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Sample records for binding domain proteins

  1. The BRCT domain is a phospho-protein binding domain.

    Science.gov (United States)

    Yu, Xiaochun; Chini, Claudia Christiano Silva; He, Miao; Mer, Georges; Chen, Junjie

    2003-10-24

    The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.

  2. Ligand Binding Domain Protein in Tetracycline-Inducible Expression

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  3. Computational design of binding proteins to EGFR domain II.

    Directory of Open Access Journals (Sweden)

    Yoon Sup Choi

    Full Text Available We developed a process to produce novel interactions between two previously unrelated proteins. This process selects protein scaffolds and designs protein interfaces that bind to a surface patch of interest on a target protein. Scaffolds with shapes complementary to the target surface patch were screened using an exhaustive computational search of the human proteome and optimized by directed evolution using phage display. This method was applied to successfully design scaffolds that bind to epidermal growth factor receptor (EGFR domain II, the interface of EGFR dimerization, with high reactivity toward the target surface patch of EGFR domain II. One potential application of these tailor-made protein interactions is the development of therapeutic agents against specific protein targets.

  4. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    Science.gov (United States)

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-02-21

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 μM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

  5. Differential binding of heavy chain variable domain 3 antigen binding fragments to protein A chromatography resins.

    Science.gov (United States)

    Bach, Julia; Lewis, Nathaniel; Maggiora, Kathy; Gillespie, Alison J; Connell-Crowley, Lisa

    2015-08-28

    This work examines the binding of 15 different VH3 IgGs and their corresponding F(ab')2 fragments to two different protein A chromatography resins: MabSelect(®), which utilizes a recombinant protein A ligand, and MabSelect SuRe(®) (SuRe), which utilizes a tetrameric Z domain ligand. The results show that VH3 F(ab')2 fragments can exhibit a variety of binding behaviours for the two resins. Contrary to previously published data, a subset of these molecules show strong interaction with the Z domain of SuRe(®). Furthermore, the results show that sequence variability of residue 57 in the VH3 heavy chain CDR2 domain correlates with binding behaviour on MabSelect(®) and SuRe(®). Site-directed mutagenesis of this residue confers gain or loss of VH3 F(ab')2 binding to these resins in 3 mAbs, demonstrating that it plays a key role in both recombinant protein A and Z domain interaction. A fourth mAb with a longer CDR2 loop was not affected by mutation of residue 57, indicating that CDR2 domain length may alter the binding interface and lead to the involvement of other residues in protein A binding. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

    OpenAIRE

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-01-01

    Membrane-associated guanylate kinases (MAGUK) family proteins contain an inactive guanylate kinase (GK) domain, whose function has been elusive. Here, this domain is revealed as a new type of phospho-peptide-binding module, in which the GMP-binding site has evolved to accommodate phospho-serines or -threonines.

  7. Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

    Directory of Open Access Journals (Sweden)

    Waqasuddin Khan

    Full Text Available Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58.Next, we trained a bidirectional recurrent neural network (BRNN using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72 showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors.

  8. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

    DEFF Research Database (Denmark)

    Jensen, P Y; Bonander, N; Møller, L B

    1999-01-01

    spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two...... and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains...... is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration...

  9. RNA-Binding Domain Proteins in Kinetoplastids: a Comparative Analysis†

    Science.gov (United States)

    De Gaudenzi, Javier; Frasch, Alberto C.; Clayton, Christine

    2005-01-01

    RNA-binding proteins are important in many aspects of RNA processing, function, and destruction. One class of such proteins contains the RNA recognition motif (RRM), which consists of about 90 amino acid residues, including the canonical RNP1 octapeptide: (K/R)G(F/Y)(G/A)FVX(F/Y). We used a variety of homology searches to classify all of the RRM proteins of the three kinetoplastids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. All three organisms have similar sets of RRM-containing protein orthologues, suggesting common posttranscriptional processing and regulatory pathways. Of the 75 RRM proteins identified in T. brucei, only 13 had clear homologues in other eukaryotes, although 8 more could be given putative functional assignments. A comparison with the 18 RRM proteins of the obligate intracellular parasite Encephalitozoon cuniculi revealed just 3 RRM proteins which appear to be conserved at the primary sequence level throughout eukaryotic evolution: poly(A) binding protein, the rRNA-processing protein MRD1, and the nuclear cap binding protein. PMID:16339728

  10. Facilitating identification of minimal protein binding domains by cross-linking mass spectrometry

    NARCIS (Netherlands)

    Liu, Qingyang; Remmelzwaal, Sanne; Heck, Albert J R; Akhmanova, Anna; Liu, Fan

    2017-01-01

    Characterization of protein interaction domains is crucial for understanding protein functions. Here we combine cross-linking mass spectrometry (XL-MS) with deletion analysis to accurately locate minimal protein interaction domains. As a proof of concept, we investigated in detail the binding

  11. The CRM domain: An RNA binding module derived from an ancient ribosome-associated protein

    Science.gov (United States)

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P.

    2007-01-01

    The CRS1–YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved “GxxG” loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes. PMID:17105995

  12. The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

    Science.gov (United States)

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

    2007-01-01

    The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

  13. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

    Science.gov (United States)

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-11-25

    Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

  14. A small cellulose binding domain protein (CBD1) is highly variable in the nonbinding amino terminus

    Science.gov (United States)

    The small cellulose binding domain protein CBD1 is tightly bound to the cellulosic cell wall of the plant pathogenic stramenophile Phytophthora infestans. Transgene expression of the protein in plants has also demonstrated binding to plant cell walls. A study was undertaken using 47 isolates of P. ...

  15. Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module

    DEFF Research Database (Denmark)

    Salcini, A E; Confalonieri, S; Doria, M

    1997-01-01

    EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct...

  16. STRUCTURAL CHARACTERIZATION OF THE RNA BINDING DOMAIN OF HUMAN STEM LOOP BINDING PROTEIN

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    Maruthi Kashyap

    2013-12-01

    Full Text Available A gene encoding the RNA binding domain (RBD of human stem loop binding protein (SLBP was cloned in pET 28a vector and over-expressed in E. coli codon plus cells. The over-expressed SLBP-RBD carried no tag and aggregated as inclusion bodies in the cell lysate. Inclusion bodies were semi-purified to >85% purity by establishing a method involving detergent washing and subsequently denatured in 8 M urea. Refolding of the denatured RBD was carried out by step dialysis in decreasing concentrations of urea and L-arginine. Refolded SLBP-RBD was analyzed using size exclusion chromatography that revealed its monomeric nature and folded state. Uniformly 15N and 15N,13C labeled SLBP-RBD was prepared at concentrations for solution NMR studies. Approximately, 60% of the sequence specific backbone resonance assignments have been achieved through standard triple resonance NMR experiments. Analyses of secondary chemical shifts reveal presence of a small helical secondary structural elements and large intrinsically disordered regions.

  17. The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.; Gomes Guimaraes, B.

    2008-01-01

    Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstroms resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

  18. Proteins containing the UBA domain are able to bind to multi-ubiquitin chains

    DEFF Research Database (Denmark)

    Wilkinson, C R; Seeger, M; Hartmann-Petersen, R

    2001-01-01

    The UBA domain is a motif found in a variety of proteins, some of which are associated with the ubiquitin-proteasome system. We describe the isolation of a fission-yeast gene, mud1+, which encodes a UBA domain containing protein that is able to bind multi-ubiquitin chains. We show that the UBA...... domain is responsible for this activity. Two other proteins containing this motif, the fission-yeast homologues of Rad23 and Dsk2, are also shown to bind multi-ubiquitin chains via their UBA domains. These two proteins are implicated, along with the fission-yeast Pus1(S5a/Rpn10) subunit of the 26 S...

  19. Prediction of small molecule binding property of protein domains with Bayesian classifiers based on Markov chains.

    Science.gov (United States)

    Bulashevska, Alla; Stein, Martin; Jackson, David; Eils, Roland

    2009-12-01

    Accurate computational methods that can help to predict biological function of a protein from its sequence are of great interest to research biologists and pharmaceutical companies. One approach to assume the function of proteins is to predict the interactions between proteins and other molecules. In this work, we propose a machine learning method that uses a primary sequence of a domain to predict its propensity for interaction with small molecules. By curating the Pfam database with respect to the small molecule binding ability of its component domains, we have constructed a dataset of small molecule binding and non-binding domains. This dataset was then used as training set to learn a Bayesian classifier, which should distinguish members of each class. The domain sequences of both classes are modelled with Markov chains. In a Jack-knife test, our classification procedure achieved the predictive accuracies of 77.2% and 66.7% for binding and non-binding classes respectively. We demonstrate the applicability of our classifier by using it to identify previously unknown small molecule binding domains. Our predictions are available as supplementary material and can provide very useful information to drug discovery specialists. Given the ubiquitous and essential role small molecules play in biological processes, our method is important for identifying pharmaceutically relevant components of complete proteomes. The software is available from the author upon request.

  20. High-affinity single-domain binding proteins with a binary-code interface.

    Science.gov (United States)

    Koide, Akiko; Gilbreth, Ryan N; Esaki, Kaori; Tereshko, Valentina; Koide, Shohei

    2007-04-17

    High degrees of sequence and conformation complexity found in natural protein interaction interfaces are generally considered essential for achieving tight and specific interactions. However, it has been demonstrated that specific antibodies can be built by using an interface with a binary code consisting of only Tyr and Ser. This surprising result might be attributed to yet undefined properties of the antibody scaffold that uniquely enhance its capacity for target binding. In this work we tested the generality of the binary-code interface by engineering binding proteins based on a single-domain scaffold. We show that Tyr/Ser binary-code interfaces consisting of only 15-20 positions within a fibronectin type III domain (FN3; 95 residues) are capable of producing specific binding proteins (termed "monobodies") with a low-nanomolar K(d). A 2.35-A x-ray crystal structure of a monobody in complex with its target, maltose-binding protein, and mutation analysis revealed dominant contributions of Tyr residues to binding as well as striking molecular mimicry of a maltose-binding protein substrate, beta-cyclodextrin, by the Tyr/Ser binary interface. This work suggests that an interaction interface with low chemical diversity but with significant conformational diversity is generally sufficient for tight and specific molecular recognition, providing fundamental insights into factors governing protein-protein interactions.

  1. Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization.

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    Maia V Vinogradova

    Full Text Available Kinesin-like calmodulin binding protein (KCBP, a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca(2+-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca(2+ signaling since Ca(2+- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface.

  2. Ligand Binding Domain Protein in Tetracycline-Inducible Expression ...

    African Journals Online (AJOL)

    LXR agonist T-0901317 to the expression culture media and to all buffers used for lysis, and purification. The recombinant LXR β-LBD(197-461) fusion protein induced or uninduced at 15 ºC in the presence or absence of a synthetic agonist. T-0901317 and separated by SDS-PAGE and stained with Coomassie Brilliant Blue ...

  3. RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp.

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    Chumporn Soowannayan

    Full Text Available Gill-associated virus (GAV infects Penaeus monodon shrimp and is the type species okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs using His(6-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ssRNAs tested independent of their sequence. The ssRNAs included (+ and (- sense regions of the GAV genome as well as a (+ sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (dsRNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa N-terminal sequence spanning Met(11 to Arg(60. Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11PVRRPLPPQPPRNARLI(29 encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.

  4. Ligand binding by PDZ domains

    DEFF Research Database (Denmark)

    Chi, Celestine N.; Bach, Anders; Strømgaard, Kristian

    2012-01-01

    The postsynaptic density protein-95/disks large/zonula occludens-1 (PDZ) protein domain family is one of the most common protein-protein interaction modules in mammalian cells, with paralogs present in several hundred human proteins. PDZ domains are found in most cell types, but neuronal proteins...... as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context....

  5. 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

  6. Structural feature extraction protocol for classifying reversible membrane binding protein domains.

    Science.gov (United States)

    Källberg, Morten; Lu, Hui

    2009-01-01

    Machine learning based classification protocols for automated function annotation of protein structures have in many instances proven superior to simpler sequence based procedures. Here we present an automated method for extracting features from protein structures by construction of surface patches to be used in such protocols. The utility of the developed patch-growing procedure is exemplified by its ability to identify reversible membrane binding domains from the C1, C2, and PH families.

  7. A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain: Periplasmic Ligand Binding Protein Dret_0059

    Energy Technology Data Exchange (ETDEWEB)

    Wu, R. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Wilton, R. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Cuff, M. E. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Argonne National Laboratory, Argonne Illinois 60439; Endres, M. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Babnigg, G. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Edirisinghe, J. N. [Mathematics and Computer Science Division, Argonne National Laboratory, Argonne Illinois 60439; Computation Institute, University of Chicago, Chicago Illinois 60637; Henry, C. S. [Mathematics and Computer Science Division, Argonne National Laboratory, Argonne Illinois 60439; Computation Institute, University of Chicago, Chicago Illinois 60637; Joachimiak, A. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Argonne National Laboratory, Argonne Illinois 60439; Department of Biochemistry and Molecular Biology, University of Chicago, Chicago Illinois 60637; Schiffer, M. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Pokkuluri, P. R. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439

    2017-03-06

    We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from the Salt Lake Retba in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.

  8. A Novel Protein Interaction between Nucleotide Binding Domain of Hsp70 and p53 Motif

    Directory of Open Access Journals (Sweden)

    Asita Elengoe

    2015-01-01

    Full Text Available Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD of heat shock 70 kDa protein (PDB: 1HJO with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD simulation. Human DNA binding domain of p53 motif (SCMGGMNR retrieved from UniProt (UniProtKB: P04637 was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were −0.44 Kcal/mol and −9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy.

  9. Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein*

    Science.gov (United States)

    Grüning, Clara S. R.; Mirecka, Ewa A.; Klein, Antonia N.; Mandelkow, Eckhard; Willbold, Dieter; Marino, Stephen F.; Stoldt, Matthias; Hoyer, Wolfgang

    2014-01-01

    The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337–342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau. PMID:24966331

  10. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    Science.gov (United States)

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

  11. A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice.

    Science.gov (United States)

    Fenyk, Stepan; Campillo, Alba de San Eustaquio; Pohl, Ehmke; Hussey, Patrick J; Cann, Martin J

    2012-02-03

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.

  12. A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution

    DEFF Research Database (Denmark)

    Wong, W T; Schumacher, C; Salcini, A E

    1995-01-01

    In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heteroge......In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several...... heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic...

  13. Recognition of methylated DNA through methyl-CpG binding domain proteins

    DEFF Research Database (Denmark)

    Zou, Xueqing; Ma, Wen; Solov'yov, Ilia

    2012-01-01

    DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although...... the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines...

  14. WW domain-binding protein 2: an adaptor protein closely linked to the development of breast cancer.

    Science.gov (United States)

    Chen, Shuai; Wang, Han; Huang, Yu-Fan; Li, Ming-Li; Cheng, Jiang-Hong; Hu, Peng; Lu, Chuan-Hui; Zhang, Ya; Liu, Na; Tzeng, Chi-Meng; Zhang, Zhi-Ming

    2017-07-19

    The WW domain is composed of 38 to 40 semi-conserved amino acids shared with structural, regulatory, and signaling proteins. WW domain-binding protein 2 (WBP2), as a binding partner of WW domain protein, interacts with several WW-domain-containing proteins, such as Yes kinase-associated protein (Yap), paired box gene 8 (Pax8), WW-domain-containing transcription regulator protein 1 (TAZ), and WW-domain-containing oxidoreductase (WWOX) through its PPxY motifs within C-terminal region, and further triggers the downstream signaling pathway in vitro and in vivo. Studies have confirmed that phosphorylated form of WBP2 can move into nuclei and activate the transcription of estrogen receptor (ER) and progesterone receptor (PR), whose expression were the indicators of breast cancer development, indicating that WBP2 may participate in the progression of breast cancer. Both overexpression of WBP2 and activation of tyrosine phosphorylation upregulate the signal cascades in the cross-regulation of the Wnt and ER signaling pathways in breast cancer. Following the binding of WBP2 to the WW domain region of TAZ which can accelerate migration, invasion and is required for the transformed phenotypes of breast cancer cells, the transformation of epithelial to mesenchymal of MCF10A is activated, suggesting that WBP2 is a key player in regulating cell migration. When WBP2 binds with WWOX, a tumor suppressor, ER transactivation and tumor growth can be suppressed. Thus, WBP2 may serve as a molecular on/off switch that controls the crosstalk between E2, WWOX, Wnt, TAZ, and other oncogenic signaling pathways. This review interprets the relationship between WBP2 and breast cancer, and provides comprehensive views about the function of WBP2 in the regulation of the pathogenesis of breast cancer and endocrine therapy in breast cancer treatment.

  15. Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains

    International Nuclear Information System (INIS)

    Vallee, B.L.; Auld, D.S.; Coleman, J.E.

    1991-01-01

    The authors recognize three distinct motifs of DNA-binding zinc proteins: (i) zinc fingers, (ii) zinc clusters, and (iii) zinc twists. Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses of DNA-binding zinc proteins have not been available to serve as standards of reference for the zinc binding sites of these families of proteins. Those of the DNA-binding domains of the fungal transcription factor GAL4 and the rat glucocorticoid receptor are the first to have been determined. Both proteins contain two zinc binding sites, and in both, cysteine residues are the sole zinc ligands. In GAL4, two zinc atoms are bound to six cysteine residues which form a zinc cluster akin to that of metallothionein; the distance between the two zinc atoms of GAL4 is ∼3.5 angstrom. In the glucocorticoid receptor, each zinc atom is bound to four cysteine residues; the interatomic zinc-zinc distance is ∼13 angstrom, and in this instance, a zinc twist is represented by a helical DNA recognition site located between the two zinc atoms. Zinc clusters and zinc twists are here recognized as two distinctive motifs in DNA-binding proteins containing multiple zinc atoms. For native zinc fingers, structural data do not exist as yet; consequently, the interatomic distances between zinc atoms are not known. As further structural data become available, the structural and functional significance of these different motifs in their binding to DNA and other proteins participating in the transmission of the genetic message will become apparent

  16. Hinderin, a five-domains protein including coiled-coil motifs that binds to SMC3

    Directory of Open Access Journals (Sweden)

    Ghiselli Giancarlo

    2005-01-01

    Full Text Available Abstract Background The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions. Results In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin. Conclusions Hinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes.

  17. Structure-Based Design of a Periplasmic Binding Protein Antagonist that Prevents Domain Closure

    Energy Technology Data Exchange (ETDEWEB)

    Borrok, M. Jack; Zhu, Yimin; Forest, Katrina T.; Kiessling, Laura L.; (UW)

    2009-07-31

    Many receptors undergo ligand-induced conformational changes to initiate signal transduction. Periplasmic binding proteins (PBPs) are bacterial receptors that exhibit dramatic conformational changes upon ligand binding. These proteins mediate a wide variety of fundamental processes including transport, chemotaxis, and quorum sensing. Despite the importance of these receptors, no PBP antagonists have been identified and characterized. In this study, we identify 3-O-methyl-D-glucose as an antagonist of glucose/galactose-binding protein and demonstrate that it inhibits glucose chemotaxis in E. coli. Using small-angle X-ray scattering and X-ray crystallography, we show that this antagonist acts as a wedge. It prevents the large-scale domain closure that gives rise to the active signaling state. Guided by these results and the structures of open and closed glucose/galactose-binding protein, we designed and synthesized an antagonist composed of two linked glucose residues. These findings provide a blueprint for the design of new bacterial PBP inhibitors. Given the key role of PBPs in microbial physiology, we anticipate that PBP antagonists will have widespread uses as probes and antimicrobial agents.

  18. Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

    Directory of Open Access Journals (Sweden)

    Sofiya Fedosyuk

    2016-12-01

    Full Text Available Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83 structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240, we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.

  19. The expanded octarepeat domain selectively binds prions and disrupts homomeric prion protein interactions.

    Science.gov (United States)

    Leliveld, Sirik Rutger; Dame, Remus Thei; Wuite, Gijs J L; Stitz, Lothar; Korth, Carsten

    2006-02-10

    Insertion of additional octarepeats into the prion protein gene has been genetically linked to familial Creutzfeldt Jakob disease and hence to de novo generation of infectious prions. The pivotal event during prion formation is the conversion of the normal prion protein (PrPC) into the pathogenic conformer PrPSc, which subsequently induces further conversion in an autocatalytic manner. Apparently, an expanded octarepeat domain directs folding of PrP toward the PrPSc conformation and initiates a self-replicating conversion process. Here, based on three main observations, we have provided a model on how altered molecular interactions between wild-type and mutant PrP set the stage for familial Creutzfeldt Jakob disease with octarepeat insertions. First, we showed that wild-type octarepeat domains interact in a copper-dependent and reversible manner, a "copper switch." This interaction becomes irreversible upon domain expansion, possibly reflecting a loss of function. Second, expanded octarepeat domains of increasing length gradually form homogenous globular multimers of 11-21 nm in the absence of copper ions when expressed as soluble glutathione S-transferase fusion proteins. Third, octarepeat domain expansion causes a gain of function with at least 10 repeats selectively binding PrPSc in a denaturant-resistant complex in the absence of copper ions. Thus, the combination of both a loss and gain of function profoundly influences homomeric interaction behavior of PrP with an expanded octarepeat domain. A multimeric cluster of prion proteins carrying expanded octarepeat domains may therefore capture and incorporate spontaneously arising short-lived PrPSc-like conformers, thereby providing a matrix for their conversion.

  20. Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

    DEFF Research Database (Denmark)

    Jensen, R B; Lykke-Andersen, K; Frandsen, G I

    2000-01-01

    Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2...... containing the ZAC-C2 domain bind anionic phospholipids non-specifically, with some variance in Ca2+ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1-174) for phosphatidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact...

  1. A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies.

    Directory of Open Access Journals (Sweden)

    Kevin A Henry

    Full Text Available Staphylococcal protein A (SpA and streptococcal protein G (SpG affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species.

  2. De novo design and engineering of functional metal and porphyrin-binding protein domains

    Science.gov (United States)

    Everson, Bernard H.

    In this work, I describe an approach to the rational, iterative design and characterization of two functional cofactor-binding protein domains. First, a hybrid computational/experimental method was developed with the aim of algorithmically generating a suite of porphyrin-binding protein sequences with minimal mutual sequence information. This method was explored by generating libraries of sequences, which were then expressed and evaluated for function. One successful sequence is shown to bind a variety of porphyrin-like cofactors, and exhibits light- activated electron transfer in mixed hemin:chlorin e6 and hemin:Zn(II)-protoporphyrin IX complexes. These results imply that many sophisticated functions such as cofactor binding and electron transfer require only a very small number of residue positions in a protein sequence to be fixed. Net charge and hydrophobic content are important in determining protein solubility and stability. Accordingly, rational modifications were made to the aforementioned design procedure in order to improve its overall success rate. The effects of these modifications are explored using two `next-generation' sequence libraries, which were separately expressed and evaluated. Particular modifications to these design parameters are demonstrated to effectively double the purification success rate of the procedure. Finally, I describe the redesign of the artificial di-iron protein DF2 into CDM13, a single chain di-Manganese four-helix bundle. CDM13 acts as a functional model of natural manganese catalase, exhibiting a kcat of 0.08s-1 under steady-state conditions. The bound manganese cofactors have a reduction potential of +805 mV vs NHE, which is too high for efficient dismutation of hydrogen peroxide. These results indicate that as a high-potential manganese complex, CDM13 may represent a promising first step toward a polypeptide model of the Oxygen Evolving Complex of the photosynthetic enzyme Photosystem II.

  3. Convergence of Domain Architecture, Structure, and Ligand Affinity in Animal and Plant RNA-Binding Proteins.

    Science.gov (United States)

    Dias, Raquel; Manny, Austin; Kolaczkowski, Oralia; Kolaczkowski, Bryan

    2017-06-01

    Reconstruction of ancestral protein sequences using phylogenetic methods is a powerful technique for directly examining the evolution of molecular function. Although ancestral sequence reconstruction (ASR) is itself very efficient, downstream functional, and structural studies necessary to characterize when and how changes in molecular function occurred are often costly and time-consuming, currently limiting ASR studies to examining a relatively small number of discrete functional shifts. As a result, we have very little direct information about how molecular function evolves across large protein families. Here we develop an approach combining ASR with structure and function prediction to efficiently examine the evolution of ligand affinity across a large family of double-stranded RNA binding proteins (DRBs) spanning animals and plants. We find that the characteristic domain architecture of DRBs-consisting of 2-3 tandem double-stranded RNA binding motifs (dsrms)-arose independently in early animal and plant lineages. The affinity with which individual dsrms bind double-stranded RNA appears to have increased and decreased often across both animal and plant phylogenies, primarily through convergent structural mechanisms involving RNA-contact residues within the β1-β2 loop and a small region of α2. These studies provide some of the first direct information about how protein function evolves across large gene families and suggest that changes in molecular function may occur often and unassociated with major phylogenetic events, such as gene or domain duplications. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein

    NARCIS (Netherlands)

    van der Wolk, J.P.W.; Klose, M; de Wit, Janny; Blaauwen, T.den; Freudl, R; Driessen, A.J.M.

    1995-01-01

    The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain

  5. Optimizing the protein switch: altering nuclear import and export signals, and ligand binding domain

    Science.gov (United States)

    Kakar, Mudit; Davis, James R.; Kern, Steve E.; Lim, Carol S.

    2007-01-01

    Ligand regulated localization controllable protein constructs were optimized in this study. Several constructs were made from a classical nuclear export signal (HIV-rev, MAPKK, or progesterone receptor) in combination with a SV40 T-antigen type nuclear import signal. Different ligand binding domains (LBDs from glucocorticoid receptor or progesterone receptor) were also tested for their ability to impart control over localization of proteins. This study was designed to create constructs which are cytoplasmic in the absence of ligand and nuclear in the presence of ligand, and also to regulate the amount of protein translocating to the nucleus on ligand induction. The balance between the strengths of import and export signals was critical for overall localization of proteins. The amount of protein entering the nucleus was also affected by the dose of ligand (10-100nM). However, the overall import characteristics were determined by the strengths of localization signals and the inherent localization properties of the LBD used. This study established that the amount of protein present in a particular compartment can be regulated by the use of localization signals of various strengths. These optimized localization controllable protein constructs can be used to correct for diseases due to aberrant localization of proteins. PMID:17574289

  6. Zn-binding AZUL domain of human ubiquitin protein ligase Ube3A

    Energy Technology Data Exchange (ETDEWEB)

    Lemak, Alexander; Yee, Adelinda [University of Toronto, and Northeast Structural Genomics Consortium, Ontario Cancer Institute, Campbell Family Cancer Research Institute and Department of Medical Biophysics (Canada); Bezsonova, Irina, E-mail: bezsonova@uchc.edu [University of Connecticut Health Center, Department of Molecular Microbial and Structural Biology (United States); Dhe-Paganon, Sirano, E-mail: sirano.dhepaganon@utoronto.ca [University of Toronto, Structural Genomics Consortium (Canada); Arrowsmith, Cheryl H., E-mail: carrow@uhnresearch.ca [University of Toronto, and Northeast Structural Genomics Consortium, Ontario Cancer Institute, Campbell Family Cancer Research Institute and Department of Medical Biophysics (Canada)

    2011-09-15

    Ube3A (also referred to as E6AP for E6 Associated Protein) is a E3 ubiquitin-protein ligase implicated in the development of Angelman syndrome by controlling degradation of synaptic protein Arc and oncogenic papilloma virus infection by controlling degradation of p53. This article describe the solution NMR structure of the conserved N-terminal domain of human Ube3A (residues 24-87) that contains two residues (Cys44 and Arg62) found to be mutated in patients with Angelman syndrome. The structure of this domain adopts a novel Zn-binding fold we called AZUL (Amino-terminal Zn-finger of Ube3a Ligase). The AZUL domain has a helix-loop-helix architecture with a Zn ion coordinated by four Cys residues arranged in Cys-X{sub 4}-Cys-X{sub 4}-Cys-X{sub 28}-Cys motif. Three of the Zn-bound residues are located in a 23-residue long and well structured loop that connects two {alpha}-helicies.

  7. Zn-binding AZUL domain of human ubiquitin protein ligase Ube3A

    International Nuclear Information System (INIS)

    Lemak, Alexander; Yee, Adelinda; Bezsonova, Irina; Dhe-Paganon, Sirano; Arrowsmith, Cheryl H.

    2011-01-01

    Ube3A (also referred to as E6AP for E6 Associated Protein) is a E3 ubiquitin-protein ligase implicated in the development of Angelman syndrome by controlling degradation of synaptic protein Arc and oncogenic papilloma virus infection by controlling degradation of p53. This article describe the solution NMR structure of the conserved N-terminal domain of human Ube3A (residues 24-87) that contains two residues (Cys44 and Arg62) found to be mutated in patients with Angelman syndrome. The structure of this domain adopts a novel Zn-binding fold we called AZUL (Amino-terminal Zn-finger of Ube3a Ligase). The AZUL domain has a helix-loop-helix architecture with a Zn ion coordinated by four Cys residues arranged in Cys-X 4 -Cys-X 4 -Cys-X 28 -Cys motif. Three of the Zn-bound residues are located in a 23-residue long and well structured loop that connects two α-helicies.

  8. The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain

    International Nuclear Information System (INIS)

    Lechner, Mark S.; Schultz, David C.; Negorev, Dmitri; Maul, Gerd G.; Rauscher, Frank J.

    2005-01-01

    The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization

  9. Binding of curcumin and its long chain derivatives to the activator binding domain of novel protein kinase C.

    Science.gov (United States)

    Majhi, Anjoy; Rahman, Ghazi M; Panchal, Shyam; Das, Joydip

    2010-02-15

    Protein kinase C (PKC) is a family of serine/threonine kinases that play a central role in cellular signal transduction. The second messenger diacylglycerol having two long carbon chains acts as the endogenous ligand for the PKCs. Polyphenol curcumin, the active constituent of Curcuma longa is an anti-cancer agent and modulates PKC activity. To develop curcumin derivatives as effective PKC activators, we synthesized several long chain derivatives of curcumin, characterized their absorption and fluorescence properties and studied their interaction with the activator binding second cysteine-rich C1B subdomain of PKCdelta, PKCepsilon and PKCtheta. Curcumin (1) and its C16 long chain analog (4) quenched the intrinsic fluorescence of PKCdeltaC1B, PKCepsilonC1B and PKCthetaC1B in a manner similar to that of PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). The EC(50)s of the curcumin derivatives for fluorescence quenching varied in the range of 4-11 microM, whereas, EC(50)s for TPA varied in the range of 3-6 microM. Fluorescence emission maxima of 1 and 4 were blue shifted and the fluorescence anisotropy values were increased in the presence of the C1B domains in a manner similar to that shown by the fluorescent analog of TPA, sapintoxin-D, confirming that they were bound to the proteins. Molecular docking of 1 and 4 with novel PKC C1B revealed that both the molecules form hydrogen bonds with the protein residues. The present result shows that curcumin and its long chain derivatives bind to the C1B subdomain of novel PKCs and can be further modified structurally to improve its binding and activity. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. Methyl-CpG binding domain proteins inhibit interspecies courtship and promote aggression in Drosophila.

    Science.gov (United States)

    Gupta, Tarun; Morgan, Hannah R; Andrews, Jonathan C; Brewer, Edmond R; Certel, Sarah J

    2017-07-14

    Reproductive isolation and speciation are driven by the convergence of environmental and genetic variation. The integration of these variation sources is thought to occur through epigenetic marks including DNA methylation. Proteins containing a methyl-CpG-binding domain (MBD) bind methylated DNA and interpret epigenetic marks, providing a dynamic yet evolutionarily adapted cellular output. Here, we report the Drosophila MBD-containing proteins, dMBD-R2 and dMBD2/3, contribute to reproductive isolation and survival behavioral strategies. Drosophila melanogaster males with a reduction in dMBD-R2 specifically in octopamine (OA) neurons exhibit courtship toward divergent interspecies D. virilis and D. yakuba females and a decrease in conspecific mating success. Conspecific male-male courtship is increased between dMBD-R2-deficient males while aggression is reduced. These changes in adaptive behavior are separable as males with a hypermethylated OA neuronal genome exhibited a decrease in aggression without altering male-male courtship. These results suggest Drosophila MBD-containing proteins are required within the OA neural circuitry to inhibit interspecies and conspecific male-male courtship and indicate that the genetically hard-wired neural mechanisms enforcing behavioral reproductive isolation include the interpretation of the epigenome.

  11. Roles of phosphorylation and helix propensity in the binding of the KIX domain of CREB-binding protein by constitutive (c-Myb) and inducible (CREB) activators.

    Science.gov (United States)

    Zor, Tsaffrir; Mayr, Bernhard M; Dyson, H Jane; Montminy, Marc R; Wright, Peter E

    2002-11-01

    cAMP-response element-binding protein (CREB)-binding protein (CBP) is a general transcriptional co-activator that mediates interactions between transcription factors and the basal transcription machinery. To obtain insights into the mechanism by which the KIX domain of CBP can recognize the transactivation domains of many different transcription factors, we have used NMR and biochemical analyses to study the interactions of KIX with the transactivation domain from the constitutive activator c-Myb and with the kinase-inducible transactivation domain (KID) from CREB. NMR chemical shift mapping shows that both activation domains bind to the same surface of KIX. In the unbound state, both the phosphorylated KID and c-Myb activation domains are only partly structured, and binding to KIX is coupled with folding to form an amphipathic helix. Helix-destabilizing mutations significantly impair binding, whereas mutations that increase the intrinsic secondary structure content of the free phosphorylated KID peptide have only a small influence on binding affinity. Low affinity but specific binding of unphosphorylated KID to KIX was measured by ITC and was also observed in Western blot assays and by a fluorescence resonance energy transfer experiment in living cells. The large increase in the affinity for phosphorylated KID is due to favorable intermolecular interactions involving the phosphate moiety. After induction by phosphorylation, CREB is able to compete effectively with other transcriptional activators for binding to CBP.

  12. Guanine nucleotide-binding protein (Gα) endocytosis by a cascade of ubiquitin binding domain proteins is required for sustained morphogenesis and proper mating in yeast.

    Science.gov (United States)

    Dixit, Gauri; Baker, Rachael; Sacks, Carly M; Torres, Matthew P; Dohlman, Henrik G

    2014-05-23

    Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Non-canonical binding interactions of the RNA recognition motif (RRM domains of P34 protein modulate binding within the 5S ribonucleoprotein particle (5S RNP.

    Directory of Open Access Journals (Sweden)

    Anyango D Kamina

    Full Text Available RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP and P34 binds to 5S ribosomal RNA (rRNA and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM domains. We generated truncated P34 proteins to determine these domains' interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP.

  14. Ligand binding to the PDZ domains of postsynaptic density protein 95

    DEFF Research Database (Denmark)

    Toto, Angelo; Pedersen, Søren W; Karlsson, O Andreas

    2016-01-01

    Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 ...

  15. Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain.

    Science.gov (United States)

    Prashek, Jennifer; Bouyain, Samuel; Fu, Mingui; Li, Yong; Berkes, Dusan; Yao, Xiaolan

    2017-08-25

    De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this study we show that isolated PH and START domains interact with each other. The crystal structure of a PH-START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH-START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine-rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain

    Energy Technology Data Exchange (ETDEWEB)

    Prashek, Jennifer; Bouyain, Samuel; Fu, Mingui; Li, Yong; Berkes, Dusan; Yao, Xiaolan

    2017-06-26

    De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this study we show that isolated PH and START domains interact with each other. The crystal structure of a PH–START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH–START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine–rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.

  17. A Novel, Highly Stable Fold of the Immunoglobulin Binding Domain of Streptococcal Protein G

    Science.gov (United States)

    Gronenborn, Angela M.; Filpula, David R.; Essig, Nina Z.; Achari, Aniruddha; Whitlow, Marc; Wingfield, Paul T.; Marius Clore, G.

    1991-08-01

    The high-resolution three-dimensional structure of a single immunoglobulin binding domain (B1, which comprises 56 residues including the NH_2-terminal Met) of protein G from group G Streptococcus has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1058 experimental restraints. The average atomic root-mean-square distribution about the mean coordinate positions is 0.27 angstrom (overset{circ}{mathrm A}) for the backbone atoms, 0.65 overset{circ}{mathrm A} for all atoms, and 0.39 overset{circ}{mathrm A} for atoms excluding disordered surface side chains. The structure has no disulfide bridges and is composed of a four-stranded β sheet, on top of which lies a long helix. The central two strands (β 1 and β 4), comprising the NH_2- and COOH-termini, are parallel, and the outer two strands (β 2 and β 3) are connected by the helix in a +3x crossover. This novel topology (-1, +3x, -1), coupled with an extensive hydrogen-bonding network and a tightly packed and buried hydrophobic core, is probably responsible for the extreme thermal stability of this small domain (reversible melting at 87^circC).

  18. Characterization of the hormone-binding domain of the chicken c-erbA/thyroid hormone receptor protein

    DEFF Research Database (Denmark)

    Muñoz, A; Zenke, M; Gehring, U

    1988-01-01

    To identify and characterize the hormone-binding domain of the thyroid hormone receptor, we analyzed the ligand-binding capacities of proteins representing chimeras between the normal receptor and P75gag-v-erbA, the retrovirus-encoded form deficient in binding ligand. Our results show that several...... mutations present in the carboxy-terminal half of P75gag-v-erbA co-operate in abolishing hormone binding, and that the ligand-binding domain resides in a position analogous to that of steroid receptors. Furthermore, a point mutation that is located between the putative DNA and ligand-binding domains of P75......gag-v-erbA and that renders it biologically inactive fails to affect hormone binding by the c-erbA protein. These results suggest that the mutation changed the ability of P75gag-v-erbA to affect transcription since it also had no effect on DNA binding. Our data also suggest that hormone...

  19. Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS

    Energy Technology Data Exchange (ETDEWEB)

    Teplova, Marianna; Farazi, Thalia A.; Tuschl, Thomas; Patel, Dinshaw J.

    2015-09-08

    Abstract

    RNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutationsin vivo.

  20. Multimerization of the mouse TATA-binding protein (TBP) driven by its C-terminal conserved domain.

    Science.gov (United States)

    Kato, K; Makino, Y; Kishimoto, T; Yamauchi, J; Kato, S; Muramatsu, M; Tamura, T

    1994-04-11

    The conformational states of the mouse TATA-binding protein (TBP) in solution were studied. A histidine tag and a factor Xa recognition site-carrying mouse TBP was expressed in E. coli, highly purified, and its fundamental functions as a TBP were demonstrated. We analyzed the molecular states of mouse TBP by gel filtration and glycerol gradient sedimentation, and found that TBP forms heterogeneous multimers in solution. Direct binding of TBP molecules to each other was proven by the far-Western procedure. Analyses using TBPs truncated at the N- and C-termini demonstrated that the functionally important C-terminal domain was responsible for homomultimer formation, and the N-terminal domain enhances multimerization. Furthermore, it was found that the TATA sequence dissociates homomultimers, and only monomeric TBP binds to the TATA-box. We suggest that TBP shares structural motifs in the C-terminal conserved domain for intermolecular interaction and TATA-binding.

  1. Conformational control of the binding of the transactivation domain of the MLL protein and c-Myb to the KIX domain of CREB.

    Directory of Open Access Journals (Sweden)

    Elif Nihal Korkmaz

    Full Text Available The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL transcription factor lead to the biologically active ternary MLL∶KIX∶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD simulations for the MLL∶KIX∶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX∶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX∶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events.

  2. Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Gao, Xiaoli [Department of Biochemistry and Molecular Biology, Michigan State University (United States); Michael Garavito, R., E-mail: garavito@msu.edu [Department of Biochemistry and Molecular Biology, Michigan State University (United States)

    2011-04-22

    Highlights: {yields} Crystal structure of the intracellular domain of (pro)renin receptor (PRR-IC) as MBP fusion protein at 2.0 A (maltose-free) and 2.15 A (maltose-bound). {yields} MBP fusion protein is a dimer in crystals in the presence and absence of maltose. {yields} PRR-IC domain is responsible for the dimerization of the fusion protein. {yields} Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermolecular interactions, suggesting a role for the PRR-IC domain in PRR dimerization. -- Abstract: The (pro)renin receptor (PRR) is an important component of the renin-angiotensin system (RAS), which regulates blood pressure and cardiovascular function. The integral membrane protein PRR contains a large extracellular domain ({approx}310 amino acids), a single transmembrane domain ({approx}20 amino acids) and an intracellular domain ({approx}19 amino acids). Although short, the intracellular (IC) domain of the PRR has functionally important roles in a number of signal transduction pathways activated by (pro)renin binding. Meanwhile, together with the transmembrane domain and a small portion of the extracellular domain ({approx}30 amino acids), the IC domain is also involved in assembly of V{sub 0} portion of the vacuolar proton-translocating ATPase (V-ATPase). To better understand structural and multifunctional roles of the PRR-IC, we report the crystal structure of the PRR-IC domain as maltose-binding protein (MBP) fusion proteins at 2.0 A (maltose-free) and 2.15 A (maltose-bound). In the two separate crystal forms having significantly different unit-cell dimensions and molecular packing, MBP-PRR-IC fusion protein was found to be a dimer, which is different with the natural monomer of native MBP. The PRR-IC domain appears as a relatively flexible loop and is responsible for the dimerization of MBP fusion protein. Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermonomer interactions, suggesting a role for the PRR

  3. The NEAT Domain-Containing Proteins of Clostridium perfringens Bind Heme.

    Science.gov (United States)

    Choo, Jocelyn M; Cheung, Jackie K; Wisniewski, Jessica A; Steer, David L; Bulach, Dieter M; Hiscox, Thomas J; Chakravorty, Anjana; Smith, A Ian; Gell, David A; Rood, Julian I; Awad, Milena M

    2016-01-01

    The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on C. perfringens gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for in vitro growth. Analysis of the C. perfringens strain 13 genome sequence identified a putative heme acquisition system encoded by an iron-regulated surface gene region that we have named the Cht (Clostridium perfringens heme transport) locus. This locus comprises eight genes that are co-transcribed and includes genes that encode NEAT domain-containing proteins (ChtD and ChtE) and a putative sortase (Srt). The ChtD, ChtE and Srt proteins were shown to be expressed in JIR325 cells grown under iron-limited conditions and were localized to the cell envelope. Moreover, the NEAT proteins, ChtD and ChtE, were found to bind heme. Both chtDE and srt mutants were constructed, but these mutants were not defective in hemoglobin or ferric chloride utilization. They were, however, attenuated for virulence when tested in a mouse myonecrosis model, although the virulence phenotype could not be restored via complementation and, as is common with such systems, secondary mutations were identified in these strains. In summary, this study provides evidence for the functional redundancies that occur in the heme transport pathways of this life threatening pathogen.

  4. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Keryer-Bibens, Cecile, E-mail: cecile.keryer-bibens@univ-rennes1.fr [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Legagneux, Vincent; Namanda-Vanderbeken, Allen [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Cosson, Bertrand [UPMC Universite de Paris 06, UMR 7150, Equipe Traduction Cycle Cellulaire et Developpement, Station Biologique de Roscoff, 29682 Roscoff (France); CNRS, UMR 7150, Station Biologique de Roscoff, 29682 Roscoff (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Paillard, Luc [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France); Poncet, Didier [Virologie Moleculaire et Structurale, UMR CNRS, 2472, INRA, 1157, 91198 Gif sur Yvette (France); Osborne, H. Beverley, E-mail: beverley.osborne@univ-rennes1.fr [Universite de Rennes 1, IFR 140, Institut de Genetique et Developpement de Rennes, 35000 Rennes (France); CNRS, UMR 6061, equipe Expression Genetique et Developpement, 35000 Rennes (France); Universite Europeenne de Bretagne, 35000 Rennes (France)

    2009-12-11

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

  5. AtMBD6, a methyl CpG binding domain protein, maintains gene ...

    Indian Academy of Sciences (India)

    DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome fromtransposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG bindingdomain proteins are members of a class of proteins that bind tomethylated ...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-02-15

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

  7. AtMBD6, a methyl CpG binding domain protein, maintains gene ...

    Indian Academy of Sciences (India)

    ... targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved inRNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 alsointeracts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM ...

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been...... of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite...

  9. Evaluation of Cu(i) binding to the E2 domain of the amyloid precursor protein - a lesson in quantification of metal binding to proteins via ligand competition.

    Science.gov (United States)

    Young, Tessa R; Wedd, Anthony G; Xiao, Zhiguang

    2018-01-24

    The extracellular domain E2 of the amyloid precursor protein (APP) features a His-rich metal-binding site (denoted as the M1 site). In conjunction with surrounding basic residues, the site participates in interactions with components of the extracellular matrix including heparins, a class of negatively charged polysaccharide molecules of varying length. This work studied the chemistry of Cu(i) binding to APP E2 with the probe ligands Bcs, Bca, Fz and Fs. APP E2 forms a stable Cu(i)-mediated ternary complex with each of these anionic ligands. The complex with Bca was selected for isolation and characterization and was demonstrated, by native ESI-MS analysis, to have the stoichiometry E2 : Cu(i) : Bca = 1 : 1 : 1. Formation of these ternary complexes is specific for the APP E2 domain and requires Cu(i) coordination to the M1 site. Mutation of the M1 site was consistent with the His ligands being part of the E2 ligand set. It is likely that interactions between the negatively charged probe ligands and a positively charged patch on the surface of APP E2 are one aspect of the generation of the stable ternary complexes. Their formation prevented meaningful quantification of the affinity of Cu(i) binding to the M1 site with these probe ligands. However, the ternary complexes are disrupted by heparin, allowing reliable determination of a picomolar Cu(i) affinity for the E2/heparin complex with the Fz or Bca probe ligands. This is the first documented example of the formation of stable ternary complexes between a Cu(i) binding protein and a probe ligand. The ready disruption of the complexes by heparin identified clear 'tell-tale' signs for diagnosis of ternary complex formation and allowed a systematic review of conditions and criteria for reliable determination of affinities for metal binding via ligand competition. This study also provides new insights into a potential correlation of APP functions regulated by copper binding and heparin interaction.

  10. AtMBD6, a methyl CpG binding domain protein, maintains gene ...

    Indian Academy of Sciences (India)

    2017-01-13

    , Johnson CA, Laherty CD, Turner BM, Eisenman. RN and Bird A 1998 Transcriptional repression by the methyl-. CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393 386-389. Pendle AF, Clark ...

  11. Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters.

    Science.gov (United States)

    Ziegler, André; Seelig, Joachim

    2004-01-01

    The positively charged protein transduction domain of the HIV-1 TAT protein (TAT-PTD; residues 47-57 of TAT) rapidly translocates across the plasma membrane of living cells. This property is exploited for the delivery of proteins, drugs, and genes into cells. The mechanism of this translocation is, however, not yet understood. Recent theories for translocation suggest binding of the protein transduction domain (PTD) to extracellular glycosaminoglycans as a possible mechanism. We have studied the binding equilibrium between TAT-PTD and three different glycosaminoglycans with high sensitivity isothermal titration calorimetry and provide the first quantitative thermodynamic description. The polysulfonated macromolecules were found to exhibit multiple identical binding sites for TAT-PTD with only small differences between the three species as far as the thermodynamic parameters are concerned. Heparan sulfate (HS, molecular weight, 14.2 +/- 2 kDa) has 6.3 +/- 1.0 independent binding sites for TAT-PTD which are characterized by a binding constant K0 = (6.0 +/- 0.6) x 10(5) M(-1) and a reaction enthalpy deltaHpep0 = -4.6 +/- 1.0 kcal/mol at 28 degrees C. The binding affinity, deltaGpep0, is determined to equal extent by enthalpic and entropic contributions. The HS-TAT-PTD complex formation entails a positive heat capacity change of deltaCp0 = +135 cal/mol peptide, which is characteristic of a charge neutralization reaction. This is in contrast to hydrophobic binding reactions which display a large negative heat capacity change. The stoichiometry of 6-7 TAT-PTD molecules per HS corresponds to an electric charge neutralization. Light scattering data demonstrate a maximum scattering intensity at this stoichiometric ratio, the intensity of which depends on the order of mixing of the two components. The data suggest cross-linking and/or aggregation of HS-TAT-PTD complexes. Two other glycosaminoglycans, namely heparin and chondroitin sulfate B, were also studied with isothermal

  12. Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

    International Nuclear Information System (INIS)

    Delorme, Caroline; Joshi, Monika; Allingham, John S.

    2012-01-01

    Highlights: ► The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. ► The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. ► The MBP–Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance, we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the “ATP state” of the mechanochemical cycle. This site differs from the Kar3 neck–core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.

  13. In-depth study of DNA binding of Cys2His2 finger domains in testis zinc-finger protein.

    Directory of Open Access Journals (Sweden)

    Chun-Chi Chou

    Full Text Available Previously, we identified that both fingers 1 and 2 in the three Cys2His2 zinc-finger domains (TZD of testis zinc-finger protein specifically bind to its cognate DNA; however, finger 3 is non-sequence-specific. To gain insights into the interaction mechanism, here we further investigated the DNA-binding characteristics of TZD bound to non-specific DNAs and its finger segments bound to cognate DNA. TZD in non-specific DNA binding showed smaller chemical shift perturbations, as expected. However, the direction of shift perturbation, change of DNA imino-proton NMR signal, and dynamics on the 15N backbone atom significantly differed between specific and non-specific binding. Using these unique characteristics, we confirmed that the three single-finger segments (TZD1, TZD2 and TZD3 and the two-finger segment (TZD23 non-specifically bind to the cognate DNA. In comparison, the other two-finger segment (TZD12 binding to the cognate DNA features simultaneous non-specific and semi-specific binding, both slowly exchanged in terms of NMR timescale. The process of TZD binding to the cognate DNA is likely stepwise: initially TZD non-specifically binds to DNA, then fingers 1 and 2 insert cooperatively into the major groove of DNA by semi-specific binding, and finally finger 3 non-specifically binds to DNA, which promotes the specific binding on fingers 1 and 2 and stabilizes the formation of a specific TZD-DNA complex.

  14. FRKAS: knowledge acquisition using a fuzzy rule base approach to insight of DNA-binding domains/proteins.

    Science.gov (United States)

    Huang, Hui-Ling; Chang, Fang-Lin; Ho, Shinn-Jang; Shu, Li-Sun; Huang, Wen-Lin; Ho, Shinn-Ying

    2013-03-01

    Numerous prediction methods of DNA-binding domains/proteins were proposed by identifying informative features and designing effective classifiers. These researches reveal that the DNA-protein binding mechanism is complicated and existing accurate predictors such as support vector machine (SVM) with position specific scoring matrices (PSSMs) are regarded as black-box methods which are not easily interpretable for biologists. In this study, we propose an ensemble fuzzy rule base classifier consisting of a set of interpretable fuzzy rule classifiers (iFRCs) using informative physicochemical properties as features. In designing iFRCs, feature selection, membership function design, and fuzzy rule base generation are all simultaneously optimized using an intelligent genetic algorithm (IGA). IGA maximizes prediction accuracy, minimizes the number of features selected, and minimizes the number of fuzzy rules to generate an accurate and concise fuzzy rule base. Benchmark datasets of DNA-binding domains are used to evaluate the proposed ensemble classifier of 30 iFRCs. Each iFRC has a mean test accuracy of 77.46%, and the ensemble classifier has a test accuracy of 83.33%, where the method of SVM with PSSMs has the accuracy of 82.81%. The physicochemical properties of the first two ranks according to their contribution are positive charge and Van Der Waals volume. Charge complementarity between protein and DNA is thought to be important in the first step of recognition between protein and DNA. The amino acid residues of binding peptides have larger Van Der Waals volumes and positive charges than those of non-binding ones. The proposed knowledge acquisition method by establishing a fuzzy rule-based classifier can also be applicable to predict and analyze other protein functions from sequences.

  15. The MAM (meprin/A5-protein/PTPmu) domain is a homophilic binding site promoting the lateral dimerization of receptor-like protein-tyrosine phosphatase mu.

    Science.gov (United States)

    Cismasiu, Valeriu B; Denes, Stefan A; Reiländer, Helmut; Michel, Hartmut; Szedlacsek, Stefan E

    2004-06-25

    The MAM (meprin/A5-protein/PTPmu) domain is present in numerous proteins with diverse functions. PTPmu belongs to the MAM-containing subclass of protein-tyrosine phosphatases (PTP) able to promote cell-to-cell adhesion. Here we provide experimental evidence that the MAM domain is a homophilic binding site of PTPmu. We demonstrate that the MAM domain forms oligomers in solution and binds to the PTPmu ectodomain at the cell surface. The presence of two disulfide bridges in the MAM molecule was evidenced and their integrity was found to be essential for MAM homophilic interaction. Our data also indicate that PTPmu ectodomain forms oligomers and mediates the cellular adhesion, even in the absence of MAM domain homophilic binding. Reciprocally, MAM is able to interact homophilically in the absence of ectodomain trans binding. The MAM domain therefore contains independent cis and trans interaction sites and we predict that its main role is to promote lateral dimerization of PTPmu at the cell surface. This finding contributes to the understanding of the signal transduction mechanism in MAM-containing PTPs.

  16. Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding

    Science.gov (United States)

    Shi, Yunhua; Mowery, Richard A; Ashley, Jonathan; Hentz, Michelle; Ramirez, Alejandro J; Bilgicer, Basar; Slunt-Brown, Hilda; Borchelt, David R; Shaw, Bryan F

    2012-01-01

    The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of “gel shifting” has perplexed biochemists and biologists for decades. We identify a mechanism for “gel shifting” that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS-PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues ∼80–101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80–101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS-PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1-SDS complexes. The high negative charge of domain 80–101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse-human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic). PMID:22692797

  17. De novo design of RNA-binding proteins with a prion-like domain related to ALS/FTD proteinopathies.

    Science.gov (United States)

    Mitsuhashi, Kana; Ito, Daisuke; Mashima, Kyoko; Oyama, Munenori; Takahashi, Shinichi; Suzuki, Norihiro

    2017-12-04

    Aberrant RNA-binding proteins form the core of the neurodegeneration cascade in spectrums of disease, such as amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Six ALS-related molecules, TDP-43, FUS, TAF15, EWSR1, heterogeneous nuclear (hn)RNPA1 and hnRNPA2 are RNA-binding proteins containing candidate mutations identified in ALS patients and those share several common features, including harboring an aggregation-prone prion-like domain (PrLD) containing a glycine/serine-tyrosine-glycine/serine (G/S-Y-G/S)-motif-enriched low-complexity sequence and rich in glutamine and/or asparagine. Additinally, these six molecules are components of RNA granules involved in RNA quality control and become mislocated from the nucleus to form cytoplasmic inclusion bodies (IBs) in the ALS/FTD-affected brain. To reveal the essential mechanisms involved in ALS/FTD-related cytotoxicity associated with RNA-binding proteins containing PrLDs, we designed artificial RNA-binding proteins harboring G/S-Y-G/S-motif repeats with and without enriched glutamine residues and nuclear-import/export-signal sequences and examined their cytotoxicity in vitro. These proteins recapitulated features of ALS-linked molecules, including insoluble aggregation, formation of cytoplasmic IBs and components of RNA granules, and cytotoxicity instigation. These findings indicated that these artificial RNA-binding proteins mimicked features of ALS-linked molecules and allowed the study of mechanisms associated with gain of toxic functions related to ALS/FTD pathogenesis.

  18. Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev

    Science.gov (United States)

    Cooper, Christopher D. O.; Newman, Joseph A.; Aitkenhead, Hazel; Allerston, Charles K.; Gileadi, Opher

    2015-01-01

    Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40–200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors. PMID:25866208

  19. Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

    Energy Technology Data Exchange (ETDEWEB)

    Dahms, Sven O., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Mayer, Magnus C. [Freie Universität Berlin, Thielallee 63, 14195 Berlin (Germany); Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow (Germany); Roeser, Dirk [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Multhaup, Gerd [McGill University Montreal, Montreal, Quebec H3G 1Y6 (Canada); Than, Manuel E., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany)

    2015-03-01

    Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.

  20. Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein

    DEFF Research Database (Denmark)

    Richardson, Roy; Denis, Clyde L; Zhang, Chongxu

    2012-01-01

    Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from...... Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins...

  1. Maturation and activity of sterol regulatory element binding protein 1 is inhibited by acyl-CoA binding domain containing 3.

    Directory of Open Access Journals (Sweden)

    Yong Chen

    Full Text Available Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3, a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN. Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis.

  2. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Goupille, Olivier [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Penglong, Tipparat [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Thalassemia Research Center, Mahidol University (Thailand); Kadri, Zahra; Granger-Locatelli, Marine [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Fucharoen, Suthat [Thalassemia Research Center, Mahidol University (Thailand); Maouche-Chrétien, Leila [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); INSERM, Paris (France); Prost, Stéphane [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Leboulch, Philippe [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Thalassemia Research Center, Mahidol University (Thailand); Chrétien, Stany, E-mail: stany.chretien@cea.fr [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); INSERM, Paris (France)

    2016-04-15

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. - Highlights: • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into white adipocytes. • JQ1 affected clonal cell expansion and abolished lipid accumulation. • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into brown adipocytes. • JQ1 treatment did not lead to the conversion of white adipose tissue into brown adipose tissue. • JQ1 decreased STAT5 expression, but STAT5B{sup ca} expression did not restore adipogenesis.

  3. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis

    International Nuclear Information System (INIS)

    Goupille, Olivier; Penglong, Tipparat; Kadri, Zahra; Granger-Locatelli, Marine; Fucharoen, Suthat; Maouche-Chrétien, Leila; Prost, Stéphane; Leboulch, Philippe; Chrétien, Stany

    2016-01-01

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. - Highlights: • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into white adipocytes. • JQ1 affected clonal cell expansion and abolished lipid accumulation. • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into brown adipocytes. • JQ1 treatment did not lead to the conversion of white adipose tissue into brown adipose tissue. • JQ1 decreased STAT5 expression, but STAT5B ca expression did not restore adipogenesis.

  4. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Parent, Kristin N., E-mail: kparent@msu.edu [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Tang, Jinghua; Cardone, Giovanni [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Gilcrease, Eddie B. [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Janssen, Mandy E.; Olson, Norman H. [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Casjens, Sherwood R., E-mail: sherwood.casjens@path.utah.edu [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Baker, Timothy S., E-mail: tsb@ucsd.edu [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); University of California, San Diego, Division of Biological Sciences, La Jolla, CA, 92093 (United States)

    2014-09-15

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer.

  5. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    International Nuclear Information System (INIS)

    Parent, Kristin N.; Tang, Jinghua; Cardone, Giovanni; Gilcrease, Eddie B.; Janssen, Mandy E.; Olson, Norman H.; Casjens, Sherwood R.; Baker, Timothy S.

    2014-01-01

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer

  6. Glutamine rich and basic region/leucine zipper (bZIP) domains stabilize cAMP-response element-binding protein (CREB) binding to chromatin.

    Science.gov (United States)

    Mayr, Bernhard M; Guzman, Ernesto; Montminy, Marc

    2005-04-15

    We have examined the dynamics of cAMP-response element-binding protein (CREB) binding to chromatin in live cells using fluorescence recovery after photobleaching (FRAP). CREB was found to bind to target sites with a residence time of 100 s, and exposure to a cAMP agonist had no effect on these kinetics. In addition to the basic region/leucine zipper (bZIP) domain, a glutamine-rich trans-activation domain in CREB called Q2 also appeared to be critical for promoter occupancy. Indeed, mutations in Q2 that reduced residence time by FRAP assay disrupted target gene activation via CREB in cells exposed to a cAMP agonist. Notably, insertion of the glutamine-rich B trans-activation domain of SP1 into a mutant CREB polypeptide lacking Q2 stabilized CREB occupancy and rescued target gene activation. These results suggest a novel mechanism by which the family of glutamine-rich activators promotes cellular gene expression.

  7. Enhancer-binding proteins with a forkhead-associated domain and the sigma(54) regulon in Myxococcus xanthus fruiting body development

    DEFF Research Database (Denmark)

    Jelsbak, Lars; Givskov, Michael Christian; Kaiser, D.

    2005-01-01

    In response to starvation, Myxococcus xanthus initiates a developmental program that results in the formation of spore-filled, multicellular fruiting bodies. Many developmentally regulated genes in M. xanthus are transcribed from sigma(54) promoters, and these genes require enhancer......-binding proteins. Here we report the finding of an unusual group of 12 genes encoding sigma(54)-dependent enhancer-binding proteins containing a forkhead-associated (FHA) domain as their N-terminal sensory domain. FHA domains in other proteins recognize phosphothreonine residues. An insertion mutation in one...

  8. Enhancer-binding proteins with a forkhead-associated domain and the sigma(54) regulon in Myxococcus xanthus fruiting body development

    DEFF Research Database (Denmark)

    Jelsbak, Lars; Givskov, Michael Christian; Kaiser, D.

    2005-01-01

    of these genes, Mx4885, caused a cell autonomous aggregation and sporulation defect. In-frame deletion mutants showed that the FHA domain is necessary for proper Mx4885 function. The altered pattern of developmental gene expression in the mutant implied that Mx4885 is on the pathway of response...... donor cell. Because FHA domains respond to phosphothreonine-containing proteins, these results suggest a regulatory link to the abundant Ser/Thr protein kinases in M. xanthus.......-binding proteins. Here we report the finding of an unusual group of 12 genes encoding sigma(54)-dependent enhancer-binding proteins containing a forkhead-associated (FHA) domain as their N-terminal sensory domain. FHA domains in other proteins recognize phosphothreonine residues. An insertion mutation in one...

  9. Solution structure of the Arabidopsis thaliana telomeric repeat-binding protein DNA binding domain: a new fold with an additional C-terminal helix.

    Science.gov (United States)

    Sue, Shih-Che; Hsiao, Hsin-Hao; Chung, Ben C-P; Cheng, Ying-Hsien; Hsueh, Kuang-Lung; Chen, Chung Mong; Ho, Chia Hsing; Huang, Tai-Huang

    2006-02-10

    The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1(464-560)), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1(464-560) binds to a 13-mer DNA duplex containing a single repeat of an A.thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K(D) approximately 10(-6)-10(-7) M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1(464-560) is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1(464-560) induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs.

  10. Identification of pseudomurein cell wall binding domains.

    Science.gov (United States)

    Steenbakkers, Peter J M; Geerts, Wim J; Ayman-Oz, Nilgün A; Keltjens, Jan T

    2006-12-01

    Methanothermobacter thermautotrophicus is a methanogenic Gram-positive microorganism with a cell wall consisting of pseudomurein. Currently, no information is available on extracellular pseudomurein biology and so far only two prophage pseudomurein autolysins, PeiW and PeiP, have been reported. In this paper we show that PeiW and PeiP contain two different N-terminal pseudomurein cell wall binding domains. This finding was used to identify a novel domain, PB007923, on the M. thermautotrophicus genome present in 10 predicted open reading frames. Three homologues were identified in the Methanosphaera stadtmanae genome. Binding studies of fusion constructs of three separate PB007923 domains to green fluorescent protein revealed that it also constituted a cell wall binding domain. Both prophage domains and the PB007923 domain bound to the cell walls of Methanothermobacter species and fluorescence microscopy showed a preference for the septal region. Domain specificities were revealed by binding studies with other pseudomurein-containing archaea. Localized binding was observed for M. stadtmanae and Methanobrevibacter species, while others stained evenly. The identification of the first pseudomurein cell wall binding domains reveals the dynamics of the pseudomurein cell wall and provides marker proteins to study the extracellular pseudomurein biology of M. thermautotrophicus and of other pseudomurein-containing archaea.

  11. Inhibition of tumor metastasis by a growth factor receptor bound protein 2 Src homology 2 domain-binding antagonist.

    Science.gov (United States)

    Giubellino, Alessio; Gao, Yang; Lee, Sunmin; Lee, Min-Jung; Vasselli, James R; Medepalli, Sampath; Trepel, Jane B; Burke, Terrence R; Bottaro, Donald P

    2007-07-01

    Metastasis, the primary cause of death in most forms of cancer, is a multistep process whereby cells from the primary tumor spread systemically and colonize distant new sites. Blocking critical steps in this process could potentially inhibit tumor metastasis and dramatically improve cancer survival rates; however, our understanding of metastasis at the molecular level is still rudimentary. Growth factor receptor binding protein 2 (Grb2) is a widely expressed adapter protein with roles in epithelial cell growth and morphogenesis, as well as angiogenesis, making it a logical target for anticancer drug development. We have previously shown that a potent antagonist of Grb2 Src homology-2 domain-binding, C90, blocks growth factor-driven cell motility in vitro and angiogenesis in vivo. We now report that C90 inhibits metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. These results support the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establish a critical role for Grb2 Src homology-2 domain-mediated interactions in this process.

  12. Mapping of a Microbial Protein Domain Involved in Binding and Activation of the TLR2/TLR1 Heterodimer 1

    Science.gov (United States)

    Liang, Shuang; Hosur, Kavita B.; Lu, Shanyun; Nawar, Hesham F.; Weber, Benjamin R.; Tapping, Richard I.; Connell, Terry D.; Hajishengallis, George

    2009-01-01

    LT-IIb-B5, a doughnut-shaped oligomeric protein from enterotoxigenic Escherichia coli, is known to activate the TLR2/TLR1 heterodimer (TLR2/1). We investigated the molecular basis of the LT-IIb-B5 interaction with TLR2/1 in order to define the structure-function relationship of LT-IIb-B5 and, moreover, to gain an insight into how TLR2/1 recognizes large, non-acylated protein ligands that cannot fit within its lipid-binding pockets, as previously shown for the Pam3CSK4 lipopeptide. We first identified four critical residues in the upper region of the LT-IIb-B5 pore: Corresponding point mutants (M69E, A70D, L73E, S74D) were defective in binding TLR2 or TLR1 and could not activate antigen-presenting cells, despite retaining full ganglioside-binding capacity. Point mutations in the TLR2/1 dimer interface, as determined in the crystallographic structure of the TLR2/1-Pam3CSK4 complex, resulted in diminished activation by both Pam3CSK4 and LT-IIb-B5. Docking analysis of the LT-IIb-B5 interaction with this apparently “predominant” activation conformation of TLR2/1 revealed that LT-IIb-B5 may primarily contact the convex surface of the TLR2 central domain. Although the TLR1/LT-IIb-B5 interface is relatively smaller, the leucine-rich repeat motifs 9–12 in the central domain of TLR1 were found to be critical for cooperative TLR2-induced cell activation by LT-IIb-B5. Moreover, the putative LT-IIb-B5 binding site overlaps partially with that of Pam3CSK4; consistent with this, Pam3CSK4 suppressed TLR2 binding of LT-IIb-B5, albeit not as potently as self-competitive inhibition. In conclusion, we identified the upper pore region of LT-IIb-B5 as a TLR2/1 interactive domain, which contacts the heterodimeric receptor at a site that is distinct from, though overlaps with, that of Pam3CSK4. PMID:19234193

  13. Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8.

    Science.gov (United States)

    Jagannathan, Indu; Culver, Gloria M

    2003-07-04

    Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.

  14. Alcohol binding in the C1 (C1A + C1B) domain of protein kinase C epsilon

    Science.gov (United States)

    Pany, Satyabrata; Das, Joydip

    2015-01-01

    Background Alcohol regulates the expression and function of protein kinase C epsilon (PKCε). In a previous study we identified an alcohol binding site in the C1B, one of the twin C1 subdomains of PKCε. Methods In this study, we investigated alcohol binding in the entire C1 domain (combined C1A and C1B) of PKCε. Fluorescent phorbol ester, SAPD and fluorescent diacylglycerol (DAG) analog, dansyl-DAG were used to study the effect of ethanol, butanol, and octanol on the ligand binding using fluorescence resonance energy transfer (FRET). To identify alcohol binding site(s), PKCεC1 was photolabeled with 3-azibutanol and 3-azioctanol, and analyzed by mass spectrometry. The effects of alcohols and the azialcohols on PKCε were studied in NG108-15 cells. Results In the presence of alcohol, SAPD and dansyl-DAG showed different extent of FRET, indicating differential effects of alcohol on the C1A and C1B subdomains. Effects of alcohols and azialcohols on PKCε in NG108-15 cells were comparable. Azialcohols labeled Tyr-176 of C1A and Tyr-250 of C1B. Inspection of the model structure of PKCεC1 reveals that these residues are 40 Å apart from each other indicating that these residues form two different alcohol binding sites. Conclusions The present results provide evidence for the presence of multiple alcohol-binding sites on PKCε and underscore the importance of targeting this PKC isoform in developing alcohol antagonists. PMID:26210390

  15. Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains*

    Science.gov (United States)

    Barnes, Mark; van Rensburg, Gerrit; Li, Wai-Ming; Mehmood, Kashif; Mackedenski, Sebastian; Chan, Ching-Man; King, Dustin T.; Miller, Andrew L.; Lee, Chow H.

    2015-01-01

    The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function. PMID:25389298

  16. The C-terminal domain of the Arabidopsis AtMBD7 protein confers strong chromatin binding activity

    International Nuclear Information System (INIS)

    Zemach, Assaf; Paul, Laju K.; Stambolsky, Perry; Efroni, Idan; Rotter, Varda; Grafi, Gideon

    2009-01-01

    The Arabidopsis MBD7 (AtMBD7) - a naturally occurring poly MBD protein - was previously found to be functional in binding methylated-CpG dinucleotides in vitro and localized to highly methylated chromocenters in vivo. Furthermore, AtMBD7 has significantly lower mobility within the nucleus conferred by cooperative activity of its three MBD motifs. Here we show that besides the MBD motifs, AtMBD7 possesses a strong chromatin binding domain located at its C-terminus designated sticky-C (StkC). Mutational analysis showed that a glutamic acid residue near the C-terminus is essential though not sufficient for the StkC function. Further analysis demonstrated that this motif can render nuclear proteins highly immobile both in plant and animal cells, without affecting their native subnuclear localization. Thus, the C-terminal, StkC motif plays an important role in fastening AtMBD7 to its chromosomal, CpG-methylated sites. It may be possible to utilize this motif for fastening nuclear proteins to their chromosomal sites both in plant and animal cells for research and gene therapy applications.

  17. Protein-binding elements establish in the oocyte the primary imprint of the Prader-Willi/Angelman syndromes domain.

    Science.gov (United States)

    Kaufman, Yotam; Heled, Maya; Perk, Jonathan; Razin, Aharon; Shemer, Ruth

    2009-06-23

    Imprinting of the PWS/AS 2.4 Mb domain in the human is controlled by a paternally active imprinting center (PWS-IC). PWS-IC on the maternal allele is methylated and inactivated by an 880-bp sequence (AS-IC) located 30 kb upstream. In this communication, we report the identification of 7 cis acting elements within AS-IC. The elements: DMR, DNS, 2 OCTA sequences, SOX, E1, and E2 bind specific proteins that form at least 2 protein complexes. Using variants of an imprinted transgene, mutated at the elements each at a time, we show that (i) all 7 elements are involved in the methylation and inactivation of the maternal PWS-IC; (ii) the OCTA and SOX elements that bind a protein complex, and the E1 and E2 elements, function in establishing the primary imprint that constitutes an active and unmethylated AS-IC in the oocyte; (iii) DNS and DMR bind a multiprotein complex that may facilitate interaction between AS-IC and PWS-IC, mediating the inactivation in cis of PWS-IC; and (iv) all 7 elements participate in maintaining an unmethylated PWS-IC in the oocyte, which is essential for its maternal methylation later in development. Altogether, the above observations imply that the cis acting elements on AS-IC display diverse functions in establishing the imprints at both AS-IC and PWS-IC in the oocyte. A postulated epigenetic mark imprints the PWS-IC in the oocyte and maintains its inactive status during development before it is translated into maternal methylation.

  18. Genetic variability and natural selection at the ligand domain of the Duffy binding protein in Brazilian Plasmodium vivax populations.

    Science.gov (United States)

    Sousa, Taís N; Tarazona-Santos, Eduardo M; Wilson, Daniel J; Madureira, Ana P; Falcão, Paula R K; Fontes, Cor J F; Gil, Luiz H S; Ferreira, Marcelo U; Carvalho, Luzia H; Brito, Cristiana F A

    2010-11-22

    Plasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBPII), which is the most variable segment of the protein. To test whether diversifying natural selection has shaped the nucleotide diversity of PvDBPII in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBPII, and T- and B-cell epitopes were localized on the 3-D structure. The results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBPII, and (ii) PvDBPII appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium. This study shows that some polymorphisms of PvDBPII are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion.

  19. Expansion of protein domain repeats.

    Directory of Open Access Journals (Sweden)

    Asa K Björklund

    2006-08-01

    Full Text Available Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein-protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.

  20. A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis.

    Science.gov (United States)

    Karamysheva, Zemfira N; Surovtseva, Yulia V; Vespa, Laurent; Shakirov, Eugene V; Shippen, Dorothy E

    2004-11-12

    Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis.

  1. Loss of sialic acid binding domain redirects protein σ1 to enhance M cell-directed vaccination.

    Directory of Open Access Journals (Sweden)

    Dagmara Zlotkowska

    Full Text Available Ovalbumin (OVA genetically fused to protein sigma 1 (pσ1 results in tolerance to both OVA and pσ1. Pσ1 binds in a multi-step fashion, involving both protein- and carbohydrate-based receptors. To assess the relative pσ1 components responsible for inducing tolerance and the importance of its sialic binding domain (SABD for immunization, modified OVA-pσ1, termed OVA-pσ1(short, was deleted of its SABD, but with its M cell targeting moiety intact, and was found to be immunostimulatory and enhanced CD4(+ and CD8(+ T cell proliferation. When used to nasally immunize mice given with and without cholera toxin (CT adjuvant, elevated SIgA and serum IgG responses were induced, and OVA-pσ1(s was more efficient for immunization than native OVA+CT. The immune antibodies (Abs were derived from elevated Ab-forming cells in the upper respiratory tissues and submaxillary glands and were supported by mixed Th cell responses. Thus, these studies show that pσ1(s can be fused to vaccines to effectively elicit improved SIgA responses.

  2. FhCaBP1 (FH22): A Fasciola hepatica calcium-binding protein with EF-hand and dynein light chain domains.

    Science.gov (United States)

    Cheung, Sarah; Thomas, Charlotte M; Timson, David J

    2016-11-01

    FH22 has been previously identified as a calcium-binding protein from the common liver fluke, Fasciola hepatica. It is part of a family of at least four proteins in this organism which combine an EF-hand containing N-terminal domain with a C-terminal dynein light chain-like domain. Here we report further biochemical properties of FH22, which we propose should be renamed FhCaBP1 for consistency with other family members. Molecular modelling predicted that the two domains are linked by a flexible region and that the second EF-hand in the N-terminal domain is most likely the calcium ion binding site. Native gel electrophoresis demonstrated that the protein binds both calcium and manganese ions, but not cadmium, magnesium, strontium, barium, cobalt, copper(II), iron (II), nickel, zinc, lead or potassium ions. Calcium ion binding alters the conformation of the protein and increases its stability towards thermal denaturation. FhCaBP1 is a dimer in solution and calcium ions have no detectable effect on the protein's ability to dimerise. FhCaBP1 binds to the calmodulin antagonists trifluoperazine and chlorpromazine. Overall, the FhCaBP1's biochemical properties are most similar to FhCaBP2 a fact consistent with the close sequence and predicted structural similarity between the two proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Phosphorylation of the PCNA binding domain of the large subunit of replication factor C by Ca2+/calmodulin-dependent protein kinase II inhibits DNA synthesis

    DEFF Research Database (Denmark)

    Maga, G; Mossi, R; Fischer, R

    1997-01-01

    Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and DNA repair. It is a molecular matchmaker required for loading of the proliferating cell nuclear antigen (PCNA) sliding clamp onto double-strand DNA and for PCNA-dependent DNA synthesis by DNA polymerases...... delta and epsilon. The DNA and PCNA binding domains of the large 140 kDa subunit of human RF-C have been recently cloned [Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messier, H., Khastilba. S., Hübscher, U., & Fotedar, A. (1996) EMBO J. 15, 4423-4433]. Here we show...... that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF-C...

  4. FhCaBP2: a Fasciola hepatica calcium-binding protein with EF-hand and dynein light chain domains.

    Science.gov (United States)

    Thomas, Charlotte M; Timson, David J

    2015-09-01

    FhCaBP2 is a Fasciola hepatica protein which belongs to a family of helminth calcium-binding proteins which combine an N-terminal domain containing two EF-hand motifs and a C-terminal dynein light chain-like (DLC-like) domain. Its predicted structure showed two globular domains joined by a flexible linker. Recombinant FhCaBP2 interacted reversibly with calcium and manganese ions, but not with magnesium, barium, strontium, copper (II), colbalt (II), iron (II), nickel, lead or potassium ions. Cadmium (II) ions appeared to bind non-site-specifically and destabilize the protein. Interaction with either calcium or magnesium ions results in a conformational change in which the protein's surface becomes more hydrophobic. The EF-hand domain alone was able to interact with calcium and manganese ions; the DLC-like domain was not. Alteration of a residue (Asp-58 to Ala) in the second EF-hand motif in this domain abolished ion-binding activity. This suggests that the second EF-hand is the one responsible for ion-binding. FhCaBP2 homodimerizes and the extent of dimerization was not affected by calcium ions or by the aspartate to alanine substitution in the second EF-hand. The isolated EF-hand and DLC-like domains are both capable of homodimerization. FhCaBP2 interacted with the calmodulin antagonists trifluoperazine, chlorpromazine, thiamylal and W7. Interestingly, while chlorpromazine and thiamylal interacted with the EF-hand domain (as expected), trifluoperazine and W7 bound to the DLC-like domain. Overall, FhCaBP2 has distinct biochemical properties compared with other members of this protein family from Fasciola hepatica, a fact which supports the hypothesis that these proteins have different physiological roles.

  5. The expanded octarepeat domain selectively binds prions and disrupts homomeric prion protein interactions

    NARCIS (Netherlands)

    Leliveld, S. R.; Dame, R.T.; Wuite, G.J.L.; Stitz, L.; Korth, C.

    2006-01-01

    Insertion of additional octarepeats into the prion protein gene has been genetically linked to familial Creutzfeldt Jakob disease and hence to de novo generation of infectious prions. The pivotal event during prion formation is the conversion of the normal prion protein (PrP

  6. The F-Actin Binding Protein Cortactin Regulates the Dynamics of the Exocytotic Fusion Pore through its SH3 Domain

    Science.gov (United States)

    González-Jamett, Arlek M.; Guerra, María J.; Olivares, María J.; Haro-Acuña, Valentina; Baéz-Matus, Ximena; Vásquez-Navarrete, Jacqueline; Momboisse, Fanny; Martinez-Quiles, Narcisa; Cárdenas, Ana M.

    2017-01-01

    Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells. PMID:28522963

  7. The nature of the hydrophobic n-alkanol binding site within the C1 domains of protein kinase Calpha.

    Science.gov (United States)

    Slater, Simon J; Malinowski, Steve A; Stubbs, Christopher D

    2004-06-15

    The activator-binding sites within the C1 domains of protein kinase C (PKC) are also able to bind alcohols and anesthetics. In this study, the nature of the interaction of these agents with the hydrophobic region within the C1 domains was investigated and a structure-activity relationship for the alcohol effects was obtained. The effects of a series of n-alkanols on PKCalpha activity, determined using an in vitro assay system that lacked lipids, were found to be a nonlinear function of the chain length. In the absence of phorbol ester or diacylglycerol, 1-octanol potently activated PKCalpha in a concentration-dependent manner, while 1-heptanol was completely without effect, despite differing by one methylene unit. The minimal structural requirement for the activating effect corresponded to R-CH(OH)-(CH(2))(n)-CH(3), where R = H or an alkyl group and n >or= 6. Consistent with this, 2-octanol, for which n = 5, was without effect on the activity, even though this alcohol is only marginally less hydrophobic than 1-octanol, whereas 2-nonanol, for which n = 6, was able to produce activity. Importantly, it was found that PKCalpha was activated to a greater extent by R-2-nonanol than by the S enantiomer. The potentiation of phorbol ester-induced, membrane-associated PKCalpha activity by long-chain n-alkanols reported previously (Slater, S. J., Kelly, M. B., Larkin, J. D., Ho, C, Mazurek, A, Taddeo, F. J., Yeager, M. D., Stubbs, C. D. (1997) J. Biol. Chem. 272, 6167-6173), was also found here for nonmembrane associated PKC, indicating that this effect is an intrinsic property of the enzyme rather than a result of membrane perturbation. Overall, the results suggest that the alcohol-binding sites within the C1 domains of PKCalpha contain spatially distinct hydrophilic and hydrophobic regions that impose a high degree of structural specificity on the interactions of alcohols and other anesthetic compounds, as well as diacylglycerols and phorbol esters.

  8. Structural Analysis of a Complex between Small Ubiquitin-like Modifier 1 (SUMO1) and the ZZ Domain of CREB-binding Protein (CBP/p300) Reveals a New Interaction Surface on SUMO

    DEFF Research Database (Denmark)

    Diehl, Carl; Akke, Mikael; Bekker-Jensen, Simon

    2016-01-01

    We have recently discovered that the ZZ zinc finger domain represents a novel small ubiquitin-like modifier (SUMO) binding motif. In this study we identify the binding epitopes in the ZZ domain of CBP (CREB-binding protein) and SUMO1 using NMR spectroscopy. The binding site on SUMO1 represents a ...

  9. Abnormal swelling of the peritrophic membrane in Eri silkworm gut caused by MLX56 family defense proteins with chitin-binding and extensin domains.

    Science.gov (United States)

    Konno, Kotaro; Shimura, Sachiko; Ueno, Chihiro; Arakawa, Toru; Nakamura, Masatoshi

    2018-03-01

    MLX56 family defense proteins, MLX56 and its close homolog LA-b, are chitin-binding defense proteins found in mulberry latex that show strong growth-inhibitions against caterpillars when fed at concentrations as low as 0.01%. MLX56 family proteins contain a unique structure with an extensin domain surrounded by two hevein-like chitin-binding domains, but their defensive modes of action remain unclear. Here, we analyzed the effects of MLX56 family proteins on the peritrophic membrane (PM), a thin and soft membrane consisting of chitin that lines the midgut lumen of insects. We observed an abnormally thick (>1/5 the diameter of midgut) hard gel-like membrane consisted of chitin and MLX56 family proteins, MLX56 and LA-b, in the midgut of the Eri silkworms, Samia ricini, fed a diet containing MLX56 family proteins, MLX56 and LA-b. When polyoxin AL, a chitin-synthesis-inhibitor, was added to the diet containing MLX56 family proteins, the toxicity of MLX56 family proteins disappeared and PM became thinner and fragmented. These results suggest that MLX56 family proteins, through their chitin-binding domains, bind to the chitin framework of PM, then through their extensin-domain (gum arabic-like structure), which functions as swelling agent, expands PM into an abnormally thick membrane that inhibits the growth of insects. This study shows that MLX56 family proteins are plant defense lectins with a totally unique mode of action, and reveals the functions of extensin domains and arabinogalactan proteins as swelling (gel-forming) agents of plants. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes.

    Science.gov (United States)

    Maurer, Lisa M; Tomasini-Johansson, Bianca R; Ma, Wenjiang; Annis, Douglas S; Eickstaedt, Nathan L; Ensenberger, Martin G; Satyshur, Kenneth A; Mosher, Deane F

    2010-12-24

    The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with K(d) values of 5.2 and 59 nM to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the (2)FNI module had little effect, whereas mAb 7D5 to the (4)FNI module in the fibrin-binding region, 5C3 to the (9)FNI module in the gelatin-binding region, or L8 to the G-strand of (1)FNIII module adjacent to (9)FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by β-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to (2)FNI-(5)FNI of the fibrin-binding domain and (8)FNI-(9)FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel β-zipper in which FUD interacts with the E-strands of (2)FNI-(5)FNI and (8)FNI-(9)FNI.

  11. Production of recombinant PvDBPII, receptor binding domain of Plasmodium vivax Duffy binding protein, and evaluation of immunogenicity to identify an adjuvant formulation for vaccine development.

    Science.gov (United States)

    Bhardwaj, Rukmini; Shakri, Ahmad Rushdi; Hans, Dhiraj; Gupta, Pankaj; Fernandez-Becerra, Carmen; Del Portillo, Hernando A; Pandey, Gaurav; Chitnis, Chetan E

    2017-08-01

    Plasmodium vivax is dependent on interaction with the Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes. The P. vivax Duffy binding protein (PvDBP) mediates interaction of P. vivax merozoites with DARC. The DARC receptor-binding domain lies in a conserved N-terminal cysteine-rich region of PvDBP referred to as region II (PvDBPII). PvDBPII is an attractive vaccine candidate since antibodies raised against PvDBPII block erythrocyte invasion by P. vivax. Here, we describe methods to produce recombinant PvDBPII in its correctly folded conformation. A synthetic gene optimized for expression of PvDBPII in Escherichia coli and fed batch fermentation process based on exponential feeding strategy was used to achieve high levels of expression of recombinant PvDBPII. Recombinant PvDBPII was isolated from inclusion bodies, refolded by rapid dilution and purified by ion exchange chromatography. Purified recombinant PvDBPII was characterized for identity, purity and functional activity using standardized release assays. Recombinant PvDBPII formulated with various human compatible adjuvants including glycosylpyranosyl lipid A-stable emulsion (GLA-SE) and alhydrogel was used for immunogenicity studies in small animals to downselect a suitable formulation for clinical development. Sera collected from immunized animals were tested for recognition of PvDBPII and inhibition of PvDBPII-DARC binding. GLA-SE formulations of PvDBPII yielded higher ELISA and binding inhibition titres compared to PvDBPII formulated with alhydrogel. These data support further development of a recombinant vaccine for P. vivax based on PvDBPII formulated with GLA-SE. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. AtMBD6, a methyl CpG binding domain protein, maintains gene ...

    Indian Academy of Sciences (India)

    2017-01-13

    Jan 13, 2017 ... transcriptional gene expression. FEBS J. 272 2118-2131. Matragkou C, Papachristou H, Karetsou Z, Papadopoulos G, Papa- marcaki T, Vizirianakis IS, Tsiftsoglou AS and Choli-. Papadopoulou T 2009 On the intracellular trafficking of mouse. S5 ribosomal protein from cytoplasm to nucleoli. J. Mol. Biol. 392.

  13. Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA

    DEFF Research Database (Denmark)

    Nielsen, Morten S; Gustafsen, Camilla; Madsen, Peder

    2007-01-01

    established that the AP-1 adaptor complex is essential to SorLA's transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged...

  14. Microbial starch-binding domains as a tool for targeting proteins to granules during starch biosynthesis

    NARCIS (Netherlands)

    Ji, Q.; Vincken, J.P.; Suurs, L.C.J.M.; Visser, R.G.F.

    2003-01-01

    Modification of starch biosynthesis pathways holds an enormous potential for tailoring granules or polymers with new functionalities. In this study, we explored the possibility of engineering artificial granule-bound proteins, which can be incorporated in the granule during biosynthesis. The

  15. Solution structure and DNA-binding properties of the winged helix domain of the meiotic recombination HOP2 protein.

    Science.gov (United States)

    Moktan, Hem; Guiraldelli, Michel F; Eyster, Craig A; Zhao, Weixing; Lee, Chih-Ying; Mather, Timothy; Camerini-Otero, R Daniel; Sung, Patrick; Zhou, Donghua H; Pezza, Roberto J

    2014-05-23

    The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Solution Structure and DNA-binding Properties of the Winged Helix Domain of the Meiotic Recombination HOP2 Protein*

    Science.gov (United States)

    Moktan, Hem; Guiraldelli, Michel F.; Eyster, Craig A.; Zhao, Weixing; Lee, Chih-Ying; Mather, Timothy; Camerini-Otero, R. Daniel; Sung, Patrick; Zhou, Donghua H.; Pezza, Roberto J.

    2014-01-01

    The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination. PMID:24711446

  17. Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)*

    Science.gov (United States)

    Makkinje, Anthony; Vanden Borre, Pierre; Near, Richard I.; Patel, Prayag S.; Lerner, Adam

    2012-01-01

    The focal adhesion adapter protein p130cas regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130cas. AND-34/BCAR3, one of three NSP family members, binds the p130cas carboxyl terminus, adjacent to a bipartite p130cas Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130cas. Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130cas complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130cas to bind the Src SH3 domain through an RPLPSPP motif in the p130cas SBD. Although our prior work identified phosphorylation of the serine within the p130cas RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130cas. The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130cas complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130cas substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130cas. Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130cas and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130cas complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130cas SBD. PMID:22711540

  18. Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

    DEFF Research Database (Denmark)

    Ammendrup-Johnsen, Ina; Thorsen, Thor Seneca; Gether, Ulrik

    2012-01-01

    PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing...... site for PKCα. Mutation of Ser77 reduced the level of PKCα-mediated phosphorylation ~50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical...... lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution....

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

    NARCIS (Netherlands)

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

    1991-01-01

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

  20. 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.

  1. The stem rust resistance gene Rpg5 encodes a protein with nucleotide-binding-site, leucine-rich, and protein kinase domains.

    Science.gov (United States)

    Brueggeman, R; Druka, A; Nirmala, J; Cavileer, T; Drader, T; Rostoks, N; Mirlohi, A; Bennypaul, H; Gill, U; Kudrna, D; Whitelaw, C; Kilian, A; Han, F; Sun, Y; Gill, K; Steffenson, B; Kleinhofs, A

    2008-09-30

    We isolated the barley stem rust resistance genes Rpg5 and rpg4 by map-based cloning. These genes are colocalized on a 70-kb genomic region that was delimited by recombination. The Rpg5 gene consists of an unusual structure encoding three typical plant disease resistance protein domains: nucleotide-binding site, leucine-rich repeat, and serine threonine protein kinase. The predicted RPG5 protein has two putative transmembrane sites possibly involved in membrane binding. The gene is expressed at low but detectable levels. Posttranscriptional gene silencing using VIGS resulted in a compatible reaction with a normally incompatible stem rust pathogen. Allele sequencing also validated the candidate Rpg5 gene. Allele and recombinant sequencing suggested that the probable rpg4 gene encoded an actin depolymerizing factor-like protein. Involvement of actin depolymerizing factor genes in nonhost resistance has been documented, but discovery of their role in gene-for-gene interaction would be novel and needs to be further substantiated.

  2. Perturbation of discrete sites on a single protein domain with RNA aptamers: targeting of different sides of the TATA-binding protein (TBP).

    Science.gov (United States)

    Hohmura, Ken I; Shi, Hua; Hirayoshi, Kazunori

    2013-01-01

    Control of interactions among proteins is critical in the treatment of diseases, but the specificity required is not easily incorporated into small molecules. Macromolecules could be more suitable as antagonists in this situation, and RNA aptamers have become particularly promising. Here we describe a novel selection procedure for RNA aptamers against a protein that constitutes a single structural domain, the Drosophila TATA-binding protein (TBP). In addition to the conventional filter partitioning method with free TBP as target, we performed another experiment, in which the TATA-bound form of TBP was targeted. Aptamers generated by both selections were able to bind specifically to TBP, but the two groups showed characteristics which were clearly different in terms of their capability to compete with TATA-DNA, their effects on the TATA-bound form of TBP, and their effects on in vitro transcription. The method used to generate these two groups of aptamers can be used with other targets to direct aptamer specificity to discrete sites on the surface of a protein.

  3. Structural Insights into Immune Recognition of the Severe Acute Respiratory Syndrome Coronavirus S Protein Receptor Binding Domain

    Energy Technology Data Exchange (ETDEWEB)

    Pak, J.; Sharon, C; Satkunarajah, M; Thierry, C; Cameron, C; Kelvin, D; Seetharaman, J; Cochrane, A; Plummer, F; et. al.

    2009-01-01

    The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for host cell attachment and fusion of the viral and host cell membranes. Within S the receptor binding domain (RBD) mediates the interaction with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV host cell receptor. Both S and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Reported here is the X-ray crystal structure of the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The RBD-F26G19 Fab complex represents the first example of the structural characterization of an antibody elicited by an immune response to SARS-CoV or any fragment of it. The structure reveals that the RBD surface recognized by F26G19 overlaps significantly with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction.

  4. Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

    Directory of Open Access Journals (Sweden)

    Saray Santamaría-Hernando

    Full Text Available Proteins of the animal heme peroxidase (ANP superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20, where it was found to be involved in Ca(2+ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+ binding with a K(D of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821 is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of

  5. An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells.

    Science.gov (United States)

    Hung, Jan-Jong; Hsieh, Meng-Ti; Young, Ming-Jer; Kao, Chuan-Liang; King, Chwan-Chuen; Chang, Wen

    2004-01-01

    Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.

  6. Mapping of interaction domains of putative telomere-binding proteins AtTRB1 and AtPOT1b from Arabidopsis thaliana.

    Science.gov (United States)

    Schrumpfová, Petra Procházková; Kuchar, Milan; Palecek, Jan; Fajkus, Jirí

    2008-04-30

    We previously searched for interactions between plant telomere-binding proteins and found that AtTRB1, from the single-myb-histone (Smh) family, interacts with the Arabidopsis POT1-like-protein, AtPOT1b, involved in telomere capping. Here we identify domains responsible for that interaction. We also map domains in AtTRB1 responsible for interactions with other Smh-family-members. Our results show that the N-terminal OB-fold-domain of AtPOT1b mediates the interaction with AtTRB1. This domain is characteristic for POT1- proteins and is involved with binding the G-rich-strand of telomeric DNA. AtPOT1b also interacts with AtTRB2 and AtTRB3. The central histone-globular-domain of AtTRB1 is involved with binding to AtTRB2 and 3, as well as to AtPOT1b. AtTRB1-heterodimers with other Smh-family-members are more stable than AtTRB1-homodimers. Our results reveal interaction networks of plant telomeres.

  7. Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

    Science.gov (United States)

    Ray, Swagat; Anderson, Emma C

    2016-03-03

    The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

  8. Multimerization of the mouse TATA-binding protein (TBP) driven by its C-terminal conserved domain.

    OpenAIRE

    Kato, K; Makino, Y; Kishimoto, T; Yamauchi, J; Kato, S; Muramatsu, M; Tamura, T

    1994-01-01

    The conformational states of the mouse TATA-binding protein (TBP) in solution were studied. A histidine tag and a factor Xa recognition site-carrying mouse TBP was expressed in E. coli, highly purified, and its fundamental functions as a TBP were demonstrated. We analyzed the molecular states of mouse TBP by gel filtration and glycerol gradient sedimentation, and found that TBP forms heterogeneous multimers in solution. Direct binding of TBP molecules to each other was proven by the far-Weste...

  9. The E3 Ubiquitin Ligase- and Protein Phosphatase 2A (PP2A)-binding Domains of the Alpha4 Protein Are Both Required for Alpha4 to Inhibit PP2A Degradation

    Energy Technology Data Exchange (ETDEWEB)

    LeNoue-Newton, Michele; Watkins, Guy R.; Zou, Ping; Germane, Katherine L.; McCorvey, Lisa R.; Wadzinski, Brian E.; Spiller, Benjamin W. (Vanderbilt)

    2012-04-30

    Protein phosphatase 2A (PP2A) is regulated through a variety of mechanisms, including post-translational modifications and association with regulatory proteins. Alpha4 is one such regulatory protein that binds the PP2A catalytic subunit (PP2Ac) and protects it from polyubiquitination and degradation. Alpha4 is a multidomain protein with a C-terminal domain that binds Mid1, a putative E3 ubiquitin ligase, and an N-terminal domain containing the PP2Ac-binding site. In this work, we present the structure of the N-terminal domain of mammalian Alpha4 determined by x-ray crystallography and use double electron-electron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. Structurally, Alpha4 differs from its yeast homolog, Tap42, in two important ways: (1) the position of the helix containing the PP2Ac-binding residues is in a more open conformation, showing flexibility in this region; and (2) Alpha4 contains a ubiquitin-interacting motif. The effects of wild-type and mutant Alpha4 on PP2Ac ubiquitination and stability were examined in mammalian cells by performing tandem ubiquitin-binding entity precipitations and cycloheximide chase experiments. Our results reveal that both the C-terminal Mid1-binding domain and the PP2Ac-binding determinants are required for Alpha4-mediated protection of PP2Ac from polyubiquitination and degradation.

  10. The Inhibitor of Apoptosis Protein-Binding Domain of Smac Is Not Essential for Its Proapoptotic Activity

    OpenAIRE

    Roberts, Darren L.; Merrison, Wendy; MacFarlane, Marion; Cohen, Gerald M.

    2001-01-01

    Smac/DIABLO, a recently identified inhibitor of apoptosis protein (IAP)-binding protein, is released from the mitochondria during apoptosis and reportedly potentiates apoptosis by relieving the inhibition of IAPs on caspases. We now describe the molecular characterization of Smac β, an alternatively spliced form of Smac, which lacks the mitochondrial-targeting sequence found in Smac and has a cortical distribution in both human embryonic kidney 293 and breast epithelial tumor MCF-7 cells. Sma...

  11. The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

    Directory of Open Access Journals (Sweden)

    Geldner Niko

    2005-02-01

    Full Text Available Abstract Background Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Results Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7. Conclusion Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

  12. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  13. A fusion protein of the synthetic IgG-binding domain and aequorin: Expression and purification from E. coli cells and its application.

    Science.gov (United States)

    Inouye, Satoshi; Sahara-Miura, Yuiko

    2017-09-01

    Aequorin is a Ca 2+ -binding photoprotein that is a complex of apoaequorin (apoAQ) and 2-peroxycoelenterazine. In this study, the fusion protein (ZZ-apoAQ) composed of the synthetic IgG-binding domain (ZZ domain) derived from Staphylococcus aureus protein A and apoAQ was expressed into the periplasmic space of Escherichia coli cells. ZZ-apoAQ was highly purified using Ni-chelate affinity chromatography followed by IgG affinity chromatography. ZZ-AQ was prepared from purified ZZ-apoAQ by incubation with coelenterazine and was characterized, including its luminescence properties. ZZ-AQ could be used as a reporter for detecting IgG and the measurable range of IgG coated on a 96-well plate was 1-1000 ng/mL. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

    International Nuclear Information System (INIS)

    He Yuxian; Zhou Yusen; Liu Shuwen; Kou Zhihua; Li Wenhui; Farzan, Michael; Jiang Shibo

    2004-01-01

    The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS

  15. Acyl CoA Binding Domain Containing 3 (ACBD3) Protein in Huntington’s Disease 
Human Skin Fibroblasts

    Czech Academy of Sciences Publication Activity Database

    Kratochvílová, H.; Rodinová, M.; Sládková, J.; Klempíř, J.; Lišková, Irena; Motlík, Jan; Zeman, J.; Hansíková, H.; Tesařová, M.

    2015-01-01

    Roč. 78, Suppl. 2 (2015), s. 34-38 ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. Liblice, 08.11.2015-10.11.2015] R&D Projects: GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : Huntington’s disease * Acyl-CoA binding domain containing 3 protein * human skin fibroblasts Subject RIV: FH - Neurology Impact factor: 0.209, year: 2015

  16. Strategy for Treating Motor Neuron Diseases Using a Fusion Protein of Botulinum Toxin Binding Domain and Streptavidin for Viral Vector Access: Work in Progress

    Directory of Open Access Journals (Sweden)

    Uma Balasubramanian

    2010-12-01

    Full Text Available Although advances in understanding of the pathogenesis of amyotrophic lateral sclerosis (ALS and spinal muscular atrophy (SMA have suggested attractive treatment strategies, delivery of agents to motor neurons embedded within the spinal cord is problematic. We have designed a strategy based on the specificity of botulinum toxin, to direct entry of viral vectors carrying candidate therapeutic genes into motor neurons. We have engineered and expressed fusion proteins consisting of the binding domain of botulinum toxin type A fused to streptavidin (SAv. This fusion protein will direct biotinylated viral vectors carrying therapeutic genes into motor nerve terminals where they can enter the acidified endosomal compartments, be released and undergo retrograde transport, to deliver the genes to motor neurons. Both ends of the fusion proteins are shown to be functionally intact. The binding domain end binds to mammalian nerve terminals at neuromuscular junctions, ganglioside GT1b (a target of botulinum toxin, and a variety of neuronal cells including primary chick embryo motor neurons, N2A neuroblastoma cells, NG108-15 cells, but not to NG CR72 cells, which lack complex gangliosides. The streptavidin end binds to biotin, and to a biotinylated Alexa 488 fluorescent tag. Further studies are in progress to evaluate the delivery of genes to motor neurons in vivo, by the use of biotinylated viral vectors.

  17. The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors

    International Nuclear Information System (INIS)

    Piccioli, Zachary; McKee, Courtney H.; Leszczynski, Anna; Onder, Zeynep; Hannah, Erin C.; Mamoor, Shahan; Crosby, Lauren; Moroianu, Junona

    2010-01-01

    We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7 39-98 localized mostly to the nucleus. The GST-11E7 and GST-11cE7 39-98 were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.

  18. An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium

    International Nuclear Information System (INIS)

    Myre, Michael A.; O'Day, Danton H.

    2005-01-01

    Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD ( 171 EDVSRFIKGKLLQKQQKIYKDLERF 195 ) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues 48 KKSYQDPEIIAHSRPRK 64 that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to 48 EF 49 abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the 48 EF 49 construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium

  19. The HCM-linked W792R mutation in cardiac myosin binding protein-C reduces C6 FnIII domain stability.

    Science.gov (United States)

    Smelter, Dan F; De Lange, Willem J; Cai, Wenxuan; Ge, Ying; Ralphe, John C

    2018-02-16

    Cardiac myosin binding protein-C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue (mECT) constructs. Based on complete conservation of tryptophan at this location in FnIII domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII-domain, destabilizing the mutant protein. Adenoviral transduction of the wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance but not equivalent protein levels with W792R compared to WT controls. mECT constructs expressing W792R demonstrated abnormal contractile kinetics compared to WT that were nearly identical to cMyBP-C deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability, and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of haploinsufficiency.

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

    Science.gov (United States)

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

    1998-01-01

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

  1. Integral UBL domain proteins: a family of proteasome interacting proteins

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2004-01-01

    The family of ubiquitin-like (UBL) domain proteins (UDPs) comprises a conserved group of proteins involved in a multitude of different cellular activities. However, recent studies on UBL-domain proteins indicate that these proteins appear to share a common property in their ability to interact......-domain proteins catalyse the formation of ubiquitin-protein conjugates, whereas others appear to target ubiquitinated proteins for degradation and interact with chaperones. Hence, by binding to the 26S proteasome the UBL-domain proteins seem to tailor and direct the basic proteolytic functions of the particle...

  2. The orientation of the C-terminal domain of the Saccharomyces cerevisiae Rap1 protein is determined by its binding to DNA

    Science.gov (United States)

    Matot, Béatrice; Le Bihan, Yann-Vaï; Lescasse, Rachel; Pérez, Javier; Miron, Simona; David, Gabriel; Castaing, Bertrand; Weber, Patrick; Raynal, Bertrand; Zinn-Justin, Sophie; Gasparini, Sylvaine; Le Du, Marie-Hélène

    2012-01-01

    Rap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere. PMID:22139930

  3. Electrostatics effects on Ca(2+) binding and conformational changes in EF-hand domains: Functional implications for EF-hand proteins.

    Science.gov (United States)

    Ababou, Abdessamad; Zaleska, Mariola

    2015-12-01

    Mutations of Gln41 and Lys75 with nonpolar residues in the N-terminal domain of calmodulin (N-Cam) revealed the importance of solvation energetics in conformational change of Ca(2+) sensor EF-hand domains. While in general these domains have polar residues at these corresponding positions yet the extent of their conformational response to Ca(2+) binding and their Ca(2+) binding affinity can be different from N-Cam. Consequently, here we address the charge state of the polar residues at these positions. The results show that the charge state of these polar residues can affect substantially the conformational change and the Ca(2+) binding affinity of our N-Cam variants. Since all the variants kept their conformational activity in the presence of Ca(2+) suggests that the differences observed among them mainly originate from the difference in their molecular dynamics. Hence we propose that the molecular dynamics of Ca(2+) sensor EF-hand domains is a key factor in the multifunctional aspect of EF-hand proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Functional Differences between the N-Terminal Domains of Mouse and Human Myosin Binding Protein-C

    Directory of Open Access Journals (Sweden)

    Justin F. Shaffer

    2010-01-01

    Full Text Available The N-terminus of cMyBP-C can activate actomyosin interactions in the absence of Ca2+, but it is unclear which domains are necessary. Prior studies suggested that the Pro-Ala rich region of human cMyBP-C activated force in permeabilized human cardiomyocytes, whereas the C1 and M-domains of mouse cMyBP-C activated force in permeabilized rat cardiac trabeculae. Because the amino acid sequence of the P/A region differs between human and mouse cMyBP-C isoforms (46% identity, we investigated whether species-specific differences in the P/A region could account for differences in activating effects. Using chimeric fusion proteins containing combinations of human and mouse C0, Pro-Ala, and C1 domains, we demonstrate here that the human P/A and C1 domains activate actomyosin interactions, whereas the same regions of mouse cMyBP-C are less effective. These results suggest that species-specific differences between homologous cMyBP-C isoforms confer differential effects that could fine-tune cMyBP-C function in hearts of different species.

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

    Science.gov (United States)

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

    2015-12-17

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

  6. Structural and histone binding ability characterizations of human PWWP domains.

    Directory of Open Access Journals (Sweden)

    Hong Wu

    Full Text Available 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 β-barrel core, an insertion motif between the second and third β-strands and a C-terminal α-helix bundle. Both the canonical β-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.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

  7. Loss of Expression of Human Spectrin Src Homology Domain Binding Protein 1 is Associated with 10p Loss in Human Prostatic Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Jill A. Macoska

    2001-01-01

    Full Text Available The gene encoding human spectrin Src homology domain binding protein 1, or Hssh3bpl, which is a marker of macropinocytic vesicles and a potential regulator of macropinocytosis, co-localizes to a YAC containing chromosome 10p sequences at loci D10S89 and D10S111 that are frequently deleted in prostate tumors. Expression of Hssh3bp1 was evaluated at the protein level in 17 paired normal and malignant prostate tumor samples using the monoclonal antibody 2G8 to Hssh3bpl. These experiments demonstrated that 4/6 tumors (67% with 10p deletion failed to express Hssh3bp1 protein compared to 5/11 (46% tumors with intact 10p. Thus, loss of Hssh3bp1 expression is concordant with allelic loss of adjacent 10p sequences in human prostate tumors. In addition, two prostate tumor cell lines contain an exon skipping mutation in the Hssh3bp1 gene that leads to the abnormal splicing of the mRNA and loss of a portion of Abl tyrosine kinase SH3 domain binding site in the protein. These data are consistent with a role for Hssh3bp1 as a candidate tumor suppressor gene inactivated during prostate tumorigenesis.

  8. The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells

    Energy Technology Data Exchange (ETDEWEB)

    Gough, Mallory, E-mail: m.gough1@lancaster.ac.uk; Blanthorn-Hazell, Sophee, E-mail: s.blanthorn-hazell@lancaster.ac.uk; Delury, Craig, E-mail: c.delury@lancaster.ac.uk; Parkin, Edward, E-mail: e.parkin@lancaster.ac.uk

    2014-10-31

    Highlights: • Copper levels are elevated in the tumour microenvironment. • APP mitigates copper-induced growth inhibition of DU145 prostate cancer (PCa) cells. • The APP intracellular domain is a prerequisite; soluble forms have no effect. • The E1 CuBD of APP is also a prerequisite. • APP copper binding potentially mitigates copper-induced PCa cell growth inhibition. - Abstract: Copper plays an important role in the aetiology and growth of tumours and levels of the metal are increased in the serum and tumour tissue of patients affected by a range of cancers including prostate cancer (PCa). The molecular mechanisms that enable cancer cells to proliferate in the presence of elevated copper levels are, therefore, of key importance in our understanding of tumour growth progression. In the current study, we have examined the role played by the amyloid precursor protein (APP) in mitigating copper-induced growth inhibition of the PCa cell line, DU145. A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored. Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein. APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment. Targeting the expression of such proteins may contribute to therapeutic strategies for the treatment of cancers.

  9. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

    Directory of Open Access Journals (Sweden)

    Tatjana P Kristensen

    2014-03-01

    Full Text Available The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural

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

    Science.gov (United States)

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

    2016-02-09

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

  11. An External Loop Region of Domain III of Dengue Virus Type 2 Envelope Protein Is Involved in Serotype-Specific Binding to Mosquito but Not Mammalian Cells

    OpenAIRE

    Hung, Jan-Jong; Hsieh, Meng-Ti; Young, Ming-Jer; Kao, Chuan-Liang; King, Chwan-Chuen; Chang, Wen

    2004-01-01

    Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin c...

  12. Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

    Directory of Open Access Journals (Sweden)

    Evaristus Chibunna Mbanefo

    Full Text Available BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6 M and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation, and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

  13. Structural dynamics and ssDNA binding activity of the three N-terminal domains of the large subunit of Replication Protein A from small angle X-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Pretto, Dalyir I.; Tsutakawa, Susan; Brosey, Chris A.; Castillo, Amalchi; Chagot, Marie-Eve; Smith, Jarrod A.; Tainer, John A.; Chazin, Walter J.

    2010-03-11

    Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial inter-domain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module.

  14. Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver.

    Science.gov (United States)

    Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S; Sun, Baodong

    2016-08-05

    A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. PTEN-PDZ domain interactions: Binding of PTEN to PDZ domains of PTPN13.

    NARCIS (Netherlands)

    Sotelo, N.S.; Schepens, J.T.G.; Valiente, M.; Hendriks, W.J.A.J.; Pulido, R.

    2015-01-01

    Protein modular interactions mediated by PDZ domains are essential for the establishment of functional protein networks controlling diverse cellular functions. The tumor suppressor PTEN possesses a C-terminal PDZ-binding motif (PDZ-BM) that is recognized by a specific set of PDZ domains from

  16. Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites.

    Science.gov (United States)

    Friedrich, Nikolas; Santos, Joana M; Liu, Yan; Palma, Angelina S; Leon, Ester; Saouros, Savvas; Kiso, Makoto; Blackman, Michael J; Matthews, Stephen; Feizi, Ten; Soldati-Favre, Dominique

    2010-01-15

    Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for alpha2-3- over alpha2-6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to alpha2-9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6'sulfo-sialyl Lewis(x) might have implications for tissue tropism.

  17. Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

    Science.gov (United States)

    Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

    2015-01-01

    Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

  18. Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

    Science.gov (United States)

    Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

    2015-01-09

    Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

    Science.gov (United States)

    Iwaya, Naoko; Takasu, Hirotoshi; Goda, Natsuko; Shirakawa, Masahiro; Tanaka, Toshiki; Hamada, Daizo; Hiroaki, Hidekazu

    2013-05-01

    The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

  20. Thermodynamics of complex structures formed between single-stranded DNA oligomers and the KH domains of the far upstream element binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Kaushik; Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)

    2016-05-28

    The noncovalent interaction between protein and DNA is responsible for regulating the genetic activities in living organisms. The most critical issue in this problem is to understand the underlying driving force for the formation and stability of the complex. To address this issue, we have performed atomistic molecular dynamics simulations of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein (FBP) complexed with two single-stranded DNA (ss-DNA) oligomers in aqueous media. Attempts have been made to calculate the individual components of the net entropy change for the complexation process by adopting suitable statistical mechanical approaches. Our calculations reveal that translational, rotational, and configurational entropy changes of the protein and the DNA components have unfavourable contributions for this protein-DNA association process and such entropy lost is compensated by the entropy gained due to the release of hydration layer water molecules. The free energy change corresponding to the association process has also been calculated using the Free Energy Perturbation (FEP) method. The free energy gain associated with the KH4–DNA complex formation has been found to be noticeably higher than that involving the formation of the KH3–DNA complex.

  1. UBA domain containing proteins in fission yeast

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Semple, Colin A M; Ponting, Chris P

    2003-01-01

    characterised on both the functional and structural levels. One example of a widespread ubiquitin binding module is the ubiquitin associated (UBA) domain. Here, we discuss the approximately 15 UBA domain containing proteins encoded in the relatively small genome of the fission yeast Schizosaccharomyces pombe...

  2. Novel Structure and Unexpected RNA-Binding Ability of the C-Terminal Domain of Herpes Simplex Virus 1 Tegument Protein UL21

    Energy Technology Data Exchange (ETDEWEB)

    Metrick, Claire M.; Heldwein, Ekaterina E. (Tufts-MED)

    2016-04-06

    Proteins forming the tegument layers of herpesviral virions mediate many essential processes in the viral replication cycle, yet few have been characterized in detail. UL21 is one such multifunctional tegument protein and is conserved among alphaherpesviruses. While UL21 has been implicated in many processes in viral replication, ranging from nuclear egress to virion morphogenesis to cell-cell spread, its precise roles remain unclear. Here we report the 2.7-Å crystal structure of the C-terminal domain of herpes simplex virus 1 (HSV-1) UL21 (UL21C), which has a unique α-helical fold resembling a dragonfly. Analysis of evolutionary conservation patterns and surface electrostatics pinpointed four regions of potential functional importance on the surface of UL21C to be pursued by mutagenesis. In combination with the previously determined structure of the N-terminal domain of UL21, the structure of UL21C provides a 3-dimensional framework for targeted exploration of the multiple roles of UL21 in the replication and pathogenesis of alphaherpesviruses. Additionally, we describe an unanticipated ability of UL21 to bind RNA, which may hint at a yet unexplored function.

    IMPORTANCEDue to the limited genomic coding capacity of viruses, viral proteins are often multifunctional, which makes them attractive antiviral targets. Such multifunctionality, however, complicates their study, which often involves constructing and characterizing null mutant viruses. Systematic exploration of these multifunctional proteins requires detailed road maps in the form of 3-dimensional structures. In this work, we determined the crystal structure of the C-terminal domain of UL21, a multifunctional tegument protein that is conserved among alphaherpesviruses. Structural analysis pinpointed surface areas of potential functional importance that provide a starting point for mutagenesis. In addition, the unexpected RNA-binding ability of UL21 may expand its functional repertoire

  3. Spatial regulation of the KH domain RNA-binding protein Rnc1 mediated by a Crm1-independent nuclear export system in Schizosaccharomyces pombe.

    Science.gov (United States)

    Satoh, Ryosuke; Matsumura, Yasuhiro; Tanaka, Akitomo; Takada, Makoto; Ito, Yuna; Hagihara, Kanako; Inari, Masahiro; Kita, Ayako; Fukao, Akira; Fujiwara, Toshinobu; Hirai, Shinya; Tani, Tokio; Sugiura, Reiko

    2017-05-01

    RNA-binding proteins (RBPs) play important roles in the posttranscriptional regulation of gene expression, including mRNA stability, transport and translation. Fission yeast rnc1 + encodes a K Homology (KH)-type RBP, which binds and stabilizes the Pmp1 MAPK phosphatase mRNA thereby suppressing the Cl - hypersensitivity of calcineurin deletion and MAPK signaling mutants. Here, we analyzed the spatial regulation of Rnc1 and discovered a putative nuclear export signal (NES) Rnc1 , which dictates the cytoplasmic localization of Rnc1 in a Crm1-independent manner. Notably, mutations in the NES Rnc1 altered nucleocytoplasmic distribution of Rnc1 and abolished its function to suppress calcineurin deletion, although the Rnc1 NES mutant maintains the ability to bind Pmp1 mRNA. Intriguingly, the Rnc1 NES mutant destabilized Pmp1 mRNA, suggesting the functional importance of the Rnc1 cytoplasmic localization. Mutation in Rae1, but not Mex67 deletion or overproduction, induced Rnc1 accumulation in the nucleus, suggesting that Rnc1 is exported from the nucleus to the cytoplasm via the mRNA export pathway involving Rae1. Importantly, mutations in the Rnc1 KH-domains abolished the mRNA-binding ability and induced nuclear localization, suggesting that Rnc1 may be exported from the nucleus together with its target mRNAs. Collectively, the functional Rae1-dependent mRNA export system may influence the cytoplasmic localization and function of Rnc1. © 2017 John Wiley & Sons Ltd.

  4. The human parvovirus B19 non-structural protein 1 N-terminal domain specifically binds to the origin of replication in the viral DNA.

    Science.gov (United States)

    Tewary, Sunil Kumar; Zhao, Haiyan; Deng, Xuefeng; Qiu, Jianming; Tang, Liang

    2014-01-20

    The non-structural protein 1 (NS1) of human parvovirus B19 plays a critical role in viral DNA replication. Previous studies identified the origin of replication in the viral DNA, which contains four DNA elements, namely NSBE1 to NSBE4, that are required for optimal viral replication (Guan et al., 2009). Here we have demonstrated in vitro that the NS1 N-terminal domain (NS1N) binds to the origin of replication in a sequence-specific, length-dependent manner that requires NSBE1 and NSBE2, while NSBE3 and NSBE4 are dispensable. Mutagenesis analysis has identified nucleotides in NSBE1 and NSBE2 that are critical for NS1N binding. These results suggest that NS1 binds to the NSBE1-NSBE2 region in the origin of replication, while NSBE3 and NSBE4 may provide binding sites for potential cellular factors. Such a specialized nucleoprotein complex may enable NS1 to nick the terminal resolution site and separate DNA strands during replication. © 2013 Published by Elsevier Inc.

  5. A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication.

    Science.gov (United States)

    Ohsaki, Eriko; Ueda, Keiji

    2017-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Distant relationships amongst protein domains

    Indian Academy of Sciences (India)

    ncbs

    3. Small domains. The 'pleckstrin homology' (PH) domain is a domain of about 100 residues that occurs in a wide range of proteins involved in intracellular signaling or as constituents of the cytoskeleton.

  7. Both TEAD-binding and WW domains are required for the growth stimulation and oncogenic transformation activity of yes-associated protein.

    Science.gov (United States)

    Zhao, Bin; Kim, Joungmok; Ye, Xin; Lai, Zhi-Chun; Guan, Kun-Liang

    2009-02-01

    The Yes-associated protein (YAP) transcription coactivator is a candidate human oncogene and a key regulator of organ size. It is phosphorylated and inhibited by the Hippo tumor suppressor pathway. TEAD family transcription factors were recently shown to play a key role in mediating the biological functions of YAP. Here, we show that the WW domain of YAP has a critical role in inducing a subset of YAP target genes independent of or in cooperation with TEAD. Mutation of the WW domains diminishes the ability of YAP to stimulate cell proliferation and oncogenic transformation. Inhibition of YAP oncogenic-transforming activity depends on intact serine residues 127 and 381, two sites that could be phosphorylated by the Hippo pathway. Furthermore, genetic experiments in Drosophila support that WW domains of YAP and Yki, the fly YAP homologue, have an important role in stimulating tissue growth. Our data suggest a model in which YAP induces gene expression and exerts its biological functions by interacting with transcription factors through both the TEAD-binding and WW domains.

  8. Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION.

    Science.gov (United States)

    Cooper, Christopher D O; Newman, Joseph A; Aitkenhead, Hazel; Allerston, Charles K; Gileadi, Opher

    2015-05-29

    Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Mapping small molecule binding data to structural domains.

    Science.gov (United States)

    Kruger, Felix A; Rostom, Raghd; Overington, John P

    2012-01-01

    Large-scale bioactivity/SAR Open Data has recently become available, and this has allowed new analyses and approaches to be developed to help address the productivity and translational gaps of current drug discovery. One of the current limitations of these data is the relative sparsity of reported interactions per protein target, and complexities in establishing clear relationships between bioactivity and targets using bioinformatics tools. We detail in this paper the indexing of targets by the structural domains that bind (or are likely to bind) the ligand within a full-length protein. Specifically, we present a simple heuristic to map small molecule binding to Pfam domains. This profiling can be applied to all proteins within a genome to give some indications of the potential pharmacological modulation and regulation of all proteins. In this implementation of our heuristic, ligand binding to protein targets from the ChEMBL database was mapped to structural domains as defined by profiles contained within the Pfam-A database. Our mapping suggests that the majority of assay targets within the current version of the ChEMBL database bind ligands through a small number of highly prevalent domains, and conversely the majority of Pfam domains sampled by our data play no currently established role in ligand binding. Validation studies, carried out firstly against Uniprot entries with expert binding-site annotation and secondly against entries in the wwPDB repository of crystallographic protein structures, demonstrate that our simple heuristic maps ligand binding to the correct domain in about 90 percent of all assessed cases. Using the mappings obtained with our heuristic, we have assembled ligand sets associated with each Pfam domain. Small molecule binding has been mapped to Pfam-A domains of protein targets in the ChEMBL bioactivity database. The result of this mapping is an enriched annotation of small molecule bioactivity data and a grouping of activity classes

  10. Experimental verification, and domain definition, of structural alerts for protein binding: epoxides, lactones, nitroso, nitros, aldehydes and ketones.

    Science.gov (United States)

    Nelms, M D; Cronin, M T D; Schultz, T W; Enoch, S J

    2013-01-01

    This study outlines how a combination of in chemico and Tetrahymena pyriformis data can be used to define the applicability domain of selected structural alerts within the profilers of the OECD QSAR Toolbox. Thirty-three chemicals were profiled using the OECD and OASIS profilers, enabling the applicability domain of six structural alerts to be defined, the alerts being: epoxides, lactones, nitrosos, nitros, aldehydes and ketones. Analysis of the experimental data showed the applicability domains for the epoxide, nitroso, aldehyde and ketone structural alerts to be well defined. In contrast, the data showed the applicability domains for the lactone and nitro structural alerts needed modifying. The accurate definition of the applicability domain for structural alerts within in silico profilers is important due to their use in the chemical category in predictive and regulatory toxicology. This study highlights the importance of utilizing multiple profilers in category formation.

  11. An MHC-I cytoplasmic domain/HIV-1 Nef fusion protein binds directly to the mu subunit of the AP-1 endosomal coat complex.

    Directory of Open Access Journals (Sweden)

    Rajendra Kumar Singh

    2009-12-01

    Full Text Available The down-regulation of the major histocompatibility complex class I (MHC-I from the surface of infected cells by the Nef proteins of primate immunodeficiency viruses likely contributes to pathogenesis by providing evasion of cell-mediated immunity. HIV-1 Nef-induced down-regulation involves endosomal trafficking and a cooperative interaction between the cytoplasmic domain (CD of MHC-I, Nef, and the clathrin adaptor protein complex-1 (AP-1. The CD of MHC-I contains a key tyrosine within the sequence YSQA that is required for down-regulation by Nef, but this sequence does not conform to the canonical AP-binding tyrosine-based motif Yxxphi, which mediates binding to the medium (micro subunits of AP complexes. We previously proposed that Nef allows the MHC-I CD to bind the mu subunit of AP-1 (micro1 as if it contained a Yxxphimotif.Here, we show that a direct interaction between the MHC-I CD/Nef and micro1 plays a primary role in the down-regulation of MHC-I: GST pulldown assays using recombinant proteins indicated that most of the MHC-I CD and Nef residues that are required for the down-regulation in human cells contribute to direct interactions with a truncated version of micro1. Specifically, the tyrosine residue of the YSQA sequence in the MHC-I CD as well as Nef residues E62-65 and P78 each contributed to the interaction between MHC-I CD/Nef and micro1 in vitro, whereas Nef M20 had little to no role. Conversely, residues F172/D174 and V392/L395 of the binding pocket on micro1 for Yxxphi motifs were required for a robust interaction.These data indicate that the MHC-I cytoplasmic domain, Nef, and the C-terminal two thirds of the mu subunit of AP-1 are sufficient to constitute a biologically relevant interaction. The data also reveal an unexpected role for a hydrophobic pocket in micro1 for interaction with MHC-I CD/Nef.

  12. A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Medina, Caren; Boissinot, Sylvaine [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France); Chapuis, Sophie [Institut de Biologie Moléculaire des Plantes, Laboratoire propre du CNRS conventionné avec l’Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg (France); Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France); Revers, Frédéric [INRA, Université de Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, 33882 Villenave d’Ornon (France); Ziegler-Graff, Véronique [Institut de Biologie Moléculaire des Plantes, Laboratoire propre du CNRS conventionné avec l’Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg (France); Brault, Véronique, E-mail: veronique.brault@colmar.inra.fr [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France)

    2015-12-15

    Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74 kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RT{sub Cter}) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RT{sub Cter}. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. - Highlights: • The C-terminal domain of TuYV-RT is required for long-distance movement. • CIPK7 from Arabidopsis interacts with RT{sub Cter} in yeast and in plants. • CIPK7 overexpression increases virus titer locally but not virus systemic movement. • CIPK7 localizes to plasmodesmata. • CIPK7 could be a defense protein regulating virus export.

  13. Involvement of an RNA binding protein containing Alba domain in the stage-specific regulation of beta-amastin expression in Trypanosoma cruzi.

    Science.gov (United States)

    Pérez-Díaz, Leticia; Silva, Tais Caroline; Teixeira, Santuza M R

    2017-01-01

    Amastins are surface glycoproteins, first identified in amastigotes of T. cruzi but later found to be expressed in several Leishmania species, as well as in T. cruzi epimastigotes. Amastins are encoded by a diverse gene family that can be grouped into four subfamilies named α, β, γ, and δ amastins. Differential expression of amastin genes results from regulatory mechanisms involving changes in mRNA stability and/or translational control. Although distinct regulatory elements were identified in the 3' UTR of T. cruzi and Leishmania amastin mRNAs, RNA binding proteins involved with amastin gene regulation have only being characterized in L. infantum where an Alba-domain protein (LiAlba20) able to bind to the 3' UTR of a δ-amastin mRNA was identified. Here we investigated the role of TcAlba30, the LiAlba20 homologue in T. cruzi, in the post transcriptional regulation of amastin genes. TcAlba30 transcripts are present in all stages of the T. cruzi life cycle. RNA immunoprecipitation assays using a transfected cell line expressing a cMyc tagged TcAlba30 revealed that TcAlba30 can interact with β-amastin mRNA. In addition, over-expression of TcAlba30 in epimastigotes resulted in 50% decreased levels of β-amastin mRNAs compared to wild type parasites. Since luciferase assays indicated the presence of regulatory elements in the 3' UTR of β-amastin mRNA and reduced levels of luciferase mRNA were found in parasites over expressing TcAlba30, we conclude that TcAlba30 acts as a T. cruzi RNA binding protein involved in the negative control of β-amastin expression through interactions with its 3'UTR. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Domain-based small molecule binding site annotation

    Directory of Open Access Journals (Sweden)

    Dumontier Michel

    2006-03-01

    Full Text Available Abstract Background Accurate small molecule binding site information for a protein can facilitate studies in drug docking, drug discovery and function prediction, but small molecule binding site protein sequence annotation is sparse. The Small Molecule Interaction Database (SMID, a database of protein domain-small molecule interactions, was created using structural data from the Protein Data Bank (PDB. More importantly it provides a means to predict small molecule binding sites on proteins with a known or unknown structure and unlike prior approaches, removes large numbers of false positive hits arising from transitive alignment errors, non-biologically significant small molecules and crystallographic conditions that overpredict ion binding sites. Description Using a set of co-crystallized protein-small molecule structures as a starting point, SMID interactions were generated by identifying protein domains that bind to small molecules, using NCBI's Reverse Position Specific BLAST (RPS-BLAST algorithm. SMID records are available for viewing at http://smid.blueprint.org. The SMID-BLAST tool provides accurate transitive annotation of small-molecule binding sites for proteins not found in the PDB. Given a protein sequence, SMID-BLAST identifies domains using RPS-BLAST and then lists potential small molecule ligands based on SMID records, as well as their aligned binding sites. A heuristic ligand score is calculated based on E-value, ligand residue identity and domain entropy to assign a level of confidence to hits found. SMID-BLAST predictions were validated against a set of 793 experimental small molecule interactions from the PDB, of which 472 (60% of predicted interactions identically matched the experimental small molecule and of these, 344 had greater than 80% of the binding site residues correctly identified. Further, we estimate that 45% of predictions which were not observed in the PDB validation set may be true positives. Conclusion By

  15. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    International Nuclear Information System (INIS)

    Carmen Herranz, Ma; Sanchez-Navarro, Jesus-Angel; Sauri, Ana; Mingarro, Ismael; Pallas, Vicente

    2005-01-01

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed

  16. Methyl-binding domain protein-based DNA isolation from human blood serum combines DNA analyses and serum-autoantibody testing

    Directory of Open Access Journals (Sweden)

    Jungbauer Christof

    2011-09-01

    Full Text Available Abstract Background Circulating cell free DNA in serum as well as serum-autoantibodies and the serum proteome have great potential to contribute to early cancer diagnostics via non invasive blood tests. However, most DNA preparation protocols destroy the protein fraction and therefore do not allow subsequent protein analyses. In this study a novel approach based on methyl binding domain protein (MBD is described to overcome the technical difficulties of combining DNA and protein analysis out of one single serum sample. Methods Serum or plasma samples from 98 control individuals and 54 breast cancer patients were evaluated upon silica membrane- or MBD affinity-based DNA isolation via qPCR targeting potential DNA methylation markers as well as by protein-microarrays for tumor-autoantibody testing. Results In control individuals, an average DNA level of 22.8 ± 25.7 ng/ml was detected applying the silica membrane based protocol and 8.5 ± 7.5 ng/ml using the MBD-approach, both values strongly dependent on the serum sample preparation methods used. In contrast to malignant and benign tumor serum samples, cell free DNA concentrations were significantly elevated in sera of metastasizing breast cancer patients. Technical evaluation revealed that serum upon MBD-based DNA isolation is suitable for protein-array analyses when data are consistent to untreated serum samples. Conclusion MBD affinity purification allows DNA isolations under native conditions retaining the protein function, thus for example enabling combined analyses of DNA methylation and autoantigene-profiles from the same serum sample and thereby improving minimal invasive diagnostics.

  17. Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.

    Science.gov (United States)

    Tsfasman, Tatyana; Kost, Vladimir; Markushin, Stanislav; Lotte, Vera; Koptiaeva, Irina; Bogacheva, Elena; Baratova, Ludmila; Radyukhin, Victor

    2015-12-02

    The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    /LRP was essentially abolished following deletion of residues 404-430, and pretreatment of CHO cells with the peptide comprising aa 402-423 inhibited the binding of LpL-(313-448). We conclude that the C-terminal folding domain of human LpL has a site for binding to heparin and to HSPG, presumably involving amino acids...

  19. RNA-protein binding motifs mining with a new hybrid deep learning based cross-domain knowledge integration approach

    DEFF Research Database (Denmark)

    Pan, Xiaoyong; Shen, Hong Bin

    2017-01-01

    space using multiple layers of learning blocks, where the shared representations across different domains are integrated. To validate our iDeep method, we performed experiments on 31 large-scale CLIP-seq datasets, and our results show that by integrating multiple sources of data, the average AUC can...

  20. Identification and functional characterization of the Arabidopsis Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain

    NARCIS (Netherlands)

    Julkowska, M.M.; McLoughlin, F.; Galvan-Ampudia, C.S.; Rankenberg, J.M.; Kawa, D.; Klimecka, M.; Haring, M.A.; Munnik, T.; Kooijman, E.E.; Testerink, C.

    2015-01-01

    Phosphatidic acid (PA) is an important signalling lipid involved in various stress-induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA-binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate

  1. One repeat of the cell wall binding domain is sufficient for anchoring the Lactobacillus acidophilus surface layer protein

    NARCIS (Netherlands)

    Smit, E.; Pouwels, P.H.

    2002-01-01

    The N-terminal repeat (SAC1) of the S-protein of Lactobacillus acidophilus bound efficiently and specifically to cell wall fragments (CWFs) when fused to green fluorescent protein, whereas the C-terminal repeat (SAC2) did not. Treatment of CWFs with hydrofluoric acid, but not phenol, prevented

  2. PTEN-PDZ domain interactions: binding of PTEN to PDZ domains of PTPN13.

    Science.gov (United States)

    Sotelo, Natalia S; Schepens, Jan T G; Valiente, Miguel; Hendriks, Wiljan J A J; Pulido, Rafael

    2015-05-01

    Protein modular interactions mediated by PDZ domains are essential for the establishment of functional protein networks controlling diverse cellular functions. The tumor suppressor PTEN possesses a C-terminal PDZ-binding motif (PDZ-BM) that is recognized by a specific set of PDZ domains from scaffolding and regulatory proteins. Here, we review the current knowledge on PTEN-PDZ domain interactions and tumor suppressor networks, describe methodology suitable to analyze these interactions, and report the binding of PTEN and the PDZ domain-containing protein tyrosine phosphatase PTPN13. Yeast two-hybrid and GST pull-down analyses showed that PTEN binds to PDZ2/PTPN13 domain in a manner that depends on the specific PTPN13 PDZ domain arrangement involving the interdomain region between PDZ1 and PDZ2. Furthermore, a specific binding profile of PTEN to PDZ2/PTPN13 domain was observed by mutational analysis of the PTEN PDZ-BM. Our results disclose a PDZ-mediated physical interaction of PTEN and PTPN13 with potential relevance in tumor suppression and cell homeostasis. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Molecular Evolution of the Oxygen-Binding Hemerythrin Domain.

    Directory of Open Access Journals (Sweden)

    Claudia Alvarez-Carreño

    Full Text Available The evolution of oxygenic photosynthesis during Precambrian times entailed the diversification of strategies minimizing reactive oxygen species-associated damage. Four families of oxygen-carrier proteins (hemoglobin, hemerythrin and the two non-homologous families of arthropodan and molluscan hemocyanins are known to have evolved independently the capacity to bind oxygen reversibly, providing cells with strategies to cope with the evolutionary pressure of oxygen accumulation. Oxygen-binding hemerythrin was first studied in marine invertebrates but further research has made it clear that it is present in the three domains of life, strongly suggesting that its origin predated the emergence of eukaryotes.Oxygen-binding hemerythrins are a monophyletic sub-group of the hemerythrin/HHE (histidine, histidine, glutamic acid cation-binding domain. Oxygen-binding hemerythrin homologs were unambiguously identified in 367/2236 bacterial, 21/150 archaeal and 4/135 eukaryotic genomes. Overall, oxygen-binding hemerythrin homologues were found in the same proportion as single-domain and as long protein sequences. The associated functions of protein domains in long hemerythrin sequences can be classified in three major groups: signal transduction, phosphorelay response regulation, and protein binding. This suggests that in many organisms the reversible oxygen-binding capacity was incorporated in signaling pathways. A maximum-likelihood tree of oxygen-binding hemerythrin homologues revealed a complex evolutionary history in which lateral gene transfer, duplications and gene losses appear to have played an important role.Hemerythrin is an ancient protein domain with a complex evolutionary history. The distinctive iron-binding coordination site of oxygen-binding hemerythrins evolved first in prokaryotes, very likely prior to the divergence of Firmicutes and Proteobacteria, and spread into many bacterial, archaeal and eukaryotic species. The later evolution of the

  4. Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha

    DEFF Research Database (Denmark)

    Lim, Ssang-Taek; Longley, Robert L; Couchman, John R

    2003-01-01

    alpha. Full-length PKC alpha weakly interacted with 4V by yeast two-hybrid assays, but PKC alpha constructs that lack the pseudosubstrate region or constructs of the whole catalytic domain interacted more strongly. A mutated 4V sequence (4V(YF): LGKKPIFKK) did not interact with PKC alpha, indicating...

  5. Roles of Arg-97 and Phe-113 in regulation of distal ligand binding to heme in the sensor domain of Ec DOS protein. Resonance Raman and mutation study.

    Science.gov (United States)

    El-Mashtoly, Samir F; Nakashima, Satoru; Tanaka, Atsunari; Shimizu, Toru; Kitagawa, Teizo

    2008-07-04

    The direct oxygen sensor protein isolated from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of O(2), CO, or NO to a reduced heme significantly enhances the PDE activity toward 3',5'-cyclic diguanylic acid. We report stationary and time-resolved resonance Raman spectra of the wild-type and several mutants (Glu-93 --> Ile, Met-95 --> Ala, Arg-97 --> Ile, Arg-97 --> Ala, Arg-97 --> Glu, Phe-113 --> Leu, and Phe-113 --> Thr) of the heme-containing PAS domain of Ec DOS. For the CO- and NO-bound forms, both the hydrogen-bonded and non-hydrogen-bonded conformations were found, and in the former Arg-97 forms a hydrogen bond with the heme-bound external ligand. The resonance Raman results revealed significant interactions of Arg-97 and Phe-113 with a ligand bound to the sixth coordination site of the heme and profound structural changes in the heme propionates upon dissociation of CO. Mutation of Phe-113 perturbed the PDE activities, and the mutation of Arg-97 and Phe-113 significantly influenced the transient binding of Met-95 to the heme upon photodissociation of CO. This suggests that the electrostatic interaction of Arg-97 and steric interaction of Phe-113 are crucial for regulating the competitive recombination of Met-95 and CO to the heme. On the basis of these results, we propose a model for the role of the heme propionates in communicating the heme structural changes to the protein moiety.

  6. Evaluation of post-acrosomal sheath WW domain binding protein expression in spermatozoa from infertile men with varicocele

    Directory of Open Access Journals (Sweden)

    Nasim Ghazavi-Khorasgani

    2017-09-01

    Conclusion: Expression of PAWP as a protein involved in spermatogenesis and fertilization process, has decreased in infertile men with varicocele. In addition, sperm DNA integrity was disrupted in these individuals that can be considered as a main etiology of infertility.

  7. STRUCTURE AND FUNCTION OF PALLADIN’S ACTIN BINDING DOMAIN

    Science.gov (United States)

    Beck, Moriah R.; Dixon, Richard D.S.; Goicoechea, Silvia M.; Murphy, Grant S.; Brungardt, Joseph G.; Beam, Matthew T.; Srinath, Pavan; Patel, Julie; Mohiuddin, Jahan; Otey, Carol A.; Campbell, Sharon L.

    2013-01-01

    Here we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct F-actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and crosslinking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin crosslinking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo. PMID:23806659

  8. Neutron Crystallography Detects Differences in Protein Dynamics: Structure of the PKG II Cyclic Nucleotide Binding Domain in Complex with an Activator.

    Science.gov (United States)

    Gerlits, Oksana; Campbell, James C; Blakeley, Matthew P; Kim, Choel; Kovalevsky, Andrey

    2018-03-27

    As one of the main receptors of a second messenger, cGMP, cGMP-dependent protein kinase (PKG) isoforms I and II regulate distinct physiological processes. The design of isoform-specific activators is thus of great biomedical importance and requires detailed structural information about PKG isoforms bound with activators, including accurate positions of hydrogen atoms and a description of the hydrogen bonding and water architecture. Here, we determined a 2.2 Å room-temperature joint X-ray/neutron (XN) structure of the human PKG II carboxyl cyclic nucleotide binding (CNB-B) domain bound with a potent PKG II activator, 8-pCPT-cGMP. The XN structure directly visualizes intermolecular interactions and reveals changes in hydrogen bonding patterns upon comparison to the X-ray structure determined at cryo-temperatures. Comparative analysis of the backbone hydrogen/deuterium exchange patterns in PKG II:8-pCPT-cGMP and previously reported PKG Iβ:cGMP XN structures suggests that the ability of these agonists to activate PKG is related to how effectively they quench dynamics of the cyclic nucleotide binding pocket and the surrounding regions.

  9. Nephronectin binds to heparan sulfate proteoglycans via its MAM domain.

    Science.gov (United States)

    Sato, Yuya; Shimono, Chisei; Li, Shaoliang; Nakano, Itsuko; Norioka, Naoko; Sugiura, Nobuo; Kimata, Koji; Yamada, Masashi; Sekiguchi, Kiyotoshi

    2013-04-24

    Nephronectin is a basement membrane protein comprising five N-terminal epidermal growth factor (EGF)-like repeats, a central linker segment containing an Arg-Gly-Asp (RGD) motif and a C-terminal meprin-A5 protein-receptor protein tyrosine phosphatase μ (MAM) domain. Nephronectin has been shown to interact with α8β1 integrin through the central linker segment, but its interactions with other molecules remain to be elucidated. Here, we examined the binding of nephronectin to a panel of glycosaminoglycan (GAG) chains. Nephronectin bound strongly to heparin and chondroitin sulfate (CS)-E and moderately to heparan sulfate (HS), but failed to bind to CS-A, CS-C, CS-D, dermatan sulfate and hyaluronic acid. Deletion of the MAM domain severely impaired the binding of nephronectin to heparin but not CS-E, whereas deletion of the EGF-like repeats reduced its binding to CS-E but not heparin, suggesting that nephronectin interacts with CS-E and heparin through the EGF-like repeats and MAM domain, respectively. Consistent with these results, nephronectin bound to agrin and perlecan, which are heparan sulfate proteoglycans (HSPGs) in basement membranes, in HS-dependent manners. Site-directed mutagenesis of the MAM domain revealed that multiple basic amino acid residues in the putative loop regions were involved in the binding of the MAM domain to agrin. The binding of nephronectin to basement membrane HSPGs was further confirmed by in situ nephronectin overlay assays using mouse frozen tissue sections. Taken together, these findings indicate that nephronectin is capable of binding to HSPGs in basement membranes via the MAM domain, and thereby raise the possibility that interactions with basement membrane HSPGs may be involved in the deposition of nephronectin onto basement membranes. Copyright © 2013 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  10. Extended HSR/CARD domain mediates AIRE binding to DNA

    Energy Technology Data Exchange (ETDEWEB)

    Maslovskaja, Julia, E-mail: julia.maslovskaja@ut.ee; Saare, Mario; Liiv, Ingrid; Rebane, Ana; Peterson, Pärt

    2015-12-25

    Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved in AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.

  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. Functional characterization of domains in AtTRB1, a putative telomere-binding protein in Arabidopsis thaliana

    Czech Academy of Sciences Publication Activity Database

    Mozgová, I.; Procházková Schrumpfová, P.; Hofr, C.; Fajkus, Jiří

    2008-01-01

    Roč. 69, - (2008), s. 1814-1819 ISSN 0031-9422 R&D Projects: GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : plant * telomere * single -myb-histone protein Subject RIV: BO - Biophysics Impact factor: 2.946, year: 2008

  13. Novel binders derived from an albumin-binding domain scaffold targeting human prostate secretory protein 94 (PSP94)

    Czech Academy of Sciences Publication Activity Database

    Marečková, Lucie; Petroková, Hana; Osička, Radim; Kuchař, Milan; Malý, Petr

    2015-01-01

    Roč. 6, č. 10 (2015), s. 774-779 ISSN 1674-800X Institutional support: RVO:86652036 ; RVO:61388971 Keywords : prostate secretory protein * prostate cancer * oncomarker Subject RIV: EB - Genetics ; Molecular Biology; EB - Genetics ; Molecular Biology (MBU-M) Impact factor: 3.817, year: 2015

  14. Novel high-affinity binders of human interferon gamma derived from albumin-binding domain of protein G

    Czech Academy of Sciences Publication Activity Database

    Ahmad, Jawid Nazir; Li, J.; Biedermannová, Lada; Kuchař, Milan; Šípová, Hana; Semerádtová, A.; Černý, Jiří; Petroková, Hana; Mikulecký, Pavel; Polínek, Jiří; Staněk, Ondřej; Vondrášek, Jiří; Homola, Jiří; Malý, J.; Osička, Radim; Šebo, Peter; Malý, Petr

    2012-01-01

    Roč. 80, č. 3 (2012), s. 774-789 ISSN 0887-3585 R&D Projects: GA AV ČR KAN200520702; GA ČR GAP305/10/2184 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50520701; CEZ:AV0Z20670512 Keywords : recombinant ligand * protein scaffold * computational design Subject RIV: CE - Biochemistry Impact factor: 3.337, year: 2012

  15. Activation of Fetal γ-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains

    Directory of Open Access Journals (Sweden)

    Mir A Hossain

    2016-01-01

    Full Text Available Reactivation of γ-globin expression has been shown to ameliorate disease phenotypes associated with mutations in the adult β-globin gene, including sickle cell disease. Specific mutations in the promoter of the γ-globin genes are known to prevent repression of the genes in the adult and thus lead to hereditary persistence of fetal hemoglobin. One such hereditary persistence of fetal hemoglobin is associated with a sequence located 567 bp upstream of the Gγ-globin gene which assembles a GATA-containing repressor complex. We generated two synthetic zinc-finger DNA-binding domains (ZF-DBDs targeting this sequence. The -567Gγ ZF-DBDs associated with high affinity and specificity with the target site in the γ-globin gene promoter. We delivered the -567Gγ ZF-DBDs directly to primary erythroid cells. Exposure of these cells to the recombinant -567Gγ ZF-DBDs led to increased expression of the γ-globin gene. Direct protein delivery of ZF-DBDs that compete with transcription regulatory proteins will have broad implications for modulating gene expression in analytical or therapeutic settings.

  16. Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Chunmao [State Key Laboratory of Pathogen and Biosecurity, Beijng Institute of Microbiology and Infectious Disease, No. 20 Dongda Street, Fengtai District, Beijing 100071 (China); Yu, You [Key Laboratory for Protein Sciences of Ministry of Education, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 100084, Beijing (China); Yang, Maojun, E-mail: maojunyang@tsinghua.edu.cn [Key Laboratory for Protein Sciences of Ministry of Education, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 100084, Beijing (China); Jiang, Yongqiang, E-mail: jiangyq@bmi.ac.cn [State Key Laboratory of Pathogen and Biosecurity, Beijng Institute of Microbiology and Infectious Disease, No. 20 Dongda Street, Fengtai District, Beijing 100071 (China)

    2015-10-23

    Fhb is a surface virulence protein from Streptococcus suis, which could aid bacterial evasion of host innate immune defense by recruiting complement regulator factor H to inactivate C3b deposited on bacterial surface in blood. Here we successfully expressed and purified the N terminal domain of Fhb (N-Fhb) and obtained crystals of the N-Fhb by sitting-drop vapor diffusion method with a resolution of 1.50 Å. The crystals belong to space group C2 with unit cell parameters a = 127.1 Å, b = 77.3 Å, c = 131.6 Å, α = 90°, β = 115.9°, γ = 90°. The structure of N-Fhb was determined by SAD method and the core structure of N-Fhb is a β sandwich. We speculated that binding of Fhb to human factor H may be mainly mediated by surface amino acids with negative charges. - Highlights: • We expressed N-Fhb as the soluble protein in Escherichia coli. • Crystals of N-Fhb were grown by sitting drop vapor diffusion method. • Crystals of N-Fhb could diffracted to 1.5 Å. • The core structure of N-Fhb was a β sandwich. • A part of the surface of N-Fhb was rich with negative charges.

  17. High-mobility group nucleosome-binding domain 2 protein inhibits the invasion of Klebsiella pneumoniae into mouse lungs in vivo.

    Science.gov (United States)

    Zheng, Shuang; Ren, Laibin; Li, Heng; Shen, Xiaofei; Yang, Xiaolong; Li, Na; Wang, Xinyuan; Guo, Xiaojuan; Wang, Xiaoying; Huang, Ning

    2015-07-01

    Since bacterial invasion into host cells is a critical step in the infection process and the predominance of multiple-antibiotic-resistant Klebsiella (K.) pneumoniae strains, using molecular agents to interfere with K. pneumoniae invasion is an attractive approach for the prevention of infection and suppress the immune inflammatory response. In previous studies by our group, high-mobility group nucleosome-binding domain 2 (HMGN2) protein was shown to exhibit anti-bacterial activity in vitro. The objective of the present study was to investigate the effects of HMGN2 protein on the invasion of K. pneumoniae 03183 in vivo. The results showed that pre-treatment with 128 µg/ml HMGN2 significantly reduced K. pneumoniae 03183 invasion into mouse lungs and increased the mRNA expression of CXCL1 and LCN2 within 2 h. Immunohistochemical staining showed that F-actin expression was significantly decreased, and fluorescence microscopy and western blot analysis further demonstrated that HMGN2 significantly blocked K. pneumoniae 03183-induced actin polymerization. These changes implied that HMGN2 may provide protection against K. pneumoniae 03183 infection in vivo.

  18. Prevention of adverse events of interferon γ gene therapy by gene delivery of interferon γ-heparin-binding domain fusion protein in mice

    Directory of Open Access Journals (Sweden)

    Mitsuru Ando

    2014-01-01

    Full Text Available Sustained gene delivery of interferon (IFN γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight. These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer. To achieve this, we selected the heparin-binding domain (HBD of extracellular superoxide dismutase as a molecule to anchor IFNγ to the cell surface. We designed three IFNγ derivatives, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, each of which had 1, 2, or 3 HBDs, respectively. Each plasmid-encoding fusion proteins was delivered to the liver, a model target in this study, by hydrodynamic tail vein injection. The serum concentration of IFNγ-HBD2 and IFNγ-HBD3 after gene delivery was lower than that of IFNγ or IFNγ-HBD1. Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2. Gene delivery of IFNγ-HBD2-suppressed tumor growth in the liver as efficiently as that of IFNγ with much less symptoms of adverse effects. These results indicate that the adverse events of IFNγ gene transfer can be prevented by gene delivery of IFNγ-HBD2, a fusion protein with high cell surface affinity.

  19. Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis

    International Nuclear Information System (INIS)

    Zhang, Chunmao; Yu, You; Yang, Maojun; Jiang, Yongqiang

    2015-01-01

    Fhb is a surface virulence protein from Streptococcus suis, which could aid bacterial evasion of host innate immune defense by recruiting complement regulator factor H to inactivate C3b deposited on bacterial surface in blood. Here we successfully expressed and purified the N terminal domain of Fhb (N-Fhb) and obtained crystals of the N-Fhb by sitting-drop vapor diffusion method with a resolution of 1.50 Å. The crystals belong to space group C2 with unit cell parameters a = 127.1 Å, b = 77.3 Å, c = 131.6 Å, α = 90°, β = 115.9°, γ = 90°. The structure of N-Fhb was determined by SAD method and the core structure of N-Fhb is a β sandwich. We speculated that binding of Fhb to human factor H may be mainly mediated by surface amino acids with negative charges. - Highlights: • We expressed N-Fhb as the soluble protein in Escherichia coli. • Crystals of N-Fhb were grown by sitting drop vapor diffusion method. • Crystals of N-Fhb could diffracted to 1.5 Å. • The core structure of N-Fhb was a β sandwich. • A part of the surface of N-Fhb was rich with negative charges.

  20. Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2.

    Directory of Open Access Journals (Sweden)

    Jennifer H Stuart

    2016-12-01

    Full Text Available The type I interferon (IFN response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3 complex to the interferon stimulated response element (ISRE. Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.

  1. Bioinformatic analysis and molecular modelling of human ameloblastin suggest a two-domain intrinsically unstructured calcium-binding protein

    Czech Academy of Sciences Publication Activity Database

    Vymětal, Jiří; Slabý, I.; Spahr, A.; Vondrášek, Jiří; Lyngstadaas, S. P.

    2008-01-01

    Roč. 116, č. 2 (2008), s. 124-134 ISSN 0909-8836 R&D Projects: GA ČR GA203/05/0009; GA ČR GA203/06/1727; GA MŠk LC512 Grant - others:EU(XE) QLK3-CT-2001-00090 Institutional research plan: CEZ:AV0Z40550506 Keywords : ameloblastin * bioinformatic modelling * calcium * intrinsically unstructured protein Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.957, year: 2008

  2. Molecular cloning and functional analysis of nucleotide-binding oligomerization domain-containing protein 1 in rainbow trout, Oncorhynchus mykiss.

    Science.gov (United States)

    Jang, Ju Hye; Kim, Hyun; Kim, Yu Jin; Cho, Ju Hyun

    2016-04-01

    NOD1 has important roles in innate immunity as sensor of microbial components derived from bacterial peptidoglycan. In this study, we identified genes encoding components of the NOD1 signaling pathway, including NOD1 (OmNOD1) and RIP2 (OmRIP2) from rainbow trout, Oncorhynchus mykiss, and investigated whether OmNOD1 has immunomodulating activity in a rainbow trout hepatoma cell line RTH-149 treated with NOD1-specific ligand (iE-DAP). The deduced amino acid sequence of OmNOD1 contained conserved CARD, NOD and LRR domains. Loss-of-function and gain-of-function experiments indicated that OmNOD1 is involved in the expression of pro-inflammatory cytokines. Silencing of OmNOD1 in RTH-149 cells treated with iE-DAP decreased the expression of IL-1β, IL-6, IL-8 and TNF-α. Conversely, overexpression of OmNOD1 resulted in up-regulation of IL-1β, IL-6, IL-8 and TNF-α expression. In addition, RIP2 inhibitor (gefitinib) significantly decreased the expression of these pro-inflammatory cytokines induced by iE-DAP in RTH-149 cells. These findings highlight the important role of NOD1 signaling pathway in fish in eliciting innate immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. IGF binding proteins.

    Science.gov (United States)

    Bach, Leon A

    2017-12-18

    Insulin-like growth factor binding proteins (IGFBPs) 1-6 bind IGFs but not insulin with high affinity. They were initially identified as serum carriers and passive inhibitors of IGF actions. However, subsequent studies showed that, although IGFBPs inhibit IGF actions in many circumstances, they may also potentiate these actions. IGFBPs are widely expressed in most tissues, and they are flexible endocrine and autocrine/paracrine regulators of IGF activity, which is essential for this important physiological system. More recently, individual IGFBPs have been shown to have IGF-independent actions. Mechanisms underlying these actions include (i) interaction with non-IGF proteins in compartments including the extracellular space and matrix, the cell surface and intracellularly; (ii) interaction with and modulation of other growth factor pathways including EGF, TGF- and VEGF; and (iii) direct or indirect transcriptional effects following nuclear entry of IGFBPs. Through these IGF-dependent and IGF-independent actions, IGFBPs modulate essential cellular processes including proliferation, survival, migration, senescence, autophagy and angiogenesis. They have been implicated in a range of disorders including malignant, metabolic, neurological and immune diseases. A more complete understanding of their cellular roles may lead to the development of novel IGFBP-based therapeutic opportunities.

  4. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα-encoding (GNAS genomic imprinting domain are associated with performance traits

    Directory of Open Access Journals (Sweden)

    Mullen Michael P

    2011-01-01

    Full Text Available Abstract Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486 were located upstream of the GNAS gene, while one SNP (rs41694646 was located in the second intron of the GNAS gene. The final SNP (rs41694656 was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646 is associated (P ≤ 0.05 with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf and gestation length. Association (P ≤ 0.01 with direct calving difficulty (i.e. due to calf size and maternal calving difficulty (i.e. due to the maternal pelvic width size was also observed at the rs

  5. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding (GNAS) genomic imprinting domain are associated with performance traits

    Science.gov (United States)

    2011-01-01

    Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs) spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS) domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486) were located upstream of the GNAS gene, while one SNP (rs41694646) was located in the second intron of the GNAS gene. The final SNP (rs41694656) was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646) is associated (P ≤ 0.05) with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf) and gestation length. Association (P ≤ 0.01) with direct calving difficulty (i.e. due to calf size) and maternal calving difficulty (i.e. due to the maternal pelvic width size) was also observed at the rs43101491 SNP. Following

  6. Structural elucidation of transmembrane domain zero (TMD0) of EcdL: A multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporter protein revealed by atomistic simulation.

    Science.gov (United States)

    Bera, Krishnendu; Rani, Priyanka; Kishor, Gaurav; Agarwal, Shikha; Kumar, Antresh; Singh, Durg Vijay

    2017-09-20

    ATP-Binding cassette (ABC) transporters play an extensive role in the translocation of diverse sets of biologically important molecules across membrane. EchnocandinB (antifungal) and EcdL protein of Aspergillus rugulosus are encoded by the same cluster of genes. Co-expression of EcdL and echinocandinB reflects tightly linked biological functions. EcdL belongs to Multidrug Resistance associated Protein (MRP) subfamily of ABC transporters with an extra transmembrane domain zero (TMD0). Complete structure of MRP subfamily comprising of TMD0 domain, at atomic resolution is not known. We hypothesized that the transportation of echonocandinB is mediated via EcdL protein. Henceforth, it is pertinent to know the topological arrangement of TMD0, with other domains of protein and its possible role in transportation of echinocandinB. Absence of effective template for TMD0 domain lead us to model by I-TASSER, further structure has been refined by multiple template modelling using homologous templates of remaining domains (TMD1, NBD1, TMD2, NBD2). The modelled structure has been validated for packing, folding and stereochemical properties. MD simulation for 0.1 μs has been carried out in the biphasic environment for refinement of modelled protein. Non-redundant structures have been excavated by clustering of MD trajectory. The structural alignment of modelled structure has shown Z-score -37.9; 31.6, 31.5 with RMSD; 2.4, 4.2, 4.8 with ABC transporters; PDB ID 4F4C, 4M1 M, 4M2T, respectively, reflecting the correctness of structure. EchinocandinB has been docked to the modelled as well as to the clustered structures, which reveals interaction of echinocandinB with TMD0 and other TM helices in the translocation path build of TMDs.

  7. Informing the Human Plasma Protein Binding of Environmental Chemicals by Machine Learning in the Pharmaceutical Space: Applicability Domain and Limits of Predictability

    Science.gov (United States)

    The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores th...

  8. Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

    Science.gov (United States)

    Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

    2001-07-01

    The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

  9. Ubiquitin domain proteins in disease

    DEFF Research Database (Denmark)

    Klausen, Louise Kjær; Schulze, Andrea; Seeger, Michael

    2007-01-01

    The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite...... and cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com)....

  10. Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.

    Science.gov (United States)

    Sarafanov, Andrey G; Makogonenko, Evgeny M; Pechik, Igor V; Radtke, Klaus-Peter; Khrenov, Alexey V; Ananyeva, Natalya M; Strickland, Dudley K; Saenko, Evgueni L

    2006-02-14

    Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484-509. Isolated A2 was subjected to alanine-scanning mutagenesis followed by expression of a set of mutants in a baculovirus system. In competition and surface plasmon resonance assays, affinities of A2 mutants K466A, R471A, R484A, S488A, R489A, R490A, H497A, and K499A for LRP were found to be decreased by 2-4-fold. This correlated with 1.3-1.5-fold decreases in the degree of LRP-mediated internalization of the mutants in cell culture. Combining these mutations into pairs led to cumulative effects, i.e., 7-13-fold decrease in affinity for LRP and 1.6-2.2-fold decrease in the degree of LRP-mediated internalization in cell culture. We conclude that the residues mentioned above play a key role in formation of the A2 binding epitope for LRP. Experiments in mice revealed an approximately 4.5 times shorter half-life for A2 in the circulation in comparison with that of fVIII. The half-lives of A2 mutant R471A/R484A or A2 co-injected with receptor-associated protein, a classical ligand of LRP, were prolonged by approximately 1.9 and approximately 3.5 times, respectively, compared to that of A2. This further confirms the importance of the mutated residues for interaction of A2 with LRP and suggests the existence of an LRP-dependent mechanism for removing A2 as a product of dissociation of activated fVIII from the circulation.

  11. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus

    2005-01-01

    and phosphatases, specific sets of ubiquitinating/deubiquitinating enzymes control the degree of ubiquitination. A large number of ubiquitin-binding proteins act at different steps in the downstream pathways, followed by the ubiquitinated protein. Different families of ubiquitin-binding proteins have been...... described. UBA (ubiquitin-associated) domain-containing proteins is the largest family and includes members involved in different cell processes. The smaller groups of UIM (ubiquitin-interacting motif), GAT [GGA (Golgi-associated gamma-adaptin homologous) and Tom1 (target of Myb 1)], CUE (coupling...

  12. The IntFOLD server: an integrated web resource for protein fold recognition, 3D model quality assessment, intrinsic disorder prediction, domain prediction and ligand binding site prediction.

    Science.gov (United States)

    Roche, Daniel B; Buenavista, Maria T; Tetchner, Stuart J; McGuffin, Liam J

    2011-07-01

    The IntFOLD server is a novel independent server that integrates several cutting edge methods for the prediction of structure and function from sequence. Our guiding principles behind the server development were as follows: (i) to provide a simple unified resource that makes our prediction software accessible to all and (ii) to produce integrated output for predictions that can be easily interpreted. The output for predictions is presented as a simple table that summarizes all results graphically via plots and annotated 3D models. The raw machine readable data files for each set of predictions are also provided for developers, which comply with the Critical Assessment of Methods for Protein Structure Prediction (CASP) data standards. The server comprises an integrated suite of five novel methods: nFOLD4, for tertiary structure prediction; ModFOLD 3.0, for model quality assessment; DISOclust 2.0, for disorder prediction; DomFOLD 2.0 for domain prediction; and FunFOLD 1.0, for ligand binding site prediction. Predictions from the IntFOLD server were found to be competitive in several categories in the recent CASP9 experiment. The IntFOLD server is available at the following web site: http://www.reading.ac.uk/bioinf/IntFOLD/.

  13. Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    Directory of Open Access Journals (Sweden)

    Baptiste Monsion

    2018-02-01

    Full Text Available Double-stranded RNA (dsRNA plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.

  14. LIM-Only Protein 4 (LMO4 and LIM Domain Binding Protein 1 (LDB1 Promote Growth and Metastasis of Human Head and Neck Cancer (LMO4 and LDB1 in Head and Neck Cancer.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Simonik

    Full Text Available Squamous cell carcinoma of the head and neck (HNSCC accounts for more than 300,000 deaths worldwide per year as a consequence of tumor cell invasion of adjacent structures or metastasis. LIM-only protein 4 (LMO4 and LIM-domain binding protein 1 (LDB1, two directly interacting transcriptional adaptors that have important roles in normal epithelial cell differentiation, have been associated with increased metastasis, decreased differentiation, and shortened survival in carcinoma of the breast. Here, we implicate two LDB1-binding proteins, single-stranded binding protein 2 (SSBP2 and 3 (SSBP3, in controlling LMO4 and LDB1 protein abundance in HNSCC and in regulating specific tumor cell functions in this disease. First, we found that the relative abundance of LMO4, LDB1, and the two SSBPs correlated very significantly in a panel of human HNSCC cell lines. Second, expression of these proteins in tumor primaries and lymph nodes involved by metastasis were concordant in 3 of 3 sets of tissue. Third, using a Matrigel invasion and organotypic reconstruct assay, CRISPR/Cas9-mediated deletion of LDB1 in the VU-SCC-1729 cell line, which is highly invasive of basement membrane and cellular monolayers, reduced tumor cell invasiveness and migration, as well as proliferation on tissue culture plastic. Finally, inactivation of the LDB1 gene in these cells decreased growth and vascularization of xenografted human tumor cells in vivo. These data show that LMO4, LDB1, and SSBP2 and/or SSBP3 regulate metastasis, proliferation, and angiogenesis in HNSCC and provide the first evidence that SSBPs control LMO4 and LDB1 protein abundance in a cancer context.

  15. Dystrophin contains multiple independent membrane-binding domains.

    Science.gov (United States)

    Zhao, Junling; Kodippili, Kasun; Yue, Yongping; Hakim, Chady H; Wasala, Lakmini; Pan, Xiufang; Zhang, Keqing; Yang, Nora N; Duan, Dongsheng; Lai, Yi

    2016-09-01

    Dystrophin is a large sub-sarcolemmal protein. Its absence leads to Duchenne muscular dystrophy (DMD). Binding to the sarcolemma is essential for dystrophin to protect muscle from contraction-induced injury. It has long been thought that membrane binding of dystrophin depends on its cysteine-rich (CR) domain. Here, we provide in vivo evidence suggesting that dystrophin contains three additional membrane-binding domains including spectrin-like repeats (R)1-3, R10-12 and C-terminus (CT). To systematically study dystrophin membrane binding, we split full-length dystrophin into ten fragments and examined subcellular localizations of each fragment by adeno-associated virus-mediated gene transfer. In skeletal muscle, R1-3, CR domain and CT were exclusively localized at the sarcolemma. R10-12 showed both cytosolic and sarcolemmal localization. Importantly, the CR-independent membrane binding was conserved in murine and canine muscles. A critical function of the CR-mediated membrane interaction is the assembly of the dystrophin-associated glycoprotein complex (DGC). While R1-3 and R10-12 did not restore the DGC, surprisingly, CT alone was sufficient to establish the DGC at the sarcolemma. Additional studies suggest that R1-3 and CT also bind to the sarcolemma in the heart, though relatively weak. Taken together, our study provides the first conclusive in vivo evidence that dystrophin contains multiple independent membrane-binding domains. These structurally and functionally distinctive membrane-binding domains provide a molecular framework for dystrophin to function as a shock absorber and signaling hub. Our results not only shed critical light on dystrophin biology and DMD pathogenesis, but also provide a foundation for rationally engineering minimized dystrophins for DMD gene therapy. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA Proteins Alter Amyloid-β Precursor Protein (APP Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1.

    Directory of Open Access Journals (Sweden)

    Bjoern von Einem

    Full Text Available Proteolytic processing of amyloid-β precursor protein (APP by beta-site APP cleaving enzyme 1 (BACE1 is the initial step in the production of amyloid beta (Aβ, which accumulates in senile plaques in Alzheimer's disease (AD. Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα, sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.

  17. Modular protein domains

    National Research Council Canada - National Science Library

    Cesareni, Giovanni

    2005-01-01

    ... encodes not only sequence, but somehow explicitly specifies folding, structure, and biological function as well. How, then, can one learn to read this 'language of proteins'? One of the most powerful approaches to 'cracking the protein code' has involved sequence comparisons between and within species, a task now greatly simplified by the ever...

  18. A large-scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand-binding domains of taste receptor.

    Science.gov (United States)

    Yamashita, Atsuko; Nango, Eriko; Ashikawa, Yuji

    2017-11-01

    Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo- or heteromeric multimer protein complexes. Successful recombinant production of such proteins is often achieved by co-expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large-scale expression of hetero-multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high-expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large-scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes. © 2017 The Protein Society.

  19. Engineering bispecificity into a single albumin-binding domain.

    Directory of Open Access Journals (Sweden)

    Johan Nilvebrant

    Full Text Available Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-α (TNF-α was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-α as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-α and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-α was analyzed. This analysis revealed an affinity for TNF-α below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-α and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed.

  20. The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity.

    Science.gov (United States)

    Henke, Sarah K; Cronan, John E

    2016-11-01

    Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (Soares da Costa et al. (2014) Mol Microbiol 91: 110-120). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity. © 2016 John Wiley & Sons Ltd.

  1. Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity

    OpenAIRE

    Chen, M; Sun, S; Chan, CCS; Zhao, G; Du, L; Jiang, S; Zheng, BJ; Zhou, Y; He, Y; Guo, H; Liu, Z

    2009-01-01

    Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease. The potential recurrence of the disease from animal reservoirs highlights the significance of development of safe and efficient vaccines to prevent a future SARS epidemic. In this study, we expressed the recombinant receptor-binding domain (rRBD) in mammalian (293T) cells, insect (Sf9) cells, and E. coli, respectively, and compared their immunogenicity and protection against SARS-CoV infection in an established m...

  2. Use of a collagen membrane loaded with recombinant human bone morphogenetic protein-2 with collagen-binding domain for vertical guided bone regeneration.

    Science.gov (United States)

    Lai, Chun-Hua; Zhou, Lei; Wang, Zhong-Lei; Lu, Hai-Bin; Gao, Yan

    2013-07-01

    Vertical bone regeneration of severe atrophic alveolar ridges remains a challenging procedure in implant dentistry. The aim of this study, accordingly, is to use a rabbit vertical guided bone regeneration model to evaluate whether using a collagen membrane (CM) loaded with small doses of recombinant human bone morphogenetic protein-2 with collagen-binding domain (rhBMP-2/CBD) would enhance two-way vertical bone regeneration. In each of eight rabbits, four titanium cylinders were screwed in perforated slits made into the external cortical bones of the calvaria. The following four treatment modalities were randomly allocated: 1) cylinders filled with mineralized bone matrix and covered with CM/rhBMP-2/CBD; 2) cylinders filled with mineralized bone matrix and covered with CM/rhBMP-2; 3) cylinders filled with mineralized bone matrix and covered with CM alone; or 4) cylinders filled with mineralized bone matrix without a membrane cover. After 6 weeks, the new bones were examined by histologic analysis. Slender new bone trabeculae were observed in the superficial layer of the titanium cylinders covered with CM/rhBMP-2/CBD, and higher degrees of bone were observed in this group compared with the other three groups. The average area fraction of newly formed bone was significantly more in the CM/rhBMP-2/CBD group compared with the CM/rhBMP-2, CM, or the no membrane control groups (all P bone formation not only from the surface of the native bone, but also from the superficial structures. The augmented new bone, therefore, is improved in both quantity and quality.

  3. The S-layer protein of Lactobacillus acidophilus ATCC 4356 : identification and characterisation of domains responsible for S-protein assembly and cell wall binding

    NARCIS (Netherlands)

    Smit, E.; Oling, F.; Demel, R.; Martinez, B.; Pouwels, P.H.

    2001-01-01

    Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (SA-protein) of 43 kDa. SA-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net

  4. The S-layer protein of Lactobacillus acidophilus ATCC 4356 : Identification and characterisation of domains responsible for S-protein assembly and cell wall binding

    NARCIS (Netherlands)

    Smit, E; Oling, F; Demel, R; Martinez, B; Pouwels, PH

    2001-01-01

    Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S-A-protein) of 43 kDa. S-A-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net

  5. Helical propensity in an intrinsically disordered protein accelerates ligand binding

    DEFF Research Database (Denmark)

    Iesmantavicius, Vytautas; Dogan, Jakob; Jemth, Per

    2014-01-01

    domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure......Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well-ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation...... the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins....

  6. Complementation between HIV integrase proteins mutated in different domains

    NARCIS (Netherlands)

    D.C. van Gent (Dik); C. Vink (Cornelis); A.A. Groeneger; R.H. Plassterk

    1993-01-01

    textabstractHIV integrase (IN) cleaves two nucleotides off the 3' end of viral DNA and integrates viral DNA into target DNA. Previously, three functional domains in the HIV IN protein have been identified: (i) the central catalytic domain, (ii) the C-terminal DNA binding domain,

  7. Protein binding of psychotropic agents

    International Nuclear Information System (INIS)

    Hassan, H.A.

    1990-01-01

    Based upon fluorescence measurements, protein binding of some psychotropic agents (chlorpromazine, promethazine, and trifluoperazine) to human IgG and HSA was studied in aqueous cacodylate buffer, PH7. The interaction parameters determined from emission quenching of the proteins. The interaction parameters determined include the equilibrium constant (K), calculated from equations derived by Borazan and coworkers, the number of binding sites (n) available to the monomer molecules on a single protein molecule. The results revealed a high level of affinity, as reflected by high values of K, and the existence of specific binding sites, since a limited number of n values are obtained. 39 tabs.; 37 figs.; 83 refs

  8. Structural and functional analysis of the YAP-binding domain of human TEAD2

    OpenAIRE

    Tian, Wei; Yu, Jianzhong; Tomchick, Diana R.; Pan, Duojia; Luo, Xuelian

    2010-01-01

    The Hippo pathway controls organ size and suppresses tumorigenesis in metazoans by blocking cell proliferation and promoting apoptosis. The TEAD1-4 proteins (which contain a DNA-binding domain but lack an activation domain) interact with YAP (which lacks a DNA-binding domain but contains an activation domain) to form functional heterodimeric transcription factors that activate proliferative and prosurvival gene expression programs. The Hippo pathway inhibits the YAP-TEAD hybrid transcription ...

  9. A specific interdomain interaction preserves the structural and binding properties of the ModA protein from the phytopathogen Xanthomonas citri domain interaction and transport in ModA.

    Science.gov (United States)

    Santacruz-Perez, Carolina; Pegos, Vanessa Rodrigues; Honorato, Rodrigo V; Verli, Hugo; Lindahl, Erik; Barbosa, João Alexandre Ribeiro Gonçalves; Balan, Andrea

    2013-11-01

    The periplasmic-binding proteins in ATP-binding cassette systems (ABC Transporters) are responsible for the capture and delivery of ligands to their specific transporters, triggering a series of ATP-driven conformational changes that leads to the transport of the ligand. Structurally consisting of two lobes, the proteins change conformation after interaction with the ligand. The structure of the molybdate-binding protein (ModA) from Xanthomonas citri, bound to molybdate, was previously solved by our group and an interdomain interaction, mediated by a salt bridge between K127 and D59, apparently supports the binding properties and keeps the domains closed. To determinate the importance of this interaction, we built two ModA mutants, K127S and D59A, and analysed their functional and structural properties. Based on a set of spectroscopic experiments, crystallisation trials, structure determination and molecular dynamics (MD) simulations, we showed that the salt bridge is essential to maintain the structure and binding properties. Additionally, the MD simulations revealed that this mutant adopted a more compact structure that packed down the ligand-binding pocket. From the closed bound to open structure, the positioning of the helices forming the dipole and the salt bridge are essential to induce an intermediate state. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Crystal Structure of the Ubiquitin-like Domain-CUT Repeat-like Tandem of Special AT-rich Sequence Binding Protein 1 (SATB1) Reveals a Coordinating DNA-binding Mechanism*

    Science.gov (United States)

    Wang, Zheng; Yang, Xue; Guo, Shuang; Yang, Yin; Su, Xun-Cheng; Shen, Yuequan; Long, Jiafu

    2014-01-01

    SATB1 is essential for T-cell development and growth and metastasis of multitype tumors and acts as a global chromatin organizer and gene expression regulator. The DNA binding ability of SATB1 plays vital roles in its various biological functions. We report the crystal structure of the N-terminal module of SATB1. Interestingly, this module contains a ubiquitin-like domain (ULD) and a CUT repeat-like (CUTL) domain (ULD-CUTL tandem). Detailed biochemical experiments indicate that the N terminus of SATB1 (residues 1–248, SATB1(1–248)), including the extreme 70 N-terminal amino acids, and the ULD-CUTL tandem bind specifically to DNA targets. Our results show that the DNA binding ability of full-length SATB1 requires the contribution of the CUTL domain, as well as the CUT1-CUT2 tandem domain and the homeodomain. These findings may reveal a multiple-domain-coordinated mechanism whereby SATB1 recognizes DNA targets. PMID:25124042

  11. Crystal structure of the ubiquitin-like domain-CUT repeat-like tandem of special AT-rich sequence binding protein 1 (SATB1) reveals a coordinating DNA-binding mechanism.

    Science.gov (United States)

    Wang, Zheng; Yang, Xue; Guo, Shuang; Yang, Yin; Su, Xun-Cheng; Shen, Yuequan; Long, Jiafu

    2014-10-03

    SATB1 is essential for T-cell development and growth and metastasis of multitype tumors and acts as a global chromatin organizer and gene expression regulator. The DNA binding ability of SATB1 plays vital roles in its various biological functions. We report the crystal structure of the N-terminal module of SATB1. Interestingly, this module contains a ubiquitin-like domain (ULD) and a CUT repeat-like (CUTL) domain (ULD-CUTL tandem). Detailed biochemical experiments indicate that the N terminus of SATB1 (residues 1-248, SATB1((1-248))), including the extreme 70 N-terminal amino acids, and the ULD-CUTL tandem bind specifically to DNA targets. Our results show that the DNA binding ability of full-length SATB1 requires the contribution of the CUTL domain, as well as the CUT1-CUT2 tandem domain and the homeodomain. These findings may reveal a multiple-domain-coordinated mechanism whereby SATB1 recognizes DNA targets. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. A new method for measuring scouring efficiency of natural fibers based on the cellulose-binding domain-beta-glucuronidase fused protein.

    Science.gov (United States)

    Degani, Ofir; Gepstein, Shimon; Dosoretz, Carlos G

    2004-02-05

    Cellulose-binding domains (CBDs) are characterized by their ability to strongly bind to different forms of cellulose. This study examined the use of a recombinant CBD fused to the reporter enzyme beta-glucuronidase (CBD-GUS) to determine the extent of removal of the water-repellent waxy component of cotton fiber cuticles following hydrolytic treatment, i.e., scouring. The CBD-GUS test displayed higher sensitivity and repeatability than conventional water absorb techniques applied in the textile industry. Increases in the levels of CBD-GUS bound to the exposed cellulose correlated to increases in the fabric's hydrophilicity as a function of the severity of the scouring treatment applied, clearly indicating that the amount of bound enzyme increases proportionally with the amount of available binding sites. The binding of CBD-GUS also gave measurable and repeatable results when used on untreated or raw fabrics in comparison with conventional water drop techniques. The quantitative response of the reaction as bound enzyme activity was optimized for fully wettable fabrics. A minimal free enzyme concentration-to-swatch weight ratio of 75:1 was found to be necessary to ensure enzyme saturation (i.e., a linear response), corresponding to a free enzyme-to-bound enzyme ratio of at least 3:5.

  13. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C. (Scripps); (UW)

    2011-11-02

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  14. Two domains of RD3 antifreeze protein diffuse independently.

    Science.gov (United States)

    Holland, Nolan B; Nishimiya, Yoshiyuki; Tsuda, Sakae; Sönnichsen, Frank D

    2008-06-03

    Antifreeze proteins (AFPs) make up a class of structurally diverse proteins that help to protect many organisms from freezing temperatures by inhibiting ice crystal growth at temperatures below the colligative freezing point. AFPs are typically small proteins with a relatively flat, slightly hydrophobic binding region that matches the lattice structure of a specific ice crystal plane. The only known two-domain AFP is RD3 from the Antarctic eel pout. It consists of two nearly identical type III domains connected by a nine-residue linker. This protein exhibits higher activity than the single-domain protein at low concentrations. The initial solution structure of RD3 revealed that the domains were aligned so that the binding regions were nearly coplanar, effectively doubling the surface area for binding. A more recent report suggests that the domains may not be aligned in solution but rather diffuse independently. To resolve the issue, we have measured the NMR residual dipolar couplings using alignment media of stretched gels and filamentous phage to determine the relative orientation of the domains. We find that the two domains of RD3 are free to move relative to each other, within the constraint of the flexible nine-residue linker. Our data show that there is no strongly preferred alignment in solution. Furthermore, the flexibility and length of the linker are sufficient to allow the two domains to have their binding faces in the same orientation and coplanar for simultaneous binding to an ice crystal surface.

  15. binding protein (HABP1)

    Indian Academy of Sciences (India)

    Unknown

    adsorbed on carbon coated copper grid (400 mesh) for. 5 min at room temperature. The grids were subsequently .... and inhibition by GAGs and DMA were determined on polystyrene wells of microtitre plates (Costar, ... for binding inhibition assays was carried out by mixing equal volumes of the conjugate and the inhibitor at ...

  16. binding protein (HABP1)

    Indian Academy of Sciences (India)

    Unknown

    of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity ... site is seen to correspond to the carbohydrate-binding site in E-selectin, which has similarity in the ... adsorbed on carbon coated copper grid (400 mesh) for. 5 min at room temperature.

  17. An alternative function of C-type lectin comprising low-density lipoprotein receptor domain from Fenneropenaeus merguiensis to act as a binding receptor for viral protein and vitellogenin.

    Science.gov (United States)

    Kwankaew, Pattamaporn; Praparatana, Rachanida; Runsaeng, Phanthipha; Utarabhand, Prapaporn

    2018-03-01

    A diversity of C-type lectins (CTLs) was coming reported and they are known to participate in invertebrate innate immunity by act as pattern recognition receptor (PRR). In the present study, a unique CTL containing low-density lipoprotein receptor (LDLR) domain from Fenneropenaeus merguiensis (designated as FmLdlr) was cloned. Its sequence contained a single LDLR domain and one carbohydrate recognition domain (CRD) with a QAP motif putative for galactose-specific binding. The expression of FmLdlr was detected only in hemocytes of healthy shrimp. Its expression was significantly up-regulated by Vibrio parahaemolyticus or white spot syndrome virus (WSSV) challenge. The knockdown by FmLdlr dsRNA resulted in severe gene down-regulation. The gene silencing with pathogenic co-inoculation led to reduction of the median lethal time and increasing in the cumulative mortality including the remained WSSV in WSSV co-challenge group. Recombinant proteins of FmLdlr and two domains could agglutinate various bacterial strains which LDLR domain revealed the lowest activity. Only FmLdlr and CRD could enhance phagocytosis and encapsulation by hemocytes. Both FmLdlr and CRD except LDLR domain exhibited the antibacterial activity by inhibiting the growth of pathogenic V. parahaemolyticus in cultured medium and disk diffusion assay. Only FmLdlr and CRD could bind to WSSV proteins, envelope VP28, tegument VP39A and also capsid VP15, which FmLdlr had the higher binding affinity than that of CRD. Altogether, we concluded that FmLdlr contributed in shrimp immune defense through the main action of CRD in capable of bacterial agglutination, enhancing the phagocytosis and encapsulation, antimicrobial activity and binding to viral proteins. Interestingly, ELISA approach revealed that LDLR domain displayed the highest binding affinity to vitellogenin than whole molecule and CRD. We signified a new function of FmLdlr that it might presumably act as a receptor for vitellogenin transportation in

  18. Role of exposed aromatic residues in substrate-binding of CBM family 5 chitin-binding domain of alkaline chitinase.

    Science.gov (United States)

    Uni, Fumiya; Lee, Sunmi; Yatsunami, Rie; Fukui, Toshiaki; Nakamura, Satoshi

    2009-01-01

    Chitinase J (ChiJ) from alkaliphilic Bacillus sp. strain J813 has a multidomain structure containing a catalytic domain (CatD), a fibronectin type III like domain (FnIIID) and a chitin-binding domain (ChBD). It has been shown that the ChBD binds to an insoluble chitin and enhances its degradation by the CatD. Further binding study of the ChBD was performed with a glutathione-S-transferase fusion protein. This fusion protein showed binding abilities to insoluble chitin and chitosan. Two surface-exposed aromatic residues (Trp541 and Trp542) were found in the tertiary-structure model of ChBD and targeted for mutational analysis. Single and double mutations of the two aromatic residues decreased the chitin- and chitosan-binding abilities. It was revealed that these residues would be important for substrate-binding of the ChBD.

  19. Computational studies on receptor-ligand interactions between novel buffalo (Bubalus bubalis) nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants and muramyl dipeptide (MDP).

    Science.gov (United States)

    Brahma, Biswajit; Patra, Mahesh Chandra; Mishra, Purusottam; De, Bidhan Chandra; Kumar, Sushil; Maharana, Jitendra; Vats, Ashutosh; Ahlawat, Sonika; Datta, Tirtha Kumar; De, Sachinandan

    2016-04-01

    Nucleotide binding and oligomerization domain 2 (NOD2), a member of intracellular NOD-like receptors (NLRs) family, recognizes the bacterial peptidoglycan, muramyl dipeptide (MDP) and initiates host immune response. The precise ligand recognition mechanism of NOD2 has remained elusive, although studies have suggested leucine rich repeat (LRR) region of NOD2 as the possible binding site of MDP. In this study, we identified multiple transcripts of NOD2 gene in buffalo (buNOD2) and at least five LRR variants (buNOD2-LRRW (wild type), buNOD2-LRRV1-V4) were found to be expressed in buffalo peripheral blood mononuclear cells. The newly identified buNOD2 transcripts were shorter in lengths as a result of exon-skipping and frame-shift mutations. Among the variants, buNOD2-LRRW, V1, and V3 were expressed more frequently in the animals studied. A comparative receptor-ligand interaction study through modeling of variants, docking, and molecular dynamics simulation revealed that the binding affinity of buNOD2-LRRW towards MDP was greater than that of the shorter variants. The absence of a LRR segment in the buNOD2 variants had probably affected their affinity toward MDP. Notwithstanding a high homology among the variants, the amino acid residues that interact with MDP were located on different LRR motifs. The binding free energy calculation revealed that the amino acids Arg850(LRR4) and Glu932(LRR7) of buNOD2-LRRW, Lys810(LRR3) of buNOD2-LRRV1, and Lys830(LRR3) of buNOD2-LRRV3 largely contributed towards MDP recognition. The knowledge of MDP recognition and binding modes on buNOD2 variants could be useful to understand the regulation of NOD-mediated immune response as well as to develop next generation anti-inflammatory compounds. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Understanding the role of BAR and SH3 domain-containing proteins in fungi

    NARCIS (Netherlands)

    Gkourtsa, A.

    2017-01-01

    This thesis addresses the role of SH3 and BAR domain-containing proteins in different fungal species. SH3 domains are small modules that mediate protein-protein interactions and BAR domains are dimerization domains with membrane binding and bending properties. It is known that the ScRvs167 protein

  1. Segmental, Domain-Selective Perdeuteration and Small-Angle Neutron Scattering for Structural Analysis of Multi-Domain Proteins.

    Science.gov (United States)

    Sonntag, Miriam; Jagtap, Pravin Kumar Ankush; Simon, Bernd; Appavou, Marie-Sousai; Geerlof, Arie; Stehle, Ralf; Gabel, Frank; Hennig, Janosch; Sattler, Michael

    2017-08-01

    Multi-domain proteins play critical roles in fine-tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi-domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA-1 using Sortase A mediated protein ligation. We show that domain-selective perdeuteration combined with contrast-matched small-angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi-domain proteins and changes induced by ligand binding. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Protein phosphatase 2a (PP2A binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3

    Directory of Open Access Journals (Sweden)

    Jones Candace A

    2011-10-01

    Full Text Available Abstract Background Striatin, a putative protein phosphatase 2A (PP2A B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM, which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3 protein, the mammalian Mps one binder (MOB homolog, Mob3/phocein, the mammalian sterile 20-like (Mst kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases. Results To help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3. Conclusions Striatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via

  3. Phosphorylation of the chromatin binding domain of KSHV LANA.

    Directory of Open Access Journals (Sweden)

    Crystal Woodard

    Full Text Available The Kaposi sarcoma associated herpesvirus (KSHV latency associated nuclear antigen (LANA is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1-329 that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3-21. Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.

  4. The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

    Science.gov (United States)

    Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

    2014-10-10

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    Directory of Open Access Journals (Sweden)

    Marc Lenoir

    2015-10-01

    Full Text Available The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH and Tec homology (TH domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  6. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    Science.gov (United States)

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-10-23

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  7. A KH-Domain RNA-Binding Protein Interacts with FIERY2/CTD Phosphatase-Like 1 and Splicing Factors and Is Important for Pre-mRNA Splicing in Arabidopsis

    KAUST Repository

    Chen, Tao

    2013-10-17

    Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH)-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5), as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II) carboxyl terminal domain (CTD) phosphatase-like 1 (FRY2/CPL1) both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR)-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In fry2 mutants, HOS5 failed to be localized in nuclear speckles but was found mainly in the nucleoplasm. hos5 mutants were impaired in mRNA export and accumulated a significant amount of mRNA in the nuclei, particularly under salt stress conditions. Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges. © 2013 Chen et al.

  8. A KH-domain RNA-binding protein interacts with FIERY2/CTD phosphatase-like 1 and splicing factors and is important for pre-mRNA splicing in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Tao Chen

    Full Text Available Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5, as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II carboxyl terminal domain (CTD phosphatase-like 1 (FRY2/CPL1 both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In fry2 mutants, HOS5 failed to be localized in nuclear speckles but was found mainly in the nucleoplasm. hos5 mutants were impaired in mRNA export and accumulated a significant amount of mRNA in the nuclei, particularly under salt stress conditions. Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges.

  9. Novel mutation in ATP-binding domain of ABCD1 gene in ...

    Indian Academy of Sciences (India)

    TMD) and nucleotide binding domain (NBD). (b) Multiple protein sequence alignment of ALDP of various species indicating conserved nature of arginine at position 617 in Homo sapiens. (c) Comparisons of native and mutated models of ALDP.

  10. A minimum of three motifs is essential for optimal binding of pseudomurein cell wall-binding domain of Methanothermobacter thermautotrophicus.

    Directory of Open Access Journals (Sweden)

    Ganesh Ram R Visweswaran

    Full Text Available We have biochemically and functionally characterized the pseudomurein cell wall-binding (PMB domain that is present at the C-terminus of the Surface (S-layer protein MTH719 from Methanothermobacter thermautotrophicus. Chemical denaturation of the protein with guanidinium hydrochloride occurred at 3.8 M. A PMB-GFP fusion protein not only binds to intact pseudomurein of methanogenic archaea, but also to spheroplasts of lysozyme-treated bacterial cells. This binding is pH dependent. At least two of the three motifs that are present in the domain are necessary for binding. Limited proteolysis revealed a possible cleavage site in the spacing sequence between motifs 1 and 2 of the PMB domain, indicating that the motif region itself is protected from proteases.

  11. In vitro and in vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B type I (SR-BI) to the PDZ1 Domain of its Cytoplasmic Adaptor Protein PDZK1

    Energy Technology Data Exchange (ETDEWEB)

    O Kocher; G Birrane; K Tsukamoto; S Fenske; A Yesilaltay; R Pal; K Daniels; J Ladias; M Krieger

    2011-12-31

    The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 ?M, respectively, similar to 2.6 ?M measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.

  12. Src binds cortactin through an SH2 domain cystine-mediated linkage

    Science.gov (United States)

    Evans, Jason V.; Ammer, Amanda G.; Jett, John E.; Bolcato, Chris A.; Breaux, Jason C.; Martin, Karen H.; Culp, Mark V.; Gannett, Peter M.; Weed, Scott A.

    2012-01-01

    Summary Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions. PMID:23097045

  13. IgG antibodies to endothelial protein C receptor-binding Cysteine-rich interdomain region domains of Plasmodium falciparum erythrocyte membrane protein 1 are acquired early in life in individuals exposed to malaria

    DEFF Research Database (Denmark)

    Turner, Louise; Lavstsen, Thomas; Mmbando, Bruno P

    2015-01-01

    Severe malaria syndromes are precipitated by Plasmodium falciparum parasites binding to endothelial receptors on the vascular lining. This binding is mediated by members of the highly variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. We have previously identified a subset of Pf...

  14. The Inner Centromere Protein (INCENP) Coil Is a Single α-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis.

    Science.gov (United States)

    Samejima, Kumiko; Platani, Melpomeni; Wolny, Marcin; Ogawa, Hiromi; Vargiu, Giulia; Knight, Peter J; Peckham, Michelle; Earnshaw, William C

    2015-08-28

    The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ∼ 32-nm-long single α-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ∼ 80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible "dog leash," allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins

    Directory of Open Access Journals (Sweden)

    Cesareni Gianni

    2008-07-01

    Full Text Available Abstract Background The human POB1/REPS2 (Partner of RalBP1 protein is highly conserved in mammals where it has been suggested to function as a molecular scaffold recruiting proteins involved in vesicular traffic and linking them to the actin cytoskeleton remodeling machinery. More recently POB1/REPS2 was found highly expressed in androgen-dependent prostate cancer cell lines, while one of its isoforms (isoform 2 is down regulated during prostate cancer progression. Results In this report we characterize the central proline rich domain of POB1/REPS2 and we describe for the first time its functional role in receptor endocytosis. We show that the ectopic expression of this domain has a dominant negative effect on the endocytosis of activated epidermal growth factor receptor (EGFR while leaving transferrin receptor endocytosis unaffected. By a combination of different approaches (phage display, bioinformatics predictions, peptide arrays, mutagenic analysis, in vivo co-immunoprecipitation, we have identified two closely spaced binding motifs for 14-3-3 and for the SH3 of the proteins Amphiphysin II and Grb2. Differently from wild type, proline rich domains that are altered in these motifs do not inhibit EGFR endocytosis, suggesting that these binding motifs play a functional role in this process. Conclusion Our findings are relevant to the characterization of the molecular mechanism underlying the involvement of POB1/REPS2, SH3 and 14-3-3 proteins in receptor endocytosis, suggesting that 14-3-3 could work by bridging the EGF receptor and the scaffold protein POB1/REPS2.

  16. 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

    2016-01-01

    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

  17. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains

    DEFF Research Database (Denmark)

    Behrendt, N; Ronne, E; Dano, K

    1996-01-01

    The urokinase plasminogen activator receptor (uPAR) is a membrane protein comprised of three extracellular domains. In order to study the importance of this domain organization in the ligand-binding process of the receptor we subjected a recombinant, soluble uPAR (suPAR) to specific proteolytic...

  18. The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates.

    Science.gov (United States)

    Whitaker, Neal; Chen, Yuqing; Jakubowski, Simon J; Sarkar, Mayukh K; Li, Feng; Christie, Peter J

    2015-07-01

    Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD), and a sequence-variable helical bundle called the all-alpha domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Here, we defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer, whereas production of the AAD in otherwise wild-type donor strains diminished the transfer of cognate but not heterologous substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished the transfer of cognate DNA substrates, although strikingly, the VirD4-AADPcfC chimera (VirD4 with the PcfC AAD) supported the transfer of a mobilizable plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 and PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Our findings support a model in which the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. For conjugative transfer of mobile DNA elements, members of the VirD4/TraG/TrwB receptor superfamily bind cognate DNA substrates through mechanisms that are largely undefined. Here

  19. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, Thomas J.; Parker, Michael W.; (SVIMR-A); (Chugai); (Melbourne)

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  20. When is protein binding important?

    Science.gov (United States)

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. Copyright © 2013 Wiley Periodicals, Inc.

  1. Starch‐binding domains in the CBM45 family – low‐affinity domains from glucan, water dikinase and α‐amylase involved in plastidial starch metabolism

    DEFF Research Database (Denmark)

    Glaring, Mikkel Andreas; Baumann, Martin; Abou Hachem, Maher

    2011-01-01

    Starch‐binding domains are noncatalytic carbohydrate‐binding modules that mediate binding to granular starch. The starch‐binding domains from the carbohydrate‐binding module family 45 (CBM45, ) are found as N‐terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing...... organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45‐type domains, the Solanum tuberosumα‐glucan, water dikinase and the Arabidopsis thaliana plastidial α‐amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry...

  2. End binding proteins are obligatory dimers.

    Directory of Open Access Journals (Sweden)

    Indrani Sen

    Full Text Available End binding (EB proteins are responsible for the recruitment of an array of microtubule plus-end tracking proteins (+TIPs to growing microtubules ends. EBs encompass an N-terminal calponin homology domain that confers microtubule tip tracking activity to the protein. The C-terminal domain of EBs contains a coiled coil that mediates the parallel dimerization of EB monomers. This part of the protein is also responsible for partner binding. While dimerization is not essential for microtubule tip tracking by EBs it is a prerequisite for +TIP partner binding. The concentration of EBs in cells has been estimated to be in the range of hundreds of nanomoles. In contrast, in in vitro single molecule experiments EB concentrations of subnanomoles are employed. From a mechanistic point of view it is important to assess the oligomerization state of EBs at physiologically and experimentally relevant protein concentrations, in particular if the goal of a study is to model the behavior of EB-dependent dynamic +TIP networks. Here we have determined the stability of the EB1 and EB3 dimers using multi-angle light scattering and fluorescence analytical ultracentrifugation. We show that these EBs form stable dimers and do not dissociate even at very low nanomolar concentrations. The dimers remained stable at both room temperature as well as at the physiologically relevant temperature of 37°C. Together, our results reveal that EBs are obligatory dimers, a conclusion that has implications for the mechanistic understanding of these key proteins involved in the orchestration of dynamic protein networks at growing microtubule ends.

  3. A Fusion Protein of the p53 Transaction Domain and the p53-Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors.

    Science.gov (United States)

    Chen, Rong; Zhou, Jingjing; Qin, Lingyun; Chen, Yao; Huang, Yongqi; Liu, Huili; Su, Zhengding

    2017-06-27

    In nearly half of cancers, the anticancer activity of p53 protein is often impaired by the overexpressed oncoprotein Mdm2 and its homologue, MdmX, demanding efficient therapeutics to disrupt the aberrant p53-MdmX/Mdm2 interactions to restore the p53 activity. While many potent Mdm2-specific inhibitors have already undergone clinical investigations, searching for MdmX-specific inhibitors has become very attractive, requiring a more efficient screening strategy for evaluating potential scaffolds or leads. In this work, considering that the intrinsic fluorescence residue Trp23 in the p53 transaction domain (p53p) plays an important role in determining the p53-MdmX/Mdm2 interactions, we constructed a fusion protein to utilize this intrinsic fluorescence signal to monitor high-throughput screening of a compound library. The fusion protein was composed of the p53p followed by the N-terminal domain of MdmX (N-MdmX) through a flexible amino acid linker, while the whole fusion protein contained a sole intrinsic fluorescence probe. The fusion protein was then evaluated using fluorescence spectroscopy against model compounds. Our results revealed that the variation of the fluorescence signal was highly correlated with the concentration of the ligand within 65 μM. The fusion protein was further evaluated with respect to its feasibility for use in high-throughput screening using a model compound library, including controls. We found that the imidazo-indole scaffold was a bona fide scaffold for template-based design of MdmX inhibitors. Thus, the p53p-N-MdmX fusion protein we designed provides a convenient and efficient tool for high-throughput screening of new MdmX inhibitors. The strategy described in this work should be applicable for other protein targets to accelerate drug discovery.

  4. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    International Nuclear Information System (INIS)

    Roehrig, John T.; Butrapet, Siritorn; Liss, Nathan M.; Bennett, Susan L.; Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E.; Blair, Carol D.; Huang, Claire Y.-H.

    2013-01-01

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants

  5. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Roehrig, John T., E-mail: jtr1@cdc.gov [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Butrapet, Siritorn; Liss, Nathan M. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Bennett, Susan L. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  6. Activity of a bacterial cell envelope stress response is controlled by the interaction of a protein binding domain with different partners.

    Science.gov (United States)

    Flores-Kim, Josué; Darwin, Andrew J

    2015-05-01

    The bacterial phage shock protein (Psp) system is a highly conserved cell envelope stress response required for virulence in Yersinia enterocolitica and Salmonella enterica. In non-inducing conditions the transcription factor PspF is inhibited by an interaction with PspA. In contrast, PspA associates with the cytoplasmic membrane proteins PspBC during inducing conditions. This has led to the proposal that PspBC exists in an OFF state, which cannot recruit PspA, or an ON state, which can. However, nothing was known about the difference between these two states. Here, we provide evidence that it is the C-terminal domain of Y. enterocolitica PspC (PspC(CT)) that interacts directly with PspA, both in vivo and in vitro. Site-specific photocross-linking revealed that this interaction occurred only during Psp-inducing conditions in vivo. Importantly, we have also discovered that PspC(CT) can interact with the C-terminal domain of PspB (PspC(CT)·PspB(CT)). However, the PspC(CT)·PspB(CT) and PspC(CT)·PspA interactions were mutually exclusive in vitro. Furthermore, in vivo, PspC(CT) contacted PspB(CT) in the OFF state, whereas it contacted PspA in the ON state. These findings provide the first description of the previously proposed PspBC OFF and ON states and reveal that the regulatory switch is centered on a PspC(CT) partner-switching mechanism. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Directory of Open Access Journals (Sweden)

    Spaan Willy JM

    2009-05-01

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

  8. GTP binding to the ROC domain of DAP-kinase regulates its function through intramolecular signalling

    Science.gov (United States)

    Carlessi, Rodrigo; Levin-Salomon, Vered; Ciprut, Sara; Bialik, Shani; Berissi, Hanna; Albeck, Shira; Peleg, Yoav; Kimchi, Adi

    2011-01-01

    Death-associated protein kinase (DAPk) was recently suggested by sequence homology to be a member of the ROCO family of proteins. Here, we show that DAPk has a functional ROC (Ras of complex proteins) domain that mediates homo-oligomerization and GTP binding through a defined P-loop motif. Upon binding to GTP, the ROC domain negatively regulates the catalytic activity of DAPk and its cellular effects. Mechanistically, GTP binding enhances an inhibitory autophosphorylation at a distal site that suppresses kinase activity. This study presents a new mechanism of intramolecular signal transduction, by which GTP binding operates in cis to affect the catalytic activity of a distal domain in the protein. PMID:21738225

  9. Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

    Science.gov (United States)

    Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

    2016-11-01

    Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

  10. Inferring domain-domain interactions from protein-protein interactions with formal concept analysis.

    Directory of Open Access Journals (Sweden)

    Susan Khor

    Full Text Available Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where the score of a domain-pair is the ratio between observed and expected frequencies because the protein-protein interaction network is sparse. As such, many protein-pairs will be non-interacting and domain-pairs with promiscuous domains will be penalized. This domain promiscuity challenge to the problem of inferring reliable domain-domain interactions from protein-protein interactions has been recognized, and a number of work-arounds have been proposed. This paper reports on an application of Formal Concept Analysis to this problem. It is found that the relationship between formal concepts provides a natural way for rare domains to elevate the rank of promiscuous domain-pairs and enrich highly ranked domain-pairs with reliable domain-domain interactions. This piggybacking of promiscuous domain-pairs onto less promiscuous domain-pairs is possible only with concept lattices whose attribute-labels are not reduced and is enhanced by the presence of proteins that comprise both promiscuous and rare domains.

  11. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Varnum, Susan M.

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

  12. Accurate prediction of peptide binding sites on protein surfaces.

    Directory of Open Access Journals (Sweden)

    Evangelia Petsalaki

    2009-03-01

    Full Text Available Many important protein-protein interactions are mediated by the binding of a short peptide stretch in one protein to a large globular segment in another. Recent efforts have provided hundreds of examples of new peptides binding to proteins for which a three-dimensional structure is available (either known experimentally or readily modeled but where no structure of the protein-peptide complex is known. To address this gap, we present an approach that can accurately predict peptide binding sites on protein surfaces. For peptides known to bind a particular protein, the method predicts binding sites with great accuracy, and the specificity of the approach means that it can also be used to predict whether or not a putative or predicted peptide partner will bind. We used known protein-peptide complexes to derive preferences, in the form of spatial position specific scoring matrices, which describe the binding-site environment in globular proteins for each type of amino acid in bound peptides. We then scan the surface of a putative binding protein for sites for each of the amino acids present in a peptide partner and search for combinations of high-scoring amino acid sites that satisfy constraints deduced from the peptide sequence. The method performed well in a benchmark and largely agreed with experimental data mapping binding sites for several recently discovered interactions mediated by peptides, including RG-rich proteins with SMN domains, Epstein-Barr virus LMP1 with TRADD domains, DBC1 with Sir2, and the Ago hook with Argonaute PIWI domain. The method, and associated statistics, is an excellent tool for predicting and studying binding sites for newly discovered peptides mediating critical events in biology.

  13. The Polypyrimidine Tract-Binding Protein Affects Coronavirus RNA Accumulation Levels and Relocalizes Viral RNAs to Novel Cytoplasmic Domains Different from Replication-Transcription Sites ▿

    Science.gov (United States)

    Sola, Isabel; Galán, Carmen; Mateos-Gómez, Pedro A.; Palacio, Lorena; Zúñiga, Sonia; Cruz, Jazmina L.; Almazán, Fernando; Enjuanes, Luis

    2011-01-01

    The coronavirus (CoV) discontinuous transcription mechanism is driven by long-distance RNA-RNA interactions between transcription-regulating sequences (TRSs) located at the 5′ terminal leader (TRS-L) and also preceding each mRNA-coding sequence (TRS-B). The contribution of host cell proteins to CoV transcription needs additional information. Polypyrimidine tract-binding protein (PTB) was reproducibly identified in association with positive-sense RNAs of transmissible gastroenteritis coronavirus (TGEV) TRS-L and TRS-B by affinity chromatography and mass spectrometry. A temporal regulation of PTB cytoplasmic levels was observed during infection, with a significant increase from 7 to 16 h postinfection being inversely associated with a decrease in viral replication and transcription. Silencing the expression of PTB with small interfering RNA in two cell lines (Huh7 and HEK 293T) led to a significant increase of up to 4-fold in mRNA levels and virus titer, indicating a negative effect of PTB on CoV RNA accumulation. During CoV infection, PTB relocalized from the nucleus to novel cytoplasmic structures different from replication-transcription sites in which stress granule markers T-cell intracellular antigen-1 (TIA-1) and TIA-1-related protein (TIAR) colocalized. PTB was detected in these modified stress granules in TGEV-infected swine testis cells but not in stress granules induced by oxidative stress. Furthermore, viral genomic and subgenomic RNAs were detected in association with PTB and TIAR. These cytoplasmic ribonucleoprotein complexes might be involved in posttranscriptional regulation of virus gene expression. PMID:21411518

  14. Modeling protein network evolution under genome duplication and domain shuffling

    Directory of Open Access Journals (Sweden)

    Isambert Hervé

    2007-11-01

    Full Text Available Abstract Background Successive whole genome duplications have recently been firmly established in all major eukaryote kingdoms. Such exponential evolutionary processes must have largely contributed to shape the topology of protein-protein interaction (PPI networks by outweighing, in particular, all time-linear network growths modeled so far. Results We propose and solve a mathematical model of PPI network evolution under successive genome duplications. This demonstrates, from first principles, that evolutionary conservation and scale-free topology are intrinsically linked properties of PPI networks and emerge from i prevailing exponential network dynamics under duplication and ii asymmetric divergence of gene duplicates. While required, we argue that this asymmetric divergence arises, in fact, spontaneously at the level of protein-binding sites. This supports a refined model of PPI network evolution in terms of protein domains under exponential and asymmetric duplication/divergence dynamics, with multidomain proteins underlying the combinatorial formation of protein complexes. Genome duplication then provides a powerful source of PPI network innovation by promoting local rearrangements of multidomain proteins on a genome wide scale. Yet, we show that the overall conservation and topology of PPI networks are robust to extensive domain shuffling of multidomain proteins as well as to finer details of protein interaction and evolution. Finally, large scale features of direct and indirect PPI networks of S. cerevisiae are well reproduced numerically with only two adjusted parameters of clear biological significance (i.e. network effective growth rate and average number of protein-binding domains per protein. Conclusion This study demonstrates the statistical consequences of genome duplication and domain shuffling on the conservation and topology of PPI networks over a broad evolutionary scale across eukaryote kingdoms. In particular, scale

  15. ATP binding to p97/VCP D1 domain regulates selective recruitment of adaptors to its proximal N-domain.

    Directory of Open Access Journals (Sweden)

    Wei Sheng Chia

    Full Text Available p97/Valosin-containing protein (VCP is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD. It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.

  16. Mutation of the elongin C binding domain of human respiratory syncytial virus non-structural protein 1 (NS1 results in degradation of NS1 and attenuation of the virus

    Directory of Open Access Journals (Sweden)

    Headlam Madeleine J

    2011-05-01

    Full Text Available Abstract Background Human respiratory syncytial virus (RSV is an important cause of lower respiratory tract disease in the paediatic population, immunocompromised individuals and the elderly worldwide. However, despite global efforts over the past several decades there are no commercially available vaccines. RSV encodes 2 non-structural proteins, NS1 and NS2, that are type I interferon antagonists. RSV restricts type I interferon signaling and the expression of antiviral genes by degrading STAT2. It has been proposed that NS1 binds to elongin C to form a ubiquitin ligase (E3 complex that targets STAT2 for ubiquitination and proteosomal degradation. Results Here, we have engineered a live recombinant RSV in which the 3 consensus amino acids of the NS1 elongin C binding domain have been replaced with alanine (NS1F-ELCmut. Mutation of this region of NS1 resulted in attenuation of RSV replication in A549 cells to levels similar to that observed when the NS1 gene is completely deleted (ΔNS1. This mutation also resulted in moderate attenuation in Vero cells. Attenuation was correlated to intracellular degradation of the mutated NS1 protein. Time course analysis showed that mutant NS1 protein accumulated in cytoplasmic bodies that contained the lysosomal marker LAMP1. However lack of cleavage of LC3 suggested that autophagy was not involved. Induction of IFN-β mRNA expression also was observed in association with the degradation of NS1 protein and attenuation of viral growth. Conclusions These results indicate that the elongin C binding region of NS1 is crucial for survival of the protein and that disruption of this region results in the degradation of NS1 and restriction of RSV replication.

  17. The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Lorentsen, R H; Jacobsen, C

    1998-01-01

    Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon...

  18. SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

    Science.gov (United States)

    Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

    2016-10-20

    RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

    2005-01-01

    to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...... to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of (125)I-labeled folate binding protein (FBP) to sections of kidney proximal tubule, known...

  20. SCOWLP classification: Structural comparison and analysis of protein binding regions

    Directory of Open Access Journals (Sweden)

    Anders Gerd

    2008-01-01

    Full Text Available Abstract Background Detailed information about protein interactions is critical for our understanding of the principles governing protein recognition mechanisms. The structures of many proteins have been experimentally determined in complex with different ligands bound either in the same or different binding regions. Thus, the structural interactome requires the development of tools to classify protein binding regions. A proper classification may provide a general view of the regions that a protein uses to bind others and also facilitate a detailed comparative analysis of the interacting information for specific protein binding regions at atomic level. Such classification might be of potential use for deciphering protein interaction networks, understanding protein function, rational engineering and design. Description Protein binding regions (PBRs might be ideally described as well-defined separated regions that share no interacting residues one another. However, PBRs are often irregular, discontinuous and can share a wide range of interacting residues among them. The criteria to define an individual binding region can be often arbitrary and may differ from other binding regions within a protein family. Therefore, the rational behind protein interface classification should aim to fulfil the requirements of the analysis to be performed. We extract detailed interaction information of protein domains, peptides and interfacial solvent from the SCOWLP database and we classify the PBRs of each domain family. For this purpose, we define a similarity index based on the overlapping of interacting residues mapped in pair-wise structural alignments. We perform our classification with agglomerative hierarchical clustering using the complete-linkage method. Our classification is calculated at different similarity cut-offs to allow flexibility in the analysis of PBRs, feature especially interesting for those protein families with conflictive binding regions

  1. Endolysin of bacteriophage BFK20: evidence of a catalytic and a cell wall binding domain.

    Science.gov (United States)

    Gerova, Martina; Halgasova, Nora; Ugorcakova, Jana; Bukovska, Gabriela

    2011-08-01

    A gene product of ORF24' was identified on the genome of corynephage BFK20 as a putative phage endolysin. The protein of endolysin BFK20 (gp24') has a modular structure consisting of an N-terminal amidase_2 domain (gp24CD) and a C-terminal cell wall binding domain (gp24BD). The C-terminal domain is unrelated to any of the known cell wall binding domains of phage endolysins. The whole endolysin gene and the sequences of its N-terminal and C-terminal domains were cloned; proteins were expressed in Escherichia coli and purified to homogeneity. The lytic activities of endolysin and its catalytic domain were demonstrated on corynebacteria and bacillus substrates. The binding activity of cell wall binding domain alone and in fusion with green fluorescent protein (gp24BD-GFP) were shown by specific binding assays to the cell surface of BFK20 host Brevibacterium flavum CCM 251 as well as those of other corynebacteria. 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  2. Polymeric competitive protein binding adsorbents for radioassay

    International Nuclear Information System (INIS)

    Adams, R.J.

    1976-01-01

    Serum protein comprising specific binding proteins such as antibodies, B 12 intrinsic factor, thyroxin binding globulin and the like may be copolymerized with globulin constituents of serum by the action of ethylchloroformate to form readily packed insoluble precipitates which, following purification as by washing, are eminently suited for employment as competitive binding protein absorbents in radioassay procedures. 10 claims, no drawings

  3. A brave new world of RNA-binding proteins.

    Science.gov (United States)

    Hentze, Matthias W; Castello, Alfredo; Schwarzl, Thomas; Preiss, Thomas

    2018-01-17

    RNA-binding proteins (RBPs) are typically thought of as proteins that bind RNA through one or multiple globular RNA-binding domains (RBDs) and change the fate or function of the bound RNAs. Several hundred such RBPs have been discovered and investigated over the years. Recent proteome-wide studies have more than doubled the number of proteins implicated in RNA binding and uncovered hundreds of additional RBPs lacking conventional RBDs. In this Review, we discuss these new RBPs and the emerging understanding of their unexpected modes of RNA binding, which can be mediated by intrinsically disordered regions, protein-protein interaction interfaces and enzymatic cores, among others. We also discuss the RNA targets and molecular and cellular functions of the new RBPs, as well as the possibility that some RBPs may be regulated by RNA rather than regulate RNA.

  4. SH3b Cell wall binding domains can enhance anti-staphylococcal activity of endolysin lytic domains.

    Science.gov (United States)

    Bacteriophage endolysins are peptidoglycan hydrolases and a potential new source of antimicrobials. A large subset of these proteins contain a C-terminal SH3b_5 cell wall binding domain that has been shown [for some] to be essential for accurate cell wall recognition and subsequent staphylolytic ac...

  5. CNE, a collagen-binding protein of Streptococcus equi.

    Science.gov (United States)

    Lannergård, Jonas; Frykberg, Lars; Guss, Bengt

    2003-05-16

    Streptococcus equi subspecies equi is an important horse pathogenic bacterium causing a serious disease called strangles. Using bioinformatics we identified a gene denoted cne (gene encoding collagen-binding protein from S. equi) coding for a novel potential virulence factor of this species called protein CNE. The protein is composed of 657 amino acids and has the typical features found in cell surface-anchored proteins in Gram-positive bacteria. CNE displays amino acid sequence similarities to the previously well-studied collagen-binding protein CNA from Staphylococcus aureus, a proven virulence factor in septic arthritis. Based on similarity to CNA the structure of the mature CNE protein can be divided into an N-terminal A domain and a C-terminal B domain. The highest similarity between CNA and CNE is found in the A domains. The A domain in CNA is known to be the collagen-binding domain. Two parts of cne were amplified using polymerase chain reaction (PCR) and ligated into an expression vector, and recombinant CNE proteins were produced in Escherichia coli. The purified CNE proteins were shown to display collagen-binding activity in a Western ligand blot and to inhibit collagen binding to cells of subsp. equi and to CNE-coated microtitre wells. Furthermore, the A domain of CNE was sufficient for binding collagen, and was shown to compete for the same site on collagen as CNA in inhibition studies. Using PCR, the cne gene was detected in all studied strains of subsp. equi and S. equi subsp. zooepidemicus.

  6. Actin binding proteins and spermiogenesis

    Science.gov (United States)

    Mruk, Dolores D

    2011-01-01

    Drebrin E, an actin-binding protein lacking intrinsic activity in the regulation of actin dynamics (e.g., polymerization, capping, nucleation, branching, cross-linking, bundling and severing), is known to recruit actin regulatory proteins to a specific cellular site. Herein, we critically evaluate recent findings in the field which illustrate that drebrin E works together with two other actin-binding proteins, namely Arp3 (actin-related protein 3, a component of the Arp2/3 complex that simultaneously controls actin nucleation for polymerization and branching of actin filaments) and Eps8 (epidermal growth factor receptor pathway substrate 8 that controls capping of the barbed ends of actin filaments, as well as actin filament bundling) to regulate the homeostasis of F-actin filament bundles at the ectoplasmic specialization (ES), a testis-specific atypical adherens junction (AJ) in the seminiferous epithelium. This is mediated by the strict temporal and spatial expression of these three actin-binding proteins at the apical and basal ES at the Sertoli cell-spermatid (step 8–19) and Sertoli-Sertoli cell interface, respectively, during the seminiferous epithelial cycle of spermatogenesis. In this Commentary, we put forth a possible model by which drebrin E may be acting as a platform upon which proteins (e.g., Arp3) that are needed to alter the conformation of actin filament bundles at the ES can be recruited to the site, thus facilitating changes in cell shape and cell position in the epithelium during spermiogenesis and spermiation. In short, drebrin E may be acting as a “logistic” distribution center to manage different regulatory proteins at the apical ES, thereby regulating the dynamics of actin filament bundles and modulating the plasticity of the apical ES. This would allow adhesion to be altered continuously throughout the epithelial cycle to accommodate spermatid movement in the seminiferous epithelium during spermiogenesis and spermiation. We also

  7. Ligand photo-isomerization triggers conformational changes in iGluR2 ligand binding domain.

    Directory of Open Access Journals (Sweden)

    Tino Wolter

    Full Text Available Neurological glutamate receptors bind a variety of artificial ligands, both agonistic and antagonistic, in addition to glutamate. Studying their small molecule binding properties increases our understanding of the central nervous system and a variety of associated pathologies. The large, oligomeric multidomain membrane protein contains a large and flexible ligand binding domains which undergoes large conformational changes upon binding different ligands. A recent application of glutamate receptors is their activation or inhibition via photo-switchable ligands, making them key systems in the emerging field of optochemical genetics. In this work, we present a theoretical study on the binding mode and complex stability of a novel photo-switchable ligand, ATA-3, which reversibly binds to glutamate receptors ligand binding domains (LBDs. We propose two possible binding modes for this ligand based on flexible ligand docking calculations and show one of them to be analogues to the binding mode of a similar ligand, 2-BnTetAMPA. In long MD simulations, it was observed that transitions between both binding poses involve breaking and reforming the T686-E402 protein hydrogen bond. Simulating the ligand photo-isomerization process shows that the two possible configurations of the ligand azo-group have markedly different complex stabilities and equilibrium binding modes. A strong but slow protein response is observed after ligand configuration changes. This provides a microscopic foundation for the observed difference in ligand activity upon light-switching.

  8. Identification of binding peptides of the ADAM15 disintegrin domain ...

    Indian Academy of Sciences (India)

    Madhsudhan

    Karkkainen et al. 2006). In this study, the recombinant ADAM15 disintegrin domain (RADD) was expressed in E. coli, and 4 specific binding peptides of RADD were obtained by positive panning and subtractive selection using a phage display 12-mer.

  9. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...... diffract X-rays to at least 2.0 A resolution. A complete diffraction data set has been collected to 2.7 A resolution. The crystals of TN, obtained by the vapour-diffusion reverse salting-in method at 280 K, are rhombohedral, space group R3, with the hexagonal axes a = b = 89.1, c = 75.8 A, and diffract...

  10. FHA domains as phospho-threonine binding modules in cell signaling.

    Science.gov (United States)

    Hammet, Andrew; Pike, Brietta L; McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

    2003-01-01

    Forkhead-associated (FHA) domains are present in >200 diverse proteins in all phyla from bacteria to mammals and seem to be particularly prevalent in proteins with cell cycle control functions. Recent work from several laboratories has considerably improved our understanding of the structure and function of these domains that were virtually unknown a few years ago, and the first disease associations of FHA domains have now emerged. FHA domains form 11-stranded beta-sandwiches that contain some 100-180 amino acid residues with a high degree of sequence diversity. FHA domains act as phosphorylation-dependent protein-protein interaction modules that preferentially bind to phospho-threonine residues in their targets. Interestingly, point mutations in the human CHK2 gene that lead to single-residue amino acid substitutions in the FHA domain of this cell cycle checkpoint kinase have been found to cause a subset of cases of the Li-Fraumeni multi-cancer syndrome.

  11. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang (Cornell); (UMM-MED); (Colorado)

    2011-09-28

    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

  12. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.

    Science.gov (United States)

    Reinhardt, H Christian; Yaffe, Michael B

    2013-09-01

    Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.

  13. Multiple graph regularized protein domain ranking

    KAUST Repository

    Wang, Jim Jing-Yan

    2012-11-19

    Background: Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods.Results: To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods.Conclusion: The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications. 2012 Wang et al; licensee BioMed Central Ltd.

  14. Multiple graph regularized protein domain ranking.

    Science.gov (United States)

    Wang, Jim Jing-Yan; Bensmail, Halima; Gao, Xin

    2012-11-19

    Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods. To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods. The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications.

  15. Multiple graph regularized protein domain ranking

    Directory of Open Access Journals (Sweden)

    Wang Jim

    2012-11-01

    Full Text Available Abstract Background Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods. Results To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods. Conclusion The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications.

  16. Biosensors engineered from conditionally stable ligand-binding domains

    Energy Technology Data Exchange (ETDEWEB)

    Church, George M.; Feng, Justin; Mandell, Daniel J.; Baker, David; Fields, Stanley; Jester, Benjamin Ward; Tinberg, Christine Elaine

    2017-09-19

    Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

  17. MARs Wars: heterogeneity and clustering of DNA-binding domains in the nuclear matrix

    Directory of Open Access Journals (Sweden)

    Ioudinkova E. S.

    2009-12-01

    Full Text Available Aim. CO326 is a chicken nuclear scaffold/matrix attachment region (MAR associated with the nuclear matrix in several types of chicken cells. It contains a binding site for a sequence-specific DNA-binding protein, F326. We have studied its interaction with the nuclear matrix. Methods. We have used an in vitro MAR assay with isolated matrices from chicken HD3 cells. Results. We have found that an oligonucleotide binding site for the F326 inhibits binding of the CO326 to the nuclear matrix. At the same time, the binding of heterologous MARs is enhanced. Conclusions. Taken together, these data suggest that there exist several classes of MARs and MAR-binding domains and that the MAR-binding proteins may be clustered in the nuclear matrix.

  18. Engineered Lactobacillus rhamnosus GG expressing IgG-binding domains of protein G: Capture of hyperimmune bovine colostrum antibodies and protection against diarrhea in a mouse pup rotavirus infection model.

    Science.gov (United States)

    Günaydın, Gökçe; Zhang, Ran; Hammarström, Lennart; Marcotte, Harold

    2014-01-16

    Rotavirus-induced diarrhea causes more than 500,000 deaths annually in the world, and although vaccines are being made available, new effective treatment strategies should still be considered. Purified antibodies derived from hyperimmune bovine colostrum (HBC), from cows immunized with rotavirus, were previously used for treatment of rotavirus diarrhea in children. A combination of HBC antibodies and a probiotic strain of Lactobacillus (L. rhamnosus GG) was also found to be more effective than HBC alone in reducing diarrhea in a mouse model of rotavirus infection. In order to further improve this form of treatment, L. rhamnosus GG was engineered to display surface expressed IgG-binding domains of protein G (GB1, GB2, and GB3) which capture HBC-derived IgG antibodies (HBC-IgG) and thus target rotavirus. The expression of IgG-binding domains on the surface of the bacteria as well as their binding to HBC-IgG and to rotavirus (simian strain RRV) was demonstrated by Western blot, flow cytometry, and electron microscopy. The prophylactic effect of engineered L. rhamnosus GG and anti-rotaviral activity of HBC antibodies was evaluated in a mouse pup model of RRV infection. The combination therapy with engineered L. rhamnosus GG (PG3) and HBC was significantly more effective in reducing the prevalence, severity, and duration of diarrhea in comparison to HBC alone or a combination of wild-type L. rhamnosus GG and HBC. The new therapy reduces the effective dose of HBC between 10 to 100-fold and may thus decrease treatment costs. This antibody capturing platform, tested here for the first time in vivo, could potentially be used to target additional gastrointestinal pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    Science.gov (United States)

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors and , and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein-protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.

  20. CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems

    Directory of Open Access Journals (Sweden)

    Kira eMakarova

    2014-04-01

    Full Text Available CRISPR-Cas adaptive immunity systems of bacteria and archaea insert fragments of virus or plasmid DNA as spacer sequences into CRISPR repeat loci. Processed transcripts encompassing these spacers guide the cleavage of the cognate foreign DNA or RNA. Most CRISPR-Cas loci, in addition to recognized cas genes, also include genes that are not directly implicated in spacer acquisition, CRISPR transcript processing or interference. Here we comprehensively analyze sequences, structures and genomic neighborhoods of one of the most widespread groups of such genes that encode proteins containing a predicted nucleotide-binding domain with a Rossmann-like fold, which we denote CARF (CRISPR-associated Rossmann fold. Several CARF protein structures have been determined but functional characterization of these proteins is lacking. The CARF domain is most frequently combined with a C-terminal winged helix-turn-helix DNA-binding domain and effector domains most of which are predicted to possess DNase or RNase activity. Divergent CARF domains are also found in RtcR proteins, sigma-54 dependent regulators of the rtc RNA repair operon. CARF genes frequently co-occur with those coding for proteins containing the WYL domain with the Sm-like SH3 β-barrel fold, which is also predicted to bind ligands. CRISPR-Cas and possibly other defense systems are predicted to be transcriptionally regulated by multiple ligand-binding proteins containing WYL and CARF domains which sense modified nucleotides and nucleotide derivatives generated during virus infection. We hypothesize that CARF domains also transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids, resulting, respectively, in immunity complementing the CRISPR-Cas action or in dormancy/programmed cell death.

  1. Interaction of calmodulin with the calmodulin binding domain of the plasma membrane Ca2+ pump

    International Nuclear Information System (INIS)

    Vorherr, T.; James, P.; Krebs, J.; Carafoli, E.; McCormick, D.J.; Penniston, J.T.; Enyedi, A.

    1990-01-01

    Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca 2+ pump were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its first 15 or 20 amino acids, and to its C-terminal 14 amino acids. The first three peptides interacted with calmodulin. The K value was similar to that of the intact enzyme in the 28 and 20 amino acid peptides, but increased substantially in the shorter 15 amino acid peptide. The 14 amino acid peptide corresponding to the C-terminal portion of the domain failed to bind calmodulin. 2D NMR experiments on the 20 amino acid peptides have indicated that the interaction occurred with the C-terminal half of calmodulin. A tryptophan that is conserved in most calmodulin binding domains of proteins was replaced by other amino acids, giving rise to modified peptides which had lower affinity for calmodulin. An 18 amino acid peptide corresponding to an acidic sequence immediately N-terminal to the calmodulin binding domain which is likely to be a Ca 2+ binding site in the pump was also synthesized. Circular dichroism experiments have shown that it interacted with calmodulin binding domain, supporting the suggestion that the latter, or a portion of it, may act as a natural inhibitor of the pump

  2. Structural and functional analysis of the YAP-binding domain of human TEAD2.

    Science.gov (United States)

    Tian, Wei; Yu, Jianzhong; Tomchick, Diana R; Pan, Duojia; Luo, Xuelian

    2010-04-20

    The Hippo pathway controls organ size and suppresses tumorigenesis in metazoans by blocking cell proliferation and promoting apoptosis. The TEAD1-4 proteins (which contain a DNA-binding domain but lack an activation domain) interact with YAP (which lacks a DNA-binding domain but contains an activation domain) to form functional heterodimeric transcription factors that activate proliferative and prosurvival gene expression programs. The Hippo pathway inhibits the YAP-TEAD hybrid transcription factors by phosphorylating and promoting cytoplasmic retention of YAP. Here we report the crystal structure of the YAP-binding domain (YBD) of human TEAD2. TEAD2 YBD adopts an immunoglobulin-like beta-sandwich fold with two extra helix-turn-helix inserts. NMR studies reveal that the TEAD-binding domain of YAP is natively unfolded and that TEAD binding causes localized conformational changes in YAP. In vitro binding and in vivo functional assays define an extensive conserved surface of TEAD2 YBD as the YAP-binding site. Therefore, our studies suggest that a short segment of YAP adopts an extended conformation and forms extensive contacts with a rigid surface of TEAD. Targeting a surface-exposed pocket of TEAD might be an effective strategy to disrupt the YAP-TEAD interaction and to reduce the oncogenic potential of YAP.

  3. Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals

    DEFF Research Database (Denmark)

    Janeček, Štefan; Svensson, Birte; MacGregor, E. Ann

    2011-01-01

    glycoside hydrolase (GH) families GH13, GH14, GH15, GH31, GH57 and GH77, as well as in a number of regulatory enzymes, e.g., phosphoglucan, water dikinase-3, genethonin-1, laforin, starch-excess protein-4, the β-subunit of AMP-activated protein kinase and its homologues from sucrose non-fermenting-1 protein...

  4. Integral UBL domain proteins: a family of proteasome interacting proteins

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2004-01-01

    The family of ubiquitin-like (UBL) domain proteins (UDPs) comprises a conserved group of proteins involved in a multitude of different cellular activities. However, recent studies on UBL-domain proteins indicate that these proteins appear to share a common property in their ability to interact wi...

  5. Acyl-coenzyme A binding protein (ACBP)

    DEFF Research Database (Denmark)

    Kragelund, B B; Knudsen, J; Poulsen, F M

    1999-01-01

    Acyl-coenzyme A binding proteins are known from a large group of eukaryote species and to bind a long chain length acyl-CoA ester with very high affinity. Detailed biochemical mapping of ligand binding properties has been obtained as well as in-depth structural studies on the bovine apo-protein a...

  6. DENN Domain Proteins: Regulators of Rab GTPases*

    Science.gov (United States)

    Marat, Andrea L.; Dokainish, Hatem; McPherson, Peter S.

    2011-01-01

    The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways. PMID:21330364

  7. DENN domain proteins: regulators of Rab GTPases.

    Science.gov (United States)

    Marat, Andrea L; Dokainish, Hatem; McPherson, Peter S

    2011-04-22

    The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways.

  8. Identification of two functional PCNA-binding domains in human DNA polymerase κ.

    Science.gov (United States)

    Yoon, Jung-Hoon; Acharya, Narottam; Park, Jeseong; Basu, Debashree; Prakash, Satya; Prakash, Louise

    2014-07-01

    Previously, we have shown that human DNA polymerase (Pol) η has two functional PCNA-binding motifs, PIP1 and PIP2, and that a C-terminal deletion of Polη that lacks the ubiquitin-binding UBZ domain and the PIP2 domain but retains the PIP1 domain promotes normal levels of translesion synthesis (TLS) opposite a cis-syn TT dimer in human cells. Here, we identify two PIP domains in Polκ and show that TLS occurs normally in human fibroblast cells in which the pip1 or pip2 mutant Polκ is expressed, but mutational inactivation of both PIP domains renders Polκ nonfunctional in TLS opposite the thymine glycol lesion. Thus, the two PIP domains of Polκ function redundantly in TLS opposite this DNA lesion in human cells. However, and surprisingly, whereas mutational inactivation of the PIP1 domain completely inhibits the stimulation of DNA synthesis by Polκ in the presence of proliferating cell nuclear antigen (PCNA), replication factor C, and replication protein A, mutations in PIP2 have no adverse effect on PCNA-dependent DNA synthesis. This raises the possibility that activation of Polκ PIP2 as a PCNA-binding domain occurs during TLS in human cells and that protein-protein interactions and post-transcriptional modifications are involved in such activation. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  9. Structural principles governing domain motions in proteins

    NARCIS (Netherlands)

    Hayward, S

    1999-01-01

    With the use of a recently developed method, twenty-four proteins for which two or more X-ray conformers are known have been analyzed to reveal structural principles that govern domain motions in proteins. In all 24 cases, the domain motion is a rotation about a physical axis created through local

  10. Biophysical and Structural Characterization of the Thioredoxin-binding Domain of Protein Kinase ASK1 and Its Interaction with Reduced Thioredoxin

    Czech Academy of Sciences Publication Activity Database

    Košek, Dalibor; Kylarová, Salome; Pšenáková, Katarína; Řežábková, L.; Herman, P.; Večeř, J.; Obšilová, Veronika; Obšil, T.

    2014-01-01

    Roč. 289, č. 35 (2014), s. 24463-24474 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA14-10061S Institutional support: RVO:67985823 Keywords : ASK1 * thioredoxin * AUC * SAXS * coiled - coiled domain Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  11. Achieving peptide binding specificity and promiscuity by loops: case of the forkhead-associated domain.

    Science.gov (United States)

    Huang, Yu-Ming M; Chang, Chia-En A

    2014-01-01

    The regulation of a series of cellular events requires specific protein-protein interactions, which are usually mediated by modular domains to precisely select a particular sequence from diverse partners. However, most signaling domains can bind to more than one peptide sequence. How do proteins create promiscuity from precision? Moreover, these complex interactions typically occur at the interface of a well-defined secondary structure, α helix and β sheet. However, the molecular recognition primarily controlled by loop architecture is not fully understood. To gain a deep understanding of binding selectivity and promiscuity by the conformation of loops, we chose the forkhead-associated (FHA) domain as our model system. The domain can bind to diverse peptides via various loops but only interact with sequences containing phosphothreonine (pThr). We applied molecular dynamics (MD) simulations for multiple free and bound FHA domains to study the changes in conformations and dynamics. Generally, FHA domains share a similar folding structure whereby the backbone holds the overall geometry and the variety of sidechain atoms of multiple loops creates a binding surface to target a specific partner. FHA domains determine the specificity of pThr by well-organized binding loops, which are rigid to define a phospho recognition site. The broad range of peptide recognition can be attributed to different arrangements of the loop interaction network. The moderate flexibility of the loop conformation can help access or exclude binding partners. Our work provides insights into molecular recognition in terms of binding specificity and promiscuity and helpful clues for further peptide design.

  12. Stability and Sugar Recognition Ability of Ricin-Like Carbohydrate Binding Domains

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Jianzhuang [ORNL; Nellas, Ricky B [ORNL; Glover, Mary M [ORNL; Shen, Tongye [ORNL

    2011-01-01

    Lectins are a class of proteins known for their novel binding to saccharides. Understanding this sugar recognition process can be crucial in creating structure-based designs of proteins with various biological roles. We focus on the sugar binding of a particular lectin, ricin, which has two -trefoil carbohydrate-binding domains (CRDs) found in several plant protein toxins. The binding ability of possible sites of ricin-like CRD has been puzzling. The apo and various (multiple) ligand-bound forms of the sugar-binding domains of ricin were studied by molecular dynamics simulations. By evaluating structural stability, hydrogen bond dynamics, flexibility, and binding energy, we obtained a detailed picture of the sugar recognition of the ricin-like CRD. Unlike what was previously believed, we found that the binding abilities of the two known sites are not independent of each other. The binding ability of one site is positively affected by the other site. While the mean positions of different binding scenarios are not altered significantly, the flexibility of the binding pockets visibly decreases upon multiple ligand binding. This change in flexibility seems to be the origin of the binding cooperativity. All the hydrogen bonds that are strong in the monoligand state are also strong in the double-ligand complex, although the stability is much higher in the latter form due to cooperativity. These strong hydrogen bonds in a monoligand state are deemed to be the essential hydrogen bonds. Furthermore, by examining the structural correlation matrix, the two domains are structurally one entity. Galactose hydroxyl groups, OH4 and OH3, are the most critical parts in both site 1 and site 2 recognition.

  13. Multifunctionality and mechanism of ligand binding in a mosquito antiinflammatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Calvo, Eric; Mans, Ben J.; Ribeiro, José M.C.; Andersen, John F.; (NIH)

    2009-04-07

    The mosquito D7 salivary proteins are encoded by a multigene family related to the arthropod odorant-binding protein (OBP) superfamily. Forms having either one or two OBP domains are found in mosquito saliva. Four single-domain and one two-domain D7 proteins from Anopheles gambiae and Aedes aegypti (AeD7), respectively, were shown to bind biogenic amines with high affinity and with a stoichiometry of one ligand per protein molecule. Sequence comparisons indicated that only the C-terminal domain of AeD7 is homologous to the single-domain proteins from A. gambiae, suggesting that the N-terminal domain may bind a different class of ligands. Here, we describe the 3D structure of AeD7 and examine the ligand-binding characteristics of the N- and C-terminal domains. Isothermal titration calorimetry and ligand complex crystal structures show that the N-terminal domain binds cysteinyl leukotrienes (cysLTs) with high affinities (50-60 nM) whereas the C-terminal domain binds biogenic amines. The lipid chain of the cysLT binds in a hydrophobic pocket of the N-terminal domain, whereas binding of norepinephrine leads to an ordering of the C-terminal portion of the C-terminal domain into an alpha-helix that, along with rotations of Arg-176 and Glu-268 side chains, acts to bury the bound ligand.

  14. CAP binding proteins associated with the nucleus.

    OpenAIRE

    Patzelt, E; Blaas, D; Kuechler, E

    1983-01-01

    Cap binding proteins of HeLa cells were identified by photo-affinity labelling using the cap analogue gamma-[32P]-[4-(benzoyl-phenyl)methylamido]-7-methylguanosine-5'- triphosphate. Photoreaction with whole cell homogenates resulted in specific labelling of five major polypeptides. The small molecular weight polypeptide appeared to be identical to the 24 000 to 26 000 dalton cap binding protein previously identified in initiation factors. A cap binding protein of 37 000 dalton was found in in...

  15. Zinc induces structural reorganization of gelatin binding domain from human fibronectin and affects collagen binding.

    Science.gov (United States)

    Graille, Marc; Pagano, Maurice; Rose, Thierry; Ravaux, Michèle Reboud; van Tilbeurgh, Herman

    2010-06-09

    Fibronectin is a modular extracellular matrix protein involved in cell adhesion, cell motility, wound healing, and maintenance of cell morphology. It is composed of multiple repeats of three distinct modules: F(I), F(II), and F(III). Various combinations of these modules create fragments able to interact with different constituents of the extracellular matrix. Here, we present the 2.5-A resolution crystal structure of its 45-kDa gelatin-binding domain (GBD; 6F(I)-1F(II)-2F(II)-7F(I)-8F(I)-9F(I)), which also corresponds to the C-terminal half of the migration stimulating factor, a Fn splice variant expressed in human breast cancers. GBD forms a very compact zinc-mediated homodimer, in stark contrast with previous structures of fibronectin fragments. Most remarkably, 8F(I) no longer adopts the canonical F(I) fold but is composed of two long strands that associate with 7F(I) and 9F(I) into a large beta-sheet superdomain. Binding studies in solution confirmed that Zn induces conformational rearrangements and causes loss of binding of Fn-GBD to high-affinity collagen peptides. These data suggest the Zn may play a regulatory role for the cellular functions of fibronectin.

  16. Molecular determinants for the complex binding specificity of the PDZ domain in PICK1

    DEFF Research Database (Denmark)

    Madsen, Kenneth L; Beuming, Thijs; Niv, Masha Y

    2005-01-01

    polarization. Our results showed that the PICK1 PDZ domain binds the type II sequence presented by the human dopamine transporter (-WLKV) with an almost 15-fold and >100-fold higher affinity than the type I sequences presented by protein kinase Calpha (-QSAV) and the beta(2)-adrenergic receptor (-DSLL......), respectively. Mutational analysis of Lys(83) in the alphaB1 position of the PDZ domain suggested that this residue mimics the function of hydrophobic residues present in this position in regular type II PDZ domains. The PICK1 PDZ domain was moreover found to prefer small hydrophobic residues in the C......-terminal P(0) position of the ligand. Molecular modeling predicted a rank order of (Val > Ile > Leu) that was verified experimentally with up to a approximately 16-fold difference in binding affinity between a valine and a leucine in P(0). The results define the structural basis for the unusual binding...

  17. New method of detecting hydrophobic interaction between C-terminal binding domain and biomacromolecules.

    Science.gov (United States)

    Huang, JiaFeng; Wu, RiBang; Wu, CuiLing; Liu, Dan; Zhang, Jiang; Liao, BinQiang; Lei, Ming; Xiao, Xiao; Ma, ChangBei; He, HaiLun

    2018-01-10

    The C-terminal domains of proteases play crucial roles in hydrolysis, substrate adsorption and targeted binding. Identifying and characterizing interactions between C-terminal domains and biomacromolecules can help to examine the diversity as well as the substrate-binding ability of C-terminal domains and to explore novel functions. The bacterial pre-peptidase C-terminal (PPC) domain is a typical C-terminal domain normally found at the C-terminus of bacterial secreted proteases. In this work, we successfully demonstrated that 8-anilinonaphthalene-1-sulfonic acid (ANS) could be used to rapidly determine the interactions between this C-terminal domain and biomacromolecules. The time-resolved ANS fluorescence of PPC and collagen interaction could be used for quantitative analysis of the collagen-binding capability based on the slope of the time-scanning curve. Using this method, we found that PPC domains had an obvious affinity to fibrillar proteins but had little or no capacity to bind polysaccharides or linear DNAs. Docking studies proved that collagen bound to the same hydrophobic site of PPC as the ANS probe, causing a decrease in the emission intensity. This method is simple and cost effective and provides an effective detection technique to analyze the interaction between this C-terminal domain and biomolecules. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Conserved SMP domains of the ERMES complex bind phospholipids and mediate tether assembly.

    Science.gov (United States)

    AhYoung, Andrew P; Jiang, Jiansen; Zhang, Jiang; Khoi Dang, Xuan; Loo, Joseph A; Zhou, Z Hong; Egea, Pascal F

    2015-06-23

    Membrane contact sites (MCS) between organelles are proposed as nexuses for the exchange of lipids, small molecules, and other signals crucial to cellular function and homeostasis. Various protein complexes, such as the endoplasmic reticulum-mitochondrial encounter structure (ERMES), function as dynamic molecular tethers between organelles. Here, we report the reconstitution and characterization of subcomplexes formed by the cytoplasm-exposed synaptotagmin-like mitochondrial lipid-binding protein (SMP) domains present in three of the five ERMES subunits--the soluble protein Mdm12, the endoplasmic reticulum (ER)-resident membrane protein Mmm1, and the mitochondrial membrane protein Mdm34. SMP domains are conserved lipid-binding domains found exclusively in proteins at MCS. We show that the SMP domains of Mdm12 and Mmm1 associate into a tight heterotetramer with equimolecular stoichiometry. Our 17-Å-resolution EM structure of the complex reveals an elongated crescent-shaped particle in which two Mdm12 subunits occupy symmetric but distal positions at the opposite ends of a central ER-anchored Mmm1 homodimer. Rigid body fitting of homology models of these SMP domains in the density maps reveals a distinctive extended tubular structure likely traversed by a hydrophobic tunnel. Furthermore, these two SMP domains bind phospholipids and display a strong preference for phosphatidylcholines, a class of phospholipids whose exchange between the ER and mitochondria is essential. Last, we show that the three SMP-containing ERMES subunits form a ternary complex in which Mdm12 bridges Mmm1 to Mdm34. Our findings highlight roles for SMP domains in ERMES assembly and phospholipid binding and suggest a structure-based mechanism for the facilitated transport of phospholipids between organelles.

  19. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  20. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

    Science.gov (United States)

    Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  1. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

    Science.gov (United States)

    Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2016-01-15

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. The identification of FANCD2 DNA binding domains reveals nuclear localization sequences.

    Science.gov (United States)

    Niraj, Joshi; Caron, Marie-Christine; Drapeau, Karine; Bérubé, Stéphanie; Guitton-Sert, Laure; Coulombe, Yan; Couturier, Anthony M; Masson, Jean-Yves

    2017-08-21

    Fanconi anemia (FA) is a recessive genetic disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. The FA pathway consists of at least 21 FANC genes (FANCA-FANCV), and the encoded protein products interact in a common cellular pathway to gain resistance against DNA interstrand crosslinks. After DNA damage, FANCD2 is monoubiquitinated and accumulates on chromatin. FANCD2 plays a central role in the FA pathway, using yet unidentified DNA binding regions. By using synthetic peptide mapping and DNA binding screen by electromobility shift assays, we found that FANCD2 bears two major DNA binding domains predominantly consisting of evolutionary conserved lysine residues. Furthermore, one domain at the N-terminus of FANCD2 bears also nuclear localization sequences for the protein. Mutations in the bifunctional DNA binding/NLS domain lead to a reduction in FANCD2 monoubiquitination and increase in mitomycin C sensitivity. Such phenotypes are not fully rescued by fusion with an heterologous NLS, which enable separation of DNA binding and nuclear import functions within this domain that are necessary for FANCD2 functions. Collectively, our results enlighten the importance of DNA binding and NLS residues in FANCD2 to activate an efficient FA pathway. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Tenascin C promiscuously binds growth factors via its fifth fibronectin type III-like domain.

    Directory of Open Access Journals (Sweden)

    Laura De Laporte

    Full Text Available Tenascin C (TNC is an extracellular matrix protein that is upregulated during development as well as tissue remodeling. TNC is comprised of multiple independent folding domains, including 15 fibronectin type III-like (TNCIII domains. The fifth TNCIII domain (TNCIII5 has previously been shown to bind heparin. Our group has shown that the heparin-binding fibronectin type III domains of fibronectin (FNIII, specifically FNIII12-14, possess affinity towards a large number of growth factors. Here, we show that TNCIII5 binds growth factors promiscuously and with high affinity. We produced recombinant fragments of TNC representing the first five TNCIII repeats (TNCIII1-5, as well as subdomains, including TNCIII5, to study interactions with various growth factors. Multiple growth factors of the platelet-derived growth factor (PDGF family, the fibroblast growth factor (FGF family, the transforming growth factor beta (TGF-β superfamily, the insulin-like growth factor binding proteins (IGF-BPs, and neurotrophins were found to bind with high affinity to this region of TNC, specifically to TNCIII5. Surface plasmon resonance was performed to analyze the kinetics of binding of TNCIII1-5 with TGF-β1, PDGF-BB, NT-3, and FGF-2. The promiscuous yet high affinity of TNC for a wide array of growth factors, mediated mainly by TNCIII5, may play a role in multiple physiological and pathological processes involving TNC.

  4. Thermal unfolding of a Ca- and Lanthanide-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Fahmy, Karim [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Goettfert, M. [Technische Univ. Dresden (Germany); Knoeppel, J.

    2017-06-01

    The MIIA (metal ion-induced autocleavage)-domain of the protein Vic001052 from the pathogen Vibrio coralliilyticus, comprises 173 amino acids and exhibits Ca-dependent autoproteolytic activity. It shows homology to nodulation proteins which are secreted by Rhizobiacea into plant host cells where they exert Ca-dependent functions. We have studied the structural and energetic aspects of metal protein interactions of the MIIA domain which appear attractive for engineering metal-binding synthetic peptides. Using a non-cleavable MIIA domain construct, we detected very similar structural changes upon binding to Ca{sup 2+} and Eu{sup 3+}. The thermal denaturation of the Ca-bound state was studied by circular dichroism spectroscopy. The metal-bound folded state unfolds reversibly into an unstructured metal-free state similar to the metal-free state at room temperature.

  5. Role of SUMO in RNF4-mediated promyelocytic leukemia protein (PML) degradation: sumoylation of PML and phospho-switch control of its SUMO binding domain dissected in living cells.

    Science.gov (United States)

    Percherancier, Yann; Germain-Desprez, Delphine; Galisson, Frédéric; Mascle, Xavier H; Dianoux, Laurent; Estephan, Patricia; Chelbi-Alix, Mounira K; Aubry, Muriel

    2009-06-12

    Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic agent, arsenic trioxide (As2O3). Here, we established a novel bioluminescence resonance energy transfer (BRET) assay to dissect and monitor PML/SUMO interactions dynamically in living cells upon addition of therapeutic agents. Using this sensitive and quantitative SUMO BRET assay that distinguishes PML sumoylation from SBD-mediated PML/SUMO non-covalent interactions, we probed the respective roles of covalent and non-covalent PML/SUMO interactions in PML degradation and interaction with RNF4. We found that, although dispensable for As2O3-enhanced PML sumoylation and RNF4 interaction, PML SBD core sequence was required for As2O3- and RNF4-induced PML degradation. As confirmed with a phosphomimetic mutant, phosphorylation of a stretch of serine residues, contained within PML SBD was needed for PML interaction with SUMO-modified protein partners and thus for NB maturation. However, mutation of these serine residues did not impair As2O3- and RNF4-induced PML degradation, contrasting with the known role of these phosphoserine residues for casein kinase 2-promoted PML degradation. Altogether, these data suggest a model whereby sumoylation- and SBD-dependent PML oligomerization within NBs is sufficient for RNF4-mediated PML degradation and does not require the phosphorylation-dependent association of PML with other sumoylated partners.

  6. Characterization of Novel Calmodulin Binding Domains within IQ Motifs of IQGAP1

    Science.gov (United States)

    Jang, Deok-Jin; Ban, Byungkwan; Lee, Jin-A

    2011-01-01

    IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca2+-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7-3) and IQ(3.5-4.4), within IQGAP1 that were involved in Ca2+-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7-3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5-4.4) were required for Ca2+/CaM binding. Finally, we showed that IQ(2.7-3) was the main apoCaM binding domain and both IQ(2.7-3) and IQ(3.5-4.4) were required for Ca2+/CaM binding within IQ(1- 2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca2+-dependent or -independent manner. PMID:22080369

  7. Expression and Production of SH2 Domain Proteins.

    Science.gov (United States)

    Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

    2017-01-01

    The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

  8. Epigenetic modification of TLR4 promotes activation of NF-κB by regulating methyl-CpG-binding domain protein 2 and Sp1 in gastric cancer.

    Science.gov (United States)

    Kim, Tae Woo; Lee, Seon-Jin; Oh, Byung Moo; Lee, Heesoo; Uhm, Tae Gi; Min, Jeong-Ki; Park, Young-Jun; Yoon, Suk Ran; Kim, Bo-Yeon; Kim, Jong Wan; Choe, Yong-Kyung; Lee, Hee Gu

    2016-01-26

    Toll-like receptor 4 (TLR4) is important in promoting the immune response in various cancers. Recently, TLR4 is highly expressed in a stage-dependent manner in gastric cancer, but the regulatory mechanism of TLR4 expression has been not elucidated it. Here, we investigated the mechanism underlying regulation of TLR4 expression through promoter methylation and histone modification between transcriptional regulation and silencing of the TLR4 gene in gastric cancer cells. Chromatin immunoprecipitation was carried out to screen for factors related to TLR4 methylation such as MeCP2, HDAC1, and Sp1 on the TLR4 promoter. Moreover, DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) induced demethylation of the TLR4 promoter and increased H3K4 trimethylation and Sp1 binding to reactivate silenced TLR4. In contrast, although the silence of TLR4 activated H3K9 trimethylation and MeCP2 complex, combined treatment with TLR4 agonist and 5-aza-dC upregulated H3K4 trimethylation and activated with transcription factors as Sp1 and NF-κB. This study demonstrates that recruitment of the MeCP2/HDAC1 repressor complex increases the low levels of TLR4 expression through epigenetic modification of DNA and histones on the TLR4 promoter, but Sp1 activates TLR4 high expression by hypomethylation and NF-κB signaling in gastric cancer cells.

  9. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Behrendt, N; Ploug, M; Patthy, L

    1991-01-01

    with the internal repeats of u-PAR constitute the extracellular part of Ly-6 antigens and of the squid glycoprotein Sgp-2. Like u-PAR, these proteins are attached to the membrane by a glycosyl-phosphatidylinositol anchor. The hydrophilic, ligand-binding u-PAR domain identified in the present study has potential...

  10. Mechanism of mRNA-STAR domain interaction: Molecular dynamics simulations of Mammalian Quaking STAR protein.

    Science.gov (United States)

    Sharma, Monika; Anirudh, C R

    2017-10-03

    STAR proteins are evolutionary conserved mRNA-binding proteins that post-transcriptionally regulate gene expression at all stages of RNA metabolism. These proteins possess conserved STAR domain that recognizes identical RNA regulatory elements as YUAAY. Recently reported crystal structures show that STAR domain is composed of N-terminal QUA1, K-homology domain (KH) and C-terminal QUA2, and mRNA binding is mediated by KH-QUA2 domain. Here, we present simulation studies done to investigate binding of mRNA to STAR protein, mammalian Quaking protein (QKI). We carried out conventional MD simulations of STAR domain in presence and absence of mRNA, and studied the impact of mRNA on the stability, dynamics and underlying allosteric mechanism of STAR domain. Our unbiased simulations results show that presence of mRNA stabilizes the overall STAR domain by reducing the structural deviations, correlating the 'within-domain' motions, and maintaining the native contacts information. Absence of mRNA not only influenced the essential modes of motion of STAR domain, but also affected the connectivity of networks within STAR domain. We further explored the dissociation of mRNA from STAR domain using umbrella sampling simulations, and the results suggest that mRNA binding to STAR domain occurs in multi-step: first conformational selection of mRNA backbone conformations, followed by induced fit mechanism as nucleobases interact with STAR domain.

  11. Revised domain structure of ulvan lyase and characterization of the first ulvan binding domain.

    Science.gov (United States)

    Melcher, Rebecca L J; Neumann, Marten; Fuenzalida Werner, Juan Pablo; Gröhn, Franziska; Moerschbacher, Bruno M

    2017-03-22

    Biomass waste products from green algae have recently been given new life, as these polysaccharides have potential applications in industry, agriculture, and medicine. One such polysaccharide group called ulvans displays many different, potentially useful properties that arise from their structural versatility. Hence, performing structural analyses on ulvan is crucial for future applications. However, chemical reaction-based analysis methods cannot fully characterize ulvan and tend to alter its structure. Thus, better methods require well-characterized ulvan-degrading enzymes. Therefore, we analysed a previously sequenced ulvan lyase (Genebank TM reference number JN104480) and characterized its domains. We suggest that the enzyme consists of a shorter than previously described catalytic domain, a newly identified substrate binding domain, and a C-terminal type 9 secretion system signal peptide. By separately expressing the two domains in E. coli, we confirmed that the binding domain is ulvan specific, having higher affinity for ulvan than most lectins for their ligands (affinity constant: 10 5  M -1 ). To our knowledge, this is the first description of an ulvan-binding domain. Overall, identifying this new binding domain is one step towards engineering ulvan enzymes that can be used to characterize ulvan, e.g. through enzymatic/mass spectrometric fingerprinting analyses, and help unlock its full potential.

  12. A conserved NAD+binding pocket that regulates protein-protein interactions during aging.

    Science.gov (United States)

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  13. Supplementary data: Novel mutation in ATP-binding domain of ...

    Indian Academy of Sciences (India)

    Novel mutation in ATP-binding domain of ABCD1 gene in adrenoleucodystrophy. Neeraj Kumar, Krishna K. Taneja, Atul Kumar, Deepti Nayar, Bhupesh Taneja, Satindra Aneja,. Madhuri Behari, Veena Kalra and Surendra K. Bansal. J. Genet. 89, 473–477. Figure 1. Rmsd plot of native and Arg617Ser substituted models.

  14. Identification of binding peptides of the ADAM15 disintegrin domain ...

    Indian Academy of Sciences (India)

    Madhsudhan

    ADAM15 disintegrin domain (RADD) that could inhibit melanoma cell adhesion by using Escherichia coli. Second, four specific binding peptides (peptides A, B, C, and D) were selected using a phage display 12-mer peptide library. The screening protocol involved 4 rounds of positive panning on RADD and 2 rounds of ...

  15. Substrate Binding Induces Domain Movements in Orotidine 5'-Monophosphate Decarboxylase

    DEFF Research Database (Denmark)

    Harris, Pernille Hanne; Poulsen, Jens-Christian Navarro; Jensen, Kaj Frank

    2002-01-01

    ); here we present the 2.5 Å structure of the uncomplexed apo enzyme, determined from twinned crystals. A structural analysis and comparison of the two structures of the E. coli enzyme show that binding of the inhibitor is accompanied by significant domain movements of approximately 12° around a hinge...

  16. Protein kinase A regulates AKAP250 (gravin) scaffold binding to the β2-adrenergic receptor

    OpenAIRE

    Tao, Jiangchuan; Wang, Hsien-yu; Malbon, Craig C.

    2003-01-01

    A-kinase-anchoring protein 250 (AKAP250; gravin) acts as a scaffold that binds protein kinase A (PKA), protein kinase C and protein phosphatases, associating reversibly with the β2-adrenergic receptor. The receptor-binding domain of the scaffold and the regulation of the receptor–scaffold association was revealed through mutagenesis and biochemical analyses. The AKAP domain found in other members of this superfamily is essential for the scaffold–receptor interactions. Gravin constructs lackin...

  17. Structural Analysis of the Receptor Binding Domain of Botulinum Neurotoxin Serotype D

    Energy Technology Data Exchange (ETDEWEB)

    Y Zhang; G Buchko; L Qin; H Robinson; S Varnum

    2011-12-31

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65{angstrom} resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10{angstrom} relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.

  18. Distant relationships amongst protein domains

    Indian Academy of Sciences (India)

    ncbs

    Homologous sequences of individual superfamily members are aligned and amino acid exchanges at individual positions were scored for conservation. Step – 1. Identification of structural motifs. Page 7. Alignment with Homologues. Page 8. il8 like proteins. 0. 50. 100. 150. 200. 250. 1. 6. 11. 16. 21. 26. 31. 36. 41. 46. 51.

  19. Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains.

    Science.gov (United States)

    Lewis, H A; Chen, H; Edo, C; Buckanovich, R J; Yang, Y Y; Musunuru, K; Zhong, R; Darnell, R B; Burley, S K

    1999-02-15

    Nova-1 and Nova-2 are related neuronal proteins that were initially cloned using antisera obtained from patients with the autoimmune neurological disease paraneoplastic opsoclonus-myoclonus ataxia (POMA). Both of these disease gene products contain three RNA-binding motifs known as K-homology or KH domains, and their RNA ligands have been identified via binding-site selection experiments. The KH motif structure has been determined previously using NMR spectroscopy, but not using X-ray crystallography. Many proteins contain more than one KH domain, yet there is no published structural information regarding the behavior of such multimers. We have obtained the first X-ray crystallographic structures of KH-domain-containing proteins. Structures of the third KH domains (KH3) of Nova-1 and Nova-2 were determined by multiple isomorphous replacement and molecular replacement at 2.6 A and 2.0 A, respectively. These highly similar RNA-binding motifs form a compact protease-resistant domain resembling an open-faced sandwich, consisting of a three-stranded antiparallel beta sheet topped by three alpha helices. In both Nova crystals, the lattice is composed of symmetric tetramers of KH3 domains that are created by two dimer interfaces. The crystal structures of both Nova KH3 domains are similar to the previously determined NMR structures. The most significant differences among the KH domains involve changes in the positioning of one or more of the alpha helices with respect to the betasheet, particularly in the NMR structure of the KH1 domain of the Fragile X disease protein FMR-1. Loop regions in the KH domains are clearly visible in the crystal structure, unlike the NMR structures, revealing the conformation of the invariant Gly-X-X-Gly segment that is thought to participate in RNA-binding and of the variable region. The tetrameric arrangements of the Nova KH3 domains provide insights into how KH domains may interact with each other in proteins containing multiple KH motifs.

  20. Characterization of αX I-Domain Binding to Receptors for Advanced Glycation End Products (RAGE).

    Science.gov (United States)

    Buyannemekh, Dolgorsuren; Nham, Sang-Uk

    2017-05-31

    The β2 integrins are cell surface transmembrane proteins regulating leukocyte functions, such as adhesion and migration. Two members of β2 integrin, αMβ2 and αXβ2, share the leukocyte distribution profile and integrin αXβ2 is involved in antigen presentation in dendritic cells and transendothelial migration of monocytes and macrophages to atherosclerotic lesions. Receptor for advanced glycation end products (RAGE), a member of cell adhesion molecules, plays an important role in chronic inflammation and atherosclerosis. Although RAGE and αXβ2 play an important role in inflammatory response and the pathogenesis of atherosclerosis, the nature of their interaction and structure involved in the binding remain poorly defined. In this study, using I-domain as a ligand binding motif of αXβ2, we characterize the binding nature and the interacting moieties of αX I-domain and RAGE. Their binding requires divalent cations (Mg 2+ and Mn 2+ ) and shows an affinity on the sub-micro molar level: the dissociation constant of αX I-domains binding to RAGE being 0.49 μM. Furthermore, the αX I-domains recognize the V-domain, but not the C1 and C2-domains of RAGE. The acidic amino acid substitutions on the ligand binding site of αX I-domain significantly reduce the I-domain binding activity to soluble RAGE and the alanine substitutions of basic amino acids on the flat surface of the V-domain prevent the V-domain binding to αX I-domain. In conclusion, the main mechanism of αX I-domain binding to RAGE is a charge interaction, in which the acidic moieties of αX I-domains, including E244, and D249, recognize the basic residues on the RAGE V-domain encompassing K39, K43, K44, R104, and K107.

  1. The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144

    Energy Technology Data Exchange (ETDEWEB)

    Briers, Yves [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Schmelcher, Mathias; Loessner, Martin J. [Institute of Food Science and Nutrition, ETH Zuerich, Schmelzbergstrasse 7, CH-8092 Zuerich (Switzerland); Hendrix, Jelle; Engelborghs, Yves [Laboratory of Biomolecular Dynamics, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200G, B-3001 Leuven (Belgium); Volckaert, Guido [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Lavigne, Rob, E-mail: rob.lavigne@biw.kuleuven.be [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium)

    2009-05-29

    The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD{sub KZ}), originating from Pseudomonas aeruginosa bacteriophage {phi}KZ, has been examined using a fusion protein of PBD{sub KZ} and green fluorescent protein (PBD{sub KZ}-GFP). A fluorescence recovery after photobleaching analysis of bound PBD{sub KZ}-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10{sup 7} M{sup -1} for the PBD{sub KZ}-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD{sub KZ}-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

  2. Resonance assignments for the substrate binding domain of Hsp70 chaperone Ssa1 from Saccharomyces cerevisiae.

    Science.gov (United States)

    Hu, Wanhui; Wu, Huiwen; Zhang, Hong; Gong, Weibin; Perrett, Sarah

    2015-10-01

    Hsp70 chaperone proteins play crucial roles in the cell. Extensive structural and functional studies have been performed for bacterial and mammalian Hsp70s. Ssa1 from Saccharomyces cerevisiae is a member of the Hsp70 family. In vivo and biochemical studies on Ssa1 have revealed that it regulates prion propagation and the cell cycle. However, no structural data has been obtained for Ssa1 up to now. Here we report the almost complete (96 %) (1)H, (13)C, (15)N backbone and side chain NMR assignment of the 18.8 kDa Ssa1 substrate binding domain. The construct includes residues 382-554, which corresponds to the entire substrate binding domain and two following α-helices in homologous structures. The secondary structure predicted from the assigned chemical shifts is consistent with that of homologous Hsp70 substrate binding domains.

  3. Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily

    DEFF Research Database (Denmark)

    Bikker, Floris J; Ligtenberg, Antoon J M; Nazmi, Kamran

    2002-01-01

    SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment...

  4. In Situ Protein Binding Assay Using Fc-Fusion Proteins.

    Science.gov (United States)

    Padmanabhan, Nirmala; Siddiqui, Tabrez J

    2017-01-01

    This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

  5. The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

    DEFF Research Database (Denmark)

    Yatsenko, A S; Kucherenko, M M; Pantoja, M

    2009-01-01

    BACKGROUND: Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal ......BACKGROUND: Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C......-terminal PPXY motif has been established as a binding site for Dystrophin (Dys) WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. RESULTS: We now find that both WW binding sites are important for maintaining full Dg...

  6. Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains.

    Directory of Open Access Journals (Sweden)

    Nicolas Siaud

    2011-12-01

    Full Text Available The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR. Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.

  7. Elucidating the Interacting Domains of Chandipura Virus Nucleocapsid Protein

    Directory of Open Access Journals (Sweden)

    Kapila Kumar

    2013-01-01

    Full Text Available The nucleocapsid (N protein of Chandipura virus (CHPV plays a crucial role in viral life cycle, besides being an important structural component of the virion through proper organization of its interactions with other viral proteins. In a recent study, the authors had mapped the associations among CHPV proteins and shown that N protein interacts with four of the viral proteins: N, phosphoprotein (P, matrix protein (M, and glycoprotein (G. The present study aimed to distinguish the regions of CHPV N protein responsible for its interactions with other viral proteins. In this direction, we have generated the structure of CHPV N protein by homology modeling using SWISS-MODEL workspace and Accelrys Discovery Studio client 2.55 and mapped the domains of N protein using PiSQRD. The interactions of N protein fragments with other proteins were determined by ZDOCK rigid-body docking method and validated by yeast two-hybrid and ELISA. The study revealed a unique binding site, comprising of amino acids 1–30 at the N terminus of the nucleocapsid protein (N1 that is instrumental in its interactions with N, P, M, and G proteins. It was also observed that N2 associates with N and G proteins while N3 interacts with N, P, and M proteins.

  8. The BAR domain protein PICK1 regulates cell recognition and morphogenesis by interacting with Neph proteins.

    Science.gov (United States)

    Höhne, Martin; Lorscheider, Johannes; von Bardeleben, Anna; Dufner, Matthias; Scharf, M Antonia; Gödel, Markus; Helmstädter, Martin; Schurek, Eva-Maria; Zank, Sibylle; Gerke, Peter; Kurschat, Christine; Sivritas, Sema Hayriye; Neumann-Haefelin, Elke; Huber, Tobias B; Reinhardt, H Christian; Schauss, Astrid C; Schermer, Bernhard; Fischbach, Karl-Friedrich; Benzing, Thomas

    2011-08-01

    Neph proteins are evolutionarily conserved membrane proteins of the immunoglobulin superfamily that control the formation of specific intercellular contacts. Cell recognition through these proteins is essential in diverse cellular contexts such as patterning of the compound eye in Drosophila melanogaster, neuronal connectivity in Caenorhabditis elegans, and the formation of the kidney filtration barrier in mammals. Here we identify the PDZ and BAR domain protein PICK1 (protein interacting with C-kinase 1) as a Neph-interacting protein. Binding required dimerization of PICK1, was dependent on PDZ domain protein interactions, and mediated stabilization of Neph1 at the plasma membrane. Moreover, protein kinase C (PKCα) activity facilitated the interaction through releasing Neph proteins from their binding to the multidomain scaffolding protein zonula occludens 1 (ZO-1), another PDZ domain protein. In Drosophila, the Neph homologue Roughest is essential for sorting of interommatidial precursor cells and patterning of the compound eye. RNA interference-mediated knockdown of PICK1 in the Drosophila eye imaginal disc caused a Roughest destabilization at the plasma membrane and a phenotype that resembled rst mutation. These data indicate that Neph proteins and PICK1 synergistically regulate cell recognition and contact formation.

  9. Proteome compression via protein domain compositions.

    Science.gov (United States)

    Hayashida, Morihiro; Ruan, Peiying; Akutsu, Tatsuya

    2014-06-01

    In this paper, we study domain compositions of proteins via compression of whole proteins in an organism for the sake of obtaining the entropy that the individual contains. We suppose that a protein is a multiset of domains. Since gene duplication and fusion have occurred through evolutionary processes, the same domains and the same compositions of domains appear in multiple proteins, which enables us to compress a proteome by using references to proteins for duplicated and fused proteins. Such a network with references to at most two proteins is modeled as a directed hypergraph. We propose a heuristic approach by combining the Edmonds algorithm and an integer linear programming, and apply our procedure to 14 proteomes of Dictyostelium discoideum, Escherichia coli, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Oryza sativa, Danio rerio, Xenopus laevis, Gallus gallus, Mus musculus, Pan troglodytes, and Homo sapiens. The compressed size using both of duplication and fusion was smaller than that using only duplication, which suggests the importance of fusion events in evolution of a proteome. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    Culard, G.; Eon, S.; DeVuyst, G.; Charlier, M.; Spotheim-Maurizot, M.

    2003-01-01

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  11. Crystal structure of the UBR-box from UBR6/FBXO11 reveals domain swapping mediated by zinc binding.

    Science.gov (United States)

    Muñoz-Escobar, Juliana; Kozlov, Guennadi; Gehring, Kalle

    2017-10-01

    The UBR-box is a 70-residue zinc finger domain present in the UBR family of E3 ubiquitin ligases that directly binds N-terminal degradation signals in substrate proteins. UBR6, also called FBXO11, is an UBR-box containing E3 ubiquitin ligase that does not bind N-terminal signals. Here, we present the crystal structure of the UBR-box domain from human UBR6. The dimeric crystal structure reveals a unique form of domain swapping mediated by zinc coordination, where three independent protein chains come together to regenerate the topology of the monomeric UBR-box fold. Analysis of the structure suggests that the absence of N-terminal residue binding arises from the lack of an amino acid binding pocket. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  12. MicroRNA-145 ModulatesN6-Methyladenosine Levels by Targeting the 3'-Untranslated mRNA Region of theN6-Methyladenosine Binding YTH Domain Family 2 Protein.

    Science.gov (United States)

    Yang, Zhe; Li, Jiong; Feng, Guoxing; Gao, Shan; Wang, Yuan; Zhang, Shuqin; Liu, Yunxia; Ye, Lihong; Li, Yueguo; Zhang, Xiaodong

    2017-03-03

    N 6 -Methyladenosine (m 6 A) is a prevalent modification present in the mRNAs of higher eukaryotes. YTH domain family 2 (YTHDF2), an m 6 A "reader" protein, can recognize mRNA m 6 A sites to mediate mRNA degradation. However, the regulatory mechanism of YTHDF2 is poorly understood. To this end, we investigated the post-transcriptional regulation of YTHDF2. Bioinformatics analysis suggested that the microRNA miR-145 might target the 3'-untranslated region (3'-UTR) of YTHDF2 mRNA. The levels of miR-145 were negatively correlated with those of YTHDF2 mRNA in clinical hepatocellular carcinoma (HCC) tissues, and immunohistochemical staining revealed that YTHDF2 was closely associated with malignancy of HCC. Interestingly, miR-145 decreased the luciferase activities of 3'-UTR of YTHDF2 mRNA. Mutation of predicted miR-145 binding sites in the 3'-UTR of YTHDF2 mRNA abolished the miR-145-induced decrease in luciferase activity. Overexpression of miR-145 dose-dependently down-regulated YTHDF2 expression in HCC cells at the levels of both mRNA and protein. Conversely, inhibition of miR-145 resulted in the up-regulation of YTHDF2 in the cells. Dot blot analysis and immunofluorescence staining revealed that the overexpression of miR-145 strongly increased m 6 A levels relative to those in control HCC cells, and this increase could be blocked by YTHDF2 overexpression. Moreover, miR-145 inhibition strongly decreased m 6 A levels, which were rescued by treatment with a small interfering RNA-based YTHDF2 knockdown. Thus, we conclude that miR-145 modulates m 6 A levels by targeting the 3'-UTR of YTHDF2 mRNA in HCC cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules*

    Science.gov (United States)

    Atkin, Kate E.; Brentnall, Andrew S.; Harris, Gemma; Bingham, Richard J.; Erat, Michele C.; Millard, Christopher J.; Schwarz-Linek, Ulrich; Staunton, David; Vakonakis, Ioannis; Campbell, Iain D.; Potts, Jennifer R.

    2010-01-01

    Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the 8F19F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to 8F19F1 and that UR binding to 8F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn. PMID:20843804

  14. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    Science.gov (United States)

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein. © 2014 The Authors.

  15. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

  16. Bay11-7082 attenuates neuropathic pain via inhibition of nuclear factor-kappa B and nucleotide-binding domain-like receptor protein 3 inflammasome activation in dorsal root ganglions in a rat model of lumbar disc herniation

    Directory of Open Access Journals (Sweden)

    Zhang AL

    2017-02-01

    Full Text Available Ailiang Zhang, Kun Wang, Lianghua Ding, Xinnan Bao, Xuan Wang, Xubin Qiu, Jinbo Liu Spine Surgery, Third Affiliated Hospital of Soochow University, Changzhou, People’s Republic of China Abstract: Lumbar disc herniation (LDH is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB and nucleotide-binding domain-like receptor protein 3 (NLRP3 inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP was implanted in the left L5 dorsal root ganglion (DRG to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC, caspase-1, interleukin (IL-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation. Keywords: pain, NLRP3, NF-κB, dorsal root ganglion, nucleus pulposus

  17. Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose.

    Directory of Open Access Journals (Sweden)

    Ting-Ying Jiang

    Full Text Available The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21 members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.

  18. Two Unique Ligand-Binding Clamps of Rhizopus oryzae Starch Binding Domain for Helical Structure Disruption of Amylose

    Science.gov (United States)

    Jiang, Ting-Ying; Ci, Yuan-Pei; Chou, Wei-I; Lee, Yuan-Chuan; Sun, Yuh-Ju; Chou, Wei-Yao; Li, Kun-Mou; Chang, Margaret Dah-Tsyr

    2012-01-01

    The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs. PMID:22815939

  19. Are many Z-DNA binding proteins actually phospholipid-binding proteins?

    OpenAIRE

    Krishna, P; Kennedy, B P; Waisman, D M; van de Sande, J H; McGhee, J D

    1990-01-01

    We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein. We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA. In al...

  20. Fatty Acid Binding Proteins in Prostate Cancer

    National Research Council Canada - National Science Library

    Jett, Marti

    2000-01-01

    We have shown that there is a distinct pattern of fatty acid binding protein (FAEP) expression in prostate cancer vs normal cells and that finding has be confirmed in patient samples of biopsy specimens...

  1. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

    DEFF Research Database (Denmark)

    Wong, Mei Mei Jaslyn Elizabeth; Midtgaard, Søren Roi; Gysel, Kira

    2015-01-01

    LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multi......LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement...... solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers...

  2. Domain distribution and intrinsic disorder in hubs in the human protein-protein interaction network.

    Science.gov (United States)

    Patil, Ashwini; Kinoshita, Kengo; Nakamura, Haruki

    2010-08-01

    Intrinsic disorder and distributed surface charge have been previously identified as some of the characteristics that differentiate hubs (proteins with a large number of interactions) from non-hubs in protein-protein interaction networks. In this study, we investigated the differences in the quantity, diversity, and functional nature of Pfam domains, and their relationship with intrinsic disorder, in hubs and non-hubs. We found that proteins with a more diverse domain composition were over-represented in hubs when compared with non-hubs, with the number of interactions in hubs increasing with domain diversity. Conversely, the fraction of intrinsic disorder in hubs decreased with increasing number of ordered domains. The difference in the levels of disorder was more prominent in hubs and non-hubs with fewer domains. Functional analysis showed that hubs were enriched in kinase and adaptor domains acting primarily in signal transduction and transcription regulation, whereas non-hubs had more DNA-binding domains and were involved in catalytic activity. Consistent with the differences in the functional nature of their domains, hubs with two or more domains were more likely to connect distinct functional modules in the interaction network when compared with single domain hubs. We conclude that the availability of greater number and diversity of ordered domains, in addition to the tendency to have promiscuous domains, differentiates hubs from non-hubs and provides an additional means of achieving interaction promiscuity. Further, hubs with fewer domains use greater levels of intrinsic disorder to facilitate interaction promiscuity with the prevalence of disorder decreasing with increasing number of ordered domains.

  3. C2 Domains as Protein-Protein Interaction Modules in the Ciliary Transition Zone

    Directory of Open Access Journals (Sweden)

    Kim Remans

    2014-07-01

    Full Text Available RPGR-interacting protein 1 (RPGRIP1 is mutated in the eye disease Leber congenital amaurosis (LCA and its structural homolog, RPGRIP1-like (RPGRIP1L, is mutated in many different ciliopathies. Both are multidomain proteins that are predicted to interact with retinitis pigmentosa G-protein regulator (RPGR. RPGR is mutated in X-linked retinitis pigmentosa and is located in photoreceptors and primary cilia. We solved the crystal structure of the complex between the RPGR-interacting domain (RID of RPGRIP1 and RPGR and demonstrate that RPGRIP1L binds to RPGR similarly. RPGRIP1 binding to RPGR affects the interaction with PDEδ, the cargo shuttling factor for prenylated ciliary proteins. RPGRIP1-RID is a C2 domain with a canonical β sandwich structure that does not bind Ca2+ and/or phospholipids and thus constitutes a unique type of protein-protein interaction module. Judging from the large number of C2 domains in most of the ciliary transition zone proteins identified thus far, the structure presented here seems to constitute a cilia-specific module that is present in multiprotein transition zone complexes.

  4. LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin

    Energy Technology Data Exchange (ETDEWEB)

    Kawamoto, Eiji [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Okamoto, Takayuki, E-mail: okamotot@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Takagi, Yoshimi [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Honda, Goichi [Medical Affairs Department, Asahi Kasei Pharma Corporation, 1-105 Kanda Jinbo-cho, Chiyoda-ku, Tokyo 101-8101 (Japan); Suzuki, Koji [Faculty of Pharmaceutical Science, Suzuka University of Medical Science, 3500-3, Minamitamagaki-cho, Suzuka, Mie 513-8679 (Japan); Imai, Hiroshi [Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Shimaoka, Motomu, E-mail: shimaoka@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan)

    2016-05-13

    LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding of PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. - Highlights: • LFA-1 and Mac-1 integrins bind to the anti-coagulant molecule thrombomodulin. • The serine/threonine-rich domain of thrombomodulin is essential to interact with the LFA-1 and Mac-1 integrins on PBMCs. • Integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.

  5. Telomere-binding proteins of Arabidopsis thaliana.

    Science.gov (United States)

    Zentgraf, U

    1995-02-01

    The nucleoprotein structure of Arabidopsis thaliana telomeres was investigated. A protein specifically binding to telomeric sequences was characterized by gel mobility shift assays with synthetic oligonucleotides consisting of four 7 bp telomeric repeats of Arabidopsis (TTTAGGG) and crude nuclear protein extracts of Arabidopsis leaves. These DNA-protein binding studies revealed that the binding affinity of this telomere-binding protein to the G-rich single-strand as well as to the double-stranded telomeric DNA is much higher than to the C-rich single-strand. The molecular mass of the protein was identified by SDS-PAGE to be 67 kDa. The isoelectric points were determined to be 5.0, 4.85 and 4.7, respectively, indicating that either one protein with different modifications or three slightly different proteins have been isolated. An RNA component, possibly serving as a template for reverse transcription of a plant telomerase, does not mediate the DNA-protein contact because the DNA-protein interactions were not RNAse-sensitive.

  6. Carcinoembryonic antigen family receptor recognition by gonococcal Opa proteins requires distinct combinations of hypervariable Opa protein domains.

    Science.gov (United States)

    Bos, Martine P; Kao, David; Hogan, Daniel M; Grant, Christopher C R; Belland, Robert J

    2002-04-01

    Neisserial Opa proteins function as a family of adhesins that bind heparan sulfate proteoglycan (HSPG) or carcinoembryonic antigen family (CEACAM) receptors on human host cells. In order to define the CEACAM binding domain on Opa proteins, we tested the binding properties of a series of gonococcal (strain MS11) recombinants producing mutant and chimeric Opa proteins with alterations in one or more of the four surface-exposed loops. Mutagenesis demonstrated that the semivariable domain, present in the first loop, was completely dispensable for CEACAM binding. In contrast, the two hypervariable (HV) regions present in the second and third loops were essential for binding; deletion of either domain resulted in loss of receptor recognition. Deletion of the fourth loop resulted in a severe decrease in Opa expression at the cell surface and could therefore not be tested for CEACAM binding. Chimeric Opa variants, containing combinations of HV regions derived from different CEACAM binding Opa proteins, lost most of their receptor binding activity. Some chimeric variants gained HSPG binding activity. Together, our results indicate that full recognition of CEACAM receptors by Opa proteins requires a highly coordinate interplay between both HV regions. Furthermore, shuffling of HV regions may result in novel HSPG receptor binding activity.

  7. Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

    Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

  8. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

    Energy Technology Data Exchange (ETDEWEB)

    Wong, Jaslyn E. M. M. [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark); Midtgaard, Søren Roi [University of Copenhagen, Universitetsparken 5, 2100 Copenhagen (Denmark); Gysel, Kira [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark); Thygesen, Mikkel B.; Sørensen, Kasper K.; Jensen, Knud J. [University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Stougaard, Jens; Thirup, Søren; Blaise, Mickaël, E-mail: mickael.blaise@cpbs.cnrs.fr [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus (Denmark)

    2015-03-01

    The crystal and solution structures of the T. thermophilus NlpC/P60 d, l-endopeptidase as well as the co-crystal structure of its N-terminal LysM domains bound to chitohexaose allow a proposal to be made regarding how the enzyme recognizes peptidoglycan. LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

  9. Escherichia coli lipoprotein binds human plasminogen via an intramolecular domain

    Directory of Open Access Journals (Sweden)

    Tammy eGonzalez

    2015-10-01

    Full Text Available Escherichia coli lipoprotein (Lpp is a major cellular component that exists in two distinct states, bound-form and free-form. Bound-form Lpp is known to interact with the periplasmic bacterial cell wall, while free-form Lpp is localized to the bacterial cell surface. A function for surface-exposed Lpp has yet to be determined. We hypothesized that the presence of C-terminal lysines in the surface-exposed region of Lpp would facilitate binding to the host zymogen plasminogen, a protease commandeered by a number of clinically important bacteria. Recombinant Lpp was synthesized and the binding of Lpp to plasminogen, the effect of various inhibitors on this binding, and the effects of various mutations of Lpp on Lpp-plasminogen interactions were examined. Additionally, the ability of Lpp-bound plasminogen to be converted to active plasmin was analyzed. We determined that Lpp binds plasminogen via an atypical domain located near the center of mature Lpp that may not be exposed on the surface of intact E. coli according to the current localization model. Finally, we found that plasminogen bound by Lpp can be converted to active plasmin. While the consequences of Lpp binding plasminogen are unclear, these results prompt further investigation of the ability of surface exposed Lpp to interact with host molecules such as extracellular matrix components and complement regulators, and the role of these interactions in infections caused by E. coli and other bacteria.

  10. BAR domain proteins regulate Rho GTPase signaling.

    Science.gov (United States)

    Aspenström, Pontus

    2014-01-01

    BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis.

  11. Reconstituting Protein Interaction Networks Using Parameter-Dependent Domain-Domain Interactions

    Science.gov (United States)

    2013-05-07

    that approximately 80% of eukaryotic proteins and 67% of prokaryotic proteins have multiple domains [13,14]. Most annotation databases characterize...domain annotations, Domain-domain interactions, Protein-protein interaction networks Background The living cell is a dynamic, interconnected system...detailed in Methods. Here, we illustrate its application on a well- annotated single- cell organism. We created a merged set of protein-domain annotations

  12. Haptenation: Chemical Reactivity and Protein Binding

    Directory of Open Access Journals (Sweden)

    Itai Chipinda

    2011-01-01

    Full Text Available Low molecular weight chemical (LMW allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

  13. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    Science.gov (United States)

    Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

    2017-01-01

    Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

  14. Takifugu rubripes cation independent mannose 6-phosphate receptor: Cloning, expression and functional characterization of the IGF-II binding domain.

    Science.gov (United States)

    A, Ajith Kumar; Nadimpalli, Siva Kumar

    2018-01-31

    Mannose 6-phosphate/IGF-II receptor mediated lysosomal clearance of insulin-like growth factor-II is significantly associated with the evolution of placental mammals. The protein is also referred to as the IGF-II receptor. Earlier studies suggested relatively low binding affinity between the receptor and ligand in prototherian and metatherian mammals. In the present study, we cloned the IGF-II binding domain of the early vertebrate fugu fish and expressed it in bacteria. A 72000Da truncated receptor containing the IGF-II binding domain was obtained. Analysis of this protein (covering domains 11-13 of the CIMPR) for its affinity to fish and human IGF-II by ligand blot assays and ELISA showed that the expressed receptor can specifically bind to both fish and human IGF-II. Additionally, a peptide-specific antibody raised against the region of the IGF-II binding domain also was able to recognize the IGF-II binding regions of mammalian and non-mammalian cation independent MPR protein. These interactions were further characterized by Surface Plasma resonance support that the receptor binds to fish IGF-II, with a dissociation constant of 548nM. Preliminary analysis suggests that the binding mechanism as well as the affinity of the fish and human receptor for IGF-II may have varied according to different evolutionary pressures. Copyright © 2018. Published by Elsevier B.V.

  15. SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G.

    Science.gov (United States)

    Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus

    2017-01-01

    The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm -positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis , respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis . The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis.

  16. Non-Catalytic Participation of the Pin1 Peptidyl-Prolyl Isomerase Domain in Target Binding

    Directory of Open Access Journals (Sweden)

    Brendan Tooke Innes

    2013-02-01

    Full Text Available Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that has the potential to add an additional level of regulation within protein kinase mediated signaling pathways. Furthermore, there is a mounting body of evidence implicating Pin1 in the emergence of pathological phenotypes in neurodegeneration and cancer through the isomerization of a wide variety of substrates at peptidyl-prolyl bonds where the residue preceding proline is a phosphorylated serine or threonine residue (ie. pS/T-P motifs. A key step in this regulatory process is the interaction of Pin-1 with its substrates. This is a complex process since Pin1 is composed of two domains, the catalytic PPIase domain, and a type IV WW domain, both of which recognize pS/T-P motifs. The observation that the WW domain exhibits considerably higher binding affinity for pS/T-P motifs has led to predictions that the two domains may have distinct roles in mediating the actions of Pin1 on its substrates. To evaluate the participation of its individual domains in target binding, we performed GST pulldowns to monitor interactions between various forms of Pin1 and mitotic phospho-proteins that revealed two classes of Pin-1 interacting proteins, differing in their requirement for residues within the PPIase domain. From these observations, we consider models for Pin1-substrate interactions and the potential functions of the different classes of Pin1 interacting proteins. We also compare sequences that are recognized by Pin1 within its individual interaction partners to investigate the underlying basis for its different types of interactions.

  17. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each...

  18. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each ...

  19. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

    2017-07-27

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  20. Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication

    DEFF Research Database (Denmark)

    Meschi, Joseph; Crouch, Erika C; Skolnik, Paul

    2005-01-01

    The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL......) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate...... defence against HIV. A chimeric protein containing the N-terminal and collagen domains of SP-D linked to the neck and carbohydrate-recognition domains of MBL (called SP-D/MBL(neck+CRD)) had greater ability to bind to gp120 and inhibit virus replication than either SP-D or MBL. The enhanced binding of SP...

  1. The Anabaena sensory rhodopsin transducer defines a novel superfamily of prokaryotic small-molecule binding domains

    Directory of Open Access Journals (Sweden)

    De Souza Robson F

    2009-08-01

    Full Text Available Abstract The Anabaena sensory rhodopsin transducer (ASRT is a small protein that has been claimed to function as a signaling molecule downstream of the cyanobacterial sensory rhodopsin. However, orthologs of ASRT have been detected in several bacteria that lack rhodopsin, raising questions about the generality of this function. Using sequence profile searches we show that ASRT defines a novel superfamily of β-sandwich fold domains. Through contextual inference based on domain architectures and predicted operons and structural analysis we present strong evidence that these domains bind small molecules, most probably sugars. We propose that the intracellular versions like ASRT probably participate as sensors that regulate a diverse range of sugar metabolism operons or even the light sensory behavior in Anabaena by binding sugars or related metabolites. We also show that one of the extracellular versions define a predicted sugar-binding structure in a novel cell-surface lipoprotein found across actinobacteria, including several pathogens such as Tropheryma, Actinomyces and Thermobifida. The analysis of this superfamily also provides new data to investigate the evolution of carbohydrate binding modes in β-sandwich domains with very different topologies. Reviewers: This article was reviewed by M. Madan Babu and Mark A. Ragan.

  2. Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2

    Directory of Open Access Journals (Sweden)

    Lennick Michael

    2003-01-01

    Full Text Available Abstract Background Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response. Results Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75 into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFα and TNFβ and induced an unexpected biological response in a TNFR2-specific manner. Conclusions To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity.

  3. XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region.

    Science.gov (United States)

    Widlund, Per O; Stear, Jeffrey H; Pozniakovsky, Andrei; Zanic, Marija; Reber, Simone; Brouhard, Gary J; Hyman, Anthony A; Howard, Jonathon

    2011-02-15

    XMAP215/Dis1 family proteins positively regulate microtubule growth. Repeats at their N termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. Here, we take advantage of a recent crystal structure of the third TOG domain from Caenorhabditis elegans, Zyg9, and mutate key residues in each TOG domain of XMAP215 that are predicted to be important for interaction with the tubulin heterodimer. We determined the contributions of the individual TOG domains to microtubule growth. We show that the TOG domains are absolutely required to bind free tubulin and that the domains differentially contribute to XMAP215's overall affinity for free tubulin. The mutants' overall affinity for free tubulin correlates well with polymerase activity. Furthermore, we demonstrate that an additional basic region is important for targeting to the microtubule lattice and is critical for XMAP215 to function at physiological concentrations. Using this information, we have engineered a "bonsai" protein, with two TOG domains and a basic region, that has almost full polymerase activity.

  4. Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

    International Nuclear Information System (INIS)

    Martinez, Elisabeth D.; Pattabiraman, Nagarajan; Danielsen, Mark

    2005-01-01

    The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increased ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids

  5. Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System.

    Science.gov (United States)

    Pliotas, Christos; Grayer, Samuel C; Ekkerman, Silvia; Chan, Anthony K N; Healy, Jess; Marius, Phedra; Bartlett, Wendy; Khan, Amjad; Cortopassi, Wilian A; Chandler, Shane A; Rasmussen, Tim; Benesch, Justin L P; Paton, Robert S; Claridge, Timothy D W; Miller, Samantha; Booth, Ian R; Naismith, James H; Conway, Stuart J

    2017-08-15

    Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

  6. Computational search for aflatoxin binding proteins

    Science.gov (United States)

    Wang, Ying; Liu, Jinfeng; Zhang, Lujia; He, Xiao; Zhang, John Z. H.

    2017-10-01

    Aflatoxin is one of the mycotoxins that contaminate various food products. Among various aflatoxin types (B1, B2, G1, G2 and M1), aflatoxin B1 is the most important and the most toxic one. In this study, through computational screening, we found that several proteins may bind specifically with different type of aflatoxins. Combination of theoretical methods including target fishing, molecular docking, molecular dynamics (MD) simulation, MM/PBSA calculation were utilized to search for new aflatoxin B1 binding proteins. A recently developed method for calculating entropic contribution to binding free energy called interaction entropy (IE) was employed to compute the binding free energy between the protein and aflatoxin B1. Through comprehensive comparison, three proteins, namely, trihydroxynaphthalene reductase, GSK-3b, and Pim-1 were eventually selected as potent aflatoxin B1 binding proteins. GSK-3b and Pim-1 are drug targets of cancers or neurological diseases. GSK-3b is the strongest binder for aflatoxin B1.

  7. Targeting proteins to liquid-ordered domains in lipid membranes.

    Science.gov (United States)

    Stachowiak, Jeanne C; Hayden, Carl C; Sanchez, Mari Angelica A; Wang, Julia; Bunker, Bruce C; Voigt, James A; Sasaki, Darryl Y

    2011-02-15

    We demonstrate the construction of novel protein-lipid assemblies through the design of a lipid-like molecule, DPIDA, endowed with tail-driven affinity for specific lipid membrane phases and head-driven affinity for specific proteins. In studies performed on giant unilamellar vesicles (GUVs) with varying mole fractions of dipalymitoylphosphatidylcholine (DPPC), cholesterol, and diphytanoylphosphatidyl choline (DPhPC), DPIDA selectively partitioned into the more ordered phases, either solid or liquid-ordered (L(o)) depending on membrane composition. Fluorescence imaging established the phase behavior of the resulting quaternary lipid system. Fluorescence correlation spectroscopy confirmed the fluidity of the L(o) phase containing DPIDA. In the presence of CuCl(2), the iminodiacetic acid (IDA) headgroup of DPIDA forms the Cu(II)-IDA complex that exhibits a high affinity for histidine residues. His-tagged proteins were bound specifically to domains enriched in DPIDA, demonstrating the capacity to target protein binding selectively to both solid and L(o) phases. Steric pressure from the crowding of surface-bound proteins transformed the domains into tubules with persistence lengths that depended on the phase state of the lipid domains.

  8. Structure of a group C streptococcal protein that binds to fibrinogen, albumin and immunoglobulin G via overlapping modules.

    Science.gov (United States)

    Talay, S R; Grammel, M P; Chhatwal, G S

    1996-04-15

    Pathogenic streptococci express surface proteins that bind to host serum proteins. A novel multiple-ligand-binding protein has now been identified in a species belonging to serotype C streptococci. This protein binds to fibrinogen, albumin and IgG and was therefore designated FAI protein. The structure of the fai gene has been determined, and deletion analysis and expression of FAI fusion polypeptides revealed that the binding domain for fibrinogen and IgG is located within the nonrepetitive N-terminal half of the protein. A 93-amino acid peptide retained the ability to bind both proteins, whereas a 56-amino acid subpeptide only bound fibrinogen. IgG-binding activity required the complete fibrinogen-binding domain and an additional 37 amino acids C-terminal to it, and albumin-binding activity was only obtained with a polypeptide reflecting the complete surface-exposed region of FAI protein indicating that the binding sites for each ligand were located on overlapping modules. Signal sequence, C repeat region and C-terminus revealed high homology to group A streptococcal M proteins whereas the N-terminal region containing the fibrinogen/IgG-binding domains is completely different and exhibits no similarity to any other previously characterized protein. Thus FAI protein exhibits a framework structure that might have evolved in group C streptococci via fusion of unrelated sequences, thereby generating an albumin-binding domain in the functional context of multiple-ligand-binding activity.

  9. Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy

    OpenAIRE

    Shishmarev, Dmitry; Wang, Yao; Mason, Claire E.; Su, Xun-Cheng; Oakley, Aaron J.; Graham, Bim; Huber, Thomas; Dixon, Nicholas E.; Otting, Gottfried

    2013-01-01

    Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron den...

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

    International Nuclear Information System (INIS)

    Yamashita, Eiki; Nakagawa, Atsushi; Takahashi, Junichi; Tsunoda, Kin-ichi; Yamada, Seiko; Takeda, Shigeki

    2011-01-01

    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

  11. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  12. Secretory Vesicle Priming by CAPS Is Independent of Its SNARE-Binding MUN Domain

    Directory of Open Access Journals (Sweden)

    Cuc Quynh Nguyen Truong

    2014-11-01

    Full Text Available Priming of secretory vesicles is a prerequisite for their Ca2+-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca2+-dependent activator protein for secretion (CAPS also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin by MUN domains of CAPSs, and is therefore not redundant with Munc13 action.

  13. Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor*

    Science.gov (United States)

    An, Bo; Abbonante, Vittorio; Xu, Huifang; Gavriilidou, Despoina; Yoshizumi, Ayumi; Bihan, Dominique; Farndale, Richard W.; Kaplan, David L.; Balduini, Alessandra; Leitinger, Birgit; Brodsky, Barbara

    2016-01-01

    A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities. PMID:26702058

  14. DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

    Science.gov (United States)

    Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

    2017-09-01

    Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Multiple protonation equilibria in electrostatics of protein-protein binding.

    Science.gov (United States)

    Piłat, Zofia; Antosiewicz, Jan M

    2008-11-27

    All proteins contain groups capable of exchanging protons with their environment. We present here an approach, based on a rigorous thermodynamic cycle and the partition functions for energy levels characterizing protonation states of the associating proteins and their complex, to compute the electrostatic pH-dependent contribution to the free energy of protein-protein binding. The computed electrostatic binding free energies include the pH of the solution as the variable of state, mutual "polarization" of associating proteins reflected as changes in the distribution of their protonation states upon binding and fluctuations between available protonation states. The only fixed property of both proteins is the conformation; the structure of the monomers is kept in the same conformation as they have in the complex structure. As a reference, we use the electrostatic binding free energies obtained from the traditional Poisson-Boltzmann model, computed for a single macromolecular conformation fixed in a given protonation state, appropriate for given solution conditions. The new approach was tested for 12 protein-protein complexes. It is shown that explicit inclusion of protonation degrees of freedom might lead to a substantially different estimation of the electrostatic contribution to the binding free energy than that based on the traditional Poisson-Boltzmann model. This has important implications for the balancing of different contributions to the energetics of protein-protein binding and other related problems, for example, the choice of protein models for Brownian dynamics simulations of their association. Our procedure can be generalized to include conformational degrees of freedom by combining it with molecular dynamics simulations at constant pH. Unfortunately, in practice, a prohibitive factor is an enormous requirement for computer time and power. However, there may be some hope for solving this problem by combining existing constant pH molecular dynamics

  16. NMR structure of a biologically active peptide containing the RNA-binding domain of human immunodeficiency virus type 1 Tat.

    Science.gov (United States)

    Mujeeb, A; Bishop, K; Peterlin, B M; Turck, C; Parslow, T G; James, T L

    1994-01-01

    The Tat protein of human immunodeficiency virus type 1 enhances transcription by binding to a specific RNA element on nascent viral transcripts. Binding is mediated by a 10-amino acid basic domain that is rich in arginines and lysines. Here we report the three-dimensional peptide backbone structure of a biologically active 25-mer peptide that contains the human immunodeficiency virus type 1 Tat basic domain linked to the core regulatory domain of another lentiviral Tat--i.e., that from equine infectious anemia virus. Circular dichroism and two-dimensional proton NMR studies of this hybrid peptide indicate that the Tat basic domain forms a stable alpha-helix, whereas the adjacent regulatory sequence is mostly in extended form. These findings suggest that the tendency to form stable alpha-helices may be a common property of arginine- and lysine-rich RNA-binding domains. Images PMID:8058789

  17. The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

    Science.gov (United States)

    Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

    2014-12-01

    The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

  18. Structural basis of antifreeze activity of a bacterial multi-domain antifreeze protein.

    Directory of Open Access Journals (Sweden)

    Chen Wang

    Full Text Available Antifreeze proteins (AFPs enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar. Each consists of an irregular β-helix with a triangular cross-section and a long α-helix that runs parallel on one side of the β-helix. Both domains are stabilized by hydrophobic interactions. A flat plane on the same face of each domain's β-helix was identified as the ice binding site. Mutating any of the smaller residues on the ice binding site to bulkier ones decreased the antifreeze activity. The bulky side chain of Leu174 in domain A sterically hinders the binding of water molecules to the protein backbone, partially explaining why antifreeze activity by domain A is inferior to that of domain B. Our data provide a molecular basis for understanding differences in antifreeze activity between the two domains of this protein and general insight on how structural differences in the ice-binding sites affect the activity of AFPs.

  19. Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

    Science.gov (United States)

    Kandeel, Mahmoud; Kitade, Yukio

    2018-02-01

    RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, K dPolyU and K dPolyC were 15.8 and 9.3μM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and β5 in the β-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Crystallization and preliminary crystallographic characterization of the origin-binding domain of the bacteriophage λ O replication initiator

    International Nuclear Information System (INIS)

    Struble, E. B.; Gittis, A. G.; Bianchet, M. A.; McMacken, R.

    2007-01-01

    Crystallization and preliminary diffraction data of the N-terminal 19–139 fragment of the origin-binding domain of bacteriophage λ O replication initiator are reported. The bacteriophage λ O protein binds to the λ replication origin (oriλ) and serves as the primary replication initiator for the viral genome. The binding energy derived from the binding of O to oriλ is thought to help drive DNA opening to facilitate initiation of DNA replication. Detailed understanding of this process is severely limited by the lack of high-resolution structures of O protein or of any lambdoid phage-encoded paralogs either with or without DNA. The production of crystals of the origin-binding domain of λ O that diffract to 2.5 Å is reported. Anomalous dispersion methods will be used to solve this structure

  1. Structure of a novel winged-helix like domain from human NFRKB protein.

    Directory of Open Access Journals (Sweden)

    Abhinav Kumar

    Full Text Available The human nuclear factor related to kappa-B-binding protein (NFRKB is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370-495 was determined using the JCSG high-throughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.

  2. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    Energy Technology Data Exchange (ETDEWEB)

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A. (UPENN-MED)

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  3. Insulin-like growth factor binding proteins: a structural perspective

    Directory of Open Access Journals (Sweden)

    Briony eForbes

    2012-03-01

    Full Text Available Insulin-like growth factor binding proteins (IGFBP-1 to -6 bind insulin-like growth factors-I and -II (IGF-I and IGF-II with high affinity. These binding proteins maintain IGFs in the circulation and direct them to target tissues, where they promote cell growth, proliferation, differentiation and survival via the type 1 IGF receptor (IGF-1R. IGFBPs also interact with many other molecules, which not only influence their modulation of IGF action but also mediate IGF-independent activities that influence processes such as cell migration and apoptosis by influencing gene transcription.IGFBPs-1 to -6 are structurally similar proteins consisting of three distinct domains, N-terminal, Linker and C-terminal. There have been major advances in our understanding of IGFBP structure in the last decade and a half. While there is still no structure of an intact IGFBP to date, several structures of individual N- and C-domains have been solved. The structure of a complex of N-BP-4:IGF-I:C-BP-4 has also been solved, providing a detailed picture of the structural features of the IGF binding site and the mechanism of binding. Structural studies have also identified features important for interaction with extracellular matrix components and integrins. This review summarises structural studies reported so far and highlights features important for binding not only IGF but also other partners. It also highlights future directions in which structural studies will add to our knowledge of the role played by the IGFBP family in normal growth and development, as well as in disease.

  4. Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates.

    Directory of Open Access Journals (Sweden)

    Maggie P Wear

    Full Text Available Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12 cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID domains (≥ 100 amino acids and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases.

  5. Structure of the ankyrin-binding domain of alpha-Na,K-ATPase.

    Science.gov (United States)

    Zhang, Z; Devarajan, P; Dorfman, A L; Morrow, J S

    1998-07-24

    The ankyrin 33-residue repeating motif, an L-shaped structure with protruding beta-hairpin tips, mediates specific macromolecular interactions with cytoskeletal, membrane, and regulatory proteins. The association between ankyrin and alpha-Na,K-ATPase, a ubiquitous membrane protein critical to vectorial transport of ions and nutrients, is required to assemble and stabilize Na,K-ATPase at the plasma membrane. alpha-Na,K-ATPase binds both red cell ankyrin (AnkR, a product of the ANK1 gene) and Madin-Darby canine kidney cell ankyrin (AnkG, a product of the ANK3 gene) utilizing residues 142-166 (SYYQEAKSSKIMESFK NMVPQQALV) in its second cytoplasmic domain. Fusion peptides of glutathione S-transferase incorporating these 25 amino acids bind specifically to purified ankyrin (Kd = 118 +/- 50 nM). The three-dimensional structure (2.6 A) of this minimal ankyrin-binding motif, crystallized as the fusion protein, reveals a 7-residue loop with one charged hydrophilic face capping a double beta-strand. Comparison with ankyrin-binding sequences in p53, CD44, neurofascin/L1, and the inositol 1,4,5-trisphosphate receptor suggests that the valency and specificity of ankyrin binding is achieved by the interaction of 5-7-residue surface loops with the beta-hairpin tips of multiple ankyrin repeat units.

  6. AUXIN BINDING PROTEIN1: the outsider.

    Science.gov (United States)

    Sauer, Michael; Kleine-Vehn, Jürgen

    2011-06-01

    AUXIN BINDING PROTEIN1 (ABP1) is one of the first characterized proteins that bind auxin and has been implied as a receptor for a number of auxin responses. Early studies characterized its auxin binding properties and focused on rapid electrophysiological and cell expansion responses, while subsequent work indicated a role in cell cycle and cell division control. Very recently, ABP1 has been ascribed a role in modulating endocytic events at the plasma membrane and RHO OF PLANTS-mediated cytoskeletal rearrangements during asymmetric cell expansion. The exact molecular function of ABP1 is still unresolved, but its main activity apparently lies in influencing events at the plasma membrane. This review aims to connect the novel findings with the more classical literature on ABP1 and to point out the many open questions that still separate us from a comprehensive model of ABP1 action, almost 40 years after the first reports of its existence.

  7. Regulation of IGF binding protein proteolysis by pregnancy-associated plasma protein-ARegulation of IGF binding protein proteolysis by pregnancy-associated plasma protein-A

    DEFF Research Database (Denmark)

    Gaidamauskas, Ervinas

    recognised role for PAPP-A in ageing and in the development of age-related disease. PAPP-A is a secreted metalloproteinase that cleaves insulin-like growth factor binding proteins (IGFBPs). Ervinas Gaidamauskas studied the mechanism of IGF-modulated proteolysis of IGFBPs by PAPP-A and the structural......During his PhD studies, Ervinas Gaidamauskas researched the proteins pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2 in vitro. As suggested by its name, PAPP-A plays an important role in pregnancy and fetal development. Additionally, recent studies indicate a newly...... determinants for cleavage. Using small-angle X-ray scattering (SAXS), he also analysed the intermodular structural organisation of the C-terminal domain of PAPP-A involved in substrate binding. Detailed knowledge of interactions between PAPP-A and its substrates is required to understand the modulatory role...

  8. Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.

    Directory of Open Access Journals (Sweden)

    Rohini Shrivastava

    Full Text Available Ezrin, a member of the ERM (Ezrin/Radixin/Moesin protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2 or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.

  9. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article d...

  10. The chitin-binding domain of a GH-18 chitinase from Vibrio harveyi is crucial for chitin-chitinase interactions.

    Science.gov (United States)

    Suginta, Wipa; Sirimontree, Paknisa; Sritho, Natchanok; Ohnuma, Takayuki; Fukamizo, Tamo

    2016-12-01

    Vibrio harveyi chitinase A (VhChiA) is a GH-18 glycosyl hydrolase with a structure containing three distinct domains: i) the N-terminal chitin-binding domain; ii) the (α/β) 8 TIM barrel catalytic domain; and iii) the α+β insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBD Vh ChiA . The recombinant ChBD Vh ChiA was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBD Vh ChiA contained β-sheets as major structural components and fluorescence spectroscopy showed that the protein domain was folded correctly, and suitable for functional characterization. Chitin binding assays showed that ChBD Vh ChiA bound to both α- and β-chitins, with the greatest affinity for β-colloidal chitin, but barely bound to polymeric chitosan. These results identified the tandem N-acetamido functionality on chitin chains as the specific sites of enzyme-substrate interactions. The binding affinity of the isolated domain was significantly lower than that of intact VhChiA, suggesting that the catalytic domain works synergistically with the chitin-binding domain to guide the polymeric substrate into the substrate binding cleft. These data confirm the physiological role of the chitin-binding domain of the marine bacterial GH-18 chitinase A in chitin-chitinase interactions. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Diversification of HP1-like Chromo Domain Proteins in Tetrahymena thermophila.

    Science.gov (United States)

    Wiley, Emily A; Horrell, Scott; Yoshino, Alyssa; Schornak, Cara C; Bagnani, Claire; Chalker, Douglas L

    2018-01-01

    Proteins that possess a chromo domain are well-known for their roles in heterochromatin assembly and maintenance. The Heterochromatin Protein 1 (HP1) family, with a chromo domain and carboxy-terminal chromo shadow domain, targets heterochromatin through interaction with histone H3 methylated on lysine 9 (H3K9me2/3). The structural and functional diversity of these proteins observed in both fission yeast and metazoans correlate with chromatin specialization. To expand these studies, we examined chromo domain proteins in the ciliate Tetrahymena thermophila, which has functionally diverse and developmentally regulated heterochromatin domains. We identified thirteen proteins similar to HP1. Together they possess only a fraction of the possible chromo domain subtypes and most lack a recognizable chromo shadow domain. Using fluorescence microscopy to track chromatin localization of tagged proteins through the life cycle, we show evidence that in T. thermophila this family has diversified with biological roles in RNAi-directed DNA elimination, germline genome structure, and somatic heterochromatin. Those proteins with H3K27me3 binding sequence characteristics localize to chromatin in mature nuclei, whereas those with H3K9me2/3 binding characteristics localize to developing nuclei undergoing DNA elimination. Findings point to an expanded and diversified family of chromo domain proteins that parallels heterochromatin diversity in ciliates. © 2017 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.

  12. Cooperative phosphoinositide and peptide binding by PSD-95/discs large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin.

    Science.gov (United States)

    Ivarsson, Ylva; Wawrzyniak, Anna Maria; Wuytens, Gunther; Kosloff, Mickey; Vermeiren, Elke; Raport, Marie; Zimmermann, Pascale

    2011-12-30

    PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. We screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns(4,5)P(2)-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. We identified the transmembrane protein Crumbs as a Pyd PDZ2 ligand and probed the relation between peptide and PtdIns(4,5)P(2) binding. Contrary to the prevalent view on PDZ/peptide/lipid binding, we did not find competition between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns(4,5)P(2) 6-fold. Our results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns(4,5)P(2) binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals.

  13. Subcloning, expression, purification, and characterization of recombinant human leptin-binding domain.

    Science.gov (United States)

    Sandowski, Yael; Raver, Nina; Gussakovsky, Eugene E; Shochat, Suzan; Dym, Orly; Livnah, Oded; Rubinstein, Menachem; Krishna, Radha; Gertler, Arieh

    2002-11-29

    A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.

  14. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

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    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  15. The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

    Directory of Open Access Journals (Sweden)

    Deng W-M

    2009-02-01

    Full Text Available Abstract Background Dystroglycan (Dg is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. Results We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required. Conclusion Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.

  16. Binding of von Willebrand factor to collagen type III: role of specific amino acids in the collagen binding domain of vWF and effects of neighboring domains

    NARCIS (Netherlands)

    van der Plas, R. M.; Gomes, L.; Marquart, J. A.; Vink, T.; Meijers, J. C.; de Groot, P. G.; Sixma, J. J.; Huizinga, E. G.

    2000-01-01

    Binding of von Willebrand Factor (vWF) to sites of vascular injury is the first step of hemostasis. Collagen types I and III are important binding sites for vWF. We have previously determined the three-dimensional structure of the collagen binding A3 domain of vWF (Huizinga et al., Structure 1997;

  17. Change in Allosteric Network Affects Binding Affinities of PDZ Domains: Analysis through Perturbation Response Scanning

    Science.gov (United States)

    Gerek, Z. Nevin; Ozkan, S. Banu

    2011-01-01

    The allosteric mechanism plays a key role in cellular functions of several PDZ domain proteins (PDZs) and is directly linked to pharmaceutical applications; however, it is a challenge to elaborate the nature and extent of these allosteric interactions. One solution to this problem is to explore the dynamics of PDZs, which may provide insights about how intramolecular communication occurs within a single domain. Here, we develop an advancement of perturbation response scanning (PRS) that couples elastic network models with linear response theory (LRT) to predict key residues in allosteric transitions of the two most studied PDZs (PSD-95 PDZ3 domain and hPTP1E PDZ2 domain). With PRS, we first identify the residues that give the highest mean square fluctuation response upon perturbing the binding sites. Strikingly, we observe that the residues with the highest mean square fluctuation response agree with experimentally determined residues involved in allosteric transitions. Second, we construct the allosteric pathways by linking the residues giving the same directional response upon perturbation of the binding sites. The predicted intramolecular communication pathways reveal that PSD-95 and hPTP1E have different pathways through the dynamic coupling of different residue pairs. Moreover, our analysis provides a molecular understanding of experimentally observed hidden allostery of PSD-95. We show that removing the distal third alpha helix from the binding site alters the allosteric pathway and decreases the binding affinity. Overall, these results indicate that (i) dynamics plays a key role in allosteric regulations of PDZs, (ii) the local changes in the residue interactions can lead to significant changes in the dynamics of allosteric regulations, and (iii) this might be the mechanism that each PDZ uses to tailor their binding specificities regulation. PMID:21998559

  18. Different functional modes of BAR domain proteins in formation and plasticity of mammalian postsynapses.

    Science.gov (United States)

    Kessels, Michael M; Qualmann, Britta

    2015-09-01

    A plethora of cell biological processes involve modulations of cellular membranes. By using extended lipid-binding interfaces, some proteins have the power to shape membranes by attaching to them. Among such membrane shapers, the superfamily of Bin-Amphiphysin-Rvs (BAR) domain proteins has recently taken center stage. Extensive structural work on BAR domains has revealed a common curved fold that can serve as an extended membrane-binding interface to modulate membrane topologies and has allowed the grouping of the BAR domain superfamily into subfamilies with structurally slightly distinct BAR domain subtypes (N-BAR, BAR, F-BAR and I-BAR). Most BAR superfamily members are expressed in the mammalian nervous system. Neurons are elaborately shaped and highly compartmentalized cells. Therefore, analyses of synapse formation and of postsynaptic reorganization processes (synaptic plasticity) - a basis for learning and memory formation - has unveiled important physiological functions of BAR domain superfamily members. These recent advances, furthermore, have revealed that the functions of BAR domain proteins include different aspects. These functions are influenced by the often complex domain organization of BAR domain proteins. In this Commentary, we review these recent insights and propose to classify BAR domain protein functions into (1) membrane shaping, (2) physical integration, (3) action through signaling components, and (4) suppression of other BAR domain functions. © 2015. Published by The Company of Biologists Ltd.

  19. Solution structure and molecular determinants of hemoglobin binding of the first NEAT domain of IsdB in Staphylococcus aureus.

    Science.gov (United States)

    Fonner, Brittany A; Tripet, Brian P; Eilers, Brian J; Stanisich, Jessica; Sullivan-Springhetti, Rose K; Moore, Rebecca; Liu, Mengyao; Lei, Benfang; Copié, Valérie

    2014-06-24

    The human pathogen Staphylococcus aureus acquires heme iron from hemoglobin (Hb) via the action of a series of iron-regulated surface determinant (Isd) proteins. The cell wall anchored IsdB protein is recognized as the predominant Hb receptor, and is comprised of two NEAr transporter (NEAT) domains that act in concert to bind, extract, and transfer heme from Hb to downstream Isd proteins. Structural details of the NEAT 2 domain of IsdB have been investigated, but the molecular coordination between NEAT 2 and NEAT 1 to extract heme from hemoglobin has yet to be characterized. To obtain a more complete understanding of IsdB structure and function, we have solved the 3D solution structure of the NEAT 1 domain of IsdB (IsdB(N1)) spanning residues 125-272 of the full-length protein by NMR. The structure reveals a canonical NEAT domain fold and has particular structural similarity to the NEAT 1 and NEAT 2 domains of IsdH, which also interact with Hb. IsdB(N1) is also comprised of a short N-terminal helix, which has not been previously observed in other NEAT domain structures. Interestingly, the Hb binding region (loop 2 of IsdB(N1)) is disordered in solution. Analysis of Hb binding demonstrates that IsdB(N1) can bind metHb weakly and the affinity of this interaction is further increased by the presence of IsdB linker domain. IsdB(N1) loop 2 variants reveal that phenylalanine 164 (F164) of IsdB is necessary for Hb binding and rapid heme transfer from metHb to IsdB. Together, these findings provide a structural role for IsdB(N1) in enhancing the rate of extraction of metHb heme by the IsdB NEAT 2 domain.

  20. Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2

    Directory of Open Access Journals (Sweden)

    Kabir Hassan Biswas

    2015-04-01

    Full Text Available GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem (GAFab domain. In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.

  1. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    Directory of Open Access Journals (Sweden)

    Yu Zhu

    2015-11-01

    Full Text Available Chitin-binding proteins (CBPs are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton.

  2. 1H, 13C and 15N resonance assignments of the GTPase-activating (GAP) and Ral binding domains (GBD) of RLIP76 (RalBP1).

    Science.gov (United States)

    Rajasekar, Karthik V; Campbell, Louise J; Nietlispach, Daniel; Owen, Darerca; Mott, Helen R

    2012-10-01

    RLIP76 (also known as RalBP1) is an effector for Ral small G proteins. RLIP76 is a multifunctional, multi-domain protein that includes a GTPase activating domain for the Rho family (RhoGAP domain) and a GTPase binding domain (GBD) for the Ral small G proteins. The juxtaposition of these two domains (GAP and GBD) may be a strategy employed to co-ordinate regulation of Rho family and Ral-controlled signalling pathways at a crossover node. Here we present the (1)H, (15)N and (13)C NMR backbone and sidechain resonance assignments of the GAP and GBD di-domain (31 kDa).

  3. Complementarity of structure ensembles in protein-protein binding.

    Science.gov (United States)

    Grünberg, Raik; Leckner, Johan; Nilges, Michael

    2004-12-01

    Protein-protein association is often accompanied by changes in receptor and ligand structure. This interplay between protein flexibility and protein-protein recognition is currently the largest obstacle both to our understanding of and to the reliable prediction of protein complexes. We performed two sets of molecular dynamics simulations for the unbound receptor and ligand structures of 17 protein complexes and applied shape-driven rigid body docking to all combinations of representative snapshots. The crossdocking of structure ensembles increased the likelihood of finding near-native solutions. The free ensembles appeared to contain multiple complementary conformations. These were in general not related to the bound structure. We suggest that protein-protein binding follows a three-step mechanism of diffusion, free conformer selection, and refolding. This model combines previously conflicting ideas and is in better agreement with the current data on interaction forces, time scales, and kinetics.

  4. Protein Binding Capacity of Different Forages Tannin

    Science.gov (United States)

    Yusiati, L. M.; Kurniawati, A.; Hanim, C.; Anas, M. A.

    2018-02-01

    Eight forages of tannin sources(Leucaena leucocephala, Arachis hypogaea, Mimosa pudica, Morus alba L, Swietenia mahagoni, Manihot esculenta, Gliricidia sepium, and Bauhinia purpurea)were evaluated their tannin content and protein binding capacity. The protein binding capacity of tannin were determined using precipitation of bovine serum albumin (BSA). Swietenia mahagonihas higest total tannin level and condensed tannin (CT) compared with other forages (Ptannin (HT) level (Ptannin content of Swietenia mahagoni were 11.928±0.04 mg/100 mg and 9.241±0.02mg/100mg dry matter (DM) of leaves. The hydrolysable tannin content of Leucaena leucocephala was 5.338±0.03 mg/100 mg DM of leaves. Binding capacity was highest in Swietenia mahagoni and Leucaena leucocephala compared to the other forages (Ptannin in Leucaena leucocephala and Swietenia mahagoniwere1.181±0.44 and 1.217±0.60mg/mg dry matter of leaves. The present study reports that Swietenia mahagoni has highest of tannin content and Leucaena leucocephala and Swietenia mahagoni capacity of protein binding.

  5. New insights into interactions between the nucleotide‐binding domain of CFTR and keratin 8

    Science.gov (United States)

    Premchandar, Aiswarya; Kupniewska, Anna; Bonna, Arkadiusz; Faure, Grazyna; Fraczyk, Tomasz; Roldan, Ariel; Hoffmann, Brice; Faria da Cunha, Mélanie; Herrmann, Harald; Lukacs, Gergely L.

    2017-01-01

    Abstract The intermediate filament protein keratin 8 (K8) interacts with the nucleotide‐binding domain 1 (NBD1) of the cystic fibrosis (CF) transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508‐CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for CF patients bearing the ΔF508 mutation. Here, we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen–deuterium exchange coupled with mass spectrometry (HDX‐MS) on recombinant wild‐type (wt) NBD1 and ΔF508‐NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt‐NBD1 and ΔF508‐NBD1. Finally, we performed HDX‐MS analysis of the NBD1 molecules and full‐length K8, revealing hydrogen‐bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508‐NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1. PMID:27870250

  6. Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

    DEFF Research Database (Denmark)

    Pedersen, Søren W; Moran, Griffin E; Sereikaité, Vita

    2016-01-01

    PDZ domains are ubiquitous small protein domains that are mediators of numerous protein-protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling-transduction complexes. In recent years, PDZ domains have emerged as novel and exciting...... drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C-terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys...... into the mechanism of PDZ/ligand interaction....

  7. Conserved C-terminal nascent peptide binding domain of HYPK ...

    Indian Academy of Sciences (India)

    energies (in kcal/mol) (marked in black circles) for each amino acid residues of human HYPK protein. NPAA domain region has been ... Aromatic (H, F, W, Y). 1.17. 0.88. 0.77. 0.32. 0.99. 0.73 0.82. 0.51. 0.69. 0.47. 0.55. 0.18 polar (R, N, D, E, Q, H, K, S, T) 5.97. 1.50. 6.74. 1.22. 6.47. 1.59 6.58. 1.52. 6.32. 1.16. 6.43. 0.77.

  8. Light-activated DNA binding in a designed allosteric protein

    Energy Technology Data Exchange (ETDEWEB)

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R. (UC)

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  9. The SBASE protein domain library, release 8.0: a collection of annotated protein sequence segments.

    Science.gov (United States)

    Murvai, J; Vlahovicek, K; Barta, E; Pongor, S

    2001-01-01

    SBASE 8.0 is the eighth release of the SBASE library of protein domain sequences that contains 294 898 annotated structural, functional, ligand-binding and topogenic segments of proteins, cross-referenced to most major sequence databases and sequence pattern collections. The entries are clustered into over 2005 statistically validated domain groups (SBASE-A) and 595 non-validated groups (SBASE-B), provided with several WWW-based search and browsing facilities for online use. A domain-search facility was developed, based on non-parametric pattern recognition methods, including artificial neural networks. SBASE 8.0 is freely available by anonymous 'ftp' file transfer from ftp.icgeb.trieste.it. Automated searching of SBASE can be carried out with the WWW servers http://www.icgeb.trieste.it/sbase/ and http://sbase.abc. hu/sbase/.

  10. The structure of Prp40 FF1 domain and its interaction with the crn-TPR1 motif of Clf1 gives a new insight into the binding mode of FF domains.

    Science.gov (United States)

    Gasch, Alexander; Wiesner, Silke; Martin-Malpartida, Pau; Ramirez-Espain, Ximena; Ruiz, Lidia; Macias, Maria J

    2006-01-06

    The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs.

  11. The C-terminal portion of BM-40 (SPARC/osteonectin) is an autonomously folding and crystallisable domain that binds calcium and collagen IV.

    Science.gov (United States)

    Maurer, P; Hohenadl, C; Hohenester, E; Göhring, W; Timpl, R; Engel, J

    1995-10-20

    The extracellular glycoprotein BM-40 consists of three domains, an acidic domain I, a follistatin (FS)-like domain II and a calcium-binding EC domain with an EF-hand related motif. BM-40 and several other related proteins (QR1, SC1/hevin, testican and tsc-36/FRP) are members of a novel modular protein family that share the FS domain followed by an EC domain. We have expressed this pair of FS and EC domains (mutant delta I) and the calcium-binding EC domain alone (mutant delta I, II) of human BM-40 as recombinant proteins in human 293 cells. Circular dichroism demonstrated that both mutants were obtained as folded proteins with a distinct three-dimensional conformation. In addition, mutant delta I, II could be readily crystallized and diffraction patterns with a resolution limit of 2.4 A resolution were obtained. Calcium binding to this fragment was ten times weaker (Kd = 0.8 microM) than for the wild-type protein. Identical reversible increases in alpha-helicity upon calcium binding were observed for the 150-residue long mutant delta I, II and for BM-40 (286 residues). A 26-residue synthetic peptide corresponding to the EF-hand related motif exhibited much weaker calcium binding. The apparent dissociation constant decreased with increasing peptide concentration (from Kd 2.4 mM at 1 microM, to Kd 0.3 mM at 100 microM peptide concentration) and calcium binding was accompanied by dimerization of the peptide. This suggests that for strong calcium binding the EF-hand related motif has to be embedded into a larger protein domain that can form an autonomously folding protein module. The EC domain was also shown by surface plasmon resonance assay to be responsible for calcium-dependent binding to collagen IV with an affinity (Kd = 19 microM) only sixfold lower than that of intact human BM-40.

  12. Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3

    International Nuclear Information System (INIS)

    Diehl, Carl; Genheden, Samuel; Modig, Kristofer; Ryde, Ulf; Akke, Mikael

    2009-01-01

    The conformational entropy of proteins can make significant contributions to the free energy of ligand binding. NMR spin relaxation enables site-specific investigation of conformational entropy, via order parameters that parameterize local reorientational fluctuations of rank-2 tensors. Here we have probed the conformational entropy of lactose binding to the carbohydrate recognition domain of galectin-3 (Gal3), a protein that plays an important role in cell growth, cell differentiation, cell cycle regulation, and apoptosis, making it a potential target for therapeutic intervention in inflammation and cancer. We used 15 N spin relaxation experiments and molecular dynamics simulations to monitor the backbone amides and secondary amines of the tryptophan and arginine side chains in the ligand-free and lactose-bound states of Gal3. Overall, we observe good agreement between the experimental and computed order parameters of the ligand-free and lactose-bound states. Thus, the 15 N spin relaxation data indicate that the molecular dynamics simulations provide reliable information on the conformational entropy of the binding process. The molecular dynamics simulations reveal a correlation between the simulated order parameters and residue-specific backbone entropy, re-emphasizing that order parameters provide useful estimates of local conformational entropy. The present results show that the protein backbone exhibits an increase in conformational entropy upon binding lactose, without any accompanying structural changes

  13. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  14. Lytic activity of the staphylolytic Twort phage endolysin CHAP domain is enhanced by the SH3b cell wall binding domain.

    Science.gov (United States)

    Becker, Stephen C; Swift, Steven; Korobova, Olga; Schischkova, Nina; Kopylov, Pavel; Donovan, David M; Abaev, Igor

    2015-01-01

    Increases in the prevalence of antibiotic-resistant strains of Staphylococcus aureus have elicited efforts to develop novel antimicrobials to treat these drug-resistant pathogens. One potential treatment repurposes the lytic enzymes produced by bacteriophages as antimicrobials. The phage Twort endolysin (PlyTW) harbors three domains, a cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), an amidase-2 domain and a SH3b-5 cell wall binding domain (CBD). Our results indicate that the CHAP domain alone is necessary and sufficient for lysis of live S. aureus, while the amidase-2 domain is insufficient for cell lysis when provided alone. Loss of the CBD results in ∼10X reduction of enzymatic activity in both turbidity reduction and plate lysis assays compared to the full length protein. Deletion of the amidase-2 domain resulted in a protein (PlyTW Δ172-373) with lytic activity that exceeded the activity of the full length construct in both the turbidity reduction and plate lysis assays. Addition of Ca(2+) enhanced the turbidity reduction activity of both the full length protein and truncation constructs harboring the CHAP domain. Chelation by addition of EDTA or the addition of zinc inhibited the activity of all PlyTW constructs. Published by Oxford University Press on behalf of FEMS 2014. This work is written by US Government employees and is in the public domain in the US.

  15. Trp[superscript 2313]-His[superscript 2315] of Factor VIII C2 Domain Is Involved in Membrane Binding Structure of a Complex Between the C[subscript 2] Domain and an Inhibitor of Membrane Binding

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhuo; Lin, Lin; Yuan, Cai; Nicolaes, Gerry A.F.; Chen, Liqing; Meehan, Edward J.; Furie, Bruce; Furie, Barbara; Huang, Mingdong (Harvard-Med); (UAH); (Maastricht); (Chinese Aca. Sci.)

    2010-11-03

    Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 {angstrom}) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed {beta}-strand of the C2 domain, Trp{sup 2313}-His{sup 2315}. This result indicates that the Trp{sup 2313}-His{sup 2315} segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

  16. Bayesian modeling of the yeast SH3 domain interactome predicts spatiotemporal dynamics of endocytosis proteins.

    Directory of Open Access Journals (Sweden)

    Raffi Tonikian

    2009-10-01

    Full Text Available SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes.

  17. The telomere binding protein TRF2 induces chromatin compaction.

    Directory of Open Access Journals (Sweden)

    Asmaa M Baker

    2011-04-01

    Full Text Available Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

  18. Protein subfamily assignment using the Conserved Domain Database

    Directory of Open Access Journals (Sweden)

    Marchler-Bauer Aron

    2008-11-01

    Full Text Available Abstract Background Domains, evolutionarily conserved units of proteins, are widely used to classify protein sequences and infer protein function. Often, two or more overlapping domain models match a region of a protein sequence. Therefore, procedures are required to choose appropriate domain annotations for the protein. Here, we propose a method for assigning NCBI-curated domains from the Curated Domain Database (CDD that takes into account the organization of the domains into hierarchies of homologous domain models. Findings Our analysis of alignment scores from NCBI-curated domain assignments suggests that identifying the correct model among closely related models is more difficult than choosing between non-overlapping domain models. We find that simple heuristics based on sorting scores and domain-specific thresholds are effective at reducing classification error. In fact, in our test set, the heuristics result in almost 90% of current misclassifications due to missing domain subfamilies being replaced by more generic domain assignments, thereby eliminating a significant amount of error within the database. Conclusion Our proposed domain subfamily assignment rule has been incorporated into the CD-Search software for assigning CDD domains to query protein sequences and has significantly improved pre-calculated domain annotations on protein sequences in NCBI's Entrez resource.

  19. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    Science.gov (United States)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-11-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  20. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, O N; Engelholm, L H

    2000-01-01

    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

  1. Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus.

    Science.gov (United States)

    Pluym, Mark; Muryoi, Naomi; Heinrichs, David E; Stillman, Martin J

    2008-03-01

    Absorption, magnetic circular dichroism (MCD), and electrospray mass spectral (ESI-MS) data are reported for the heme binding NEAr iron Transporter (NEAT) domains of IsdA and IsdC, two proteins involved in heme scavenging by Staphylococcus aureus. The mass spectrometry data show that the NEAT domains are globular in structure and efficiently bind a single heme molecule. In this work, the IsdA NEAT domain is referred to as NEAT-A, the IsdC NEAT domain is referred to as NEAT-C, heme-free NEAT-C is NEAT-A and NEAT-C are inaccessible to small anionic ligands. Reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-A results in coordination by histidine and opens access, allowing for CO axial ligation, yielding 6-coordinate low-spin Fe(II) heme. In contrast, reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-C results in loss of the heme from the binding site of the protein due to the absence of a proximal histidine. The absorption and MCD data for NEAT-A closely match those previously reported for the whole IsdA protein, providing evidence that heme binding is primarily a property of the NEAT domain.

  2. A Folded Excited State of Ligand-Free Nuclear Coactivator Binding Domain (NCBD) Underlies Plasticity in Ligand Recognition

    DEFF Research Database (Denmark)

    Kjaergaard, Magnus; Andersen, Lisbeth; Nielsen, Lau Dalby

    2013-01-01

    Intrinsically disordered proteins are renowned for their structural plasticity when they undergo coupled folding and binding to partner proteins. The nuclear coactivator binding domain of CBP is a remarkable example of this adaptability as it folds into two different conformations depending...... experience conformational exchange. The dispersion data can be described by a global two-state exchange process between a ground state and an excited state populated to 8%. The three helices are still folded in the excited state but have a different packing from the ground state; the contact between helices...... with that of NCBD in complex with the ligand IRF-3. The energy landscape of this domain is thus proposed to resemble the fold-switching proteins that have two coexisting native states, which may serve as a starting point for binding via conformational selection....

  3. The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms.

    Science.gov (United States)

    Durocher, D; Taylor, I A; Sarbassova, D; Haire, L F; Westcott, S L; Jackson, S P; Smerdon, S J; Yaffe, M B

    2000-11-01

    Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.

  4. PROSITE, a protein domain database for functional characterization and annotation.

    Science.gov (United States)

    Sigrist, Christian J A; Cerutti, Lorenzo; de Castro, Edouard; Langendijk-Genevaux, Petra S; Bulliard, Virginie; Bairoch, Amos; Hulo, Nicolas

    2010-01-01

    PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE is largely used for the annotation of domain features of UniProtKB/Swiss-Prot entries. Among the 983 (DNA-binding) domains, repeats and zinc fingers present in Swiss-Prot (release 57.8 of 22 September 2009), 696 ( approximately 70%) are annotated with PROSITE descriptors using information from ProRule. In order to allow better functional characterization of domains, PROSITE developments focus on subfamily specific profiles and a new profile building method giving more weight to functionally important residues. Here, we describe AMSA, an annotated multiple sequence alignment format used to build a new generation of generalized profiles, the migration of ScanProsite to Vital-IT, a cluster of 633 CPUs, and the adoption of the Distributed Annotation System (DAS) to facilitate PROSITE data integration and interchange with other sources. The latest version of PROSITE (release 20.54, of 22 September 2009) contains 1308 patterns, 863 profiles and 869 ProRules. PROSITE is accessible at: http://www.expasy.org/prosite/.

  5. Novel interactions of ankyrins-G at the costameres: The muscle-specific Obscurin/Titin-Binding-related Domain (OTBD) binds plectin and filamin C

    Energy Technology Data Exchange (ETDEWEB)

    Maiweilidan, Yimingjiang; Klauza, Izabela; Kordeli, Ekaterini, E-mail: ekaterini.kordeli@inserm.fr

    2011-04-01

    Ankyrins, the adapters of the spectrin skeleton, are involved in local accumulation and stabilization of integral proteins to the appropriate membrane domains. In striated muscle, tissue-dependent alternative splicing generates unique Ank3 gene products (ankyrins-G); they share the Obscurin/Titin-Binding-related Domain (OTBD), a muscle-specific insert of the C-terminal domain which is highly conserved among ankyrin genes, and binds obscurin and titin to Ank1 gene products. We previously proposed that OTBD sequences constitute a novel domain of protein-protein interactions which confers ankyrins with specific cellular functions in muscle. Here we searched for muscle proteins binding to ankyrin-G OTBD by yeast two hybrid assay, and we found plectin and filamin C, two organizing elements of the cytoskeleton with essential roles in myogenesis, muscle cell cytoarchitecture, and muscle disease. The three proteins coimmunoprecipitate from skeletal muscle extracts and colocalize at costameres in adult muscle fibers. During in vitro myogenesis, muscle ankyrins-G are first expressed in postmitotic myocytes undergoing fusion to myotubes. In western blots of subcellular fractions from C2C12 cells, the majority of muscle ankyrins-G appear associated with membrane compartments. Occasional but not extensive co-localization at nascent costameres suggested that ankyrin-G interactions with plectin and filamin C are not involved in costamere assembly; they would rather reinforce stability and/or modulate molecular interactions in sarcolemma microdomains by establishing novel links between muscle-specific ankyrins-G and the two costameric dystrophin-associated glycoprotein and integrin-based protein complexes. These results report the first protein-protein interactions involving the ankyrin-G OTBD domain and support the hypothesis that OTBD sequences confer ankyrins with a gain of function in vertebrates, bringing further consolidation and resilience of the linkage between sarcomeres

  6. Identification of MDP (muramyl dipeptide)-binding key domains in NOD2 (nucleotide-binding and oligomerization domain-2) receptor of Labeo rohita.

    Science.gov (United States)

    Maharana, Jitendra; Swain, Banikalyan; Sahoo, Bikash R; Dikhit, Manas R; Basu, Madhubanti; Mahapatra, Abhijit S; Jayasankar, Pallipuram; Samanta, Mrinal

    2013-08-01

    In lower eukaryotes-like fish, innate immunity contributed by various pattern recognition receptor (PRR) plays an essential role in protection against diseases. Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic PRR that recognizes MDP (muramyl dipeptide) of the Gram positive and Gram negative bacteria as ligand and activates signalling to induce innate immunity. Hypothesizing a similar NOD2 signalling pathway of higher eukaryotes, the peripheral blood leucocytes (PBLs) of rohu (Labeo