domain boundaries, we attempt to retrace the evolutionary history within and between the different MCR-containing proteins. We propose that the observed tandem repeat of MCR is the result of a convergent evolution and attempt to explain why this domain is rarely found alone.

  10. Structure and evolution of the magnetochrome domains: no longer alone.

    Science.gov (United States)

    Arnoux, Pascal; Siponen, Marina I; Lefèvre, Christopher T; Ginet, Nicolas; Pignol, David

    2014-01-01

    Magnetotactic bacteria (MTB) can swim along Earth's magnetic field lines, thanks to the alignment of dedicated cytoplasmic organelles. These organelles, termed magnetosomes, are proteolipidic vesicles filled by a 35-120 nm crystal of either magnetite or greigite. The formation and alignment of magnetosomes are mediated by a group of specific genes, the mam genes, encoding the magnetosome-associated proteins. The whole process of magnetosome biogenesis can be divided into four sequential steps; (i) cytoplasmic membrane invagination, (ii) magnetosomes alignment, (iii) iron crystal nucleation and (iv) species-dependent mineral size and shape control. Since both magnetite and greigite are a mix of iron (III) and iron (II), iron redox state management within the magnetosome vesicle is a key issue. Recently, studies have started pointing out the importance of a MTB-specific c-type cytochrome domain found in several magnetosome-associated proteins (MamE, P, T, and X). This magnetochrome (MCR) domain is almost always found in tandem, and this tandem is either found alone (MamT), in combination with a PDZ domain (MamP), a domain of unknown function (MamX) or with a trypsin combined to one or two PDZ domains (MamE). By taking advantage of new genomic data available on MTB and a recent structural study of MamP, which helped define the MCR domain boundaries, we attempt to retrace the evolutionary history within and between the different MCR-containing proteins. We propose that the observed tandem repeat of MCR is the result of a convergent evolution and attempt to explain why this domain is rarely found alone. PMID:24723915

  11. Identification of a Chemical Probe for Bromo and Extra C-Terminal Bromodomain Inhibition through Optimization of a Fragment-Derived Hit

    OpenAIRE

    Fish, Paul V.; Filippakopoulos, Panagis; Bish, Gerwyn; Brennan, Paul E.; Bunnage, Mark E.; Cook, Andrew S.; Federov, Oleg; Gerstenberger, Brian S.; Jones, Hannah; Knapp, Stefan; Marsden, Brian; Nocka, Karl; Owen, Dafydd R.; Philpott, Martin; Picaud, Sarah

    2012-01-01

    The posttranslational modification of chromatin through acetylation at selected histone lysine residues is governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The significance of this subset of the epigenetic code is interrogated and interpreted by an acetyllysine-specific protein–protein interaction with bromodomain reader modules. Selective inhibition of the bromo and extra C-terminal domain (BET) family of bromodomains with a small molecule is feasible, and this...

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

    Science.gov (United States)

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

    2002-03-01

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

  13. Crystal structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Meiying; Cooper, David; Grossoehmerb, Nickolas; Yu, Minmin; Hung, Li-Wei; Cieslik, Murcin; Derewendaro, Urszula; Lesley, Scott; Wilson, Ian; Giedrocb, David; Derewenda, Zygmunt

    2009-06-06

    The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged helix (WH) DNA-binding domains and diverse C-terminal, regulatory domains, which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all a-helical regulatory domains classified into two related Pfam families: FadR{_}C and FCD. Only two crystal structures of the FadR family members, i.e. the E. coli FadR protein and the LldR from C. glutamicum, have been described to date in literature. Here we describe the crystal structure of TM0439, a GntR regulator with an FCD domain, found in the Thermotoga maritima genome. The FCD domain is similar to that of the LldR regulator, and contains a buried metal binding site. Using atomic absorption spectroscopy and Trp fluorescence, we show that the recombinant protein contains bound Ni{sup 2+} ions, but it is able to bind Zn{sup 2+} with K{sub D} < 70 nM . We conclude that Zn{sup 2+} is the likely physiological metal, where it may perform either or both structural and regulatory roles. Finally, we compare the TM0439 structure to two other FadR family structures recently deposited by Structural Genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.

  14. C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings.

    Directory of Open Access Journals (Sweden)

    Antoine Picciocchi

    Full Text Available Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+ channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.

  15. TMAO promotes fibrillization and microtubule assembly activity in the C-terminal repeat region of tau.

    Science.gov (United States)

    Scaramozzino, Francesca; Peterson, Dylan W; Farmer, Patrick; Gerig, J T; Graves, Donald J; Lew, John

    2006-03-21

    Alzheimer's disease most closely correlates with the appearance of the neurofibrillary tangles (NFTs), intracellular fibrous aggregates of the microtubule-associated protein, tau. Under native conditions, tau is an unstructured protein, and its physical characterization has revealed no clues about the three-dimensional structural determinants essential for aggregation or microtubule binding. We have found that the natural osmolyte trimethylamine N-oxide (TMAO) induces secondary structure in a C-terminal fragment of tau (tau(187)) and greatly promotes both self-aggregation and microtubule (MT) assembly activity. These processes could be distinguished, however, by a single-amino acid substitution (Tyr(310) --> Ala), which severely inhibited aggregation but had no effect on MT assembly activity. The inability of this mutant to aggregate could be completely reversed by TMAO. We propose a model in which TMAO induces partial order in tau(187), resulting in conformers that may correspond to on-pathway intermediates of either aggregation or tau-dependent MT assembly or both. These studies set the stage for future high-resolution structural characterization of these intermediates and the basis by which Tyr(310) may direct pathologic versus normal tau function. PMID:16533051

  16. Crystal structure of the R-protein of the multisubunit ATP-dependent restriction endonuclease NgoAVII

    OpenAIRE

    Tamulaitiene, Giedre; Silanskas, Arunas; Grazulis, Saulius; Zaremba, Mindaugas; Siksnys, Virginijus

    2014-01-01

    The restriction endonuclease (REase) NgoAVII is composed of two proteins, R.NgoAVII and N.NgoAVII, and shares features of both Type II restriction enzymes and Type I/III ATP-dependent restriction enzymes (see accompanying paper Zaremba et al., 2014). Here we present crystal structures of the R.NgoAVII apo-protein and the R.NgoAVII C-terminal domain bound to a specific DNA. R.NgoAVII is composed of two domains: an N-terminal nucleolytic PLD domain; and a C-terminal B3-like DNA-binding domain i...

  17. Synthesis of proposed macrocyclic binder of C-terminal domain of HIV-1 capsid protein

    Czech Academy of Sciences Publication Activity Database

    Rulíšek, Lubomír; Kožíšek, M.; Szmolková, B.; Machara, A.

    Praha: Czech Chemical Society, 2015. s. 111. [Liblice 2015. Advances in Organic, Bioorganic and Pharmaceutical Chemistry /50./. 06.11.2015-08.11.2015, Olomouc] R&D Projects: GA ČR GA13-19561S Institutional support: RVO:61388963 Keywords : HIV * drug design * molecular modelling Subject RIV: CC - Organic Chemistry

  18. Biophysical Studies of the C-terminal domains of the melibiose transporter from Escherichia coli

    OpenAIRE

    Fürst, Oliver

    2012-01-01

    En este trabajo, el objetivo principal ha sido el estudio del mecanismo del co-transporte de la permeasa de melibiosa (MelB) de Escherichia coli. Este proteina de membrana realiza co-transporte activo de varios oligosacáridos utilizando la fuerza electroquímica de cationes. Una característica remarcable de este transportador es el hecho que permite utilizar tres diferentes cationes, el protón, el sodio y el litio. Los sustratos con mayor afinidad a la proteína son el sodio y la melibiosa. ...

  19. Domain wall structure transformations in epitaxial ferrite garnet films

    International Nuclear Information System (INIS)

    Dynamic characteristics of domain walls (DW) in the process of the transformation of their structure occuring under the static field effect in the film plane are investigated. The epitaxiat ferrite-garnet film of the (YSmBa)3(FeGa)5O12 composition with parameters: 4πM=178 Gs (M=saturation magnetization), quality factor approximately equal to 10, thickness = 6.5 μm, band domain structure period = 13.5 μm has been studied. The signal of DW resonance oscillations excited by radio-frequency field with amplitude 0.01:1 Oe, oscillating along the slight magnetization axis perpendicular to the film plane has been observed. The constant magnetic field, H, in the film plane is oriented along the DW and is used not only for the transformation of DW structure but also for changing its resonance frequency by changing it effective mass. The results presented testify to the fact that the resonance methods may be successfully used not only for studying the DW dynamics but also for investigating into the domain wall structure and the process of its transformation

  20. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; Glover, J. N. Mark

    2009-01-01

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC282–202, which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer. PMID:19482939

  1. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon-helix-helix DNA-binding fold.

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J J; Glover, J N Mark

    2009-06-16

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-A X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2(82-202), which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer. PMID:19482939

  2. Asparagine 326 in the extremely C-terminal region of XRCC4 is essential for the cell survival after irradiation

    International Nuclear Information System (INIS)

    XRCC4 is one of the crucial proteins in the repair of DNA double-strand break (DSB) through non-homologous end-joining (NHEJ). As XRCC4 consists of 336 amino acids, N-terminal 200 amino acids include domains for dimerization and for association with DNA ligase IV and XLF and shown to be essential for XRCC4 function in DSB repair and V(D)J recombination. On the other hand, the role of the remaining C-terminal region of XRCC4 is not well understood. In the present study, we noticed that a stretch of ∼20 amino acids located at the extreme C-terminus of XRCC4 is highly conserved among vertebrate species. To explore its possible importance, series of mutants in this region were constructed and assessed for the functionality in terms of ability to rescue radiosensitivity of M10 cells lacking XRCC4. Among 13 mutants, M10 transfectant with N326L mutant (M10-XRCC4N326L) showed elevated radiosensitivity. N326L protein showed defective nuclear localization. N326L sequence matched the consensus sequence of nuclear export signal. Leptomycin B treatment accumulated XRCC4N326L in the nucleus but only partially rescued radiosensitivity of M10-XRCC4N326L. These results collectively indicated that the functional defects of XRCC4N326L might be partially, but not solely, due to its exclusion from nucleus by synthetic nuclear export signal. Further mutation of XRCC4 Asn326 to other amino acids, i.e., alanine, aspartic acid or glutamine did not affect the nuclear localization but still exhibited radiosensitivity. The present results indicated the importance of the extremely C-terminal region of XRCC4 and, especially, Asn326 therein. - Highlights: • Extremely C-terminal region of XRCC4 is highly conserved among vertebrate species. • XRCC4 C-terminal point mutants, R325F and N326L, are functionally deficient in terms of survival after irradiation. • N326L localizes to the cytoplasm because of synthetic nuclear export signal. • Leptomycin B restores the nuclear localization of N

  3. Asparagine 326 in the extremely C-terminal region of XRCC4 is essential for the cell survival after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Wanotayan, Rujira; Fukuchi, Mikoto; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa, E-mail: yoshim@nr.titech.ac.jp

    2015-02-20

    XRCC4 is one of the crucial proteins in the repair of DNA double-strand break (DSB) through non-homologous end-joining (NHEJ). As XRCC4 consists of 336 amino acids, N-terminal 200 amino acids include domains for dimerization and for association with DNA ligase IV and XLF and shown to be essential for XRCC4 function in DSB repair and V(D)J recombination. On the other hand, the role of the remaining C-terminal region of XRCC4 is not well understood. In the present study, we noticed that a stretch of ∼20 amino acids located at the extreme C-terminus of XRCC4 is highly conserved among vertebrate species. To explore its possible importance, series of mutants in this region were constructed and assessed for the functionality in terms of ability to rescue radiosensitivity of M10 cells lacking XRCC4. Among 13 mutants, M10 transfectant with N326L mutant (M10-XRCC4{sup N326L}) showed elevated radiosensitivity. N326L protein showed defective nuclear localization. N326L sequence matched the consensus sequence of nuclear export signal. Leptomycin B treatment accumulated XRCC4{sup N326L} in the nucleus but only partially rescued radiosensitivity of M10-XRCC4{sup N326L}. These results collectively indicated that the functional defects of XRCC4{sup N326L} might be partially, but not solely, due to its exclusion from nucleus by synthetic nuclear export signal. Further mutation of XRCC4 Asn326 to other amino acids, i.e., alanine, aspartic acid or glutamine did not affect the nuclear localization but still exhibited radiosensitivity. The present results indicated the importance of the extremely C-terminal region of XRCC4 and, especially, Asn326 therein. - Highlights: • Extremely C-terminal region of XRCC4 is highly conserved among vertebrate species. • XRCC4 C-terminal point mutants, R325F and N326L, are functionally deficient in terms of survival after irradiation. • N326L localizes to the cytoplasm because of synthetic nuclear export signal. • Leptomycin B restores the

  4. Modeling Microwave Structures in Time Domain Using Laguerre Polynomials

    OpenAIRE

    Z. Raida; Lacik, J.

    2006-01-01

    The paper is focused on time domain modeling of microwave structures by the method of moments. Two alternative schemes with weighted Laguerre polynomials are presented. Thanks to their properties, these schemes are free of late time oscillations. Further, the paper is aimed to effective and accurate evaluation of Green's functions integrals within these schemes. For this evaluation, a first- and second-order polynomial approximation is developed. The last part of the paper deals with mode...

  5. Solution structure of the first immunoglobulin domain of human myotilin

    Energy Technology Data Exchange (ETDEWEB)

    Heikkinen, Outi [University of Helsinki, Laboratory of Organic Chemistry, Department of Chemistry (Finland)], E-mail: Outi.K.Heikkinen@helsinki.fi; Permi, Perttu [University of Helsinki, Program in Structural Biology and Biophysics, Institute of Biotechnology (Finland); Koskela, Harri [University of Helsinki, Finnish Institute for Verification of the Chemical Weapons Convention (VERIFIN) (Finland); Carpen, Olli [University of Helsinki, Department of Anatomy and Neuroscience Program, Biomedicum (Finland); Ylaenne, Jari [University of Jyvaeskylae, Department of Biological and Environmental Science and Nanoscience Center (Finland); Kilpelaeinen, Ilkka [University of Helsinki, Laboratory of Organic Chemistry, Department of Chemistry (Finland)

    2009-06-15

    Myotilin is a 57 kDa actin-binding and -bundling protein that consists of a unique serine-rich amino-terminus, two Ig-domains and a short carboxy-terminus with a PDZ-binding motif. Myotilin localizes in sarcomeric Z-discs, where it interacts with several sarcomeric proteins. Point mutations in myotilin cause muscle disorders morphologically highlighted by sarcomeric disarray and aggregation. The actin-binding and dimerization propensity of myotilin has been mapped to the Ig-domains. Here we present high-resolution structure of the first Ig-domain of myotilin (MyoIg1) determined with solution state NMR spectroscopy. Nearly complete chemical shift assignments of MyoIg1 were achieved despite several missing backbone {sup 1}H-{sup 15}N-HSQC signals. The structure derived from distance and dihedral angle restraints using torsion angle dynamics was further refined using molecular dynamics. The structure of MyoIg1 exhibits I-type Ig-fold. The absence of several backbone {sup 1}H-{sup 15}N-HSQC signals can be explained by conformational exchange taking place at the hydrophobic core of the protein.

  6. Effects of sub-domain structure on initial magnetization curve and domain size distribution of stacked media

    International Nuclear Information System (INIS)

    In this paper, in order to confirm the sub-domain structure in stacked media demagnetized with in-plane field, initial magnetization curves and magnetic domain size distribution were investigated. Both experimental and simulation results showed that an initial magnetization curve for the medium demagnetized with in-plane field (MDI) initially rose faster than that for the medium demagnetized with perpendicular field (MDP). It is inferred that this is because the MDI has a larger number of domain walls than the MDP due to the existence of the sub-domains, resulting in an increase in the probability of domain wall motion. Dispersion of domain size for the MDI was larger than that for the MDP. This is because sub-domains are formed not only inside the domain but also at the domain boundary region, and they change the position of the domain boundary to affect the domain size. - Highlights: • An initial magnetization curve for MDI initially rose faster than that for MDP. • Dispersion of domain size for the MDI was larger than that for the MDP. • Experimental and simulation results can be explained by existence of sub-domains

  7. Molecular and structural characterisation of the human sodium/iodide symporter (h N.I.S.) C-terminus and the implication of this domain in the transporter regulation; Caracterisation moleculaire et structurale de l'extremite C-Terminale du co-transporteur sodium/iode humain (h N.I.S.): Implication de ce domaine dans la regulation du transporteur

    Energy Technology Data Exchange (ETDEWEB)

    Huc, S

    2007-12-15

    The human natrium iodide symporter (h N.I.S.) is an intrinsic membrane protein expressed in thyroid cells where it allows iodide uptake and accumulation. It is composed of thirteen transmembrane helices and its ninety- three amino acids long cytosolic C-terminus presents many potential post-translational regulatory sites. A first part of the PhD work has been dedicated to the expression in a bacterial system and to the purification of the cytosolic C-terminal fragment. Biochemical and structural characterisation have revealed that this C-terminus is very flexible but prone to dimerization. The fragment has also been used as a bait to test the interactions with PDZ domain proteins spotted on a membrane. Several proteins interacting with the (natrium/iodide symporter) N.I.S. C-terminus have thus been identified and the study of their implication in the protein regulation has been initiated. A second part of the work has underlined the existence of a N.I.S. fragment co-purified with the entire protein. This fragment has been found in cells in culture stably expressing N.I.S. and also in human thyroid extracts and in rodent thyroid cells. We observed that this fragment is spontaneously associated with the entire protein. It is composed of the last 131 amino acid of the protein and so comprises the last transmembrane domain and the C-terminal extremity. The expression of a truncated form of h N.I.S., lacking the last 131 amino acids, shows that this protein is not correctly addressed to the cell membrane and cells expressing this mutated symporter cannot accumulate iodide. However, our results show that the co-expression of the two N.I.S. parts, the truncated form lacking the last 131 amino acid, and the complementary C-terminal fragment, leads to cells presenting 10 % of the activity of cells expressing the whole N.I.S.. (author)

  8. Cooperative regulation of Cav1.2 channels by intracellular Mg2+, the proximal C-terminal EF-hand, and the distal C-terminal domain

    OpenAIRE

    Brunet, Sylvain; Scheuer, Todd; Catterall, William A.

    2009-01-01

    L-type Ca2+ currents conducted by Cav1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg2+ (Mgi) inhibits the ionic current of Cav1.2 channels. Because Mgi is altered in ischemia and heart failure, its regulation of Cav1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mgi on voltage-dependent inactivation (VDI) of Cav1.2 channels using Na+ as permeant ion to eliminate the effects of permeant divalent cat...

  9. Two distinct domains of protein 4.1 critical for assembly offunctional nuclei in Vitro

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Heald, Rebecca; Lee, Gloria; Nunomura, Wataru; Gimm,J. Aura; Mohandas, Narla; Chasis, Joel AnneJ. Aura; Mohandas, Narla; Chasis, Joel Anne

    2002-11-15

    Protein 4.1R, a multifunctional structural protein, acts asan adaptor in mature red cell membrane skeletons linking spectrin-actincomplexes to plasma membrane-associated proteins. In nucleated cellsprotein 4.1 is not associated exclusively with plasma membrane but isalso detected at several important subcellular locations crucial for celldivision. To identify 4.1 domains having critical functions in nuclearassembly, 4.1 domain peptides were added to Xenopus egg extract nuclearreconstitution reactions. Morphologically disorganized, replicationdeficient nuclei assembled when spectrin-actin binding domain orNuMA-binding C-terminal domain peptides were present. However, controlvariant spectrin-actin binding domain peptides incapable of bindingactin, or mutant C-terminal domain peptides with reduced NuMA binding,had no deleterious effects on nuclear reconstitution. To test if 4.1 isrequired for proper nuclear assembly, 4.1 isoforms were depleted withspectrin-actin binding or C-terminal domain-specific antibodies. Nucleiassembled in depleted extracts ha d deranged phenotypes. However, nuclearassembly could be rescued by addition of recombinant 4.1R. Our dataestablishes that protein 4.1 is essential for nuclear assembly andidentifies two distinct 4.1 domains, initially characterized incytoskeletal interactions, that have crucial and versatile functions innuclear assembly.

  10. NMR and computational evidence that high-affinity bradykinin receptor antagonists adopt C-terminal beta-turns.

    Science.gov (United States)

    Kyle, D J; Blake, P R; Smithwick, D; Green, L M; Martin, J A; Sinsko, J A; Summers, M F

    1993-05-14

    Three tetrapeptides were prepared, each corresponding to the four C-terminal amino acid residues of highly potent, second-generation bradykinin receptor antagonists. The tetrapeptides are (IA) Ser-D-Phe-Oic-Arg, (IIA) Ser-D-Tic-Oic-Arg, and (IIIA) Ser-D-Hype(trans-propyl)-Oic-Arg. Solution conformations for each were determined by incorporating interproton distance restraints, determined by 2D NMR experiments performed in water at neutral pH, into a series of distance geometry/simulated annealing model building calculations. Similarly, systematic conformational analyses were performed for each using molecular mechanics calculations. Both the NMR-derived structures, as well as the calculated structures, are shown to adopt a beta-turn as the primary conformation. Excellent agreement between the predicted structures and the NMR-derived structures is demonstrated. Aside from being the first examples of linear tetrapeptides reported to be ordered in aqueous solvent, the results presented support the hypothesis that high-affinity bradykinin receptor antagonists must adopt C-terminal beta-turn conformations. PMID:8388469

  11. Crystal Structure of the BARD1 BRCT Domains

    Energy Technology Data Exchange (ETDEWEB)

    Birrane,G.; Varma, A.; Soni, A.; Ladias, J.

    2007-01-01

    The interaction of the breast tumor suppressor BRCA1 with the protein BARD1 results in the formation of a heterodimeric complex that has ubiquitin ligase activity and plays central roles in cell cycle checkpoint control and DNA repair. Both BRCA1 and BARD1 possess a pair of tandem BRCT domains that interact in a phosphorylation-dependent manner with target proteins. We determined the crystal structure of the human BARD1 BRCT repeats (residues 568-777) at 1.9 {angstrom} resolution. The composition and structure of the BARD1 phosphoserine-binding pocket P{sub 1} are strikingly similar to those of the BRCA1 and MDC1 BRCT domains, suggesting a similar mode of interaction with the phosphate group of the ligand. By contrast, the BARD1 BRCT selectivity pocket P{sub 2} exhibits distinct structural features, including two prominent histidine residues, His685 and His686, which may be important for ligand binding. The protonation state of these histidines has a marked effect on the calculated electrostatic potential in the vicinity of P{sub 2}, raising the possibility that ligand recognition may be regulated by changes in pH. Importantly, the BARD1 BRCT structure provides insights into the mechanisms by which the cancer-associated missense mutations C645R, V695L, and S761N may adversely affect the structure and function of BARD1.

  12. A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

    Directory of Open Access Journals (Sweden)

    Yongqian Zhao

    2015-03-01

    Full Text Available Flavivirus RNA replication occurs within a replication complex (RC that assembles on ER membranes and comprises both non-structural (NS viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase and C-terminal RNA-dependent-RNA polymerase (RdRp domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3 at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV, the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

  13. Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes.

    Directory of Open Access Journals (Sweden)

    Jonathan D Steckbeck

    Full Text Available The C-terminal tail (CTT of the HIV-1 gp41 envelope (Env protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs. Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.

  14. Evolutionary origins of C-terminal (GPPn 3-hydroxyproline formation in vertebrate tendon collagen.

    Directory of Open Access Journals (Sweden)

    David M Hudson

    Full Text Available Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPPn in addition to the fully occupied A1 site at Pro986. The C-terminal (GPPn motif has five consecutive GPP triplets in α1(I, four in α2(I and three in α1(II, all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin and type II collagen (cartilage and notochord were examined by mass spectrometry. The (GPPn domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human, up to five 3-hydroxyproline residues per (GPPn motif were found in α1(I and four in α2(I, with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPPn site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.

  15. Evolutionary origins of C-terminal (GPP)n 3-hydroxyproline formation in vertebrate tendon collagen.

    Science.gov (United States)

    Hudson, David M; Werther, Rachel; Weis, MaryAnn; Wu, Jiann-Jiu; Eyre, David R

    2014-01-01

    Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)n motif has five consecutive GPP triplets in α1(I), four in α2(I) and three in α1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)n motif were found in α1(I) and four in α2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species. PMID:24695516

  16. Domain Modeling: NP_036204.2 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_036204.2 chr20 CRYSTAL STRUCTURE OF THE C-TERMINAL DOMAIN OF BCLA, THE MAJOR ANT...IGEN OF THE EXOSPORIUM OF THE BACILLUS ANTHRACIS SPORE. p2r6qa_ chr20/NP_036204.2/NP_036204.2_apo_496-632.pd...b p1wcka_ chr20/NP_036204.2/NP_036204.2_holo_496-632.pdb swppa 551H,628Y CAC 1 ...

  17. Crystal structure of the sensory domain of Escherichia coli CadC, a member of the ToxR-like protein family

    Science.gov (United States)

    Eichinger, Andreas; Haneburger, Ina; Koller, Christiane; Jung, Kirsten; Skerra, Arne

    2011-01-01

    The membrane-integral transcriptional activator CadC comprises sensory and transcriptional regulatory functions within one polypeptide chain. Its C-terminal periplasmic domain, CadCpd, is responsible for sensing of environmental pH as well as for binding of the feedback inhibitor cadaverine. Here we describe the crystal structure of CadCpd (residues 188–512) solved at a resolution of 1.8 Å via multiple wavelength anomalous dispersion (MAD) using a ReCl62− derivative. CadCpd reveals a novel fold comprising two subdomains: an N-terminal subdomain dominated by a β-sheet in contact with three α-helices and a C-terminal subdomain formed by an eleven-membered α-helical bundle, which is oriented almost perpendicular to the helices in the first subdomain. Further to the native protein, crystal structures were also solved for its variants D471N and D471E, which show functionally different behavior in pH sensing. Interestingly, in the heavy metal derivative of CadCpd used for MAD phasing a ReCl62− ion was found in a cavity located between the two subdomains. Amino acid side chains that coordinate this complex ion are conserved in CadC homologues from various bacterial species, suggesting a function of the cavity in the binding of cadaverine, which was supported by docking studies. Notably, CadCpd forms a homo-dimer in solution, which can be explained by an extended, albeit rather polar interface between two symmetry-related monomers in the crystal structure. The occurrence of several acidic residues in this region suggests protonation-dependent changes in the mode of dimerization, which could eventually trigger transcriptional activation by CadC in the bacterial cytoplasm. PMID:21308846

  18. Crystal structure of calpain-3 penta-EF-hand (PEF) domain - a homodimerized PEF family member with calcium bound at the fifth EF-hand

    Energy Technology Data Exchange (ETDEWEB)

    Partha, Sarathy K.; Ravulapalli, Ravikiran; Allingham, John S.; Campbell, Robert L.; Davies, Peter L. [Queens

    2014-08-21

    Calpains are Ca2+dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca2+ signaling in the cell. The major isoforms of this enzyme family, calpain-1 and calpain-2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C-terminal penta-EF hand (PEF) domains. Calpain-3, or p94, is a skeletal muscle-specific isoform that is genetically linked to limb-girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain-3 support the suggestion that full-length calpain-3 exists as a homodimer. Here, we report the crystallization of calpain-3's PEF domain and its crystal structure in the presence of Ca2+, which provides evidence for the homodimer architecture of calpain-3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain-3 PEF domain contains a Ca2+ bound at the EF5-hand used for homodimer association. Three of the four Ca2+-binding EF-hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca2+ signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain-3 large subunit were to try to pair with a calpain small subunit.

  19. Molecular and structural characterisation of the human sodium/iodide symporter (h N.I.S.) C-terminus and the implication of this domain in the transporter regulation

    International Nuclear Information System (INIS)

    The human natrium iodide symporter (h N.I.S.) is an intrinsic membrane protein expressed in thyroid cells where it allows iodide uptake and accumulation. It is composed of thirteen transmembrane helices and its ninety- three amino acids long cytosolic C-terminus presents many potential post-translational regulatory sites. A first part of the PhD work has been dedicated to the expression in a bacterial system and to the purification of the cytosolic C-terminal fragment. Biochemical and structural characterisation have revealed that this C-terminus is very flexible but prone to dimerization. The fragment has also been used as a bait to test the interactions with PDZ domain proteins spotted on a membrane. Several proteins interacting with the (natrium/iodide symporter) N.I.S. C-terminus have thus been identified and the study of their implication in the protein regulation has been initiated. A second part of the work has underlined the existence of a N.I.S. fragment co-purified with the entire protein. This fragment has been found in cells in culture stably expressing N.I.S. and also in human thyroid extracts and in rodent thyroid cells. We observed that this fragment is spontaneously associated with the entire protein. It is composed of the last 131 amino acid of the protein and so comprises the last transmembrane domain and the C-terminal extremity. The expression of a truncated form of h N.I.S., lacking the last 131 amino acids, shows that this protein is not correctly addressed to the cell membrane and cells expressing this mutated symporter cannot accumulate iodide. However, our results show that the co-expression of the two N.I.S. parts, the truncated form lacking the last 131 amino acid, and the complementary C-terminal fragment, leads to cells presenting 10 % of the activity of cells expressing the whole N.I.S.. (author)

  20. Assembly of Ebola virus matrix protein VP40 is regulated by latch-like properties of N and C terminal tails.

    Directory of Open Access Journals (Sweden)

    Leslie P Silva

    Full Text Available The matrix protein VP40 coordinates numerous functions in the viral life cycle of the Ebola virus. These range from the regulation of viral transcription to morphogenesis, packaging and budding of mature virions. Similar to the matrix proteins of other nonsegmented, negative-strand RNA viruses, VP40 proceeds through intermediate states of assembly (e.g. octamers but it remains unclear how these intermediates are coordinated with the various stages of the life cycle. In this study, we investigate the molecular basis of synchronization as governed by VP40. Hydrogen/deuterium exchange mass spectrometry was used to follow induced structural and conformational changes in VP40. Together with computational modeling, we demonstrate that both extreme N and C terminal tail regions stabilize the monomeric state through a direct association. The tails appear to function as a latch, released upon a specific molecular trigger such as RNA ligation. We propose that triggered release of the tails permits the coordination of late-stage events in the viral life cycle, at the inner membrane of the host cell. Specifically, N-tail release exposes the L-domain motifs PTAP/PPEY to the transport and budding complexes, whereas triggered C-tail release could improve association with the site of budding.

  1. Structural and functional analyses of beta-glucosidase 3B from Thermotoga neapolitana: a thermostable three-domain representative of glycoside hydrolase 3.

    Science.gov (United States)

    Pozzo, Tania; Pasten, Javier Linares; Karlsson, Eva Nordberg; Logan, Derek T

    2010-04-01

    Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized-that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable beta-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 A. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 A resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (alpha/beta)(8) domain similar to a triose phosphate isomerase barrel, a five-stranded alpha/beta sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second beta-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the -1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the -1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small

  2. Permanent magnet domain structures from magnetic force microscopy

    International Nuclear Information System (INIS)

    Magnetic force microscopy (MFM) offers the ability to image domain structures of permanent magnet materials in the form of bulk samples not suitable for electron microscopy, and at scales which are not possible using optical techniques. Recent developments, such as the use of very low coercivity tips and resonant phase detection MFM systems, have made this technique yet more useful. However, as with any new technique, interpretation of images should be approached with some caution. We have performed studies on bulk samples of sintered and melt-spun Nd Fe B - type materials, comparing images collected by Kerr microscopy with those from MFM. It has been possible to image the same areas of the samples using the two techniques and thereby compare and contrast the details arising from the same magnetic structures in each. From this work it is clear that it is often possible to make straight-forward interpretations of the MFM stray field maps regarding the underlying domain structure (although, of course, there may not be a unique interpretation). In addition we have used tips with different types of magnetic coatings to image the same areas of a sample, and showed that quit different image characteristics result. Of concern was the propensity for the tip coatings to partially re-magnetize in the stray fields from the samples, introducing artifacts into the images and making interpretation more difficult. With these results it has been possible to interpret high resolution MFM images of bulk permanent magnets with increased confidence, and examples of observed structures in thermally demagnetized two-phase nanocrystalline materials are interpreted in terms of interaction domains. (author)

  3. Crystal Structures of the BAR-PH and PTB Domains of Human APPL1

    Energy Technology Data Exchange (ETDEWEB)

    Li,J.; Mao, X.; Dong, L.; Liu, F.; Tong, L.

    2007-01-01

    APPL1 interacts with adiponectin receptors and other important signaling molecules. It contains a BAR and a PH domain near its N terminus, and the two domains may function as a unit (BAR-PH domain). We report here the crystal structures of the BAR-PH and PTB domains of human APPL1. The structures reveal novel features for BAR domain dimerization and for the interactions between the BAR and PH domains. The BAR domain dimer of APPL1 contains two four-helical bundles, whereas other BAR domain dimers have only three helices in each bundle. The PH domain is located at the opposite ends of the BAR domain dimer. Yeast two-hybrid assays confirm the interactions between the BAR and PH domains. Lipid binding assays show that the BAR, PH, and PTB domains can bind phospholipids. The ability of APPL1 to interact with multiple signaling molecules and phospholipids supports an important role for this adaptor in cell signaling.

  4. Imaging of Spin Dynamics in Closure Domain and Vortex Structures

    Science.gov (United States)

    Park, J. P.; Eames, P.; Engebretson, D. M.; Berezovsky, J.; Crowell, P. A.

    2003-03-01

    Time-resolved Kerr microscopy is used to study the excitations of individual micron-scale ferromagnetic thin film elements in their remnant state. Thin (18 nm) square elements with edge dimensions between 1 and 10 μm form closure domain structures with 90 degree Néel walls between domains. We identify two classes of excitations in these systems. The first corresponds to precession of the magnetization about the local demagnetizing field in each quadrant, while the second excitation is localized in the domain walls. Two modes are also identified in ferromagnetic disks with thicknesses of 60 nm and diameters from 2 μm down to 500 nm. The equilibrium state of each disk is a vortex with a singularity at the center. As in the squares, the higher frequency mode is due to precession about the internal field, but in this case the lower frequency mode corresponds to gyrotropic motion of the entire vortex. The effect of a non-zero magnetic field on the excitation spectrum of vortices is also explored. Although an applied field shifts the position of the vortex core, it has only a small effect on the excitation spectrum. These results demonstrate clearly the existence of well-defined excitations in inhomogeneously magnetized microstructures. This work was supported by NSF DMR 99-83777, the Research Corporation, the Alfred P. Sloan Foundation, the University of Minnesota MRSEC (DMR 98-09364), and the Minnesota Supercomputing Institute.

  5. Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid

    International Nuclear Information System (INIS)

    Membrane-associated decay accelerating factor (DAF) of human erythrocytes (E/sup hu/) was analyzed for a C-terminal glycolipid anchoring structure. Automated amino acid analysis of DAF following reductive radiomethylation revealed ethanolamine and glucosamine residues in proportions identical with those present in the E/sup hu/ acetylcholinesterase (AChE) anchor. Cleavage of radiomethylated 70-kilodalton (kDa) DAF with papain released the labeled ethanolamine and glucosamine and generated 61- and 55-kDa DAF products that retained all labeled Lys and labeled N-terminal Asp. Incubation of intact E/sup hu/ with phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the anchors in trypanosome membrane form variant surface glycoproteins (mfVSGs) and murine thymocyte Thy-1 antigen, released 15% of the cell-associated DAF antigen. The released 67-kDa PI-PLC DAF derivative retained its ability to decay the classical C3 convertase C4b2a but was unable to membrane-incorporate and displayed physicochemical properties similar to urine DAF, a hydrophilic DAF form that can be isolated for urine. Nitrous acid deamination cleavage of E/sup hu/ DAF at glucosamine following labeling with the lipophilic photoreagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) released the [125I]TID label in a parallel fashion as from [125I]TID-labeled AChE. Biosynthetic labeling of HeLa cells with [3H] ethanolamine resulted in rapid 3H incorporation into both 48-kDa pro-DAF and 72-kDa mature epithelial cell DAF. The findings indicate that DAF and AChE are anchored in E/sup hu/ by the same or a similar glycolipid structure and that, like VSGs, this structure is incorporated into DAF early in DAF biosynthesis prior to processing of pro-DAF in the Golgi

  6. C-terminal region of herpes simplex virus ICP8 protein needed for intranuclear localization

    International Nuclear Information System (INIS)

    The herpes simplex virus single-stranded DNA-binding protein, ICP8, localizes initially to structures in the nucleus called prereplicative sites. As replication proceeds, these sites mature into large globular structures called replication compartments. The details of what signals or proteins are involved in the redistribution of viral and cellular proteins within the nucleus between prereplicative sites and replication compartments are poorly understood; however, we showed previously that the dominant-negative d105 ICP8 does not localize to prereplicative sites and prevents the localization of other viral proteins to prereplicative sites (J. Virol. 74 (2000) 10122). Within the residues deleted in d105 (1083 to 1168), we identified a region between amino acid residues 1080 and 1135 that was predicted by computer models to contain two α-helices, one with considerable amphipathic nature. We used site-specific and random mutagenesis techniques to identify residues or structures within this region that are required for proper ICP8 localization within the nucleus. Proline substitutions in the predicted helix generated ICP8 molecules that did not localize to prereplicative sites and acted as dominant-negative inhibitors. Other substitutions that altered the charged residues in the predicted α-helix to alanine or leucine residues had little or no effect on ICP8 intranuclear localization. The predicted α-helix was dispensable for the interaction of ICP8 with the UL9 origin-binding protein. We propose that this C-terminal α-helix is required for localization of ICP8 to prereplicative sites by binding viral or cellular factors that target or retain ICP8 at specific intranuclear sites

  7. Domain Structure and Magnetoresistance in Co2MnGe Zigzag Structures

    Science.gov (United States)

    Gross, Katherine; Westerholt, Kurt; Gómez, Maria E.; Zabel, Hartmut

    We report a clear manifestation of the negative contribution to the magnetoresistance due to domain walls in Co2MnGe-Heusler submicron zigzag wires in which the domain structure, domain size and domain wall density can be well controlled. The magnetic behavior of these systems results from the interplay between the intrinsic magneto-crystalline (K4) anisotropy, growth induced uniaxial (KU = 4.7x103 J/m3) anisotropy and shape anisotropy (KS), as observed by magnetic-force microscopy (MFM) and longitudinal Kerr hysteresis loop measurements. Magnetoresistance measurements were performed by the four-point method under a field applied in the plane of the wires at a temperature of 300 K. In these structures, domain wall-creation and annihilation occur in a coherent way. As a result, clear jumps of the resistance are detected during the transition from single-domain- to multi-domain states. At room temperature a value RDW = -2.5 mΩ was obtained; this result is the same order of magnitude as other experimental and theoretical findings. The negative resistive contribution due to the domain wall is also discussed and compared with the existing theoretical models.

  8. Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping.

    Science.gov (United States)

    Yamanaka, Masaru; Nagao, Satoshi; Komori, Hirofumi; Higuchi, Yoshiki; Hirota, Shun

    2015-03-01

    Cytochrome c555 from hyperthermophilic bacteria Aquifex aeolicus (AA cyt c555 ) is a hyperstable protein belonging to the cyt c protein family, which possesses a unique long 310 -α-310 helix containing the heme-ligating Met61. Herein, we show that AA cyt c555 forms dimers by swapping the region containing the extra 310 -α-310 helix and C-terminal α-helix. The asymmetric unit of the crystal of dimeric AA cyt c555 contained two dimer structures, where the structure of the hinge region (Val53-Lys57) was different among all four protomers. Dimeric AA cyt c555 dissociated to monomers at 92 ± 1°C according to DSC measurements, showing that the dimer was thermostable. According to CD measurements, the secondary structures of dimeric AA cyt c555 were maintained at pH 2.2-11.0. CN(-) and CO bound to dimeric AA cyt c555 in the ferric and ferrous states, respectively, owing to the flexibility of the hinge region close to Met61 in the dimer, whereas these ligands did not bind to the monomer under the same conditions. In addition, CN(-) and CO bound to the oxidized and reduced dimer at neutral pH and a wide range of pH (pH 2.2-11.0), respectively, in a wide range of temperature (25-85°C), owing to the thermostability and pH tolerance of the dimer. These results show that the ligand binding character of hyperstable AA cyt c555 changes upon dimerization by domain swapping. PMID:25586341

  9. The E2-25K ubiquitin-associated (UBA) domain aids in polyubiquitin chain synthesis and linkage specificity

    International Nuclear Information System (INIS)

    Research highlights: → We examine the role of a ubiquitin-associated (UBA) domain in an E2 enzyme. → The E2-25K UBA domain directs polyubiquitin chain linkage specificity. → The E2-25K UBA domain regulates length of polyubiquitin chains synthesized. -- Abstract: E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologs represent the only known E2 enzymes which contain a C-terminal ubiquitin-associated (UBA) domain as well as the conserved catalytic ubiquitin-conjugating (UBC) domain. As an additional non-covalent binding surface for ubiquitin, the UBA domain must provide some functional specialization. We mapped the protein-protein interface involved in the E2-25K UBA/ubiquitin complex by solution nuclear magnetic resonance (NMR) spectroscopy and subsequently modeled the structure of the complex. Domain-domain interactions between the E2-25K catalytic UBC domain and the UBA domain do not induce significant structural changes in the UBA domain or alter the affinity of the UBA domain for ubiquitin. We determined that one of the roles of the C-terminal UBA domain, in the context of E2-25K, is to increase processivity in Lys48-linked polyubiquitin chain synthesis, possibly through increased binding to the ubiquitinated substrate. Additionally, we see evidence that the UBA domain directs specificity in polyubiquitin chain linkage.

  10. Influence of domain interactions on conformational mobility of the progesterone receptor detected by hydrogen/deuterium exchange mass spectrometry

    OpenAIRE

    Goswami, Devrishi; Callaway, Celetta; Pascal, Bruce D; Kumar, Raj; Edwards, Dean P.; Griffin, Patrick R.

    2014-01-01

    Structural and functional details of the N-terminal activation function 1 (AF1) of most nuclear receptors are poorly understood due to the highly dynamic intrinsically disordered nature of this domain. A hydrogen/deuterium exchange (HDX) mass spectrometry based investigation of TATA box binding protein (TBP) interaction with various domains of progesterone receptor (PR) demonstrate that agonist bound PR interaction with TBP via AF1 impacts the mobility of the C-terminal AF2. Results from HDX ...

  11. Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies.

    Science.gov (United States)

    Jiang, Guoying; Yu, Christopher; Yadav, Daniela B; Hu, Zhilan; Amurao, Annamarie; Duenas, Eileen; Wong, Marc; Iverson, Mark; Zheng, Kai; Lam, Xanthe; Chen, Jia; Vega, Roxanne; Ulufatu, Sheila; Leddy, Cecilia; Davis, Helen; Shen, Amy; Wong, Pin Y; Harris, Reed; Wang, Y John; Li, Dongwei

    2016-07-01

    Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes. PMID:27262204

  12. Synapse associated protein 102 (SAP102 binds the C-terminal part of the scaffolding protein neurobeachin.

    Directory of Open Access Journals (Sweden)

    Juliane Lauks

    Full Text Available Neurobeachin (Nbea is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea's PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.

  13. Neurological disease mutations compromise a C-terminal ion pathway in the Na(+)/K(+)-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Khandelia, Himanshu; Morth, J Preben;

    2010-01-01

    severe neurological diseases. This novel model for ion transport by the Na(+)/K(+)-ATPase is established by electrophysiological studies of C-terminal mutations in familial hemiplegic migraine 2 (FHM2) and is further substantiated by molecular dynamics simulations. A similar ion regulation is likely to...

  14. Development of a cysteine-deprived and C-terminally truncated GLP-1 receptor

    DEFF Research Database (Denmark)

    Underwood, Christina Rye; Knudsen, Lotte Bjerre; Garibay, Patrick W.;

    2013-01-01

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C-terminally trun......The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C......-terminally truncated GLP-1R. Single cysteines were initially substituted with alanine, and functionally redundant cysteines were subsequently changed simultaneously. Our results indicate that Cys174, Cys226, Cys296 and Cys403 are important for the GLP-1-mediated response, whereas Cys236, Cys329, Cys341, Cys347, Cys438...... membrane proximal part of the C-terminal is involved in receptor expression at the cell surface. The results show that seven cysteines and more than half of the C-terminal tail can be removed from GLP-1R without compromising GLP-1 binding or function....

  15. Autotransporters with GDSL passenger domains: molecular physiology and biotechnological applications.

    Science.gov (United States)

    Wilhelm, Susanne; Rosenau, Frank; Kolmar, Harald; Jaeger, Karl-Erich

    2011-07-01

    Autotransporters are large proteins produced and secreted by Gram-negative bacteria. They consist of an N-terminal passenger domain, which typically harbours enzymatic activity and exerts a virulence function, and a C-terminal membrane anchor domain. Somehow, the membrane domain facilitates the transport of the passenger domain into the extracellular space. Several autotransporters possess hydrolase passenger domains that belong to the GDSL family of lipolytic enzymes. GDSL autotransporters represent a functionally distinct family and are characterized by several features of their passenger domains; these include 1) the absence of a conserved right-handed parallel β-helix, 2) lipolytic activity, and thus the capability to hydrolyse membranes, and 3) covalent attachment to the respective C-terminal β-domain, with the hydrolase domain exposed to the exterior. The esterase EstA of Pseudomonas aeruginosa is a typical enzyme of this type. Its physiological role was studied, its potential biotechnological application has been demonstrated, and its crystal structure was solved recently. Furthermore, it is capable of displaying different classes of enzymes in a range of Gram-negative bacteria including Escherichia coli, and FACS-based high-throughput screening for enantioselective esterases could be achieved using EstA. PMID:21598370

  16. Disruption of the NHR4 domain structure in AML1-ETO abrogates SON binding and promotes leukemogenesis

    OpenAIRE

    Ahn, Eun-Young; Yan, Ming; Malakhova, Oxana A.; Lo, Miao-Chia; Boyapati, Anita; Ommen, Hans Beier; Hines, Robert; Hokland, Peter; Zhang, Dong-Er

    2008-01-01

    AML1-ETO is generated from t(8;21)(q22;q22), which is a common form of chromosomal translocation associated with development of acute myeloid leukemia (AML). Although full-length AML1-ETO alone fails to promote leukemia because of its detrimental effects on cell proliferation, an alternatively spliced isoform, AML1-ETO9a, without its C-terminal NHR3/NHR4 domains, strongly induces leukemia. However, full-length AML1-ETO is a major form of fusion product in many t(8;21) AML patients, suggesting...

  17. Antibodies against the C-terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2-cell stage

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5'-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3'-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP)(73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mouse cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn't. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.

  18. Frequency domain identification for robust large space structure control design

    Science.gov (United States)

    Yam, Y.; Bayard, D. S.; Scheid, R. E.

    1991-01-01

    A methodology is demonstrated for frequency domain identification of large space structures which systematically transforms experimental raw data into a form required for synthesizing H(infinity) controllers using modern robust control design software (e.g., Matlab Toolboxes). A unique feature of this approach is that the additive uncertainty is characterized to a specified statistic confidence rather than with hard bounds. In this study, the difference in robust performance is minimal between the two levels of confidence. In general cases, the present methodology provides a tool for performance/confidence level tradeoff studies. For simplicity, the additive uncertainty on a frequency grid is considered and the interpolation error in between grid points is neglected.

  19. Structural Basis of Actin Filament Nucleation by Tandem W Domains

    Directory of Open Access Journals (Sweden)

    Xiaorui Chen

    2013-06-01

    Full Text Available Spontaneous nucleation of actin is very inefficient in cells. To overcome this barrier, cells have evolved a set of actin filament nucleators to promote rapid nucleation and polymerization in response to specific stimuli. However, the molecular mechanism of actin nucleation remains poorly understood. This is hindered largely by the fact that actin nucleus, once formed, rapidly polymerizes into filament, thus making it impossible to capture stable multisubunit actin nucleus. Here, we report an effective double-mutant strategy to stabilize actin nucleus by preventing further polymerization. Employing this strategy, we solved the crystal structure of AMPPNP-actin in complex with the first two tandem W domains of Cordon-bleu (Cobl, a potent actin filament nucleator. Further sequence comparison and functional studies suggest that the nucleation mechanism of Cobl is probably shared by the p53 cofactor JMY, but not Spire. Moreover, the double-mutant strategy opens the way for atomic mechanistic study of actin nucleation and polymerization.

  20. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

    Energy Technology Data Exchange (ETDEWEB)

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M.F.; Downes, C. Peter; Batty, Ian H. (Toronto); (Dundee)

    2012-10-16

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

  1. Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, James M.; Korzhnev, Dmitry M.; Ceccarelli, Derek F.; Briant, Douglas J.; Zarrine-Afsar, Arash; Sicheri, Frank; Kay, Lewis E.; Pawson, Tony (Mount Sinai Hospital); (Toronto)

    2012-10-23

    The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the N- and C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.

  2. Mapping the structural and dynamical features of kinesin motor domains.

    Directory of Open Access Journals (Sweden)

    Guido Scarabelli

    Full Text Available Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound. These groups differ in the orientation of key functional elements, most notably the microtubule binding α4-α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4-α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck

  3. NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

    Science.gov (United States)

    Bayliss, Richard; Harris, Richard; Coutte, Loic; Monier, Amy; Fronzes, Remi; Christie, Peter J; Driscoll, Paul C; Waksman, Gabriel

    2007-01-30

    Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system. PMID:17244707

  4. The Human Sodium-Glucose Cotransporter (hSGLT1) Is a Disulfide-Bridged Homodimer with a Re-Entrant C-Terminal Loop

    OpenAIRE

    Sasseville, Louis J.; Michael Morin; Coady, Michael J.; Rikard Blunck; Jean-Yves Lapointe

    2016-01-01

    Na-coupled cotransporters are proteins that use the trans-membrane electrochemical gradient of Na to activate the transport of a second solute. The sodium-glucose cotransporter 1 (SGLT1) constitutes a well-studied prototype of this transport mechanism but essential molecular characteristics, namely its quaternary structure and the exact arrangement of the C-terminal transmembrane segments, are still debated. After expression in Xenopus oocytes, human SGLT1 molecules (hSGLT1) were labelled on ...

  5. A segmental labeling strategy for unambiguous determination of domain–domain interactions of large multi-domain proteins

    International Nuclear Information System (INIS)

    NMR structural determination of large multi-domain proteins is a challenging task due to significant spectral overlap with a particular difficulty in unambiguous identification of domain–domain interactions. Segmental labeling is a NMR strategy that allows for isotopically labeling one domain and leaves the other domain unlabeled. This significantly simplifies spectral overlaps and allows for quick identification of domain–domain interaction. Here, a novel segmental labeling strategy is presented for detection of inter-domain NOEs. To identify domain–domain interactions in human apolipoprotein E (apoE), a multi-domain, 299-residues α-helical protein, on-column expressed protein ligation was utilized to generate a segmental-labeled apoE samples in which the N-terminal (NT-) domain was 2H(99%)/15N-labeled whereas the C-terminal (CT-) domain was either 15N- or 15N/13C-labeled. 3-D 15N-edited NOESY spectra of these segmental-labeled apoE samples allow for direct observation of the inter-domain NOEs between the backbone amide protons of the NT-domain and the aliphatic protons of the CT-domain. This straightforward approach permits unambiguous identification of 78 inter-domain NOEs, enabling accurate definition of the relative positions of both the NT- and the CT-domains and determination of the NMR structure of apoE.

  6. Domain structure and magnetic anisotropy fluctuations in (Ga,Mn)As : effect of annealing

    OpenAIRE

    Dourlat, Alexandre; Jeudy, Vincent; Testelin, Christophe; Bernardot, Frédéric; Bernardot, Frédérick; Khazen, Khashayar; Gourdon, Catherine; Thevenard, Laura; Largeau, Ludovic; Mauguin, Olivia; Lemaître, Aristide

    2007-01-01

    International audience We investigate the effect of post-growth annealing on the magnetic domain structure and magnetization reversal process of (Ga,Mn)As epilayers grown with tensile strain on a (Ga,In)As buffer. In the case of perpendicular magnetic easy-axis, annealing drastically changes the domain structure observed at magnetization reversal. In as-grown samples, strongly anisotropic domain growth is observed. Dendritic-like domain expansion with guided branching along the directions...

  7. Improving protein structure similarity searches using domain boundaries based on conserved sequence information

    OpenAIRE

    Madej Tom; Wang Yanli; Thompson Kenneth; Bryant Stephen H

    2009-01-01

    Abstract Background The identification of protein domains plays an important role in protein structure comparison. Domain query size and composition are critical to structure similarity search algorithms such as the Vector Alignment Search Tool (VAST), the method employed for computing related protein structures in NCBI Entrez system. Currently, domains identified on the basis of structural compactness are used for VAST computations. In this study, we have investigated how alternative definit...

  8. Opitz G/BBB syndrome in Xp22: mutations in the MID1 gene cluster in the carboxy-terminal domain.

    OpenAIRE

    Gaudenz, K; Roessler, E.; Quaderi, N; B. Franco; Feldman, G.; Gasser, D L; Wittwer, B.; Horst, J; Montini, E; Opitz, J M; Ballabio, A; Muenke, M

    1998-01-01

    The MID1 gene in Xp22 codes for a novel member of proteins containing a RING finger, B-box, coiled-coil and a conserved C-terminal domain. Initially, three mutations in the C-terminal region were found in patients with Opitz G/BBB syndrome, a defect of midline development. Here we have determined the complete gene structure of the MID1 gene and have analyzed all nine exons for mutations in a set of 40 unrelated Opitz G/BBB patients. We now report six additional mutations all clustered in the ...

  9. Time-domain seismic reliability of nonlinear structures

    Indian Academy of Sciences (India)

    Achintya Haldar; Jungwon Huh; Ali Mehrabian

    2006-08-01

    A novel reliability analysis technique is presented to estimate the reliability of real structural systems. Its unique feature is that the dynamic loadings can be applied in time domain. It is a nonlinear stochastic finite element logarithm combined with the response surface method (RSM). It generates the response surface around the most probable failure point and incorporates information of the distribution of the random variables in the RSM formulation. It is verified using the Monte Carlo simulation technique, and is found to be very efficient and accurate. Most sources of nonlinearlity and uncertainty can be explicitly incorporated in the formulation. The flexibility of connections, represented by moment-relative rotation $(M–\\theta )$ curves, is addressed. After the Northridge earthquake of 1994, several improved steel connections were proposed. Structural Sesimic Design Associates (SSDA) tested several full-scale proprietory slotted web beam–column connections. The authors suggested $(M–\\theta )$ curves for this connection using actual test data. Behaviours of steel frames, assuming the connections are fully restrained, partially restrained, consisting of pre- and post-Northridge connections are evaluated and compared. Desirable features of the post-Northridge connections observed during testing are analytically confirmed. Laterally weak steel frame is then strengthened with concrete shear walls. Capabilities and the advanced nature of the method are demonstrated with the help of realistic examples.

  10. Graphene on C-terminated face of 4H-SiC observed by noncontact scanning nonlinear dielectric potentiometry

    Science.gov (United States)

    Yamasue, Kohei; Fukidome, Hirokazu; Tashima, Keiichiro; Suemitsu, Maki; Cho, Yasuo

    2016-08-01

    We studied graphene synthesized on the C-terminated face (C-face) of a 4H-SiC substrate by noncontact scanning nonlinear dielectric potentiometry. As already reported by other researchers, multilayer graphene sheets with moiré patterns were observed in our sample, which indicates the existence of rotational disorder between adjacent layers. We found that the potentials of graphene on the C-face are almost neutral and significantly smaller than those observed on the Si-terminated face (Si-face). In addition, the neutrality of potentials is not affected by various topographic features underlying the multilayer graphene sheets. These results indicate that graphene on the C-face of SiC is decoupled or screened from the underlying structures and substrate, unlike graphene on the Si-face.

  11. Protein domain organisation: adding order

    Directory of Open Access Journals (Sweden)

    Kummerfeld Sarah K

    2009-01-01

    Full Text Available Abstract Background Domains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit. Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved. Results We identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance. To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation. Conclusion Taking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected

  12. Solution structure of the dimeric cytoplasmic domain of syndecan-4

    DEFF Research Database (Denmark)

    Shin, J; Lee, W; Lee, D;

    2001-01-01

    cytoplasmic domain is dependent on its oligomeric status, the conformation of the syndecan-4 cytoplasmic domain itself is important in the understanding of its biological roles. Gel filtration results show that the syndecan-4 cytoplasmic domain (4L) itself forms a dimer stabilized by ionic interactions...... is affected by the presence of C1 and C2 regions. Therefore, we propose that although the 4V region in the full cytoplasmic domain has a tendency for strong peptide--peptide interaction, it may not be enough to overcome the repulsion of the C1 regions of syndecan-4L....

  13. Stability of plasma concentrations of N and C terminal atrial natriuretic peptides at room temperature.

    OpenAIRE

    Cleland, J G; Ward, S; Dutka, D.; Habib, F; Impallomeni, M; Morton, I J

    1996-01-01

    BACKGROUND: Plasma concentrations of atrial natriuretic peptide (ANP) are increased in patients with ventricular dysfunction and could have a diagnostic role in heart failure. ANP may be unstable after collection, however, limiting any practical diagnostic role. METHODS: Blood samples were obtained from 18 patients with various conditions. Aliquots were either processed optimally or kept as blood or plasma at room temperature for 6-72 h before processing. RESULTS: Concentrations of C-terminal...

  14. Site-directed mutations in the C-terminal extension of human alphaB-crystallin affect chaperone function and block amyloid fibril formation.

    Directory of Open Access Journals (Sweden)

    Teresa M Treweek

    Full Text Available BACKGROUND: Alzheimer's, Parkinson's and Creutzfeldt-Jakob disease are associated with inappropriate protein deposition and ordered amyloid fibril assembly. Molecular chaperones, including alphaB-crystallin, play a role in the prevention of protein deposition. METHODOLOGY/PRINCIPAL FINDINGS: A series of site-directed mutants of the human molecular chaperone, alphaB-crystallin, were constructed which focused on the flexible C-terminal extension of the protein. We investigated the structural role of this region as well as its role in the chaperone function of alphaB-crystallin under different types of protein aggregation, i.e. disordered amorphous aggregation and ordered amyloid fibril assembly. It was found that mutation of lysine and glutamic acid residues in the C-terminal extension of alphaB-crystallin resulted in proteins that had improved chaperone activity against amyloid fibril forming target proteins compared to the wild-type protein. CONCLUSIONS/SIGNIFICANCE: Together, our results highlight the important role of the C-terminal region of alphaB-crystallin in regulating its secondary, tertiary and quaternary structure and conferring thermostability to the protein. The capacity to genetically modify alphaB-crystallin for improved ability to block amyloid fibril formation provides a platform for the future use of such engineered molecules in treatment of diseases caused by amyloid fibril formation.

  15. Domain Structures in Fourth-Order Phase and Ginzburg-Landau Equations

    CERN Document Server

    Raitt, D I; Raitt, David; Riecke, Hermann

    1995-01-01

    In pattern-forming systems, competition between patterns with different wave numbers can lead to domain structures, which consist of regions with differing wave numbers separated by domain walls. For domain structures well above threshold we employ the appropriate phase equation and obtain detailed qualitative agreement with recent experiments. Close to threshold a fourth-order Ginzburg-Landau equation is used which describes a steady bifurcation in systems with two competing critical wave numbers. The existence and stability regime of domain structures is found to be very intricate due to interactions with other modes. In contrast to the phase equation the Ginzburg-Landau equation allows a spatially oscillatory interaction of the domain walls. Thus, close to threshold domain structures need not undergo the coarsening dynamics found in the phase equation far above threshold, and can be stable even without phase conservation. We study their regime of stability as a function of their (quantized) length. Domain ...

  16. C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

    Directory of Open Access Journals (Sweden)

    Angela Brieger

    Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

  17. Expression, purification, crystallization and preliminary crystallographic study of the carboxyl-terminal domain of the human voltage-gated proton channel Hv1

    International Nuclear Information System (INIS)

    Here, the C-terminal domain of the human voltage-gated proton channel Hv1 (C-Hv1) was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The voltage-gated proton channel Hv1 is essential to proton permeation and contains a voltage-sensor domain without a pore domain. It contains three predicted domains: an N-terminal acid and proline-rich domain, a transmembrane voltage-sensor domain and a C-terminal domain that is responsible for the dimeric architecture of Hv1. Here, the C-terminal domain of the human voltage-gated proton channel Hv1 (C-Hv1) was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals have a tetragonal form and diffraction data were collected to 2.5 Å resolution in-house. The crystal belongs to space group P41212, with unit-cell parameters a = b = 37.76, c = 137.52 Å. Structural determination of C-Hv1 is in progress

  18. Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1F3 and C-terminal modules of fibronectin.

    Directory of Open Access Journals (Sweden)

    Jielin Xu

    Full Text Available BACKGROUND: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7F3-(10F3. Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7F3-(10F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. METHODOLOGY/PRINCIPAL FINDINGS: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7F3-(10F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2F3-(14F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1F3 or the C-terminal modules to modules (2F3-(14F3 resulted in some activity, and addition of both (1F3 and the C-terminal modules resulted in a construct, (1F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1F3-C V0, (1F3-C V64, and (1F3-C Delta(V(15F3(10F1 were all able to support fibronectin assembly, suggesting that (1F3 through (11F1 and/or (12F1 were important for activity. Coatings in which the active parts of (1F3-C were present in different proteins were much less active than intact (1F3-C. CONCLUSIONS: These results suggest that (1F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.

  19. Molecular basis of the fructose-2,6-bisphosphatase reaction of PFKFB3: Transition state and the C-terminal function

    Energy Technology Data Exchange (ETDEWEB)

    Cavalier, Michael C.; Kim, Song-Gun; Neau, David; Lee, Yong-Hwan (Cornell); (LSU); (KAIST-D)

    2012-03-22

    The molecular basis of fructose-2,6-bisphosphatase (F-2,6-P{sub 2}ase) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho-enzyme intermediate state (PFKFB3-P {center_dot} F-6-P), in a transition state-analogous complex (PFKFB3 {center_dot} AlF{sub 4}), and in a complex with pyrophosphate (PFKFB3 {center_dot} PP{sub i}) at resolutions of 2.45, 2.2, and 2.3 {angstrom}, respectively. Trapping the PFKFB3-P {center_dot} F-6-P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3 {center_dot} AlF{sub 4} and PFKFB3 {center_dot} PP{sub i} complexes were obtained by soaking. The PFKFB3 {center_dot} AlF{sub 4} and PFKFB3 {center_dot} PP{sub i} complexes resulted in removing F-6-P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3-P formation and break down, the phosphoryl donor and the acceptor are located within {approx}5.1 {angstrom}, and the pivotal point 2-P is on the same line, suggesting an 'in-line' transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent N-P bond, breaking the 2O-P bond in the substrate. The resulting high reactivity of the leaving group, 2O of F-6-P, is neutralized by a proton donated by Glu322. Negative charges on the equatorial oxygen of the transient bipyramidal phosphorane formed during the transfer are stabilized by Arg252, His387, and Asn259. The C-terminal domain (residues 440-446) was rearranged in PFKFB3 {center_dot} PP{sub i}, implying that this domain plays a critical role in binding of substrate to and release of product from the F-2,6-P{sub 2}ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction.

  20. Structure and expression of the maize (Zea mays L. SUN-domain protein gene family: evidence for the existence of two divergent classes of SUN proteins in plants

    Directory of Open Access Journals (Sweden)

    Simmons Carl R

    2010-12-01

    Full Text Available Abstract Background The nuclear envelope that separates the contents of the nucleus from the cytoplasm provides a surface for chromatin attachment and organization of the cortical nucleoplasm. Proteins associated with it have been well characterized in many eukaryotes but not in plants. SUN (Sad1p/Unc-84 domain proteins reside in the inner nuclear membrane and function with other proteins to form a physical link between the nucleoskeleton and the cytoskeleton. These bridges transfer forces across the nuclear envelope and are increasingly recognized to play roles in nuclear positioning, nuclear migration, cell cycle-dependent breakdown and reformation of the nuclear envelope, telomere-led nuclear reorganization during meiosis, and karyogamy. Results We found and characterized a family of maize SUN-domain proteins, starting with a screen of maize genomic sequence data. We characterized five different maize ZmSUN genes (ZmSUN1-5, which fell into two classes (probably of ancient origin, as they are also found in other monocots, eudicots, and even mosses. The first (ZmSUN1, 2, here designated canonical C-terminal SUN-domain (CCSD, includes structural homologs of the animal and fungal SUN-domain protein genes. The second (ZmSUN3, 4, 5, here designated plant-prevalent mid-SUN 3 transmembrane (PM3, includes a novel but conserved structural variant SUN-domain protein gene class. Mircroarray-based expression analyses revealed an intriguing pollen-preferred expression for ZmSUN5 mRNA but low-level expression (50-200 parts per ten million in multiple tissues for all the others. Cloning and characterization of a full-length cDNA for a PM3-type maize gene, ZmSUN4, is described. Peptide antibodies to ZmSUN3, 4 were used in western-blot and cell-staining assays to show that they are expressed and show concentrated staining at the nuclear periphery. Conclusions The maize genome encodes and expresses at least five different SUN-domain proteins, of which the PM3

  1. Theoretical Methods of Domain Structures in Ultrathin Ferroelectric Films: A Review

    Directory of Open Access Journals (Sweden)

    Jianyi Liu

    2014-09-01

    Full Text Available This review covers methods and recent developments of the theoretical study of domain structures in ultrathin ferroelectric films. The review begins with an introduction to some basic concepts and theories (e.g., polarization and its modern theory, ferroelectric phase transition, domain formation, and finite size effects, etc. that are relevant to the study of domain structures in ultrathin ferroelectric films. Basic techniques and recent progress of a variety of important approaches for domain structure simulation, including first-principles calculation, molecular dynamics, Monte Carlo simulation, effective Hamiltonian approach and phase field modeling, as well as multiscale simulation are then elaborated. For each approach, its important features and relative merits over other approaches for modeling domain structures in ultrathin ferroelectric films are discussed. Finally, we review recent theoretical studies on some important issues of domain structures in ultrathin ferroelectric films, with an emphasis on the effects of interfacial electrostatics, boundary conditions and external loads.

  2. Existence of a Steady Flow of Stokes Fluid Past a Linear Elastic Structure Using Fictitious Domain

    Science.gov (United States)

    Halanay, Andrei; Murea, Cornel Marius; Tiba, Dan

    2016-06-01

    We use fictitious domain method with penalization for the Stokes equation in order to obtain approximate solutions in a fixed larger domain including the domain occupied by the structure. The coefficients of the fluid problem, excepting the penalizing term, are independent of the deformation of the structure. It is easy to check the inf-sup condition and the coercivity of the Stokes problem in the fixed domain. Subtracting the structure equations from the fictitious fluid equations in the structure domain, we obtain a weak formulation in a fixed domain, where the continuity of the stress at the interface does not appear explicitly. Existence of a solution is proved when the structure displacement is generated by a finite number of modes.

  3. Structural Studies of MJ1529, an O6-methylguanine-DNAMethyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Anne; Pelton, Jeffrey G.; Wemmer, David E.

    2006-01-10

    The structure of an O{sub 6}-methylguanine methyltransferase from the thermophile Methanococcus jannaschii has been determined using multinuclear multidimensional NMR spectroscopy. The structure is similar to homologues from other organisms that have been determined by crystallography, with some variation in the N-terminal domain. The C-terminal domain is more highly conserved in both sequence and structure. Regions of the protein show broadening reflecting conformational flexibility that is likely related to function.

  4. Experimental observation of the interaction of propagating spin waves with Néel domain walls in a Landau domain structure

    Energy Technology Data Exchange (ETDEWEB)

    Pirro, P.; Sebastian, T.; Leven, B.; Hillebrands, B. [Fachbereich Physik and Landesforschungszentrum OPTIMAS, Technische Universität Kaiserslautern, 67663 Kaiserslautern (Germany); Koyama, T. [Department of Applied Physics, Faculty of Engineering, The University of Tokyo, Bunkyo, Tokyo 113-8656 (Japan); Brächer, T. [Fachbereich Physik and Landesforschungszentrum OPTIMAS, Technische Universität Kaiserslautern, 67663 Kaiserslautern (Germany); Graduate School Materials Science in Mainz, Gottlieb-Daimler-Strasse 47, 67663 Kaiserslautern (Germany)

    2015-06-08

    The interaction of propagating dipolar spin waves with magnetic domain walls is investigated in square-shaped microstructures patterned from the Heusler compound Co{sub 2}Mn{sub 0.6}Fe{sub 0.4}Si. Using magnetic force microscopy, the reversible preparation of a Landau state with four magnetic domains separated by Néel domain walls is confirmed. A local spin-wave excitation using a microstructured antenna is realized in one of the domains. It is shown by Brillouin light scattering microscopy that the domain structure in the remanence state has a strong influence on the spin-wave excitation and propagation. The domain walls strongly reflect the spin waves and can be used as spin-wave reflectors. A comparison with micromagnetic simulations shows that the strong reflection is due to the long-range dipolar interaction which has important implications for the use of these spin waves for exerting an all-magnonic spin-transfer torque.

  5. Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae

    OpenAIRE

    Endres, Stephan; Granzin, Joachim; Tielen, Petra; Willbold, Dieter; Jaeger, Karl-Erich; Batra-Safferling, Renu; Circolone, Franco; Stadler, Andreas; Krauss, Ulrich; Drepper, Thomas; Svensson, Vera; Knieps-Grünhagen, Esther; Wirtz, Astrid; Cousin, Anneliese

    2015-01-01

    Background Light, oxygen, voltage (LOV) domains are widely distributed in plants, algae, fungi, bacteria, and represent the photo-responsive domains of various blue-light photoreceptor proteins. Their photocycle involves the blue-light triggered adduct formation between the C(4a) atom of a non-covalently bound flavin chromophore and the sulfur atom of a conserved cysteine in the LOV sensor domain. LOV proteins show considerable variation in the structure of N- and C-terminal elements which fl...

  6. 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

  7. 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.

  8. Systematic Optimization of C-Terminal Amine-Based Isotope Labeling of Substrates Approach for Deep Screening of C-Terminome.

    Science.gov (United States)

    Zhang, Yang; He, Quanze; Ye, Juanying; Li, Yanhong; Huang, Lin; Li, Qingqing; Huang, Jingnan; Lu, Jianan; Zhang, Xumin

    2015-10-20

    It is well-known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structures and understanding protein functions. Although many efforts have been made in the field during the latest 2 decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we report an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in an Escherichia coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to the number of lysine in peptides, more reproducible and with higher MASCOT scores. Moreover, the introduction of the Single-Charge Ion Inclusion (SCII) method to alleviate the charge deficiency of small peptides allowed an additional 26% increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in E. coli in a triplicate experiments using 80 μg each. Our optimized method would benefit the deep screening of C-terminome and possibly help discover some novel C-terminal modifications. Data are available via ProteomeXchange with identifier PXD002409. PMID:26361894

  9. Crystal structure studies of NADP+ dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

    International Nuclear Information System (INIS)

    Highlights: • We determined the structure of isocitrate dehydrogenase with citrate and cofactor. • The structure reveals a unique novel terminal domain involved in dimerization. • Clasp domain shows significant difference, and catalytic residues are conserved. • Oligomerization of the enzyme is quantized with subunit-subunit interactions. • Novel domain of this enzyme is classified as subfamily of the type IV. - Abstract: NADP+ dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP+ was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV

  10. Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved α-helix for Act1 binding and IL-17 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bing [Oklahoma State University, Stillwater, OK 74078 (United States); Liu, Caini; Qian, Wen [Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195 (United States); Han, Yue [Oklahoma State University, Stillwater, OK 74078 (United States); Li, Xiaoxia, E-mail: lix@ccf.org [Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195 (United States); Deng, Junpeng, E-mail: lix@ccf.org [Oklahoma State University, Stillwater, OK 74078 (United States)

    2014-05-01

    Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling. Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′{sub ins}) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′{sub ins} helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.

  11. Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved α-helix for Act1 binding and IL-17 signaling

    International Nuclear Information System (INIS)

    Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling. Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′ins) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′ins helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling

  12. Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

    OpenAIRE

    Yamanaka, Masaru; NAGAO, SATOSHI; Komori, Hirofumi; Higuchi, Yoshiki; Hirota, Shun

    2015-01-01

    Cytochrome c555 from hyperthermophilic bacteria Aquifex aeolicus (AA cyt c555) is a hyperstable protein belonging to the cyt c protein family, which possesses a unique long 310-α-310 helix containing the heme-ligating Met61. Herein, we show that AA cyt c555 forms dimers by swapping the region containing the extra 310-α-310 helix and C-terminal α-helix. The asymmetric unit of the crystal of dimeric AA cyt c555 contained two dimer structures, where the structure of the hinge region (Val53–Lys57...

  13. 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 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. PMID:27128952

  14. The methylation of the C-terminal region of hnRNPQ (NSAP1) is important for its nuclear localization

    International Nuclear Information System (INIS)

    Protein arginine methylation is an irreversible post-translational protein modification catalyzed by a family of at least nine different enzymes entitled PRMTs (protein arginine methyl transferases). Although PRMT1 is responsible for 85% of the protein methylation in human cells, its substrate spectrum has not yet been fully characterized nor are the functional consequences of methylation for the protein substrates well understood. Therefore, we set out to employ the yeast two-hybrid system in order to identify new substrate proteins for human PRMT1. We were able to identify nine different PRMT1 interacting proteins involved in different aspects of RNA metabolism, five of which had been previously described either as substrates for PRMT1 or as functionally associated with PRMT1. Among the four new identified possible protein substrates was hnRNPQ3 (NSAP1), a protein whose function has been implicated in diverse steps of mRNA maturation, including splicing, editing, and degradation. By in vitro methylation assays we were able to show that hnRNPQ3 is a substrate for PRMT1 and that its C-terminal RGG box domain is the sole target for methylation. By further studies with the inhibitor of methylation Adox we provide evidence that hnRNPQ1-3 are methylated in vivo. Finally, we demonstrate by immunofluorescence analysis of HeLa cells that the methylation of hnRNPQ is important for its nuclear localization, since Adox treatment causes its re-distribution from the nucleus to the cytoplasm

  15. Domains of States of Chemical Systems: Le Chatelier Response, Structure of the Domains and Evolution

    OpenAIRE

    Zilbergleyt, B.

    2005-01-01

    The paper investigates influence of the Le Chatelier response on the chemical system behavior under stress, the shape of its domains of states in terms of static and dynamic bifurcation diagrams, and the system proneness to evolution. The usage of maps in thermodynamics of chemical systems is discussed. Thermodynamics of chemical triggers, designed in similarity with laser, is described. Results of this work are important in context of new model of chemical equilibrium.

  16. Structure and Mutagenesis of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Stalk Domain Reveals a Four-Helix Bundle and the Role of the Stalk in Fusion Promotion

    Energy Technology Data Exchange (ETDEWEB)

    Bose, Sayantan; Welch, Brett D.; Kors, Christopher A.; Yuan, Ping; Jardetzky, Theodore S.; Lamb, Robert A. (NWU); (Stanford-MED)

    2014-10-02

    Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 {angstrom}, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

  17. Role of the Cationic C-Terminal Segment of Melittin on Membrane Fragmentation.

    Science.gov (United States)

    Therrien, Alexandre; Fournier, Alain; Lafleur, Michel

    2016-05-01

    The widespread distribution of cationic antimicrobial peptides capable of membrane fragmentation in nature underlines their importance to living organisms. In the present work, we determined the impact of the electrostatic interactions associated with the cationic C-terminal segment of melittin, a 26-amino acid peptide from bee venom (net charge +6), on its binding to model membranes and on the resulting fragmentation. In order to detail the role played by the C-terminal charges, we prepared a melittin analogue for which the four cationic amino acids in positions 21-24 were substituted with the polar residue citrulline, providing a peptide with the same length and amphiphilicity but with a lower net charge (+2). We compared the peptide bilayer affinity and the membrane fragmentation for bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS) mixtures. It is shown that neutralization of the C-terminal considerably increased melittin affinity for zwitterionic membranes. The unfavorable contribution associated with transferring the cationic C-terminal in a less polar environment was reduced, leaving the hydrophobic interactions, which drive the peptide insertion in bilayers, with limited counterbalancing interactions. The presence of negatively charged lipids (DPPS) in bilayers increased melittin binding by introducing attractive electrostatic interactions, the augmentation being, as expected, greater for native melittin than for its citrullinated analogue. The membrane fragmentation power of the peptide was shown to be controlled by electrostatic interactions and could be modulated by the charge carried by both the membrane and the lytic peptide. The analysis of the lipid composition of the extracted fragments from DPPC/DPPS bilayers revealed no lipid specificity. It is proposed that extended phase separations are more susceptible to lead to the extraction of a lipid species in a specific manner

  18. Benefits of Matching Domain Structure for Planning Software: The Right Stuff

    Science.gov (United States)

    Billman, Dorrit Owen; Arsintescu, Lucica; Feary, Michael S.; Lee, Jessica Chia-Rong; Smith, Asha Halima; Tiwary, Rachna

    2011-01-01

    We investigated the role of domain structure in software design. We compared 2 planning applications, for a Mission Control group (International Space Station), and measured users speed and accuracy. Based on our needs analysis, we identified domain structure and used this to develop new prototype software that matched domain structure better than the legacy system. We took a high-fidelity analog of the natural task into the laboratory and found (large) periformance differences, favoring the system that matched domain structure. Our task design enabled us to attribute better periormance to better match of domain structure. We ran through the whole development cycle, in miniature, from needs analysis through design, development, and evaluation. Doing so enabled inferences not just about the particular systems compared, but also provided evidence for the viability of the design process (particularly needs analysis) that we are exploring.

  19. The structure of the Ca{sup 2+}-binding , glycosylated F-spondin domain of F-spondin- A C2-domain variant in an extracellular matrix protein.

    Energy Technology Data Exchange (ETDEWEB)

    Tan, K.; Lawler, J. (Biosciences Division); (Beth Israel Deaconess Medical Center); (Harvard Medical School)

    2011-05-10

    F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin{_}N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin. We present the crystal structure of human F-spondin FS domain at 1.95{angstrom} resolution. The structure reveals a Ca{sup 2+}-binding C2 domain variant with an 8-stranded antiparallel {beta}-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain. The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca{sup 2+}- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.

  20. The structure of the Ca2+-binding, glycosylated F-spondin domain of F-spondin - A C2-domain variant in an extracellular matrix protein

    Directory of Open Access Journals (Sweden)

    Lawler Jack

    2011-05-01

    Full Text Available Abstract Background F-spondin is a multi-domain extracellular matrix (ECM protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin_N domain and the F-spondin domain (FS domain comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin. Results We present the crystal structure of human F-spondin FS domain at 1.95Å resolution. The structure reveals a Ca2+-binding C2 domain variant with an 8-stranded antiparallel β-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain. Conclusion The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca2+- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.

  1. Baryon-antibaryon asymmetry in a model with domain structure and soft CP violation

    International Nuclear Information System (INIS)

    We consider a model with an abelian gauge symmetry, a Higgs potential involving two scalar fields, and two spinor fields coupled to the scalars through Yukawa couplings. The model accomodates soft violation of charge conjugation, and a domain structure of the universe with two different types of domains, which have identical energy but are governed by different effective lagrangians. The effective lagrangian has complex c-number coefficients that become parts of effective coupling constants, and these are different in the two kinds of domains. In spite of that fact the model neither predicts any domain-dependent effects, nor any particle-antiparticle asymmetries within domains. (orig.)

  2. Surface potential domains on lamellar P3OT structures

    Energy Technology Data Exchange (ETDEWEB)

    Perez-GarcIa, B [Departamento Fisica, Facultad de Quimica (Campus Espinardo), Universidad de Murcia, E-30100 Murcia (Spain); Abad, J [Departamento Fisica, Facultad de Quimica (Campus Espinardo), Universidad de Murcia, E-30100 Murcia (Spain); Urbina, A [Departamento Electronica, TecnologIa de Computadoras y Proyectos, Universidad Politecnica de Cartagena, E-30202 Cartagena (Spain); Colchero, J [Departamento Fisica, Facultad de Quimica (Campus Espinardo), Universidad de Murcia, E-30100 Murcia (Spain); Palacios-Lidon, E [Departamento Fisica, Facultad de Quimica (Campus Espinardo), Universidad de Murcia, E-30100 Murcia (Spain)

    2008-02-13

    In this work the electrostatic properties of poly(3-octylthiophene) thin films have been studied on a nanometer scale by means of electrostatic force microscopy and Kelvin probe microscopy (KPM). The KPM images reveal that different surface contact potential domains coexist on the polymer surface. This result, together with additional capacitance measurements, indicates that the potential domains are related to the existence of dipoles due to different molecular arrangements. Finally, capacitance measurements as a function of the tip-sample bias voltage show that in all regions large band bending effects take place.

  3. Structural integrity of the PCI domain of eIF3a/TIF32 is required for mRNA recruitment to the 43S pre-initiation complexes

    Czech Academy of Sciences Publication Activity Database

    Khoshnevis, S.; Gunišová, Stanislava; Vlčková, Vladislava; Kouba, Tomáš; Neumann, P.; Beznosková, Petra; Ficner, R.; Valášek, Leoš

    2014-01-01

    Roč. 42, č. 6 (2014), s. 4123-4139. ISSN 0305-1048 R&D Projects: GA ČR GAP305/10/0335 Institutional support: RVO:61388971 Keywords : C-TERMINAL DOMAIN * START CODON SELECTION * 40S RIBOSOMAL-SUBUNIT Subject RIV: CE - Biochemistry Impact factor: 9.112, year: 2014

  4. Solution structure of the Shc SH2 domain complexed with a tyrosine-phosphorylated peptide from the T-cell receptor.

    OpenAIRE

    M. M. Zhou; Meadows, R P; Logan, T. M.; Yoon, H S; Wade, W S; Ravichandran, K S; Burakoff, S J; Fesik, S W

    1995-01-01

    She is a widely expressed adapter protein that plays an important role in signaling via a variety of cell surface receptors and has been implicated in coupling the stimulation of growth factor, cytokine, and antigen receptors to the Ras signaling pathway. She interacts with several tyrosine-phosphorylated receptors through its C-terminal SH2 domain, and one of the mechanisms of T-cell receptor-mediated Ras activation involves the interaction of the Shc SH2 domain with the tyrosine-phosphoryla...

  5. Ferromagnetic domain structures and spin configurations measured in doped manganite

    DEFF Research Database (Denmark)

    He, J.Q.; Volkov, V.V.; Beleggia, Marco; Asaka, T.; Tao, J.; Schofield, M.A.; Zhu, Y.

    2010-01-01

    We report on measurements of the spin configuration across ferromagnetic domains in La0.325Pr0.3Ca0.375MnO3 films obtained by means of low-temperature Lorentz electron microscopy with in situ magnetizing capabilities. Due to the particular crystal symmetry of the material, we observe two sets of...

  6. Dynamic Analysis of Partially Embedded Structures Considering Soil-Structure Interaction in Time Domain

    Directory of Open Access Journals (Sweden)

    Sanaz Mahmoudpour

    2011-01-01

    Full Text Available Analysis and design of structures subjected to arbitrary dy