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Sample records for recognizing protein domains

  1. Novel modular domain PB1 recognizes PC motif to mediate functional protein–protein interactions

    PubMed Central

    Ito, Takashi; Matsui, Yasushi; Ago, Tetsuro; Ota, Kazuhisa; Sumimoto, Hideki

    2001-01-01

    Modular domains mediating specific protein–protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67phox, which activates the microbicidal phagocyte NADPH oxidase. Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40phox (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67phox, which abolishes its interaction with Cdc24p, confers on cells temperature- sensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40phox, which restores the interaction with the altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules. PMID:11483497

  2. Autoantibodies from patients with primary biliary cirrhosis recognize a region within the nucleoplasmic domain of inner nuclear membrane protein LBR.

    PubMed

    Lin, F; Noyer, C M; Ye, Q; Courvalin, J C; Worman, H J

    1996-01-01

    Autoantibodies from rare patients with primary biliary cirrhosis (PBC) recognize LBR, or lamin B receptor, an integral membrane protein of the inner nuclear membrane. Human LBR has a nucleoplasmic, amino-terminal domain of 208 amino acids followed by a carboxyl-terminal domain with eight putative transmembrane segments. Autoantibodies against LBR from four patients with PBC recognized the nucleoplasmic, amino-terminal domain but not the carboxyl-terminal domain. Immunoblotting of smaller fusion proteins demonstrated that these autoantibodies recognized a conformational epitope(s) contained within the stretch of amino acids from 1 to 60. These results, combined with those of previous studies, show that autoepitopes of nuclear membrane proteins are located within their nucleocytoplasmic domains and that autoantibodies from patients with PBC predominantly react with one domain of a protein antigen. This work also provides further characterization of anti-LBR antibodies that have found utility as reagents in cell biology research.

  3. The LIM domains of hic-5 protein recognize specific DNA fragments in a zinc-dependent manner in vitro.

    PubMed Central

    Nishiya, N; Sabe, H; Nose, K; Shibanuma, M

    1998-01-01

    hic-5 protein is a member of the LIM protein family, containing four LIM domains in its C-terminal region. It is mainly localized in focal adhesions and shows striking similarity to paxillin in its LIM domains, although the function of these LIM domains has remained elusive. In the present study, we found that full-length and the C-terminal half of hic-5 protein, including four LIM domains, bound to DNA in a zinc-dependent manner in vitro . Mouse genomic fragments that specifically bound to the hic-5 protein were isolated by successive rounds of hic-5 protein-DNA complex immunoprecipitation and PCR amplification. Seven independent clones were isolated, which contained high amounts of G+A and/or a long A/T tract. A DNA binding protein blot assay revealed the specificity of the interaction between hic-5 protein and the DNA fragment. Using a series of truncated forms of the hic-5 LIM domains, each of the four LIM domains was found to contribute to DNA binding in a distinctive manner. PMID:9722648

  4. The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5′ splice site-like sequences

    SciTech Connect

    Loughlin, Fionna E.; Mansfield, Robyn E.; Vaz, Paula M.; McGrath, Aaron P.; Setiyaputra, Surya; Gamsjaeger, Roland; Chen, Eva S.; Morris, Brian J.; Guss, J. Mitchell; Mackay, Joel P.

    2009-09-02

    The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(N{sub x})AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5{prime} splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine 'ladder.' A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.

  5. Characterization of Two Monoclonal Antibodies That Recognize Linker Region and Carboxyl Terminal Domain of Coronavirus Nucleocapsid Protein

    PubMed Central

    Zhu, Yunnuan; Shi, Hongyan; Chen, Jianfei; Shi, Da; Feng, Li

    2016-01-01

    The transmissible gastroenteritis virus (TGEV) nucleocapsid (N) protein plays important roles in the replication and translation of viral RNA. The present study provides the first description of two monoclonal antibodies (mAbs) (5E8 and 3D7) directed against the TGEV N protein linker region (LKR) and carboxyl terminal domain (CTD). The mAbs 5E8 and 3D7 reacted with native N protein in western blotting and immunofluorescence assay (IFA). Two linear epitopes, 189SVEQAVLAALKKLG202 and 246VTRFYGARSSSA257, located in the LKR and CTD of TGEV N protein, respectively, were identified after truncating the protein and applying a peptide scanning technique. Using mAb 5E8, we observed that the N protein was expressed in the cytoplasm during TGEV replication and that the protein could be immunoprecipitated from TGEV-infected PK-15 cells. The mAb 5E8 can be applied for different approaches to diagnosis of TGEV infection. In addition, the antibodies represent useful tools for investigating the antigenic properties of the N protein. PMID:27689694

  6. Haemophilus influenzae protein E recognizes the C-terminal domain of vitronectin and modulates the membrane attack complex.

    PubMed

    Singh, Birendra; Jalalvand, Farshid; Mörgelin, Matthias; Zipfel, Peter; Blom, Anna M; Riesbeck, Kristian

    2011-07-01

    Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune response in lung epithelial cells. The active epithelial binding region comprising amino acids PE 84-108 also interferes with complement-mediated bacterial killing by capturing vitronectin (Vn) that prevents complement deposition and formation of the membrane attack complex (MAC). Here, the interaction between PE and Vn was characterized using site-directed mutagenesis. Protein E variants were produced both in soluble forms and in surface-expressed molecules on Escherichia coli. Mutations within PE(84-108) in the full-length molecule revealed that K85 and R86 residues were important for the Vn binding. Bactericidal activity against H. influenzae was higher in human serum pre-treated with full-length PE as compared with serum incubated with PE(K85E, R86D) , suggesting that PE quenched Vn. A series of truncated Vn molecules revealed that the C-terminal domain comprising Vn(353-363) harboured the major binding region for PE. Interestingly, MAC deposition was significantly higher on mutants devoid of PE due to a decreased Vn-binding capacity when compared with wild-type H. influenzae. Our results define a fine-tuned interaction between H. influenzae and the innate immune system, and identify the mode of control of the MAC that is important for pathogen complement evasion.

  7. An introduction to recognizing functional domains.

    PubMed

    Stormo, Gary D

    2006-10-01

    This unit provides an overview of issues involved in domain recognition in protein and DNA sequences. It opens with a discussion of the two primary methods of domain representation, namely consensus sequences and alignment matrices (e.g., the log-odds matrix). The unit continues with a brief overview of some of the resources available for identifying functional domains in nucleotide sequences (e.g., TRANSFAC). In addition, it reviews databases such as Pfam, InterPro and Blocks, which are available for protein analysis.

  8. Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains*

    PubMed Central

    Guo, Emily Z.; Xu, Zhaohui

    2015-01-01

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. PMID:25657007

  9. Distinct mechanisms of recognizing endosomal sorting complex required for transport III (ESCRT-III) protein IST1 by different microtubule interacting and trafficking (MIT) domains.

    PubMed

    Guo, Emily Z; Xu, Zhaohui

    2015-03-27

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains

    SciTech Connect

    Guo, Emily Z.; Xu, Zhaohui

    2015-02-05

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). In this paper, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. Finally, these observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode.

  11. Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains

    DOE PAGES

    Guo, Emily Z.; Xu, Zhaohui

    2015-02-05

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). In this paper, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed thatmore » IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. Finally, these observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode.« less

  12. System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors

    DOEpatents

    Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

    2013-02-05

    The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the presence of transmembrane regions in proteins, such as G-Protein Coupled Receptors (GPCR), and protein structural models generated according to the protocol. The protocol features a coarse grain sampling method, such as hydrophobicity analysis, to provide a fast and accurate procedure for predicting transmembrane regions. Methods and apparatus of the invention are useful to screen protein or polynucleotide databases for encoded proteins with transmembrane regions, such as GPCRs.

  13. Kininogen domain 3 contains regions recognized by antiphosphatidylethanolamine antibodies.

    PubMed

    Katsunuma, J; Sugi, T; Inomo, A; Matsubayashi, H; Izumi, S-I; Makino, T

    2003-01-01

    Antiphosphatidylethanolamine antibodies (APE) have been described in patients with thrombotic diseases and recurrent pregnancy loss (RPL). It has been reported that certain APE are not specific for phosphatidylethanolamine (PE) per se, but are directed to PE-binding plasma proteins, called kininogens. Our recent in vitro data suggest that APE may recognize the domain 3 (D3) region of kininogens. In this study, we have used synthetic peptides that span the D3 of kininogens in inhibition and direct binding studies to identify epitopes that are sites for binding APE. Our present data demonstrate that among 24 RPL patients who were positive for kininogen-dependent immunoglobulin (IgG) APE, 17 patients (70.8%) recognized the LDC27 peptide. We mapped the APE-binding region on D3 using plasma from a RPL patient (X) who had a high titer of IgG APE that recognized LDC27. APE of patient X recognized a 13-residue segment in LDC27, named CNA13. Leu331-Met357 (LDC27) and Cys333-Lys345 (CNA13) are located on the carboxyl-terminal portion of kininogen D3, which is known as the major kininogen heavy chain cell attachment site where it overlaps its cysteine protease inhibitory region. Because APE interferes with the balance of hemostasis in vitro, APE may therefore induce a similar condition in patients thereby causing thrombosis and RPL.

  14. Charged and Hydrophobic Surfaces on the A Chain of Shiga-Like Toxin 1 Recognize the C-Terminal Domain of Ribosomal Stalk Proteins

    PubMed Central

    McCluskey, Andrew J.; Bolewska-Pedyczak, Eleonora; Jarvik, Nick; Chen, Gang; Sidhu, Sachdev S.; Gariépy, Jean

    2012-01-01

    Shiga-like toxins are ribosome-inactivating proteins (RIP) produced by pathogenic E. coli strains that are responsible for hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 chain of Shiga-like toxin 1 (SLT-1), a representative RIP, first docks onto a conserved peptide SD[D/E]DMGFGLFD located at the C-terminus of all three eukaryotic ribosomal stalk proteins and halts protein synthesis through the depurination of an adenine base in the sarcin-ricin loop of 28S rRNA. Here, we report that the A1 chain of SLT-1 rapidly binds to and dissociates from the C-terminal peptide with a monomeric dissociation constant of 13 µM. An alanine scan performed on the conserved peptide revealed that the SLT-1 A1 chain interacts with the anionic tripeptide DDD and the hydrophobic tetrapeptide motif FGLF within its sequence. Based on these 2 peptide motifs, SLT-1 A1 variants were generated that displayed decreased affinities for the stalk protein C-terminus and also correlated with reduced ribosome-inactivating activities in relation to the wild-type A1 chain. The toxin-peptide interaction and subsequent toxicity were shown to be mediated by cationic and hydrophobic docking surfaces on the SLT-1 catalytic domain. These docking surfaces are located on the opposite face of the catalytic cleft and suggest that the docking of the A1 chain to SDDDMGFGLFD may reorient its catalytic domain to face its RNA substrate. More importantly, both the delineated A1 chain ribosomal docking surfaces and the ribosomal peptide itself represent a target and a scaffold, respectively, for the design of generic inhibitors to block the action of RIPs. PMID:22355345

  15. The RNA-binding domain of ribosomal protein L11 recognizes an rRNA tertiary structure stabilized by both thiostrepton and magnesium ion

    PubMed Central

    Blyn, Lawrence B.; Risen, Lisa M.; Griffey, Richard H.; Draper, David E.

    2000-01-01

    Antibiotics that inhibit ribosomal function may do so by one of several mechanisms, including the induction of incorrect RNA folding or prevention of protein and/or RNA conformational transitions. Thiostrepton, which binds to the ‘GTPase center’ of the large subunit, has been postulated to prevent conformational changes in either the L11 protein or rRNA to which it binds. Scintillation proximity assays designed to look at the binding of the L11 C-terminal RNA-binding domain to a 23S ribosomal RNA (rRNA) fragment, as well as the ability of thiostrepton to induce that binding, were used to demonstrate the role of Mg2+, L11 and thiostrepton in the formation and maintenance of the rRNA fragment tertiary structure. Experiments using these assays with both an Escherichia coli rRNA fragment and a thermostable variant of that RNA show that Mg2+, L11 and thiostrepton all induce the RNA to fold to an essentially identical tertiary structure. PMID:10734197

  16. Anaplasma marginale Outer Membrane Protein A Is an Adhesin That Recognizes Sialylated and Fucosylated Glycans and Functionally Depends on an Essential Binding Domain.

    PubMed

    Hebert, Kathryn S; Seidman, David; Oki, Aminat T; Izac, Jerilyn; Emani, Sarvani; Oliver, Lee D; Miller, Daniel P; Tegels, Brittney K; Kannagi, Reiji; Marconi, Richard T; Carlyon, Jason A

    2017-03-01

    Anaplasma marginale causes bovine anaplasmosis, a debilitating and potentially fatal tick-borne infection of cattle. Because A. marginale is an obligate intracellular organism, its adhesins that mediate entry into host cells are essential for survival. Here, we demonstrate that A. marginale outer membrane protein A (AmOmpA; AM854) contributes to the invasion of mammalian and tick host cells. AmOmpA exhibits predicted structural homology to OmpA of A. phagocytophilum (ApOmpA), an adhesin that uses key lysine and glycine residues to interact with α2,3-sialylated and α1,3-fucosylated glycan receptors, including 6-sulfo-sialyl Lewis x (6-sulfo-sLe(x)). Antisera against AmOmpA or its predicted binding domain inhibits A. marginale infection of host cells. Residues G55 and K58 are contributory, and K59 is essential for recombinant AmOmpA to bind to host cells. Enzymatic removal of α2,3-sialic acid and α1,3-fucose residues from host cell surfaces makes them less supportive of AmOmpA binding. AmOmpA is both an adhesin and an invasin, as coating inert beads with it confers adhesiveness and invasiveness. Recombinant forms of AmOmpA and ApOmpA competitively antagonize A. marginale infection of host cells, but a monoclonal antibody against 6-sulfo-sLe(x) fails to inhibit AmOmpA adhesion and A. marginale infection. Thus, the two OmpA proteins bind related but structurally distinct receptors. This study provides a detailed understanding of AmOmpA function, identifies its essential residues that can be targeted by blocking antibody to reduce infection, and determines that it binds to one or more α2,3-sialylated and α1,3-fucosylated glycan receptors that are unique from those targeted by ApOmpA.

  17. Solution structure of the Ubp-M BUZ domain, a highly specific protein module that recognizes the C-terminal tail of free ubiquitin.

    PubMed

    Pai, Ming-Tao; Tzeng, Shiou-Ru; Kovacs, Jeffrey J; Keaton, Mignon A; Li, Shawn S-C; Yao, Tso-Pang; Zhou, Pei

    2007-07-06

    The BUZ/Znf-UBP domain is a distinct ubiquitin-binding module found in the cytoplasmic deacetylase HDAC6, the E3 ubiquitin ligase BRAP2/IMP, and a subfamily of deubiquitinating enzymes. Here, we report the solution structure of the BUZ domain of Ubp-M, a ubiquitin-specific protease, and its interaction with ubiquitin. Unlike the BUZ domain from isopeptidase T (isoT) that contains a single zinc finger, the Ubp-M BUZ domain features three zinc-binding sites consisting of 12 residues. These zinc ligands form a pair of cross-braced ring fingers encapsulated within a third zinc finger in the primary structure. In contrast to isoT, which can form an N-terminal loop swapped dimer in the crystal state, the formation of additional zinc fingers in the Ubp-M BUZ domain restricts its N-terminal loop to intra-domain interactions. The ubiquitin-binding site of the Ubp-M BUZ domain is mapped to the highly conserved, concave surface formed by the alpha 3 helix and the central beta-sheet. We further show that this site binds to the C-terminal tail of free ubiquitin, and corresponding peptides display essentially the same binding affinities as full-length ubiquitin does for the Ubp-M BUZ domain. However, modification of the G76(Ub) carboxylate group either by a peptide or isopeptide bond abolishes BUZ-domain interaction. The unique ubiquitin-recognition mode of the BUZ domain family suggests that they may function as "sensors" of free ubiquitin in cells to achieve regulatory roles in many aspects of ubiquitin-dependent processes.

  18. Solution Structure of the Ubp-M BUZ Domain, a Highly Specific Protein Module That Recognizes the C-terminal Tail of Free Ubiquitin

    PubMed Central

    Pai, Ming-Tao; Tzeng, Shiou-Ru; Kovacs, Jeffrey J.; Keaton, Mignon A.; Li, Shawn S.-C.; Yao, Tso-Pang; Zhou, Pei

    2010-01-01

    Summary The BUZ/Znf-UBP domain is a distinct ubiquitin-binding module found in the cytoplasmic deacetylase HDAC6, the E3 ubiquitin ligase BRAP2/IMP, and a subfamily of deubiquitinating enzymes. Here we report the solution structure of the BUZ domain of Ubp-M, a ubiquitin-specific protease, and its interaction with ubiquitin. Unlike the BUZ domain from isopeptidase T (isoT) that contains a single zinc finger, the Ubp-M BUZ domain features three zinc-binding sites consisted of twelve residues. These zinc ligands form a pair of cross-braced ring fingers encapsulated within a third zinc finger in the primary structure. In contrast to isoT, which can form an N-terminal loop swapped dimer in the crystal state, the formation of additional zinc fingers in the Ubp-M BUZ domain restricts its N-terminal loop to intra-domain interactions. The ubiquitin-binding site of the Ubp-M BUZ domain is mapped to the highly conserved, concave surface formed by the α3 helix and the central β-sheet. We further show that this site binds to the C-terminal tail of free ubiquitin, and corresponding peptides display essentially the same binding affinities as full-length ubiquitin does for the Ubp-M BUZ domain. However, modification of the G76Ub carboxylate group either by a peptide- or isopeptide-bond abolishes BUZ-domain interaction. The unique ubiquitin-recognition mode of the BUZ domain family suggests that they may function as “sensors” of free ubiquitin in cells to achieve regulatory roles in many aspects of ubiquitin-dependent processes. PMID:17512543

  19. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  20. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  1. Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core.

    PubMed Central

    Morris, J F; Hromas, R; Rauscher, F J

    1994-01-01

    The myeloid zinc finger gene 1, MZF1, encodes a transcription factor which is expressed in hematopoietic progenitor cells that are committed to myeloid lineage differentiation. MZF1 contains 13 C2H2 zinc fingers arranged in two domains which are separated by a short glycine- and proline-rich sequence. The first domain consists of zinc fingers 1 to 4, and the second domain is formed by zinc fingers 5 to 13. We have determined that both sets of zinc finger domains bind DNA. Purified, recombinant MZF1 proteins containing either the first set of zinc fingers or the second set were prepared and used to affinity select DNA sequences from a library of degenerate oligonucleotides by using successive rounds of gel shift followed by PCR amplification. Surprisingly, both DNA-binding domains of MZF1 selected similar DNA-binding consensus sequences containing a core of four or five guanine residues, reminiscent of an NF-kappa B half-site: 1-4, 5'-AGTGGGGA-3'; 5-13, 5'-CGGGnGAGGGGGAA-3'. The full-length MZF1 protein containing both sets of zinc finger DNA-binding domains recognizes synthetic oligonucleotides containing either the 1-4 or 5-13 consensus binding sites in gel shift assays. Thus, we have identified the core DNA consensus binding sites for each of the two DNA-binding domains of a myeloid-specific zinc finger transcription factor. Identification of these DNA-binding sites will allow us to identify target genes regulated by MZF1 and to assess the role of MZF1 as a transcriptional regulator of hematopoiesis. Images PMID:8114711

  2. Sac phosphatase domain proteins.

    PubMed Central

    Hughes, W E; Cooke, F T; Parker, P J

    2000-01-01

    Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae. PMID:10947947

  3. Inferring Domain-Domain Interactions from Protein-Protein Interactions with Formal Concept Analysis

    PubMed Central

    Khor, Susan

    2014-01-01

    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. PMID:24586450

  4. Antibodies recognizing different domains of the polymeric immunoglobulin receptor.

    PubMed

    Solari, R; Kühn, L; Kraehenbuhl, J P

    1985-01-25

    The receptor responsible for the transepithelial transport of IgA dimer antibodies is a transmembrane glycoprotein known as membrane secretory component (SCm). During transport, the membrane anchoring domain is cleaved and the ectoplasmic domain of the receptor (SCs) remains tightly bound to the IgA dimer in exosecretions. We have produced monoclonal antibodies with distinct specificities against both cytoplasmic and ectoplasmic epitopes of rabbit SCm. One antibody (anti-SC303) reacted both with SCm and free SCs but not with SCs bound to IgA dimer (SIgA). Therefore, it recognized an epitope close to the IgA dimer binding site. The other monoclonal antibody (anti-SC166), which was unable to react with SCs, bound to the 15-kDa cytoplasmic extension of the membrane-spanning domain of the receptor. A polyclonal antibody (GaR-SC), raised in a goat against rabbit milk SCs, reacted with a subpopulation of SCs not recognized by the anti-SC303 monoclonal antibody and in addition also reacted with covalently bound sIgA. The three antibodies cross-reacted with rat SCm. We demonstrate the ability of the anti-SC166 monoclonal antibody to immunoadsorb subcellular organelles as a result of the cytoplasmic orientation of its epitope. Our data indicate that there are functional differences between the high- and low-molecular-weight families of SC in terms of IgA dimer binding.

  5. Hybrid H-2 histocompatibility gene products assign domains recognized by alloreactive T cells.

    PubMed Central

    Ozato, K; Evans, G A; Shykind, B; Margulies, D H; Seidman, J G

    1983-01-01

    In vitro recombination of cloned H-2 genes resulted in two allogeneically novel mouse transplantation antigens in which the C2 domains of H-2Ld and H-2Dd were exchanged. These genes were introduced into mouse L cells by DNA-mediated gene transfer. We have used these transformed fibroblast lines, expressing recombinant H-2 antigens at normal levels, as targets for alloreactive cytotoxic T lymphocytes (CTL). Transformed cell lines expressing native or hybrid H-2 molecules that share the N and C1 domains but not the C2 domain were lysed to an equivalent degree by primary and secondary anti-H-2Dd CTL. Hybrid and native H-2 antigens that have the same N and C1 domains were capable of reciprocally blocking specific lysis by the CTL. Thus, polymorphic determinants recognized by alloreactive T cells are located primarily in the N domain, the C1 domain, or both domains of the H-2 antigen. In contrast, determinants recognized by monoclonal antibodies are present throughout the H-2 protein, including the C2 domain. Antibodies that bind specifically to the C2 domain effectively inhibited CTL activity, possibly due to steric effects. The same antibodies do not competitively inhibit the binding of N/C1-specific antibodies. The predominant reactivity of CTL to the N and C1 domains suggests that humoral and cellular responses "see" a different spectrum of alloantigenic determinants. PMID:6188160

  6. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  7. Cellulose binding domain proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  8. The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response

    PubMed Central

    Li, Mo; Lu, Lin-Yu; Yang, Chao-Yie; Wang, Shaomeng; Yu, Xiaochun

    2013-01-01

    Poly-ADP-ribosylation is a unique post-translational modification participating in many biological processes, such as DNA damage response. Here, we demonstrate that a set of Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains recognizes poly(ADP-ribose) (PAR) both in vitro and in vivo. Among these FHA and BRCT domains, the FHA domains of APTX and PNKP interact with iso-ADP-ribose, the linkage of PAR, whereas the BRCT domains of Ligase4, XRCC1, and NBS1 recognize ADP-ribose, the basic unit of PAR. The interactions between PAR and the FHA or BRCT domains mediate the relocation of these domain-containing proteins to DNA damage sites and facilitate the DNA damage response. Moreover, the interaction between PAR and the NBS1 BRCT domain is important for the early activation of ATM during DNA damage response and ATM-dependent cell cycle checkpoint activation. Taken together, our results demonstrate two novel PAR-binding modules that play important roles in DNA damage response. PMID:23964092

  9. The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin.

    PubMed

    Sjodt, Megan; Macdonald, Ramsay; Spirig, Thomas; Chan, Albert H; Dickson, Claire F; Fabian, Marian; Olson, John S; Gell, David A; Clubb, Robert T

    2016-03-27

    Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdH(N2N3), A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix.

  10. Protein domain connectivity and essentiality

    NASA Astrophysics Data System (ADS)

    da F. Costa, L.; Rodrigues, F. A.; Travieso, G.

    2006-10-01

    Protein-protein interactions can be properly modeled as scale-free complex networks, while the lethality of proteins has been correlated with the node degrees, therefore defining a lethality-centrality rule. In this work the authors revisit this relevant problem by focusing attention not on proteins as a whole, but on their functional domains, which are ultimately responsible for their binding potential. Four networks are considered: the original protein-protein interaction network, its randomized version, and two domain networks assuming different lethality hypotheses. By using formal statistical analysis, they show that the correlation between connectivity and essentiality is higher for domains than for proteins.

  11. Structure and function of WD40 domain proteins.

    PubMed

    Xu, Chao; Min, Jinrong

    2011-03-01

    The WD40 domain exhibits a β-propeller architecture, often comprising seven blades. The WD40 domain is one of the most abundant domains and also among the top interacting domains in eukaryotic genomes. In this review, we will discuss the identification, definition and architecture of the WD40 domains. WD40 domain proteins are involved in a large variety of cellular processes, in which WD40 domains function as a protein-protein or protein-DNA interaction platform. WD40 domain mediates molecular recognition events mainly through the smaller top surface, but also through the bottom surface and sides. So far, no WD40 domain has been found to display enzymatic activity. We will also discuss the different binding modes exhibited by the large versatile family of WD40 domain proteins. In the last part of this review, we will discuss how post-translational modifications are recognized by WD40 domain proteins.

  12. CdiA Effectors Use Modular Receptor-Binding Domains To Recognize Target Bacteria

    PubMed Central

    Ruhe, Zachary C.; Nguyen, Josephine Y.; Xiong, Jing; Koskiniemi, Sanna; Beck, Christina M.; Perkins, Basil R.; Low, David A.

    2017-01-01

    ABSTRACT Contact-dependent growth inhibition (CDI) systems encode CdiA effectors, which bind to specific receptors on neighboring bacteria and deliver C-terminal toxin domains to suppress target cell growth. Two classes of CdiA effectors that bind distinct cell surface receptors have been identified, but the molecular basis of receptor specificity is not understood. Alignment of BamA-specific CdiAEC93 from Escherichia coli EC93 and OmpC-specific CdiAEC536 from E. coli 536 suggests that the receptor-binding domain resides within a central region that varies between the two effectors. In support of this hypothesis, we find that CdiAEC93 fragments containing residues Arg1358 to Phe1646 bind specifically to purified BamA. Moreover, chimeric CdiAEC93 that carries the corresponding sequence from CdiAEC536 is endowed with OmpC-binding activity, demonstrating that this region dictates receptor specificity. A survey of E. coli CdiA proteins reveals two additional effector classes, which presumably recognize distinct receptors. Using a genetic approach, we identify the outer membrane nucleoside transporter Tsx as the receptor for a third class of CdiA effectors. Thus, CDI systems exploit multiple outer membrane proteins to identify and engage target cells. These results underscore the modularity of CdiA proteins and suggest that novel effectors can be constructed through genetic recombination to interchange different receptor-binding domains and toxic payloads. PMID:28351921

  13. ECOD: An Evolutionary Classification of Protein Domains

    PubMed Central

    Kinch, Lisa N.; Pei, Jimin; Shi, Shuoyong; Kim, Bong-Hyun; Grishin, Nick V.

    2014-01-01

    Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or “fold”). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies. PMID:25474468

  14. ECOD: an evolutionary classification of protein domains.

    PubMed

    Cheng, Hua; Schaeffer, R Dustin; Liao, Yuxing; Kinch, Lisa N; Pei, Jimin; Shi, Shuoyong; Kim, Bong-Hyun; Grishin, Nick V

    2014-12-01

    Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or "fold"). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies.

  15. Diversity in protein domain superfamilies

    PubMed Central

    Das, Sayoni; Dawson, Natalie L; Orengo, Christine A

    2015-01-01

    Whilst ∼93% of domain superfamilies appear to be relatively structurally and functionally conserved based on the available data from the CATH-Gene3D domain classification resource, the remainder are much more diverse. In this review, we consider how domains in some of the most ubiquitous and promiscuous superfamilies have evolved, in particular the plasticity in their functional sites and surfaces which expands the repertoire of molecules they interact with and actions performed on them. To what extent can we identify a core function for these superfamilies which would allow us to develop a ‘domain grammar of function’ whereby a protein's biological role can be proposed from its constituent domains? Clearly the first step is to understand the extent to which these components vary and how changes in their molecular make-up modifies function. PMID:26451979

  16. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1998-02-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  17. Cellulose binding domain fusion proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  18. The OB-fold domain 1 of human POT1 recognizes both telomeric and non-telomeric DNA motifs

    PubMed Central

    Kolar, Carol; Yan, Ying; Borgstahl, Gloria E.O.; Ouellette, Michel M.

    2015-01-01

    The POT1 protein plays a critical role in telomere protection and telomerase regulation. POT1 binds single-stranded 5’-TTAGGGTTAG-3’ and forms a dimer with the TPP1 protein. The dimer is recruited to telomeres, either directly or as part of the Shelterin complex. Human POT1 contains two Oligonucleotide/Oligosaccharide Binding (OB) fold domains, OB1 and OB2, which make physical contact with the DNA. OB1 recognizes 5’-TTAGGG whereas OB2 binds to the downstream TTAG-3’. Studies of POT1 proteins from other species have shown that some of these proteins are able to recognize a broader variety of DNA ligands than expected. To explore this possibility in humans, we have used SELEX to reexamine the sequence-specificity of the protein. Using human POT1 as a selection matrix, high-affinity DNA ligands were selected from a pool of randomized single-stranded oligonucleotides. After six successive rounds of selection, two classes of high-affinity targets were obtained. The first class was composed of oligonucleotides containing a cognate POT1 binding sites (5’-TTAGGGTTAG-3’). The second and more abundant class was made of molecules that carried a novel non-telomeric consensus: 5’-TNCANNAGKKKTTAGG-3’ (where K=G/T and N=any base). Binding studies showed that these non-telomeric sites were made of an OB1-binding motif (TTAGG) and a non-telomeric motif (NT motif), with the two motifs recognized by distinct regions of the OB1 domain. POT1 interacted with these non-telomeric binding sites with high affinity and specificity, even when bound to its dimerization partner TPP1. This intrinsic ability of POT1 to recognize NT motifs raises the possibility that the protein may fulfill additional functions at certain non-telomeric locations of the genome, in perhaps gene transcription, replication, or repair. PMID:25934589

  19. Extending Protein Domain Boundary Predictors to Detect Discontinuous Domains

    PubMed Central

    Xue, Zhidong; Jang, Richard; Govindarajoo, Brandon; Huang, Yichu; Wang, Yan

    2015-01-01

    A variety of protein domain predictors were developed to predict protein domain boundaries in recent years, but most of them cannot predict discontinuous domains. Considering nearly 40% of multidomain proteins contain one or more discontinuous domains, we have developed DomEx to enable domain boundary predictors to detect discontinuous domains by assembling the continuous domain segments. Discontinuous domains are predicted by matching the sequence profile of concatenated continuous domain segments with the profiles from a single-domain library derived from SCOP and CATH, and Pfam. Then the matches are filtered by similarity to library templates, a symmetric index score and a profile-profile alignment score. DomEx recalled 32.3% discontinuous domains with 86.5% precision when tested on 97 non-homologous protein chains containing 58 continuous and 99 discontinuous domains, in which the predicted domain segments are within ±20 residues of the boundary definitions in CATH 3.5. Compared with our recently developed predictor, ThreaDom, which is the state-of-the-art tool to detect discontinuous-domains, DomEx recalled 26.7% discontinuous domains with 72.7% precision in a benchmark with 29 discontinuous-domain chains, where ThreaDom failed to predict any discontinuous domains. Furthermore, combined with ThreaDom, the method ranked number one among 10 predictors. The source code and datasets are available at https://github.com/xuezhidong/DomEx. PMID:26502173

  20. TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in Arabidopsis.

    PubMed

    Nishimura, Marc T; Anderson, Ryan G; Cherkis, Karen A; Law, Terry F; Liu, Qingli L; Machius, Mischa; Nimchuk, Zachary L; Yang, Li; Chung, Eui-Hwan; El Kasmi, Farid; Hyunh, Michael; Osborne Nishimura, Erin; Sondek, John E; Dangl, Jeffery L

    2017-03-07

    Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll-interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR-TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that "truncated" NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system.

  1. Functional domains in tetraspanin proteins.

    PubMed

    Stipp, Christopher S; Kolesnikova, Tatiana V; Hemler, Martin E

    2003-02-01

    Exciting new findings have emerged about the structure, function and biochemistry of tetraspanin proteins. Five distinct tetraspanin regions have now been delineated linking structural features to specific functions. Within the large extracellular loop of tetraspanins, there is a variable region that mediates specific interactions with other proteins, as well as a more highly conserved region that has been suggested to mediate homodimerization. Within the transmembrane region, the four tetraspanin transmembrane domains are probable sites of both intra- and inter-molecular interactions that are crucial during biosynthesis and assembly of the network of tetraspanin-linked membrane proteins known as the 'tetraspanin web'. In the intracellular juxtamembrane region, palmitoylation of cysteine residues also contributes to tetraspanin web assembly, and the C-terminal cytoplasmic tail region could provide specific functional links to cytoskeletal or signaling proteins.

  2. Stability of domain structures in multi-domain proteins

    PubMed Central

    Bhaskara, Ramachandra M.; Srinivasan, Narayanaswamy

    2011-01-01

    Multi-domain proteins have many advantages with respect to stability and folding inside cells. Here we attempt to understand the intricate relationship between the domain-domain interactions and the stability of domains in isolation. We provide quantitative treatment and proof for prevailing intuitive ideas on the strategies employed by nature to stabilize otherwise unstable domains. We find that domains incapable of independent stability are stabilized by favourable interactions with tethered domains in the multi-domain context. Stability of such folds to exist independently is optimized by evolution. Specific residue mutations in the sites equivalent to inter-domain interface enhance the overall solvation, thereby stabilizing these domain folds independently. A few naturally occurring variants at these sites alter communication between domains and affect stability leading to disease manifestation. Our analysis provides safe guidelines for mutagenesis which have attractive applications in obtaining stable fragments and domain constructs essential for structural studies by crystallography and NMR. PMID:22355559

  3. Definition of the consensus motif recognized by gamma-adaptin ear domains.

    PubMed

    Mattera, Rafael; Ritter, Brigitte; Sidhu, Sachdev S; McPherson, Peter S; Bonifacino, Juan S

    2004-02-27

    The heterotetrameric adaptor complex 1 (AP-1) and the monomeric Golgi-localized, gamma ear-containing, Arf-binding (GGA) proteins are components of clathrin coats associated with the trans-Golgi network and endosomes. The carboxyl-terminal ear domains (or gamma-adaptin ear (GAE) domains) of two gamma-adaptin subunit isoforms of AP-1 and of the GGAs are structurally similar and bind to a common set of accessory proteins. In this study, we have systematically defined a core tetrapeptide motif PsiG(P/D/E)(Psi/L/M) (where Psi is an aromatic residue), which is responsible for the interactions of accessory proteins with GAE domains. The definition of this motif has allowed us to identify novel GAE-binding partners named NECAP and aftiphilin, which also contain clathrin-binding motifs. These findings shed light on the mechanism of accessory protein recruitment to trans-Golgi network and endosomal clathrin coats.

  4. Exhaustive enumeration of protein domain families.

    PubMed

    Heger, Andreas; Holm, Liisa

    2003-05-02

    Domains are considered as the basic units of protein folding, evolution, and function. Decomposing each protein into modular domains is thus a basic prerequisite for accurate functional classification of biological molecules. Here, we present ADDA, an automatic algorithm for domain decomposition and clustering of all protein domain families. We use alignments derived from an all-on-all sequence comparison to define domains within protein sequences based on a global maximum likelihood model. In all, 90% of domain boundaries are predicted within 10% of domain size when compared with the manual domain definitions given in the SCOP database. A representative database of 249,264 protein sequences were decomposed into 450,462 domains. These domains were clustered on the basis of sequence similarities into 33,879 domain families containing at least two members with less than 40% sequence identity. Validation against family definitions in the manually curated databases SCOP and PFAM indicates almost perfect unification of various large domain families while contamination by unrelated sequences remains at a low level. The global survey of protein-domain space by ADDA confirms that most large and universal domain families are already described in PFAM and/or SMART. However, a survey of the complete set of mobile modules leads to the identification of 1479 new interesting domain families which shuffle around in multi-domain proteins. The data are publicly available at ftp://ftp.ebi.ac.uk/pub/contrib/heger/adda.

  5. The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response

    PubMed Central

    Zhang, Feng; Shi, Jiazhong; Chen, Shih-Hsun; Bian, Chunjing; Yu, Xiaochun

    2015-01-01

    Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response. PMID:26400172

  6. A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly

    PubMed Central

    Schmidt, Florian I.; Lu, Alvin; Chen, Jeff W.; Ruan, Jianbin; Tang, Catherine

    2016-01-01

    Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro–caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASCCARD interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASCPYD filaments for the first time. These data revealed that cross-linking of ASCPYD filaments via ASCCARD mediates the assembly of ASC foci. PMID:27069117

  7. The DGCR8 RNA-binding heme domain recognizes primary microRNAs by clamping the hairpin

    PubMed Central

    Quick-Cleveland, Jen; Jacob, Jose P.; Weitz, Sara H.; Shoffner, Grant; Senturia, Rachel; Guo, Feng

    2014-01-01

    SUMMARY Canonical primary microRNA transcripts (pri-miRNAs) are characterized by a ~30-bp hairpin flanked by single-stranded regions. These pri-miRNAs are recognized and cleaved by the Microprocessor complex consisting of the Drosha nuclease and its obligate RNA-binding partner DGCR8. It is not well understood how the Microprocessor specifically recognizes pri-miRNA substrates. Here we show that in addition to the well-known double-stranded RNA-binding domains, DGCR8 uses a dimeric heme-binding domain to directly contact pri-miRNAs. This RNA-binding heme domain (Rhed) directs two DGCR8 dimers to bind each pri-miRNA hairpin. The two Rhed-binding sites are located at both ends of the hairpin. The Rhed and its RNA-binding surface are important for pri-miRNA processing activity. Additionally, the heme cofactor is required for formation of processing-competent DGCR8-pri-miRNA complexes. Our study reveals a unique protein-RNA interaction central to pri-miRNA recognition. We propose a unifying model in which two DGCR8 dimers clamp a pri-miRNA hairpin using their Rheds. PMID:24910438

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

    PubMed

    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.

  9. The OB-fold domain 1 of human POT1 recognizes both telomeric and non-telomeric DNA motifs.

    PubMed

    Choi, Kyung H; Lakamp-Hawley, Amanda S; Kolar, Carol; Yan, Ying; Borgstahl, Gloria E O; Ouellette, Michel M

    2015-08-01

    The POT1 protein plays a critical role in telomere protection and telomerase regulation. POT1 binds single-stranded 5'-TTAGGGTTAG-3' and forms a dimer with the TPP1 protein. The dimer is recruited to telomeres, either directly or as part of the Shelterin complex. Human POT1 contains two Oligonucleotide/Oligosaccharide Binding (OB) fold domains, OB1 and OB2, which make physical contact with the DNA. OB1 recognizes 5'-TTAGGG whereas OB2 binds to the downstream TTAG-3'. Studies of POT1 proteins from other species have shown that some of these proteins are able to recognize a broader variety of DNA ligands than expected. To explore this possibility in humans, we have used SELEX to reexamine the sequence-specificity of the protein. Using human POT1 as a selection matrix, high-affinity DNA ligands were selected from a pool of randomized single-stranded oligonucleotides. After six successive rounds of selection, two classes of high-affinity targets were obtained. The first class was composed of oligonucleotides containing a cognate POT1 binding sites (5'-TTAGGGTTAG-3'). The second and more abundant class was made of molecules that carried a novel non-telomeric consensus: 5'-TNCANNAGKKKTTAGG-3' (where K = G/T and N = any base). Binding studies showed that these non-telomeric sites were made of an OB1-binding motif (TTAGG) and a non-telomeric motif (NT motif), with the two motifs recognized by distinct regions of the OB1 domain. POT1 interacted with these non-telomeric binding sites with high affinity and specificity, even when bound to its dimerization partner TPP1. This intrinsic ability of POT1 to recognize NT motifs raises the possibility that the protein may fulfill additional functions at certain non-telomeric locations of the genome, in perhaps gene transcription, replication, or repair. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  10. Protein structural domains: definition and prediction.

    PubMed

    Ezkurdia, Iakes; Tress, Michael L

    2011-11-01

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

  11. TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in Arabidopsis

    PubMed Central

    Anderson, Ryan G.; Cherkis, Karen A.; Law, Terry F.; Liu, Qingli L.; Machius, Mischa; Nimchuk, Zachary L.; Yang, Li; Chung, Eui-Hwan; El Kasmi, Farid; Hyunh, Michael; Sondek, John E.; Dangl, Jeffery L.

    2017-01-01

    Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll–interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR–TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that “truncated” NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system. PMID:28137883

  12. Nickel(II)-Schiff base complex recognizing domain II of bovine and human serum albumin: spectroscopic and docking studies.

    PubMed

    Ray, Aurkie; Seth, Banabithi Koley; Pal, Uttam; Basu, Samita

    2012-06-15

    It has been spectroscopically monitored that a mononuclear nickel(II)-Schiff base complex {[NiL]·CH(3)OH=NSC} exhibits greater binding affinity for bovine serum albumin (BSA) than that of its human counterpart (HSA). Moreover the modes of binding of NSC with the two serum albumins also differ significantly. Docking studies predict a relatively rare type of 'superficial binding' of NSC at domain IIB of HSA with certain mobility whereas for BSA such phenomena has not been detected. The mobile nature of NSC at domain IIB of HSA has been well correlated with the spectroscopic results. It is to be noted that thermodynamic parameters for the NSC interaction also differ for the two serum albumins. Occurrence of energy transfer between the donor (Trp of BSA and HSA) and acceptor (NSC) has been obtained by means of Förster resonance energy transfer (FRET). The protein stability on NSC binding has also been experimented by the GuHCl-induced protein unfolding studies. Interestingly it has been found that NSC-HSA interaction enhances the protein stability whereas NSC-BSA binding has no such impact. Such observations are indicative of the fact that the conformation of NSC is responsible in recognizing the two serum albumins and selectively enhancing protein stability.

  13. Nickel(II)-Schiff base complex recognizing domain II of bovine and human serum albumin: Spectroscopic and docking studies

    NASA Astrophysics Data System (ADS)

    Ray, Aurkie; Koley Seth, Banabithi; Pal, Uttam; Basu, Samita

    It has been spectroscopically monitored that a mononuclear nickel(II)-Schiff base complex {[NiL]·CH3OH = NSC} exhibits greater binding affinity for bovine serum albumin (BSA) than that of its human counterpart (HSA). Moreover the modes of binding of NSC with the two serum albumins also differ significantly. Docking studies predict a relatively rare type of 'superficial binding' of NSC at domain IIB of HSA with certain mobility whereas for BSA such phenomena has not been detected. The mobile nature of NSC at domain IIB of HSA has been well correlated with the spectroscopic results. It is to be noted that thermodynamic parameters for the NSC interaction also differ for the two serum albumins. Occurrence of energy transfer between the donor (Trp of BSA and HSA) and acceptor (NSC) has been obtained by means of Förster resonance energy transfer (FRET). The protein stability on NSC binding has also been experimented by the GuHCl-induced protein unfolding studies. Interestingly it has been found that NSC-HSA interaction enhances the protein stability whereas NSC-BSA binding has no such impact. Such observations are indicative of the fact that the conformation of NSC is responsible in recognizing the two serum albumins and selectively enhancing protein stability.

  14. Enhanced protein domain discovery using taxonomy

    PubMed Central

    Coin, Lachlan; Bateman, Alex; Durbin, Richard

    2004-01-01

    Background It is well known that different species have different protein domain repertoires, and indeed that some protein domains are kingdom specific. This information has not yet been incorporated into statistical methods for finding domains in sequences of amino acids. Results We show that by incorporating our understanding of the taxonomic distribution of specific protein domains, we can enhance domain recognition in protein sequences. We identify 4447 new instances of Pfam domains in the SP-TREMBL database using this technique, equivalent to the coverage increase given by the last 8.3% of Pfam families and to a 0.7% increase in the number of domain predictions. We use PSI-BLAST to cross-validate our new predictions. We also benchmark our approach using a SCOP test set of proteins of known structure, and demonstrate improvements relative to standard Hidden Markov model techniques. Conclusions Explicitly including knowledge about the taxonomic distribution of protein domains can enhance protein domain recognition. Our method can also incorporate other context-specific domain distributions – such as domain co-occurrence and protein localisation. PMID:15137915

  15. Multiple graph regularized protein domain ranking

    PubMed Central

    2012-01-01

    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. PMID:23157331

  16. Structural basis for the Smad5 MH1 domain to recognize different DNA sequences

    PubMed Central

    Chai, Nan; Li, Wan-Xin; Wang, Jue; Wang, Zhi-Xin; Yang, Shi-Ming; Wu, Jia-Wei

    2015-01-01

    Smad proteins are important intracellular mediators of TGF-β signalling, which transmit signals directly from cell surface receptors to the nucleus. The MH1 domain of Smad plays a key role in DNA recognition. Two types of DNA sequence were identified as Smad binding motifs: the Smad binding element (SBE) and the GC-rich sequence. Here we report the first crystal structure of the Smad5 MH1 domain in complex with the GC-rich sequence. Compared with the Smad5-MH1/SBE complex structure, the Smad5 MH1 domain contacts the GC-rich site with the same β-hairpin, but the detailed interaction modes are different. Conserved β-hairpin residues make base specific contacts with the minimal GC-rich site, 5′-GGC-3′. The assembly of Smad5-MH1 on the GC-rich DNA also results in distinct DNA conformational changes. Moreover, the crystal structure of Smad5-MH1 in complex with a composite DNA sequence demonstrates that the MH1 domain is targeted to each binding site (GC-rich or SBE) with modular binding modes, and the length of the DNA spacer affects the MH1 assembly. In conclusion, our work provides the structural basis for the recognition and binding specificity of the Smad MH1 domain with the DNA targets. PMID:26304548

  17. Cholesterol and the interaction of proteins with membrane domains.

    PubMed

    Epand, Richard M

    2006-07-01

    Cholesterol is not uniformly distributed in biological membranes. One of the factors influencing the formation of cholesterol-rich domains in membranes is the unequal lateral distribution of proteins in membranes. Certain proteins are found in cholesterol-rich domains. In some of these cases, it is as a consequence of the proteins interacting directly with cholesterol. There are several structural features of a protein that result in the protein preferentially associating with cholesterol-rich domains. One of the best documented of these is certain types of lipidations. In addition, however, there are segments of a protein that can preferentially sequester cholesterol. We discuss two examples of these cholesterol-recognition elements: the cholesterol recognition/interaction amino acid consensus (CRAC) domain and the sterol-sensing domain (SSD). The requirements for a CRAC motif are quite flexible and predict that a large number of sequences could recognize cholesterol. There are, however, certain proteins that are known to interact with cholesterol-rich domains of cell membranes that have CRAC motifs, and synthetic peptides corresponding to these segments also promote the formation of cholesterol-rich domains. Modeling studies have provided a rationale for certain requirements of the CRAC motif. The SSD is a larger protein segment comprising five transmembrane domains. The amino acid sequence YIYF is found in several SSD and in certain other proteins for which there is evidence that they interact with cholesterol-rich domains. The CRAC sequences as well as YIYF are generally found adjacent to a transmembrane helical segment. These regions appear to have a strong influence of the localization of certain proteins into domains in biological membranes. In addition to the SSD, there is also a domain found in soluble proteins, the START domain, that binds lipids. Certain proteins with START domains specifically bind cholesterol and are believed to function in

  18. J domain independent functions of J proteins.

    PubMed

    Ajit Tamadaddi, Chetana; Sahi, Chandan

    2016-07-01

    Heat shock proteins of 40 kDa (Hsp40s), also called J proteins, are obligate partners of Hsp70s. Via their highly conserved and functionally critical J domain, J proteins interact and modulate the activity of their Hsp70 partners. Mutations in the critical residues in the J domain often result in the null phenotype for the J protein in question. However, as more J proteins have been characterized, it is becoming increasingly clear that a significant number of J proteins do not "completely" rely on their J domains to carry out their cellular functions, as previously thought. In some cases, regions outside the highly conserved J domain have become more important making the J domain dispensable for some, if not for all functions of a J protein. This has profound effects on the evolution of such J proteins. Here we present selected examples of J proteins that perform J domain independent functions and discuss this in the context of evolution of J proteins with dispensable J domains and J-like proteins in eukaryotes.

  19. Proteomic analysis of Trichinella spiralis adult worm excretory-secretory proteins recognized by early infection sera.

    PubMed

    Liu, Ruo Dan; Qi, Xin; Sun, Ge Ge; Jiang, Peng; Zhang, Xi; Wang, Li Ang; Liu, Xiao Lin; Wang, Zhong Quan; Cui, Jing

    2016-11-15

    At the intestinal stage of a Trichinella spiralis (T. spiralis) infection, the excretory-secretory (ES) antigens produced by adult worms (AWs) result in an early exposure to the host's immune system and elicit the production of specific antibodies; the AW ES proteins might provide early diagnostic markers of trichinellosis. The aim of this study was to identify early serodiagnostic markers from T. spiralis AW ES antigens. T. spiralis AWs were collected at 72h post infection, and their ES antigens were analysed by SDS-PAGE and Western blot. Then, the immunoreactive bands were subjected to shotgun LC-MS/MS and bioinformatics analyses. Our results showed that only one protein band (33kDa) was recognized by the sera of mice infected with T. spiralis at 8 days after infection. The shotgun LC-MS/MS analysis identified 23 proteins that were then clustered into 10 types; these proteins had molecular weights of 28.13-71.62kDa and pI 5.05-9.20. Certain enzymes (e.g., serine protease, adult-specific deoxyribonuclease [DNase] II, peptidase S1A subfamily, and multi cystatin-like domain protein) were found to be highly represented. The functions of the 10 proteins were further analysed: of the 6 annotated proteins, 3 had serine hydrolase activity and 2 had DNase II activity. These results provide a valuable basis for identifying early diagnostic antigens and vaccine candidates for trichinellosis.

  20. Towards the definition of a chimpanzee and human conserved CD6 domain 1 epitope recognized by T1 monoclonal antibody.

    PubMed

    Alonso, Ruby; Huerta, Vivian; de Leon, Joel; Piedra, Patricia; Puchades, Yaquelin; Guirola, Osmany; Chinea, Glay; Montero, Enrique

    2008-08-01

    Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 x 10(8). These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.

  1. Amino acid signature enables proteins to recognize modified tRNA.

    PubMed

    Spears, Jessica L; Xiao, Xingqing; Hall, Carol K; Agris, Paul F

    2014-02-25

    Human tRNA(Lys3)UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNA(Lys3)UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNA(Lys3)UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N(6)-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNA(Lys3)UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA's fully modified anticodon stem and loop domain, hASL(Lys3)UUU. Peptides of this sequence specifically recognized and bound modified htRNA(Lys3)UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions.

  2. Predicting PDZ domain mediated protein interactions from structure

    PubMed Central

    2013-01-01

    Background PDZ domains are structural protein domains that recognize simple linear amino acid motifs, often at protein C-termini, and mediate protein-protein interactions (PPIs) in important biological processes, such as ion channel regulation, cell polarity and neural development. PDZ domain-peptide interaction predictors have been developed based on domain and peptide sequence information. Since domain structure is known to influence binding specificity, we hypothesized that structural information could be used to predict new interactions compared to sequence-based predictors. Results We developed a novel computational predictor of PDZ domain and C-terminal peptide interactions using a support vector machine trained with PDZ domain structure and peptide sequence information. Performance was estimated using extensive cross validation testing. We used the structure-based predictor to scan the human proteome for ligands of 218 PDZ domains and show that the predictions correspond to known PDZ domain-peptide interactions and PPIs in curated databases. The structure-based predictor is complementary to the sequence-based predictor, finding unique known and novel PPIs, and is less dependent on training–testing domain sequence similarity. We used a functional enrichment analysis of our hits to create a predicted map of PDZ domain biology. This map highlights PDZ domain involvement in diverse biological processes, some only found by the structure-based predictor. Based on this analysis, we predict novel PDZ domain involvement in xenobiotic metabolism and suggest new interactions for other processes including wound healing and Wnt signalling. Conclusions We built a structure-based predictor of PDZ domain-peptide interactions, which can be used to scan C-terminal proteomes for PDZ interactions. We also show that the structure-based predictor finds many known PDZ mediated PPIs in human that were not found by our previous sequence-based predictor and is less dependent on

  3. A thermodynamic definition of protein domains

    PubMed Central

    Porter, Lauren L.; Rose, George D.

    2012-01-01

    Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics. PMID:22635268

  4. A thermodynamic definition of protein domains.

    PubMed

    Porter, Lauren L; Rose, George D

    2012-06-12

    Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.

  5. Epitope mapping of four monoclonal antibodies recognizing the hexose core domain of Salmonella lipopolysaccharide.

    PubMed

    Luk, J M; Lind, S M; Tsang, R S; Lindberg, A A

    1991-12-05

    Four murine monoclonal antibodies reactive with distinctive regions of the hexose core domain of Salmonella lipopolysaccharide (LPS) were generated and their epitope specificities were delineated. MAST 56 (IgG1) and MAST 50 (IgG3) antibodies elicited by immunizations with Salmonella typhimurium Rb1 and Rb2 mutants, reacted selectively in enzyme immunoassay with the LPS from rough mutants. In contrast, MATy 1 (IgM) and MATy 2 (IgG2b) antibodies raised by an attenuated Salmonella typhi 620 Ty strain were reactive with LPS from both smooth and rough Salmonellae. Immunoblotting analysis showed that MATy 1 distinguished only the bottom bands (naked LPS core) among the heterogeneous LPS populations, whereas MATy 2 gave a ladder pattern (reactive with both naked and O-chain-substituted LPS cores). Differential binding specificities of MATy 1 and MATy 2 antibodies to the naked and capped LPS cores were further analyzed utilizing S. typhimurium polysaccharide fractions with different O-chain:core ratios which were obtained after separation by Sephacryl S-200 chromatography. Steric effects on the antibody reactivity by the bulky O-polysaccharide chain were detected. The use of chemically defined native and synthetic saccharides as inhibitors, in combination with the conformation of the Salmonella core oligosaccharide, permitted the definition of antigenic determinants carried in the core domain recognized by each antibody: (i) the branches I and VIII are essential for MATy 1 recognition, (ii) the backbone III-IV-V for MATy 2, (iii) the backbone II-III-IV-V for MAST 56, and (iv) the backbone plus the branch III-IV-V-VIII for MAST 50. (formula; see text)

  6. Autoantibodies that recognize functional domains of hnRNPA1 implicate molecular mimicry in the pathogenesis of neurological disease.

    PubMed

    Lee, Sang Min; Dunnavant, Floyd D; Jang, Haeman; Zunt, Joseph; Levin, Michael C

    2006-06-19

    As a model for molecular mimicry in neurological disease, we study people infected with human T-lymphotropic virus type 1 (HTLV-1) who develop HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), an immune-mediated disease of the central nervous system (CNS). In HAM/TSP, data suggests molecular mimicry is the result of cross-reactive antibodies between HTLV-1-tax and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a protein over-expressed in human CNS neurons. The hnRNP A1 epitope recognized by autoantibodies was unknown. In this study, we hypothesized that antibodies purified from HAM/TSP patients would react with functionally significant domains of hnRNP A1. Western blotting of functionally significant deletion mutants and overlapping fusion proteins using HAM/TSP IgG revealed two core epitopes within the C-terminal region of hnRNP A1. The first (aminoacids 191-SSQRGRSGSGNF-202), overlapped the RGG domain and the second (aminoacids 293-GQYFAKPRNQGG-304), with the M9 shuttling sequence, two functionally important regions of hnRNP A1. Monoclonal antibodies to HTLV-1-tax also reacted with the epitopes. These data fulfill an important criterion of molecular mimicry, namely that mimicking epitopes are not random, but include biologically significant regions of target proteins. This suggests an important role for the cross-reactive immune response between HTLV-1 and hnRNP A1 in the pathogenesis of immune-mediated neurological diseases via molecular mimicry.

  7. The architecture of the protein domain universe.

    PubMed

    Dokholyan, Nikolay V

    2005-03-14

    Understanding the design of the universe of protein structures may provide insights into protein evolution. We study the architecture of the protein domain universe, which has been found to poses peculiar scale-free properties. We examine the origin of these scale-free properties of the graph of protein domain structures (PDUG) and determine that that the PDUG is not modular, i.e. it does not consist of modules with uniform properties. Instead, we find the PDUG to be self-similar at all scales. We further characterize the PDUG architecture by studying the properties of the hub nodes that are responsible for the scale-free connectivity of the PDUG. We introduce a measure of the betweenness centrality of protein domains in the PDUG and find a power-law distribution of the betweenness centrality values. The scale-free distribution of hubs in the protein universe suggests that a set of specific statistical mechanics models, such as the self-organized criticality model, can potentially identify the principal driving forces of protein evolution. We also find a gatekeeper protein domain, removal of which partitions the largest cluster into two large sub-clusters. We suggest that the loss of such gatekeeper protein domains in the course of evolution is responsible for the creation of new fold families.

  8. NTTMUNSW BioC modules for recognizing and normalizing species and gene/protein mentions.

    PubMed

    Dai, Hong-Jie; Singh, Onkar; Jonnagaddala, Jitendra; Su, Emily Chia-Yu

    2016-01-01

    In recent years, the number of published biomedical articles has increased as researchers have focused on biological domains to investigate the functions of biological objects, such as genes and proteins. However, the ambiguous nature of genes and their products have rendered the literature more complex for readers and curators of molecular interaction databases. To address this challenge, a normalization technique that can link variants of biological objects to a single, standardized form was applied. In this work, we developed a species normalization module, which recognizes species names and normalizes them to NCBI Taxonomy IDs. Unlike most previous work, which ignored the prefix of a gene name that represents an abbreviation of the species name to which the gene belongs, the recognition results of our module include the prefixed species. The developed species normalization module achieved an overall F-score of 0.954 on an instance-level species normalization corpus. For gene normalization, two separate modules were respectively employed to recognize gene mentions and normalize those mentions to their Entrez Gene IDs by utilizing a multistage normalization algorithm developed for processing full-text articles. All of the developed modules are BioC-compatible .NET framework libraries and are publicly available from the NuGet gallery.Database URL: https://sites.google.com/site/hjdairesearch/Projects/isn-corpus.

  9. Domain structure of Lassa virus L protein.

    PubMed

    Brunotte, Linda; Lelke, Michaela; Hass, Meike; Kleinsteuber, Katja; Becker-Ziaja, Beate; Günther, Stephan

    2011-01-01

    The 200-kDa L protein of arenaviruses plays a central role in viral genome replication and transcription. This study aimed at providing evidence for the domain structure of L protein by combining bioinformatics with a stepwise mutagenesis approach using the Lassa virus minireplicon system. Potential interdomain linkers were predicted using various algorithms. The prediction was challenged by insertion of flexible sequences into the predicted linkers. Insertion of 5 or 10 amino acid residues was tolerated at seven sites (S407, G446, G467, G774, G939, S1952, and V2074 in Lassa virus AV). At two of these sites, G467 and G939, L protein could be split into an N-terminal and a C-terminal part, which were able to trans-complement each other and reconstitute a functional complex upon coexpression. Coimmunoprecipitation studies revealed physical interaction between the N- and C-terminal domains, irrespective of whether L protein was split at G467 or G939. In confocal immunofluorescence microscopy, the N-terminal domains showed a dot-like, sometimes perinuclear, cytoplasmic distribution similar to that of full-length L protein, while the C-terminal domains were homogenously distributed in cytoplasm. The latter were redistributed into the dot-like structures upon coexpression with the corresponding N-terminal domain. In conclusion, this study demonstrates two interdomain linkers in Lassa virus L protein, at G467 and G939, suggesting that L protein is composed of at least three structural domains spanning residues 1 to 467, 467 to 939, and 939 to 2220. The first domain seems to mediate accumulation of L protein into cytoplasmic dot-like structures.

  10. Identification of a Novel Sequence Motif Recognized by the Ankyrin Repeat Domain of zDHHC17/13 S-Acyltransferases*

    PubMed Central

    Lemonidis, Kimon; Sanchez-Perez, Maria C.; Chamberlain, Luke H.

    2015-01-01

    S-Acylation is a major post-translational modification affecting several cellular processes. It is particularly important for neuronal functions. This modification is catalyzed by a family of transmembrane S-acyltransferases that contain a conserved zinc finger DHHC (zDHHC) domain. Typically, eukaryote genomes encode for 7–24 distinct zDHHC enzymes, with two members also harboring an ankyrin repeat (AR) domain at their cytosolic N termini. The AR domain of zDHHC enzymes is predicted to engage in numerous interactions and facilitates both substrate recruitment and S-acylation-independent functions; however, the sequence/structural features recognized by this module remain unknown. The two mammalian AR-containing S-acyltransferases are the Golgi-localized zDHHC17 and zDHHC13, also known as Huntingtin-interacting proteins 14 and 14-like, respectively; they are highly expressed in brain, and their loss in mice leads to neuropathological deficits that are reminiscent of Huntington's disease. Here, we report that zDHHC17 and zDHHC13 recognize, via their AR domain, evolutionary conserved and closely related sequences of a [VIAP][VIT]XXQP consensus in SNAP25, SNAP23, cysteine string protein, Huntingtin, cytoplasmic linker protein 3, and microtubule-associated protein 6. This novel AR-binding sequence motif is found in regions predicted to be unstructured and is present in a number of zDHHC17 substrates and zDHHC17/13-interacting S-acylated proteins. This is the first study to identify a motif recognized by AR-containing zDHHCs. PMID:26198635

  11. Polyclonal antibodies against the TLA1 protein also recognize with high specificity the D2 reaction center protein of PSII in the green alga Chlamydomonas reinhardtii.

    PubMed

    Mitra, Mautusi; Dewez, David; García-Cerdán, Jose Gines; Melis, Anastasios

    2012-04-01

    The Chlamydomonas reinhardtii DNA-insertional transformant truncated light-harvesting antenna 1 (tla1) mutant, helped identify the novel TLA1 gene (GenBank Accession # AF534570-71) as an important genetic determinant in the chlorophyll antenna size of photosynthesis. Down-regulation in the amount of the TLA1 23 kDa protein in the cell resulted in smaller chlorophyll antenna size for both photosystems (in Tetali et al. Planta 225:813-829, 2007). Specific polyclonal antibodies, raised against the recombinant TLA1 protein, showed a cross-reaction with the predicted 23 kDa TLA1 protein in C. reinhardtii protein extracts, but also showed a strong cross-reaction with a protein band migrating to 28.5 kDa. Questions of polymorphism, or posttranslational modification of the TLA1 protein were raised as a result of the unexpected 28.5 kDa cross-reaction. Work in this paper aimed to elucidate the nature of the unexpected 28.5 kDa cross-reaction, as this was deemed to be important in terms of the functional role of the TLA1 protein in the regulation of the chlorophyll antenna size of photosynthesis. Immuno-precipitation of the 28.5 kDa protein, followed by LC-mass spectrometry, showed amino acid sequences ascribed to the psbD/D2 reaction center protein of PSII. The common antigenic determinant between TLA1 and D2 was shown to be a stretch of nine conserved amino acids V-F-L(V)LP-GNAL in the C-terminus of the two proteins, constituting a high antigenicity "GNAL" domain. Antibodies raised against the TLA1 protein containing this domain recognized both the TLA1 and the D2 protein. Conversely, antibodies raised against the TLA1 protein minus the GNAL domain specifically recognized the 23 kDa TLA1 protein and failed to recognize the 28.5 kDa D2 protein. D2 antibodies raised against an oligopeptide containing this domain also cross-reacted with the TLA1 protein. It is concluded that the 28.5 kDa cross-reaction of C. reinhardtii protein extracts with antiTLA1 antibodies is due to

  12. Discovering interacting domains and motifs in protein-protein interactions.

    PubMed

    Hugo, Willy; Sung, Wing-Kin; Ng, See-Kiong

    2013-01-01

    Many important biological processes, such as the signaling pathways, require protein-protein interactions (PPIs) that are designed for fast response to stimuli. These interactions are usually transient, easily formed, and disrupted, yet specific. Many of these transient interactions involve the binding of a protein domain to a short stretch (3-10) of amino acid residues, which can be characterized by a sequence pattern, i.e., a short linear motif (SLiM). We call these interacting domains and motifs domain-SLiM interactions. Existing methods have focused on discovering SLiMs in the interacting proteins' sequence data. With the recent increase in protein structures, we have a new opportunity to detect SLiMs directly from the proteins' 3D structures instead of their linear sequences. In this chapter, we describe a computational method called SLiMDIet to directly detect SLiMs on domain interfaces extracted from 3D structures of PPIs. SLiMDIet comprises two steps: (1) interaction interfaces belonging to the same domain are extracted and grouped together using structural clustering and (2) the extracted interaction interfaces in each cluster are structurally aligned to extract the corresponding SLiM. Using SLiMDIet, de novo SLiMs interacting with protein domains can be computationally detected from structurally clustered domain-SLiM interactions for PFAM domains which have available 3D structures in the PDB database.

  13. Proteins and cholesterol-rich domains.

    PubMed

    Epand, Richard M

    2008-01-01

    Biological membranes are composed of many molecular species of lipids and proteins. These molecules do not mix ideally. In the plane of the membrane components are segregated into domains that are enriched in certain lipids and proteins. Cholesterol is a membrane lipid that is not uniformly distributed in the membrane. Proteins play an important role in determining cholesterol distribution. Certain types of protein lipidation are known to cause the lipoprotein to sequester with cholesterol and to stabilize cholesterol-rich domains. However, proteins that are excluded from such domains also contribute to the redistribution of cholesterol. One of the motifs that favor interaction with cholesterol is the CRAC motif. The role of the CRAC motif of the gp41 fusogenic protein of HIV is discussed. The distribution of the multianionic lipid, phosphatidylinositol(4,5)bis-phosphate (PtnIns(4,5)P2), is also not uniform in cell membranes. This lipid has several functions in the cell, including a morphological role in determining the sites of attachment of the actin cytoskeleton to the plasma membrane. PtnIns(4,5)P2 is sequestered by proteins having clusters of cationic residues in their sequence. Certain proteins containing cationic clusters also contain moieties such as myristoylation or a CRAC segment that would also endow them with the ability to sequester to a cholesterol-rich domain. These proteins interact with PtnIns(4,5)P2 in a cholesterol-dependent manner forming domains that are enriched in both cholesterol and in PtnIns(4,5)P2 but can also be distinct from liquid-ordered raft-like domains.

  14. Functional innovation from changes in protein domains and their combinations.

    PubMed

    Lees, Jonathan G; Dawson, Natalie L; Sillitoe, Ian; Orengo, Christine A

    2016-06-01

    Domains are the functional building blocks of proteins. In this work we discuss how domains can contribute to the evolution of new functions. Domains themselves can evolve through various mechanisms, altering their intrinsic function. Domains can also facilitate functional innovations by combining with other domains to make novel proteins. We discuss the mechanisms by which domain and domain combinations support functional innovations. We highlight interesting examples where changes in domain combination promote changes at the domain level.

  15. Existence of Separate Domains in Lysin PlyG for Recognizing Bacillus anthracis Spores and Vegetative Cells

    PubMed Central

    Yang, Hang; Wang, Dian-Bing; Dong, Qiuhua; Zhang, Zhiping; Cui, Zongqiang; Deng, Jiaoyu; Yu, Junping

    2012-01-01

    As a potential antimicrobial, the bacteriophage lysin PlyG has been reported to specifically recognize Bacillus anthracis vegetative cells only and to kill B. anthracis vegetative cells and its germinating spores. However, how PlyG interacts with B. anthracis spores remains unclear. Herein, a 60-amino-acid domain in PlyG (residues 106 to 165), located mainly in the previously identified catalytic domain, was found able to specifically recognize B. anthracis spores but not vegetative cells. The exosporium of the spores was found to be the most probable binding target of this domain. This is the first time that a lysin for spore-forming bacteria has been found to have separate domains to recognize spores and vegetative cells, which might help in understanding the coevolution of phages with spore-forming bacteria. Besides providing new biomarkers for developing better assays for identifying B. anthracis spores, the newly found domain may be helpful in developing PlyG as a preventive antibiotic to reduce the threat of anthrax in suspected exposures to B. anthracis spores. PMID:22802245

  16. The Fragile X Mental Retardation Protein, FMRP, Recognizes G-Quartets

    ERIC Educational Resources Information Center

    Darnell, Jennifer C.; Warren, Stephen T.; Darnell, Robert B.

    2004-01-01

    Fragile X mental retardation is a disease caused by the loss of function of a single RNA-binding protein, FMRP. Identifying the RNA targets recognized by FMRP is likely to reveal much about its functions in controlling some aspects of memory and behavior. Recent evidence suggests that one of the predominant RNA motifs recognized by the FMRP…

  17. The Fragile X Mental Retardation Protein, FMRP, Recognizes G-Quartets

    ERIC Educational Resources Information Center

    Darnell, Jennifer C.; Warren, Stephen T.; Darnell, Robert B.

    2004-01-01

    Fragile X mental retardation is a disease caused by the loss of function of a single RNA-binding protein, FMRP. Identifying the RNA targets recognized by FMRP is likely to reveal much about its functions in controlling some aspects of memory and behavior. Recent evidence suggests that one of the predominant RNA motifs recognized by the FMRP…

  18. Identifying structural domains of proteins using clustering

    PubMed Central

    2012-01-01

    Background Protein structures are comprised of modular elements known as domains. These units are used and re-used over and over in nature, and usually serve some particular function in the structure. Thus it is useful to be able to break up a protein of interest into its component domains, prior to similarity searching for example. Numerous computational methods exist for doing so, but most operate only on a single protein chain and many are limited to making a series of cuts to the sequence, while domains can and do span multiple chains. Results This study presents a novel clustering-based approach to domain identification, which works equally well on individual chains or entire complexes. The method is simple and fast, taking only a few milliseconds to run, and works by clustering either vectors representing secondary structure elements, or buried alpha-carbon positions, using average-linkage clustering. Each resulting cluster corresponds to a domain of the structure. The method is competitive with others, achieving 70% agreement with SCOP on a large non-redundant data set, and 80% on a set more heavily weighted in multi-domain proteins on which both SCOP and CATH agree. Conclusions It is encouraging that a basic method such as this performs nearly as well or better than some far more complex approaches. This suggests that protein domains are indeed for the most part simply compact regions of structure with a higher density of buried contacts within themselves than between each other. By representing the structure as a set of points or vectors in space, it allows us to break free of any artificial limitations that other approaches may depend upon. PMID:23116496

  19. Inferring Evolutionary Scenarios for Protein Domain Compositions

    NASA Astrophysics Data System (ADS)

    Wiedenhoeft, John; Krause, Roland; Eulenstein, Oliver

    Essential cellular processes are controlled by functional interactions of protein domains, which can be inferred from their evolutionary histories. Methods to reconstruct these histories are challenged by the complexity of reconstructing macroevolutionary events. In this work we model these events using a novel network-like structure that represents the evolution of domain combinations, called plexus. We describe an algorithm to find a plexus that represents the evolution of a given collection of domain histories as phylogenetic trees with the minimum number of macroevolutionary events, and demonstrate its effectiveness in practice.

  20. The N- and C-terminal domains of MecA recognize different partners in the competence molecular switch.

    PubMed

    Persuh, M; Turgay, K; Mandic-Mulec, I; Dubnau, D

    1999-08-01

    ComK is a transcription factor required for the expression of competence genes in Bacillus subtilis. Binding to MecA targets ComK for degradation by the ClpCP protease. MecA therefore acts as an adapter protein recruiting a regulatory protein for proteolysis. However, when ComS is synthesized, ComK is released from binding by MecA and thereby protected from degradation. MecA binds to three protein partners during these processes: ComK, ClpC and ComS. Using limited proteolysis, we have defined N- and C-terminal structural domains of MecA and evaluated the interactions of these domains with the protein partners of MecA. Using surface plasmon resonance, we have determined that the N-terminal domain of MecA interacts with ComK and ComS and the C-terminal domain with ClpC. MecA is shown to exist as a dimer with dimerization sites on both the N- and C-terminal domains. The C-terminal domain stimulates the ATPase activity of ClpC and is degraded by the ClpCP protease, while the N-terminal domain is inactive in both of these assays. In vivo data were consistent with these findings, as comG-lacZ expression was decreased in a strain overproducing the N-terminal domain, indicating reduced ComK activity. We propose a model in which binding of ClpC to the C-terminal domain of MecA induces a conformational change enabling the N-terminal domain to bind ComK with enhanced affinity. MecA is widespread among Gram-positive organisms and may act generally as an adapter protein, targeting proteins for regulated degradation.

  1. Folding mechanism of a multiple independently-folding domain protein: double B domain of protein A.

    PubMed

    Arora, Pooja; Hammes, Gordon G; Oas, Terrence G

    2006-10-10

    The antibody binding properties of staphylococcal protein A (SpA) can be attributed to the presence of five highly homologous domains (E, D, A, B, and C). Although the folding of the B domain of protein A (BdpA) is well-characterized, the folding behavior of this domain in the context of full-length SpA in the cell remains unexplored. The sequence of the B domain is 89 and 91% identical to those of domains A and C, respectively. We have fused B domain sequences (BBdpA) as a close approximation of the A-B or B-C portion of SpA. Circular dichroism and fluorescence-detected denaturation curves of BBdpA are experimentally indistinguishable from those of BdpA. The rate constants for folding and unfolding from NMR line shape analysis for the single- and double-domain proteins are the same within experimental uncertainties (+/-20%). These results support the designation of SpA as a multiple independently-folding domain (MIFD) protein. We develop a mathematical model that describes the folding thermodynamics and kinetics of MIFD proteins. The model depicts MIFD protein folding and unfolding as a parallel network and explicitly calculates the flux through all parallel pathways. These fluxes are combined to give a complete description of the global thermodynamics and kinetics of the folding and unfolding of MIFD proteins. The global rates for complete folding and unfolding of a MIFD protein and those of the individual domains depend on the stability of the protein. We show that the global unfolding rate of a MIFD protein may be many orders of magnitude slower than that of the constituent domains.

  2. Human autoantibodies to diacyl-phosphatidylethanolamine recognize a specific set of discrete cytoplasmic domains

    PubMed Central

    Laurino, C C F C; Fritzler, M J; Mortara, R A; Silva, N P; Almeida, I C; Andrade, L E C

    2006-01-01

    The aim of this study was to characterize a novel human autoantibody–autoantigen system represented as cytoplasmic discrete speckles (CDS) in indirect immunofluorescence (IIF). A distinct CDS IIF pattern represented by 3–20 discrete speckles dispersed throughout the cytoplasm was identified among other cytoplasmic speckled IIF patterns. The cytoplasmic domains labelled by human anti-CDS-1 antibodies did not co-localize with endosome/lysosome markers EEA1 and LAMP-2, but showed partial co-localization with glycine–tryptophan bodies (GWB). CDS-1 sera did not react with several cellular extracts in immunoblotting and did not immunoprecipitate recombinant GW182 or EEA1 proteins. The typical CDS-1 IIF labelling pattern was abolished after delipidation of HEp-2 cells. Moreover, CDS-1 sera reacted strongly with a lipid component co-migrating with phosphatidylethanolamine (PE) in high performance thin-layer chromatography (HPTLC)-immunostaining of HEp-2 cell total lipid extracts. The CDS-1 major molecular targets were established by electrospray ionization–mass spectrometry (ESI-MS), HPTLC-immunostaining and chemiluminescent enzyme-linked immunosorbent assay as diacyl-PE species, containing preferentially a cis-C18 : 1 fatty acid chain at C-2 of the glycerol moiety, namely 1,2-cis-C18 : 1-PE and 1-C16 : 0-2-cis-C18 : 1-PE. The clinical association of CDS-1 sera included a variety of systemic and organ-specific autoimmune diseases but they were also observed in patients with no evidence of autoimmune disease. PMID:16487257

  3. BAR domain proteins regulate Rho GTPase signaling

    PubMed Central

    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. PMID:25483303

  4. Linking in domain-swapped protein dimers

    PubMed Central

    Baiesi, Marco; Orlandini, Enzo; Trovato, Antonio; Seno, Flavio

    2016-01-01

    The presence of knots has been observed in a small fraction of single-domain proteins and related to their thermodynamic and kinetic properties. The exchanging of identical structural elements, typical of domain-swapped proteins, makes such dimers suitable candidates to validate the possibility that mutual entanglement between chains may play a similar role for protein complexes. We suggest that such entanglement is captured by the linking number. This represents, for two closed curves, the number of times that each curve winds around the other. We show that closing the curves is not necessary, as a novel parameter G′, termed Gaussian entanglement, is strongly correlated with the linking number. Based on 110 non redundant domain-swapped dimers, our analysis evidences a high fraction of chains with a significant intertwining, that is with |G′| > 1. We report that Nature promotes configurations with negative mutual entanglement and surprisingly, it seems to suppress intertwining in long protein dimers. Supported by numerical simulations of dimer dissociation, our results provide a novel topology-based classification of protein-swapped dimers together with some preliminary evidence of its impact on their physical and biological properties. PMID:27659606

  5. Protein transduction domain delivery of therapeutic macromolecules.

    PubMed

    van den Berg, Arjen; Dowdy, Steven F

    2011-12-01

    Owing to their unprecedented selectivity, specific activity and potential for 1000+ fold amplification of signal, macromolecules, such as peptides, catalytic protein domains, complete proteins, and oligonucleotides, offer great potential as therapeutic molecules. However, therapeutic use of macromolecules is limited by their poor penetration in tissues and their inability to cross the cellular membrane. The discovery of small cationic peptides that cross the membrane, called Protein Transduction Domains (PTDs) or Cell Penetrating Peptides (CPPs), in the late 1980s opened the door to cellular delivery of large, bioactive molecules. Now, PTDs are widely used as research tools, and impressively, multiple clinical trials are testing PTD-mediated delivery of macromolecular drug conjugates in patients with a variety of diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal

    PubMed Central

    Agrawal, Anant A.; Salsi, Enea; Chatrikhi, Rakesh; Henderson, Steven; Jenkins, Jermaine L.; Green, Michael R.; Ermolenko, Dmitri N.; Kielkopf, Clara L.

    2016-01-01

    How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals. PMID:26952537

  7. Joining RDC data from flexible protein domains

    NASA Astrophysics Data System (ADS)

    Sgheri, Luca

    2010-11-01

    We study the inverse problem of determining the conformational freedom of two protein domains from residual dipolar coupling (RDC) measurements. For each paramagnetic ion attached to one of the domains we obtain a magnetic susceptibility tensor χ from the RDC of couples of atoms of that domain, and a mean paramagnetic susceptibility tensor {\\bar{\\chi }} from the RDC of couples of atoms of the other domain. The latter is an integral average of rotations of χ which depends on the conformational freedom of the two domains. In this paper we consider the case when we have data from paramagnetic ions attached separately to each of the domains. We prove that in this case not all the elements of χ and {\\bar{\\chi }} are independent. We derive the mathematical equations for the compatibility of the measurements and show how these relations can be used in the presence of noisy data to determine a compatible set of χ and {\\bar{\\chi }} with an unconstrained minimization. If available, information about the shape of the noise can be included in the target function. We show that in this case the compatible set obtained has a reduced error with respect to the noisy data.

  8. Protein function annotation using protein domain family resources.

    PubMed

    Das, Sayoni; Orengo, Christine A

    2016-01-15

    As a result of the genome sequencing and structural genomics initiatives, we have a wealth of protein sequence and structural data. However, only about 1% of these proteins have experimental functional annotations. As a result, computational approaches that can predict protein functions are essential in bridging this widening annotation gap. This article reviews the current approaches of protein function prediction using structure and sequence based classification of protein domain family resources with a special focus on functional families in the CATH-Gene3D resource.

  9. The ROQ domain of Roquin recognizes mRNA constitutive-decay element and double-stranded RNA.

    PubMed

    Tan, Dazhi; Zhou, Mi; Kiledjian, Megerditch; Tong, Liang

    2014-08-01

    A conserved stem-loop motif of the constitutive decay element (CDE) in the 3' UTR of mRNAs is recognized by the ROQ domain of Roquin, which mediates mRNA degradation. Here we report two crystal structures of the Homo sapiens ROQ domain in complex with CDE RNA. The ROQ domain has an elongated shape with three subdomains. The 19-nt Hmgxb3 CDE is bound as a stem-loop to domain III. The 23-nt TNF RNA is bound as a duplex to a separate site at the interface between domains I and II. Mutagenesis studies confirm that the ROQ domain has two separate RNA-binding sites, one for stem-loop RNA (A site) and the other for double-stranded RNA (B site). Mutation in either site perturbs the Roquin-mediated degradation of HMGXB3 and IL6 mRNAs in human cells, demonstrating the importance of both sites for mRNA decay.

  10. Recognizing Mantle Domains Related to an Extensional Cycle: the Record from Western Europe

    NASA Astrophysics Data System (ADS)

    Picazo, S.; Muntener, O.; Manatschal, G.

    2015-12-01

    Most of the studies on rifted margins have shown that the classical predictions of models assuming a "homogeneous" mantle lithosphere without some inheritance are unable to capture the observed large variety of magmatic budgets as a function of extension. More recently, new ideas and concepts have been developed to understand the evolution of the mantle lithosphere in hyper-extended magma-poor rifted margins that are mainly based on observations from the present-day Iberia-Newfoundland and ancient Alpine Tethys rifted margins and the Pyrenean systems. In contrast to the classical assumption assuming a simple, isotropic mantle lithosphere, these new models integrate observations from exposed and drilled mantle rocks and propose that the mantle lithosphere evolved and was modified during an extensional cycle from post-orogenic collapse through several periods of rifting to embryonic oceanic (ultra-) slow seafloor spreading. But it is, at present, unclear how far these ideas can be generalized and if they can explain the nature of mantle rocks observed across Western Europe and, in a more general way, at Atlantic type rifted margins. We review the available mantle data from Western Europe, i.e. ophiolite massifs, xenoliths and dredged samples, revisit the available terminology concerning mantle massifs and xenoliths and compile the available data to identify different mantle domains. We define chemical and petrological characteristics of mantle domains based on clinopyroxene and spinel compositions and compile them on present-day and paleo-geographic maps of Western Europe. Finally we link the observed distribution of mantle domains to the post-Variscan extensional cycle and link domains to processes related to the late post-Variscan extension, the rift evolution and refertilization associated to hyper-extension and the development of embryonic oceanic domains.

  11. The Evolutionary History of Protein Domains Viewed by Species Phylogeny

    PubMed Central

    Yang, Song; Bourne, Philip E.

    2009-01-01

    Background Protein structural domains are evolutionary units whose relationships can be detected over long evolutionary distances. The evolutionary history of protein domains, including the origin of protein domains, the identification of domain loss, transfer, duplication and combination with other domains to form new proteins, and the formation of the entire protein domain repertoire, are of great interest. Methodology/Principal Findings A methodology is presented for providing a parsimonious domain history based on gain, loss, vertical and horizontal transfer derived from the complete genomic domain assignments of 1015 organisms across the tree of life. When mapped to species trees the evolutionary history of domains and domain combinations is revealed, and the general evolutionary trend of domain and combination is analyzed. Conclusions/Significance We show that this approach provides a powerful tool to study how new proteins and functions emerged and to study such processes as horizontal gene transfer among more distant species. PMID:20041107

  12. Phylogenetic Analysis of Brassica rapa MATH-Domain Proteins

    PubMed Central

    Zhao, Liming; Huang, Yong; Hu, Yan; He, Xiaoli; Shen, Wenhui; Liu, Chunlin; Ruan, Ying

    2013-01-01

    The MATH (meprin and TRAF-C homology) domain is a fold of seven anti-parallel β-helices involved in protein-protein interaction. Here, we report the identification and characterization of 90 MATH-domain proteins from the Brassica rapa genome. By sequence analysis together with MATH-domain proteins from other species, the B. rapa MATH-domain proteins can be grouped into 6 classes. Class-I protein has one or several MATH domains without any other recognizable domain; Class-II protein contains a MATH domain together with a conserved BTB (Broad Complex, Tramtrack, and Bric-a-Brac ) domain; Class-III protein belongs to the MATH/Filament domain family; Class-IV protein contains a MATH domain frequently combined with some other domains; Class-V protein has a relative long sequence but contains only one MATH domain; Class-VI protein is characterized by the presence of Peptidase and UBQ (Ubiquitinylation) domains together with one MATH domain. As part of our study regarding seed development of B. rapa, six genes are screened by SSH (Suppression Subtractive Hybridization) and their expression levels are analyzed in combination with seed developmental stages, and expression patterns suggested that Bra001786, Bra03578 and Bra036572 may be seed development specific genes, while Bra001787, Bra020541 and Bra040904 may be involved in seed and flower organ development. This study provides the first characterization of the MATH domain proteins in B. rapa PMID:24179444

  13. Evaluating, Comparing, and Interpreting Protein Domain Hierarchies

    PubMed Central

    2014-01-01

    Abstract Arranging protein domain sequences hierarchically into evolutionarily divergent subgroups is important for investigating evolutionary history, for speeding up web-based similarity searches, for identifying sequence determinants of protein function, and for genome annotation. However, whether or not a particular hierarchy is optimal is often unclear, and independently constructed hierarchies for the same domain can often differ significantly. This article describes methods for statistically evaluating specific aspects of a hierarchy, for probing the criteria underlying its construction and for direct comparisons between hierarchies. Information theoretical notions are used to quantify the contributions of specific hierarchical features to the underlying statistical model. Such features include subhierarchies, sequence subgroups, individual sequences, and subgroup-associated signature patterns. Underlying properties are graphically displayed in plots of each specific feature's contributions, in heat maps of pattern residue conservation, in “contrast alignments,” and through cross-mapping of subgroups between hierarchies. Together, these approaches provide a deeper understanding of protein domain functional divergence, reveal uncertainties caused by inconsistent patterns of sequence conservation, and help resolve conflicts between competing hierarchies. PMID:24559108

  14. Binding domains of Bacillus anthracis phage endolysins recognize cell culture age-related features on the bacterial surface.

    PubMed

    Paskaleva, Elena E; Mundra, Ruchir V; Mehta, Krunal K; Pangule, Ravindra C; Wu, Xia; Glatfelter, Willing S; Chen, Zijing; Dordick, Jonathan S; Kane, Ravi S

    2015-01-01

    Bacteriolytic enzymes often possess a C-terminal binding domain that recognizes specific motifs on the bacterial surface and a catalytic domain that cleaves covalent linkages within the cell wall peptidoglycan. PlyPH, one such lytic enzyme of bacteriophage origin, has been reported to be highly effective against Bacillus anthracis, and can kill up to 99.99% of the viable bacteria. The bactericidal activity of this enzyme, however, appears to be strongly dependent on the age of the bacterial culture. Although highly bactericidal against cells in the early exponential phase, the enzyme is substantially less effective against stationary phase cells, thus limiting its application in real-world settings. We hypothesized that the binding domain of PlyPH may differ in affinity to cells in different Bacillus growth stages and may be primarily responsible for the age-restricted activity. We therefore employed an in silico approach to identify phage lysins differing in their specificity for the bacterial cell wall. Specifically we focused our attention on Plyβ, an enzyme with improved cell wall-binding ability and age-independent bactericidal activity. Although PlyPH and Plyβ have dissimilar binding domains, their catalytic domains are highly homologous. We characterized the biocatalytic mechanism of Plyβ by identifying the specific bonds cleaved within the cell wall peptidoglycan. Our results provide an example of the diversity of phage endolysins and the opportunity for these biocatalysts to be used for broad-based protection from bacterial pathogens.

  15. Independent Structural Domains in Paramyxovirus Polymerase Protein*

    PubMed Central

    Dochow, Melanie; Krumm, Stefanie A.; Crowe, James E.; Moore, Martin L.; Plemper, Richard K.

    2012-01-01

    All enzymatic activities required for genomic replication and transcription of nonsegmented negative strand RNA viruses (or Mononegavirales) are believed to be concentrated in the viral polymerase (L) protein. However, our insight into the organization of these different enzymatic activities into a bioactive tertiary structure remains rudimentary. Fragments of Mononegavirales polymerases analyzed to date cannot restore bioactivity through trans-complementation, unlike the related L proteins of segmented NSVs. We investigated the domain organization of phylogenetically diverse Paramyxovirus L proteins derived from measles virus (MeV), Nipah virus (NiV), and respiratory syncytial virus (RSV). Through a comprehensive in silico and experimental analysis of domain intersections, we defined MeV L position 615 as an interdomain candidate in addition to the previously reported residue 1708. Only position 1708 of MeV and the homologous positions in NiV and RSV L also tolerated the insertion of epitope tags. Splitting of MeV L at residue 1708 created fragments that were unable to physically interact and trans-complement, but strikingly, these activities were reconstituted by the addition of dimerization tags to the fragments. Equivalently split fragments of NiV, RSV, and MeV L oligomerized with comparable efficiency in all homo- and heterotypic combinations, but only the homotypic pairs were able to trans-complement. These results demonstrate that synthesis as a single polypeptide is not required for the Mononegavirales polymerases to adopt a proper tertiary conformation. Paramyxovirus polymerases are composed of at least two truly independent folding domains that lack a traditional interface but require molecular compatibility for bioactivity. The functional probing of the L domain architecture through trans-complementation is anticipated to be applicable to all Mononegavirales polymerases. PMID:22215662

  16. Synthetic mimetics of protein secondary structure domains

    PubMed Central

    Ross, Nathan T.; Katt, William P.; Hamilton, Andrew D.

    2010-01-01

    Proteins modulate the majority of all biological functions and are primarily composed of highly organized secondary structural elements such as helices, turns and sheets. Many of these functions are affected by a small number of key protein–protein contacts, often involving one or more of these well-defined structural elements. Given the ubiquitous nature of these protein recognition domains, their mimicry by peptidic and non-peptidic scaffolds has become a major focus of contemporary research. This review examines several key advances in secondary structure mimicry over the past several years, particularly focusing upon scaffolds that show not only promising projection of functional groups, but also a proven effect in biological systems. PMID:20123744

  17. Pathway logic modeling of protein functional domains in signal transduction.

    PubMed

    Talcott, C; Eker, S; Knapp, M; Lincoln, P; Laderoute, K

    2004-01-01

    Protein functional domains (PFDs) are consensus sequences within signaling molecules that recognize and assemble other signaling components into complexes. Here we describe the application of an approach called Pathway Logic to the symbolic modeling signal transduction networks at the level of PFDs. These models are developed using Maude, a symbolic language founded on rewriting logic. Models can be queried (analyzed) using the execution, search and model-checking tools of Maude. We show how signal transduction processes can be modeled using Maude at very different levels of abstraction involving either an overall state of a protein or its PFDs and their interactions. The key insight for the latter is our algebraic representation of binding interactions as a graph.

  18. Modular protein domains: an engineering approach toward functional biomaterials.

    PubMed

    Lin, Charng-Yu; Liu, Julie C

    2016-08-01

    Protein domains and peptide sequences are a powerful tool for conferring specific functions to engineered biomaterials. Protein sequences with a wide variety of functionalities, including structure, bioactivity, protein-protein interactions, and stimuli responsiveness, have been identified, and advances in molecular biology continue to pinpoint new sequences. Protein domains can be combined to make recombinant proteins with multiple functionalities. The high fidelity of the protein translation machinery results in exquisite control over the sequence of recombinant proteins and the resulting properties of protein-based materials. In this review, we discuss protein domains and peptide sequences in the context of functional protein-based materials, composite materials, and their biological applications.

  19. Monoclonal antibodies that recognize transcription unit proteins on newt lampbrush chromosomes

    PubMed Central

    1987-01-01

    We prepared hybridoma cell lines from mice injected with newt germinal vesicle proteins. We tested culture supernates from these cell lines for antibodies that bound to specific morphological structures in lampbrush chromosome preparations (nucleoli, loops, chromomeres, etc.). Four mAbs that recognize antigens on the lateral transcription loops are described here. We suggest that these antigens are proteins associated with nascent RNA transcripts, although they are not among the 30-40-kD "core" heterogeneous nuclear ribonucleoproteins. PMID:3308902

  20. Primary Central Nervous System (CNS) Lymphoma B Cell Receptors Recognize CNS Proteins.

    PubMed

    Montesinos-Rongen, Manuel; Purschke, Frauke G; Brunn, Anna; May, Caroline; Nordhoff, Eckhard; Marcus, Katrin; Deckert, Martina

    2015-08-01

    Primary lymphoma of the CNS (PCNSL) is a diffuse large B cell lymphoma confined to the CNS. To elucidate its peculiar organ tropism, we generated recombinant Abs (recAbs) identical to the BCR of 23 PCNSLs from immunocompetent patients. Although none of the recAbs showed self-reactivity upon testing with common autoantigens, they recognized 1547 proteins present on a large-scale protein microarray, indicating polyreactivity. Interestingly, proteins (GRINL1A, centaurin-α, BAIAP2) recognized by the recAbs are physiologically expressed by CNS neurons. Furthermore, 87% (20/23) of the recAbs, including all Abs derived from IGHV4-34 using PCNSL, recognized galectin-3, which was upregulated on microglia/macrophages, astrocytes, and cerebral endothelial cells upon CNS invasion by PCNSL. Thus, PCNSL Ig may recognize CNS proteins as self-Ags. Their interaction may contribute to BCR signaling with sustained NF-κB activation and, ultimately, may foster tumor cell proliferation and survival. These data may also explain, at least in part, the affinity of PCNSL cells for the CNS.

  1. Feature competition and domain of attraction in artificial-perceptron pattern recognizer

    NASA Astrophysics Data System (ADS)

    Hu, Chia-Lun J.

    1993-10-01

    As we reported previously, learning of a multi-layered hard-limited perceptron can be formulated into a set of simultaneous linear inequalities. Solving these inequalities under a given training set would then allow us to achieve the goal of learning in this system. If the dimension N of the input vector is much larger than the number M of different patterns to be learned, then there is considerable freedom for the system to select a proper solution of the connection matrix. In most cases, even a single layer perceptron will do the learning satisfactorily. This paper reports the results of some theoretical and experimental studies of this one-layered, hard-limited perceptron trained under the novel, one-step, noniterative learning scheme. Particularly, the analysis of some important properties of this novel learning system, such as automatic feature competition, domain of convergence, and robustness of recognition, are discussed in detail.

  2. Plasmodium vivax: a monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.

    PubMed

    Gonzalez-Ceron, L; Rodriguez, M H; Wirtz, R A; Sina, B J; Palomeque, O L; Nettel, J A; Tsutsumi, V

    1998-11-01

    The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells. Plasmodium vivax CS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with other Plasmodium species, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with all P. vivax sporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.

  3. Plant SAM-Domain Proteins Start to Reveal Their Roles.

    PubMed

    Denay, Grégoire; Vachon, Gilles; Dumas, Renaud; Zubieta, Chloe; Parcy, François

    2017-08-01

    Proteins often act in complexes assembled via protein-protein interaction domains. The sterile alpha motif (SAM) domain is one of the most prominent interaction domains in animals and is present in proteins of diverse functions. This domain allows head-to-tail closed oligomerisation or polymer formation resulting in homo- and/or heterocomplexes that have been shown to be important for proper protein localisation and function. In plants this domain is also present but has been poorly studied except for recent studies on the LEAFY floral regulator and the tRNA import component (TRIC)1/2 proteins. Here we catalogue SAM domain-containing proteins from arabidopsis (Arabidopsis thaliana), compare plant and other eukaryotic SAM domains, and perform homology modelling to probe plant SAM domain interaction capabilities. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Evolution of Protein Domain Repeats in Metazoa

    PubMed Central

    Schüler, Andreas; Bornberg-Bauer, Erich

    2016-01-01

    Repeats are ubiquitous elements of proteins and they play important roles for cellular function and during evolution. Repeats are, however, also notoriously difficult to capture computationally and large scale studies so far had difficulties in linking genetic causes, structural properties and evolutionary trajectories of protein repeats. Here we apply recently developed methods for repeat detection and analysis to a large dataset comprising over hundred metazoan genomes. We find that repeats in larger protein families experience generally very few insertions or deletions (indels) of repeat units but there is also a significant fraction of noteworthy volatile outliers with very high indel rates. Analysis of structural data indicates that repeats with an open structure and independently folding units are more volatile and more likely to be intrinsically disordered. Such disordered repeats are also significantly enriched in sites with a high functional potential such as linear motifs. Furthermore, the most volatile repeats have a high sequence similarity between their units. Since many volatile repeats also show signs of recombination, we conclude they are often shaped by concerted evolution. Intriguingly, many of these conserved yet volatile repeats are involved in host-pathogen interactions where they might foster fast but subtle adaptation in biological arms races. Key Words: protein evolution, domain rearrangements, protein repeats, concerted evolution. PMID:27671125

  5. Polyepitope protein incorporated the HIV-1 mimotope recognized by monoclonal antibody 2G12.

    PubMed

    Karpenko, Larisa I; Scherbakova, Nadezhda S; Chikaev, Anton N; Tumanova, Olga Yu; Lebedev, Leonid R; Shalamova, Lyudmila A; Pyankova, Olga G; Ryzhikov, Alexander B; Ilyichev, Alexander A

    2012-04-01

    A major goal in HIV-1 vaccine research is to develop an immunogen that can elicit broadly neutralizing antibodies that efficiently neutralize a wide range of the HIV-1 subtypes. Using biopanning procedure we have selected linear peptide VGAFGSFYRLSVLQS mimicking the structure of discontinuous binding sites of broadly neutralizing antibodies 2G12 from phage peptide library. As a protein carrier, we used the earlier designed artificial polyepitope immunogen named TBI (T- and B-cell immunogen), which comprises B-cell and T-helper epitopes from the HIV-1 Env and Gag proteins. On the base of selected peptide mimotope VGAFGSFYRLSVLQS the artificial protein TBI-2g12 was constructed and its immunogenic properties was investigated. It was shown that the TBI-2g12 as well as the original TBI induces antibodies that recognize HIV-1 proteins and TBI protein using ELISA and immunoblotting. However only anti-TBI-2g12 serum recognized the synthetic peptide mimotope VGAFGSFYRLSVLQS, whereas the antibodies against original TBI don't recognize it. The neutralization assay demonstrated that serum antibodies of the mice immunized with TBI-2g12 possess virus neutralizing activity. The addition of selected peptide leads to inhibition neutralizing activity of anti- TBI-2g12 serum. We conclude from these results that immunogen TBI-2g12 containing the selected peptide VGAFGSFYRLSVLQS elicits HIV-1 neutralizing antibodies during immunization. Our data suggest that this immunogen may be useful in designing effective HIV-vaccine candidates.

  6. Extra domains in secondary transport carriers and channel proteins.

    PubMed

    Barabote, Ravi D; Tamang, Dorjee G; Abeywardena, Shannon N; Fallah, Neda S; Fu, Jeffrey Yu Chung; Lio, Jeffrey K; Mirhosseini, Pegah; Pezeshk, Ronnie; Podell, Sheila; Salampessy, Marnae L; Thever, Mark D; Saier, Milton H

    2006-10-01

    "Extra" domains in members of the families of secondary transport carrier and channel proteins provide secondary functions that expand, amplify or restrict the functional nature of these proteins. Domains in secondary carriers include TrkA and SPX domains in DASS family members, DedA domains in TRAP-T family members (both of the IT superfamily), Kazal-2 and PDZ domains in OAT family members (of the MF superfamily), USP, IIA(Fru) and TrkA domains in ABT family members (of the APC superfamily), ricin domains in OST family members, and TrkA domains in AAE family members. Some transporters contain highly hydrophilic domains consisting of multiple repeat units that can also be found in proteins of dissimilar function. Similarly, transmembrane alpha-helical channel-forming proteins contain unique, conserved, hydrophilic domains, most of which are not found in carriers. In some cases the functions of these domains are known. They may be ligand binding domains, phosphorylation domains, signal transduction domains, protein/protein interaction domains or complex carbohydrate-binding domains. These domains mediate regulation, subunit interactions, or subcellular targeting. Phylogenetic analyses show that while some of these domains are restricted to closely related proteins derived from specific organismal types, others are nearly ubiquitous within a particular family of transporters and occur in a tremendous diversity of organisms. The former probably became associated with the transporters late in the evolutionary process; the latter probably became associated with the carriers much earlier. These domains can be located at either end of the transporter or in a central region, depending on the domain and transporter family. These studies provide useful information about the evolution of extra domains in channels and secondary carriers and provide novel clues concerning function.

  7. Proteomic analysis of endothelial cell autoantigens recognized by anti-dengue virus nonstructural protein 1 antibodies.

    PubMed

    Cheng, Hsien-Jen; Lin, Chiou-Feng; Lei, Huan-Yao; Liu, Hsiao-Sheng; Yeh, Trai-Ming; Luo, Yueh-Hsia; Lin, Yee-Shin

    2009-01-01

    We previously showed the occurrence of autoimmune responses in dengue virus (DV) infection, which has potential implications for the pathogenesis of dengue hemorrhagic syndrome. In the present study, we have used a proteomic analysis to identify several candidate proteins on HMEC-1 endothelial cells recognized by anti-DV nonstructural protein 1 (NS1) antibodies. The target proteins, including ATP synthase beta chain, protein disulfide isomerase, vimentin, and heat shock protein 60, co-localize with anti-NS1 binding sites on nonfixed HMEC-1 cells using immunohistochemical double staining and confocal microscopy. The cross-reactivity of anti-target protein antibodies with HMEC-1 cells was inhibited by NS1 protein pre-absorption. Furthermore, a cross-reactive epitope on NS1 amino acid residues 311-330 (P311-330) was predicted using homologous sequence alignment. The reactivity of dengue hemorrhagic patient sera with HMEC-1 cells was blocked by synthetic peptide P311-330 pre-absorption. Taken together, our results identify putative targets on endothelial cells recognized by anti-DV NS1 antibodies, where NS1 P311-330 possesses the shared epitope.

  8. Evolutionary versatility of eukaryotic protein domains revealed by their bigram networks

    PubMed Central

    2011-01-01

    Background Protein domains are globular structures of independently folded polypeptides that exert catalytic or binding activities. Their sequences are recognized as evolutionary units that, through genome recombination, constitute protein repertoires of linkage patterns. Via mutations, domains acquire modified functions that contribute to the fitness of cells and organisms. Recent studies have addressed the evolutionary selection that may have shaped the functions of individual domains and the emergence of particular domain combinations, which led to new cellular functions in multi-cellular animals. This study focuses on modeling domain linkage globally and investigates evolutionary implications that may be revealed by novel computational analysis. Results A survey of 77 completely sequenced eukaryotic genomes implies a potential hierarchical and modular organization of biological functions in most living organisms. Domains in a genome or multiple genomes are modeled as a network of hetero-duplex covalent linkages, termed bigrams. A novel computational technique is introduced to decompose such networks, whereby the notion of domain "networking versatility" is derived and measured. The most and least "versatile" domains (termed "core domains" and "peripheral domains" respectively) are examined both computationally via sequence conservation measures and experimentally using selected domains. Our study suggests that such a versatility measure extracted from the bigram networks correlates with the adaptivity of domains during evolution, where the network core domains are highly adaptive, significantly contrasting the network peripheral domains. Conclusions Domain recombination has played a major part in the evolution of eukaryotes attributing to genome complexity. From a system point of view, as the results of selection and constant refinement, networks of domain linkage are structured in a hierarchical modular fashion. Domains with high degree of networking

  9. Pancreatic Polypeptide Is Recognized by Two Hydrophobic Domains of the Human Y4 Receptor Binding Pocket*

    PubMed Central

    Pedragosa-Badia, Xavier; Sliwoski, Gregory R.; Dong Nguyen, Elizabeth; Lindner, Diana; Stichel, Jan; Kaufmann, Kristian W.; Meiler, Jens; Beck-Sickinger, Annette G.

    2014-01-01

    Structural characterization of the human Y4 receptor (hY4R) interaction with human pancreatic polypeptide (hPP) is crucial, not only for understanding its biological function but also for testing treatment strategies for obesity that target this interaction. Here, the interaction of receptor mutants with pancreatic polypeptide analogs was studied through double-cycle mutagenesis. To guide mutagenesis and interpret results, a three-dimensional comparative model of the hY4R-hPP complex was constructed based on all available class A G protein-coupled receptor crystal structures and refined using experimental data. Our study reveals that residues of the hPP and the hY4R form a complex network consisting of ionic interactions, hydrophobic interactions, and hydrogen binding. Residues Tyr2.64, Asp2.68, Asn6.55, Asn7.32, and Phe7.35 of Y4R are found to be important in receptor activation by hPP. Specifically, Tyr2.64 interacts with Tyr27 of hPP through hydrophobic contacts. Asn7.32 is affected by modifications on position Arg33 of hPP, suggesting a hydrogen bond between these two residues. Likewise, we find that Phe7.35 is affected by modifications of hPP at positions 33 and 36, indicating interactions between these three amino acids. Taken together, we demonstrate that the top of transmembrane helix 2 (TM2) and the top of transmembrane helices 6 and 7 (TM6–TM7) form the core of the peptide binding pocket. These findings will contribute to the rational design of ligands that bind the receptor more effectively to produce an enhanced agonistic or antagonistic effect. PMID:24375409

  10. Epitopes recognized by human T lymphocytes in the ROP2 protein antigen of Toxoplasma gondii.

    PubMed Central

    Saavedra, R; Becerril, M A; Dubeaux, C; Lippens, R; De Vos, M J; Hérion, P; Bollen, A

    1996-01-01

    The ROP2 protein of Toxoplasma gondii possesses immunological and biological properties which have led to its proposal as a vaccine candidate. To identify epitopes recognized by human T cells in the ROP2 antigen, we submitted the sequence of this protein to three reported T-cell epitope prediction algorithms. Three sequences that were predicted by all three methods were selected (sequences 197 to 216, 393 to 410, and 501 to 524), and the corresponding peptides were synthesized. The peptides were first tested in a proliferation assay with a DPw4-restricted, ROP2-specific human T-cell clone, and the peptide corresponding to residues 197 to 216 was shown to stimulate the T-cell clone. The three peptides were further tested in proliferation assays with peripheral blood mononuclear cells from a panel of T. gondii-seropositive and -seronegative individuals. We found that cells from a high proportion of the seropositive donors (64%) recognized at least one of the three peptides. The most frequently recognized ones were peptides 197 to 216 (45%) and 501 to 524 (36%). None of the seronegative donors responded to any peptide. These results show that the ROP2 antigen of T. gondii contains T-cell epitopes recognized by a high percentage of the immune population and further strengthen its potential as a vaccine candidate. PMID:8751939

  11. The expanding superfamily of gelsolin homology domain proteins.

    PubMed

    Ghoshdastider, Umesh; Popp, David; Burtnick, Leslie D; Robinson, Robert C

    2013-11-01

    The gelsolin homology (GH) domain has been found to date exclusively in actin-binding proteins. In humans, three copies of the domain are present in CapG, five copies in supervillin, and six copies each in adseverin, gelsolin, flightless I and the villins: villin, advillin and villin-like protein. Caenorhabditis elegans contains a four-GH-domain protein, GSNL-1. These architectures are predicted to have arisen from gene triplication followed by gene duplication to result in the six-domain protein. The subsequent loss of one, two or three domains produced the five-, four-, and three-domain proteins, respectively. Here we conducted BLAST and hidden Markov based searches of UniProt and NCBI databases to identify novel gelsolin domain containing proteins. The variety in architectures suggests that the GH domain has been tested in many molecular constructions during evolution. Of particular note is flightless-like I protein (FLIIL1) from Entamoeba histolytica, which combines a leucine rich repeats (LRR) domain, seven GH domains, and a headpiece domain, thus combining many of the features of flightless I with those of villin or supervillin. As such, the GH domain superfamily appears to have developed along complex routes. The distribution of these proteins was analyzed in the 343 completely sequenced genomes, mapped onto the tree of life, and phylogenetic trees of the proteins were constructed to gain insight into their evolution. © 2013 Wiley Periodicals, Inc. Copyright © 2013 Wiley Periodicals, Inc.

  12. The ER stress sensor PERK luminal domain functions as a molecular chaperone to interact with misfolded proteins

    SciTech Connect

    Wang, Peng; Li, Jingzhi; Sha, Bingdong

    2016-11-29

    PERK is one of the major sensor proteins which can detect the protein-folding imbalance generated by endoplasmic reticulum (ER) stress. It remains unclear how the sensor protein PERK is activated by ER stress. It has been demonstrated that the PERK luminal domain can recognize and selectively interact with misfolded proteins but not native proteins. Moreover, the PERK luminal domain may function as a molecular chaperone to directly bind to and suppress the aggregation of a number of misfolded model proteins. The data strongly support the hypothesis that the PERK luminal domain can interact directly with misfolded proteins to induce ER stress signaling. To illustrate the mechanism by which the PERK luminal domain interacts with misfolded proteins, the crystal structure of the human PERK luminal domain was determined to 3.2 Å resolution. Two dimers of the PERK luminal domain constitute a tetramer in the asymmetric unit. Superimposition of the PERK luminal domain molecules indicated that the β-sandwich domain could adopt multiple conformations. It is hypothesized that the PERK luminal domain may utilize its flexible β-sandwich domain to recognize and interact with a broad range of misfolded proteins.

  13. The ER stress sensor PERK luminal domain functions as a molecular chaperone to interact with misfolded proteins.

    PubMed

    Wang, Peng; Li, Jingzhi; Sha, Bingdong

    2016-12-01

    PERK is one of the major sensor proteins which can detect the protein-folding imbalance generated by endoplasmic reticulum (ER) stress. It remains unclear how the sensor protein PERK is activated by ER stress. It has been demonstrated that the PERK luminal domain can recognize and selectively interact with misfolded proteins but not native proteins. Moreover, the PERK luminal domain may function as a molecular chaperone to directly bind to and suppress the aggregation of a number of misfolded model proteins. The data strongly support the hypothesis that the PERK luminal domain can interact directly with misfolded proteins to induce ER stress signaling. To illustrate the mechanism by which the PERK luminal domain interacts with misfolded proteins, the crystal structure of the human PERK luminal domain was determined to 3.2 Å resolution. Two dimers of the PERK luminal domain constitute a tetramer in the asymmetric unit. Superimposition of the PERK luminal domain molecules indicated that the β-sandwich domain could adopt multiple conformations. It is hypothesized that the PERK luminal domain may utilize its flexible β-sandwich domain to recognize and interact with a broad range of misfolded proteins.

  14. Photoaffinity Labeling Reveals Nuclear Proteins that Uniquely Recognize Cisplatin-DNA Interstrand Cross-Links

    PubMed Central

    Zhu, Guangyu; Lippard, Stephen J.

    2009-01-01

    The DNA-binding inorganic compound cisplatin is one of the most successful anticancer drugs. The detailed mechanism by which cells recognize and process of cisplatin-DNA damage is of great interest. Although the family of proteins that bind cisplatin 1,2- and 1,3-intrastrand cross-links has been identified, much less is known about cellular protein interactions with cisplatin interstrand cross-links (ICLs). In order to address this question, a photoreactive analogue of cisplatin, PtBP6, was used to construct a DNA duplex containing a site-specific platinum ICL. This DNA probe was characterized and used in photo-cross-linking experiments to separate and identify nuclear proteins that bind to the ICL by peptide mass fingerprint analysis. Several such proteins were discovered, including PARP-1, hMutSβ, DNA ligase III, XRCC1, and PNK. The photo-cross-linking approach was independently validated by an electrophoretic mobility shift assay demonstrating hMutSβ binding to a cisplatin ICL. Proteins that recognize the platinum ICL were also identified in cisplatin resistant cells, cells halted at various phases of the cell cycle, and in different carcinoma cells. Nuclear proteins that bind to the platinum ICL differ from those binding to intrastrand cross-links, indicating different mechanisms for disruption of cellular functions. PMID:19364127

  15. Stochastic single-molecule dynamics of synaptic membrane protein domains

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Li, Yiwei; Haselwandter, Christoph A.

    2016-09-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  16. Suppression of TLR signaling by targeting TIR domain-containing proteins.

    PubMed

    Fekonja, Ota; Avbelj, Monika; Jerala, Roman

    2012-12-01

    Toll-like receptors (TLRs) recognize molecules specific to pathogens and endogenous danger signals. Binding of agonists to the ectodomain of the receptor initiates TLR activation and is followed by the association of receptor cytosolic Toll/Interleukin-1 receptor (TIR) domains with TIR domains of adapter proteins leading to the assembly of signaling cascade of protein kinases that ultimately trigger the activation of transcription factors and expression of genes involved in the immune response. Excessive activation of TIR-domain mediated signaling has been implicated in inflammatory diseases (e.g. rheumatoid arthritis, systemic lupus erythematosus, colitis) as well as in the development of cancer. Targeting receptor-adapter interactions represents a potential strategy for the therapeutic TLR/IL-1R-specific inhibition due to the unique interacting domains involved. Peptide and protein-domain binding TLR inhibitors originating from the interacting surfaces of TIR-domain containing proteins can bind to the site on their target interacting protein thereby preventing the assembly of the functional signaling complex. Here we review protein-domain, peptide and peptidomimetic inhibitors targeting TIR-domain mediated interactions and their application demonstrated on in vitro and in vivo models. Recent structural data and elucidation of the molecular mechanisms of TIR-domain mediated signaling enabled the development of peptide inhibitors from TIR domains of TLRs and adapters, MyD88 intermediary domain as well as improved protein inhibitors based on TIR domain dimerization, mimicking bacterial TIR-domain containing immunosuppressors (TCPs) which we discuss with challenges concerning the delivery and specificity of inhibitors targeting TLR adapters.

  17. Suppression of TLR Signaling by Targeting TIR domain-Containing Proteins

    PubMed Central

    Fekonja, Ota; Avbelj, Monika; Jerala, Roman

    2012-01-01

    Toll-like receptors (TLRs) recognize molecules specific to pathogens and endogenous danger signals. Binding of agonists to the ectodomain of the receptor initiates TLR activation and is followed by the association of receptor cytosolic Toll/Interleukin-1 receptor (TIR) domains with TIR domains of adapter proteins leading to the assembly of signaling cascade of protein kinases that ultimately trigger the activation of transcription factors and expression of genes involved in the immune response. Excessive activation of TIR-domain mediated signaling has been implicated in inflammatory diseases (e.g. rheumatoid arthritis, systemic lupus erythematosus, colitis) as well as in the development of cancer. Targeting receptor-adapter interactions represents a potential strategy for the therapeutic TLR/IL-1R-specific inhibition due to the unique interacting domains involved. Peptide and protein-domain binding TLR inhibitors originating from the interacting surfaces of TIR-domain containing proteins can bind to the site on their target interacting protein thereby preventing the assembly of the functional signaling complex. Here we review protein-domain, peptide and peptidomimetic inhibitors targeting TIR-domain mediated interactions and their application demonstrated on in vitro and in vivo models. Recent structural data and elucidation of the molecular mechanisms of TIR-domain mediated signaling enabled the development of peptide inhibitors from TIR domains of TLRs and adapters, MyD88 intermediary domain as well as improved protein inhibitors based on TIR domain dimerization, mimicking bacterial TIR-domain containing immunosuppressors (TCPs) which we discuss with challenges concerning the delivery and specificity of inhibitors targeting TLR adapters. PMID:23305364

  18. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation

    PubMed Central

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-01-01

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  19. Structure and function of regulator of G protein signaling homology domains.

    PubMed

    Tesmer, John J G

    2009-01-01

    All regulator of G protein signaling (RGS) proteins contain a conserved domain of approximately 130 amino acids that binds to activated heterotrimeric G protein α subunits (Gα) and accelerates their rate of GTP hydrolysis. Homologous domains are found in at least six other protein families, including a family of Rho guanine nucleotide exchange factors (RhoGEFs) and the G protein-coupled receptor kinases (GRKs). Although some of the RhoGEF and GRK RGS-like domains can also bind to activated Gα subunits, they do so in distinct ways and with much lower levels of GTPase activation. In other protein families, the domains have as of yet no obvious relationship to heterotrimeric G protein signaling. These RGS homology (RH) domains are now recognized as mediators of extraordinarily diverse protein-protein interactions. Through these interactions, they play roles that range from enzyme to molecular scaffold to signal transducing module. In this review, the atomic structures of RH domains from RGS proteins, Axins, RhoGEFs, and GRKs are compared in light of what is currently known about their functional roles.

  20. An amphiphilic region in the cytoplasmic domain of KdpD is recognized by the signal recognition particle and targeted to the Escherichia coli membrane

    PubMed Central

    Maier, Katja S; Hubich, Stefanie; Liebhart, Helga; Krauss, Susanne; Kuhn, Andreas; Facey, Sandra J

    2008-01-01

    The sensor protein KdpD of Escherichia coli is composed of a large N-terminal hydrophilic region (aa 1–400), four transmembrane regions (aa 401–498) and a large hydrophilic region (aa 499–894) at the C-terminus. KdpD requires the signal recognition particle (SRP) for its targeting to the membrane. Deletions within KdpD show that the first 50 residues are required for SRP-driven membrane insertion. A fusion protein of the green fluorescent protein (GFP) with KdpD is found localized at the membrane only when SRP is present. The membrane targeting of GFP was not observed when the first 50 KdpD residues were deleted. A truncated mutant of KdpD containing only the first 25 amino acids fused to GFP lost its ability to specifically interact with SRP, whereas a specific interaction between SRP and the first 48 amino acids of KdpD fused to GFP was confirmed by pull-down experiments. Conclusively, a small amphiphilic region of 27 residues within the amino-terminal domain of KdpD (aa 22–48) is recognized by SRP and targets the protein to the membrane. This shows that membrane proteins with a large N-terminal region in the cytoplasm can be membrane-targeted early on to allow co-translational membrane insertion of their distant transmembrane regions. PMID:18433452

  1. Protein domain assignment from the recurrence of locally similar structures

    PubMed Central

    Tai, Chin-Hsien; Sam, Vichetra; Gibrat, Jean-Francois; Garnier, Jean; Munson, Peter J.

    2010-01-01

    Domains are basic units of protein structure and essential for exploring protein fold space and structure evolution. With the structural genomics initiative, the number of protein structures in the Protein Databank (PDB) is increasing dramatically and domain assignments need to be done automatically. Most existing structural domain assignment programs define domains using the compactness of the domains and/or the number and strength of intra-domain versus inter-domain contacts. Here we present a different approach based on the recurrence of locally similar structural pieces (LSSPs) found by one-against-all structure comparisons with a dataset of 6,373 protein chains from the PDB. Residues of the query protein are clustered using LSSPs via three different procedures to define domains. This approach gives results that are comparable to several existing programs that use geometrical and other structural information explicitly. Remarkably, most of the proteins that contribute the LSSPs defining a domain do not themselves contain the domain of interest. This study shows that domains can be defined by a collection of relatively small locally similar structural pieces containing, on average, four secondary structure elements. In addition, it indicates that domains are indeed made of recurrent small structural pieces that are used to build protein structures of many different folds as suggested by recent studies. PMID:21287617

  2. The Rice Resistance Protein Pair RGA4/RGA5 Recognizes the Magnaporthe oryzae Effectors AVR-Pia and AVR1-CO39 by Direct Binding[W][OA

    PubMed Central

    Cesari, Stella; Thilliez, Gaëtan; Ribot, Cécile; Chalvon, Véronique; Michel, Corinne; Jauneau, Alain; Rivas, Susana; Alaux, Ludovic; Kanzaki, Hiroyuki; Okuyama, Yudai; Morel, Jean-Benoit; Fournier, Elisabeth; Tharreau, Didier; Terauchi, Ryohei; Kroj, Thomas

    2013-01-01

    Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer–fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain. PMID:23548743

  3. The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding.

    PubMed

    Cesari, Stella; Thilliez, Gaëtan; Ribot, Cécile; Chalvon, Véronique; Michel, Corinne; Jauneau, Alain; Rivas, Susana; Alaux, Ludovic; Kanzaki, Hiroyuki; Okuyama, Yudai; Morel, Jean-Benoit; Fournier, Elisabeth; Tharreau, Didier; Terauchi, Ryohei; Kroj, Thomas

    2013-04-01

    Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer-fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.

  4. The Structure of the Dead ringer-DNA complex reveals how AT-rich interaction domains (ARIDs) recognize DNA

    SciTech Connect

    Iwahara, Junji; Iwahara, Mizuho; Daughdrill, Gary W.; Ford, Joe J.; Clubb, Robert T.

    2002-03-01

    The AT-rich interaction domain (ARID) is a DNA-binding module found in many eukaryotic transcription factors. Using NMR Spectroscopy, we have determined the first ever three-dimensional structure of an ARID-DNA complex (mol.wt 25.7 kDa) formed by Dead ringer from Drosophila melanogaster, ARIDs recognize DNA through a novel mechanism involving major groove immobilization of a large loop that connects the helices of a non-canonical helix-turn-helix motif, and through a concomitant structural rearrangement. that produces stabilizing contacts from a B-hairpin. Dead ringer's preference for a AT-rich DNA originates from three positions within the ARID fold that form energetically significant contacts to an adenine thymine base step.

  5. Structural basis of interactions between epidermal growth factor receptor and SH2 domain proteins.

    PubMed

    Sierke, S L; Longo, G M; Koland, J G

    1993-02-26

    The structural basis of the interactions between the activated epidermal growth factor (EGF) receptor and SH2 domain proteins was investigated. The c-src SH2 domain (second domain of src homology) was expressed as a recombinant fusion protein, and an in vitro assay was developed to monitor EGF receptor/SH2 domain interactions. EGF receptor tyrosine kinase domain (TKD) forms expressed in the baculovirus/insect cell system were shown to bind to the SH2 domain when phosphorylated. These TKD/SH2 domain interactions were characterized by dissociation constants of 60-320 nM. Deletion analysis indicated that the entire SH2 domain was required for recognition of the phosphorylated TKD. The binding of a highly truncated TKD protein to the SH2 domain suggested that the sites recognized by the SH2 domain included the EGF receptor autophosphorylation site, tyr992. A phosphorylated EGF receptor peptide containing tyr992 was also shown to interact with the SH2 domain. This residue may therefore mediate interactions between the EGF receptor and tyrosine kinases in the src family.

  6. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    PubMed Central

    2014-01-01

    Background MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear. Results We analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade. Conclusions Our results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins. PMID:24468197

  7. Design of protein function leaps by directed domain interface evolution

    PubMed Central

    Huang, Jin; Koide, Akiko; Makabe, Koki; Koide, Shohei

    2008-01-01

    Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting “affinity clamp” had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution. PMID:18445649

  8. Design of protein function leaps by directed domain interface evolution.

    PubMed

    Huang, Jin; Koide, Akiko; Makabe, Koki; Koide, Shohei

    2008-05-06

    Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting "affinity clamp" had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution.

  9. Purification and Structural Analysis of LEM-Domain Proteins.

    PubMed

    Herrada, Isaline; Bourgeois, Benjamin; Samson, Camille; Buendia, Brigitte; Worman, Howard J; Zinn-Justin, Sophie

    2016-01-01

    LAP2-emerin-MAN1 (LEM)-domain proteins are modular proteins characterized by the presence of a conserved motif of about 50 residues. Most LEM-domain proteins localize at the inner nuclear membrane, but some are also found in the endoplasmic reticulum or nuclear interior. Their architecture has been analyzed by predicting the limits of their globular domains, determining the 3D structure of these domains and in a few cases calculating the 3D structure of specific domains bound to biological targets. The LEM domain adopts an α-helical fold also found in SAP and HeH domains of prokaryotes and unicellular eukaryotes. The LEM domain binds to BAF (barrier-to-autointegration factor; BANF1), which interacts with DNA and tethers chromatin to the nuclear envelope. LAP2 isoforms also share an N-terminal LEM-like domain, which binds DNA. The structure and function of other globular domains that distinguish LEM-domain proteins from each other have been characterized, including the C-terminal dimerization domain of LAP2α and C-terminal WH and UHM domains of MAN1. LEM-domain proteins also have large intrinsically disordered regions that are involved in intra- and intermolecular interactions and are highly regulated by posttranslational modifications in vivo.

  10. Production and characterization of highly tumor-specific rat monoclonal antibodies recognizing the extracellular domain of human L-type amino-acid transporter 1.

    PubMed

    Ohno, Yoshiya; Suda, Kentaro; Masuko, Kazue; Yagi, Hideki; Hashimoto, Yoshiyuki; Masuko, Takashi

    2008-05-01

    L-type large amino acid transporter (LAT) 1, the first light chain (lc) of cluster of differentiation 98 (CD98) to be identified, is associated with the heavy chain (hc) of CD98 and expressed on the surface of various tumor cells irrespective of their origin. Because LAT1 is a 12-pass membrane protein and its possible immunogenic extracellular region is very small, specific monoclonal antibodies (mAb) had not been developed. We report the successful preparation and characterization of mAb recognizing the extracellular domain of human LAT1 protein. Two mAb were selected from hybridoma clones established by fusing mouse myeloma cells and spleen cells from rats immunized against RH7777 rat hepatoma cells expressing recombinant green fluorescent protein fused to human LAT1 protein. Designated SOL22 and SOL69, these mAb specifically reacted with the extracellular domain of LAT1 on cells transfected with cDNA of LAT1, but not with cells transfected with cDNA of other CD98 lc, namely, LAT2, y(+)LAT1, y(+)LAT2, and xCT amino acid transporters. These mAb immunoprecipitated 35- and 90-kDa proteins under reducing conditions in extracts prepared from human HeLa tumor cells, indicating the existence of intermolecular disulfide bonds between cysteine residues in the 90-kDa hc and 35-kDa lc (LAT1). SOL22 and SOL69 mAb reacted with a wide variety of living unfixed human tumor cell lines, but were only weakly reactive with HEK293F human embryonic kidney cells and human peripheral blood cells. Comparative immunohistochemical analyses of normal human tissues with anti-CD98 hc and anti-LAT1 revealed LAT1 to be an excellent molecular target for antibody therapy, possibly even superior to CD98 hc.

  11. Accurate prediction of interfacial residues in two-domain proteins using evolutionary information: implications for three-dimensional modeling.

    PubMed

    Bhaskara, Ramachandra M; Padhi, Amrita; Srinivasan, Narayanaswamy

    2014-07-01

    With the preponderance of multidomain proteins in eukaryotic genomes, it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces, and the knowledge of the interacting sites is essential to our understanding of the structure-function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multidomain), we predict the interface residues corresponding to domains from the two-domain proteins. We also use information from the three-dimensional structures of individual domains of two-domain proteins to train naïve Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (∼85%) and specific (∼95%) to the domain-domain interfaces. This method is specific to multidomain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain-domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. We believe that these results can be of considerable interest toward rational protein and interaction design, apart from providing us with valuable information on the nature of interactions. © 2013 Wiley Periodicals, Inc.

  12. AIDA: ab initio domain assembly for automated multi-domain protein structure prediction and domain–domain interaction prediction

    PubMed Central

    Xu, Dong; Jaroszewski, Lukasz; Li, Zhanwen; Godzik, Adam

    2015-01-01

    Motivation: Most proteins consist of multiple domains, independent structural and evolutionary units that are often reshuffled in genomic rearrangements to form new protein architectures. Template-based modeling methods can often detect homologous templates for individual domains, but templates that could be used to model the entire query protein are often not available. Results: We have developed a fast docking algorithm ab initio domain assembly (AIDA) for assembling multi-domain protein structures, guided by the ab initio folding potential. This approach can be extended to discontinuous domains (i.e. domains with ‘inserted’ domains). When tested on experimentally solved structures of multi-domain proteins, the relative domain positions were accurately found among top 5000 models in 86% of cases. AIDA server can use domain assignments provided by the user or predict them from the provided sequence. The latter approach is particularly useful for automated protein structure prediction servers. The blind test consisting of 95 CASP10 targets shows that domain boundaries could be successfully determined for 97% of targets. Availability and implementation: The AIDA package as well as the benchmark sets used here are available for download at http://ffas.burnham.org/AIDA/. Contact: adam@sanfordburnham.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25701568

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

    SciTech Connect

    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.

  14. Overexpression of glycosylated proteins in cervical cancer recognized by the Machaerocereus eruca agglutinin.

    PubMed

    Solórzano, Carlos; Angel Mayoral, Miguel; de los Angeles Carlos, María; Berumen, Jaime; Guevara, Jorge; Raúl Chávez, Francisco; Mendoza-Hernández, Guillermo; Agundis, Concepción; Zenteno, Edgar

    2012-10-08

    In cervical cancer, glycosylation has been suggested as being involved in both its carcinogenesis and invasive capacity. In this work, we analyzed mucin type O-glycosylation in biopsies of invasive cervical cancer in FIGO stage II B through histochemistry using lectins specific for O-glycosidically linked glycans. Our results reveal that the lectin Machaerocereus eruca (MeA, specific for Gal in a Fucα1,2 (GalNAcα1,3) Galβ1,4) showed increased recognition of tumoral cells and tumoral stroma tissue compared to other lectins with similar specificity; healthy cervical tissue was negative for MeA. Trypsin treatment of recognized tissues abolished MeA's recognition;moreover, interaction of MeA was inhibited with oligosaccharides from mucin. As demonstrated by Western blot of 2-D electrophoresis, MeA recognized ten glycoproteins in the range from 122 to 42 kDa in cervical cancer lysates. The LC-ESI-MS/MS analysis of the MeAs' recognized peptides revealed that the latter matched mainly with the amino acid sequences of lamin A/C, vimentin, elongation factor 2, keratin 1, and beta actin. Our results suggest that MeA recognizes a complex of over-expressed O-glycosidically-linked proteins that play a relevant role in cervical cancer's invasive capacity. O-glycosylation participates in the disassembly of intercellular junctions favoring cancer progression.

  15. MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III.

    PubMed

    Taddese, Samuel; Jung, Michael C; Ihling, Christian; Heinz, Andrea; Neubert, Reinhard H H; Schmelzer, Christian E H

    2010-04-01

    Collagens of either soft connective or mineralized tissues are subject to continuous remodeling and turnover. Undesired cleavage can be the result of an imbalance between proteases and their inhibitors. Owing to their superhelical structure, collagens are resistant to many proteases and matrix metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated. The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly(775)-Leu(776)- in alpha-2 type I collagen and -Gly(775)-Ile(776)- in alpha-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around these typical sites contains comparatively less prolines, of which some have been proven to be only partially hydroxylated. This is of relevance since partial hydroxylation in the vicinity of a potential scissile bond may have a local effect on the conformational thermodynamics with probable consequences on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III. Copyright 2009 Elsevier B.V. All rights reserved.

  16. A kinetic study of domain swapping of Protein L.

    PubMed

    Moschen, Thomas; Tollinger, Martin

    2014-04-14

    Domain swapping of the B1 domain of Protein L isolated from Peptostreptococcus magnus can be induced by rational mutation. We show that the monomeric and the domain swapped dimeric forms of the G55A mutant of Protein L are directly observable by solution NMR spectroscopy under equilibrium conditions. The kinetics of the domain swapping process can be characterized by real-time NMR spectroscopic techniques, and the free energy landscape for domain swapping of Protein L can be probed by variation of denaturant concentration. Our data suggest that domain swapping of Protein L proceeds through a compact transition state, with an accessible surface area that is similar in size to the transition state for folding and unfolding. It is thus conceivable that domain swapping and folding of Protein L are mechanistically related at the level of their rate-limiting step(s), which might represent a branching point along the folding pathway.

  17. The architectural design of networks of protein domain architectures.

    PubMed

    Hsu, Chia-Hsin; Chen, Chien-Kuo; Hwang, Ming-Jing

    2013-08-23

    Protein domain architectures (PDAs), in which single domains are linked to form multiple-domain proteins, are a major molecular form used by evolution for the diversification of protein functions. However, the design principles of PDAs remain largely uninvestigated. In this study, we constructed networks to connect domain architectures that had grown out from the same single domain for every single domain in the Pfam-A database and found that there are three main distinctive types of these networks, which suggests that evolution can exploit PDAs in three different ways. Further analysis showed that these three different types of PDA networks are each adopted by different types of protein domains, although many networks exhibit the characteristics of more than one of the three types. Our results shed light on nature's blueprint for protein architecture and provide a framework for understanding architectural design from a network perspective.

  18. WW domain-containing proteins: retrospectives and the future.

    PubMed

    Salah, Zaidoun; Alian, Akram; Aqeilan, Rami I

    2012-01-01

    WW domains are protein modules that mediate protein-protein interactions through recognition of proline-rich peptide motifs (PRM) and phosphorylated serine/threonine-proline sites. WW domains are found in many different structural and signaling proteins that are involved in a variety of cellular processes, including RNA transcription and processing, protein trafficking and stability, receptor signaling, and control of the cytoskeleton. WW domain-containing proteins and complexes have been implicated in major human diseases including cancer as well as in major signaling cascades such as the Hippo tumor suppressor pathway, making them targets for new diagnostics and therapeutics. In this review, we discuss how WW domains provide versatile platforms that link individual proteins into physiologically important networks and the indispensible role of WW domain-containing proteins in biology and pathology, especially tumorogenesis.

  19. Anti-idiotypic protein domains selected from protein A-based affibody libraries.

    PubMed

    Eklund, Malin; Axelsson, Lars; Uhlén, Mathias; Nygren, Per-Ake

    2002-08-15

    Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.

  20. Fold of the conserved DTC domain in deltex proteins

    SciTech Connect

    Obiero, Josiah; Walker, John R.; Dhe-Paganon, Sirano

    2012-04-30

    Human Deltex 3-like (DTX3L) is a member of the Deltex family of proteins. Initially identified as a B-lymphoma and BAL-associated protein, DTX3L is an E3 ligase that regulates subcellular localization of its partner protein, BAL, by a dynamic nucleocytoplasmic trafficking mechanism. Unlike other members of the Deltex family of proteins, DTX3L lacks the highly basic N-terminal motif and the central proline-rich motif present in other Deltex proteins, and instead contains other unique N-terminal domains. The C-terminal domains are, however, homologous with other members of the Deltex family of proteins; these include a RING domain and a previously unidentified C-terminal domain. In this study, we report the high-resolution crystal structure of this previously uncharacterized C-terminal domain of human DTX3L, which we term the Deltex C-terminal domain.

  1. Fuzzy domains: new way of describing flexibility and interdependence of the protein domains.

    PubMed

    Yesylevskyy, Semen O; Kharkyanen, Valery N

    2009-03-01

    We proposed the innovative method of domain identification based on the concept of the fuzzy domains. In this method each residue of the protein can belong to several domains simultaneously with certain weights, which reflect to what extent this residue shares the motion pattern of the given domain. Our method allows describing the fuzzy boundaries between the domains and the gradual changes of the motion pattern from one domain to the other. It provides the reasonable compromise between the continuous change of the protein dynamics from one residue to the other and the discrete description of the structure in terms of small number of domains. We suggested quantitative criterion, which shows the overall degree of domain flexibility in the protein. The concept of the fuzzy domains provides an innovative way of visualization of domain flexibility, which makes the gradual transitions between the domains clearly visible and comparable to available experimental and structural data. In the future, the concept of the fuzzy domains can be used in the coarse-grained simulations of the domain dynamics in order to account for internal protein flexibility.

  2. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses.

    PubMed Central

    Lipsich, L A; Lewis, A J; Brugge, J S

    1983-01-01

    Thirteen clones of hybrid cells which synthesize antibodies directed against the Rous sarcoma virus (RSV) transforming protein, pp60src, were isolated. Mouse myeloma cells were fused with spleen cells from mice that had been immunized with purified pp60src from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were screened by immunoprecipitation of pp60src from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp60src encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellular homolog of pp60src. Antibody from clone 261 had a high affinity for the viral yes gene product, and antibodies from clones 443 and 463 recognized the transforming proteins encoded by viruses containing the related transforming genes fps and ros. Several other clones had a low affinity for the viral yes, fps, and ros gene products which could be detected by in vitro phosphorylation of the transforming proteins after immunoprecipitation with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60src and casein in an immune complex-bound reaction. Images PMID:6312092

  3. Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein

    SciTech Connect

    Buffalo, Cosmo Z.; Bahn-Suh, Adrian J.; Hirakis, Sophia P.; Biswas, Tapan; Amaro, Rommie E.; Nizet, Victor; Ghosh, Partho

    2016-09-05

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ~90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant ‘reading head’ in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M–C4BP interaction, and also inform a path towards vaccine design.

  4. FRAN and RBF-PSO as two components of a hyper framework to recognize protein folds.

    PubMed

    Abbasi, Elham; Ghatee, Mehdi; Shiri, M E

    2013-09-01

    In this paper, an intelligent hyper framework is proposed to recognize protein folds from its amino acid sequence which is a fundamental problem in bioinformatics. This framework includes some statistical and intelligent algorithms for proteins classification. The main components of the proposed framework are the Fuzzy Resource-Allocating Network (FRAN) and the Radial Bases Function based on Particle Swarm Optimization (RBF-PSO). FRAN applies a dynamic method to tune up the RBF network parameters. Due to the patterns complexity captured in protein dataset, FRAN classifies the proteins under fuzzy conditions. Also, RBF-PSO applies PSO to tune up the RBF classifier. Experimental results demonstrate that FRAN improves prediction accuracy up to 51% and achieves acceptable multi-class results for protein fold prediction. Although RBF-PSO provides reasonable results for protein fold recognition up to 48%, it is weaker than FRAN in some cases. However the proposed hyper framework provides an opportunity to use a great range of intelligent methods and can learn from previous experiences. Thus it can avoid the weakness of some intelligent methods in terms of memory, computational time and static structure. Furthermore, the performance of this system can be enhanced throughout the system life-cycle.

  5. Domain mobility in proteins: functional and evolutionary implications.

    PubMed

    Basu, Malay Kumar; Poliakov, Eugenia; Rogozin, Igor B

    2009-05-01

    A substantial fraction of eukaryotic proteins contains multiple domains, some of which show a tendency to occur in diverse domain architectures and can be considered mobile (or 'promiscuous'). These promiscuous domains are typically involved in protein-protein interactions and play crucial roles in interaction networks, particularly those contributing to signal transduction. They also play a major role in creating diversity of protein domain architecture in the proteome. It is now apparent that promiscuity is a volatile and relatively fast-changing feature in evolution, and that only a few domains retain their promiscuity status throughout evolution. Many such domains attained their promiscuity status independently in different lineages. Only recently, we have begun to understand the diversity of protein domain architectures and the role the promiscuous domains play in evolution of this diversity. However, many of the biological mechanisms of protein domain mobility remain shrouded in mystery. In this review, we discuss our present understanding of protein domain promiscuity, its evolution and its role in cellular function.

  6. Preparation and initial application of monoclonal antibodies that recognize Eimeria tenella microneme proteins 1 and 2.

    PubMed

    Liu, Qing; Chen, Zhengtao; Shi, Wenyan; Sun, Hui; Zhang, Jie; Li, Hongmei; Xiao, Yihong; Wang, Fangkun; Zhao, Xiaomin

    2014-11-01

    Microneme proteins (MICs) of Eimeria species are critical for motility of the parasite, identification and binding of host cell-surface proteins, invasion of host cells, and intracellular survival. The microneme protein 1 (EtMIC1) and 2 (EtMIC2) from Eimeria tenella have a putative function in parasite adhesion to the host cell to initiate an invasion process. Previous studies indicated that the EtMIC1 and EtMIC2 proteins form a complex that play roles during attachment to and penetration of the host cell. Numerous studies demonstrated that both the EtMIC1 and EtMIC2 are important microneme proteins which are abundantly expressed in sporozoites and schizogony stages. But the expression of EtMIC1 and EtMIC2 in the gametogony stage is unknown. To investigate the precise roles of EtMIC1 and EtMIC2 in host-parasite interactions and expressions in the gametogony stage of E. tenella, we generated five mouse monoclonal antibodies (MAbs) which recognize the EtMIC1 and EtMIC2 proteins and investigated expressions of EtMIC1 and EtMIC2 proteins in later endogenous developmental stages, particularly focused on the gametogony phase using the specific anti-EtMIC1 and anti-EtMIC2 MAbs produced in this work. Our results showed that both EtMIC1 and EtMIC2 proteins are expressed in all developmental stages including the gametogony stage. To our knowledge, this is the first report that the EtMIC1 and EtMIC2 proteins are expressed in the gametogony stage of E. tenella.

  7. Comparative Analysis of SWIRM Domain-Containing Proteins in Plants

    PubMed Central

    Gao, Yan; Yang, Songguang; Yuan, Lianyu; Cui, Yuhai; Wu, Keqiang

    2012-01-01

    Chromatin-remodeling complexes affect gene expression by using the energy of ATP hydrolysis to locally disrupt or alter the association of histones with DNA. SWIRM (Swi3p, Rsc8p, and Moira) domain is an alpha-helical domain of about 85 residues in chromosomal proteins. SWIRM domain-containing proteins make up large multisubunit complexes by interacting with other chromatin modification factors and may have an important function in plants. However, little is known about SWIRM domain-containing proteins in plants. In this study, 67 SWIRM domain-containing proteins from 6 plant species were identified and analyzed. Plant SWIRM domain proteins can be divided into three distinct types: Swi-type, LSD1-type, and Ada2-type. Generally, the SWIRM domain forms a helix-turn-helix motif commonly found in DNA-binding proteins. The genes encoding SWIRM domain proteins in Oryza sativa are widely expressed, especially in pistils. In addition, OsCHB701 and OsHDMA701 were downregulated by cold stress, whereas OsHDMA701 and OsHDMA702 were significantly induced by heat stress. These observations indicate that SWIRM domain proteins may play an essential role in plant development and plant responses to environmental stress. PMID:22924025

  8. Identification of domains and domain interface residues in multidomain proteins from graph spectral method.

    PubMed

    Sistla, Ramesh K; K V, Brinda; Vishveshwara, Saraswathi

    2005-05-15

    We present a novel method for the identification of structural domains and domain interface residues in proteins by graph spectral method. This method converts the three-dimensional structure of the protein into a graph by using atomic coordinates from the PDB file. Domain definitions are obtained by constructing either a protein backbone graph or a protein side-chain graph. The graph is constructed based on the interactions between amino acid residues in the three-dimensional structure of the proteins. The spectral parameters of such a graph contain information regarding the domains and subdomains in the protein structure. This is based on the fact that the interactions among amino acids are higher within a domain than across domains. This is evident in the spectra of the protein backbone and the side-chain graphs, thus differentiating the structural domains from one another. Further, residues that occur at the interface of two domains can also be easily identified from the spectra. This method is simple, elegant, and robust. Moreover, a single numeric computation yields both the domain definitions and the interface residues. Copyright 2005 Wiley-Liss, Inc.

  9. Modelling protein functional domains in signal transduction using Maude

    NASA Technical Reports Server (NTRS)

    Sriram, M. G.

    2003-01-01

    Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.

  10. Modelling protein functional domains in signal transduction using Maude

    NASA Technical Reports Server (NTRS)

    Sriram, M. G.

    2003-01-01

    Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.

  11. [Anti-peptide antibodies specifically recognize the L1 protein of human papilloma virus type 31].

    PubMed

    Andreev, S M; Giliazova, A V; Khaitov, M R; Kozlov, D G

    2012-01-01

    Antibodies that specifically recognize the capsid protein (L1) of human papillomavirus (HPV) are an important tool necessary for designing vaccines against HPV infection. In this work, we have predicted and synthesized peptide fragments mimicking B cell epitopes of L1 HPV type 31 (sequences 49-65, 131-145, 172-189, 349-362 and 402-414), and conjugated their to KLH and BSA to generate the L1-31-specific anti-peptide antibodies in mice. Variants of recombinant L1-31, including full-size and mutants with C-terminal single amino acid changes and deletions and full-size L1-16 were produced in the yeast using monitoring with L1 HPV16-specific monoclonal antibody. Testing of anti-peptide antisera in ELISA showed that antibodies to peptides 49-65 and 172-189 were capable to recognize specifically L1-31 protein, but not L1-16 one. Such antibodies may be used for assay of L1-31 production in various expression systems.

  12. Do proteins facilitate the formation of cholesterol-rich domains?

    PubMed

    Epand, Richard M

    2004-11-03

    Both biological and model membranes can exhibit the formation of domains. A brief review of some of the diverse methodologies used to identify the presence of domains in membranes is given. Some of these domains are enriched in cholesterol. The segregation of lipids into cholesterol-rich domains can occur in both pure lipid systems as well as membranes containing peptides and proteins. Peptides and proteins can promote the formation of cholesterol-rich domains not only by preferentially interacting with cholesterol and being sequestered into these regions of the membrane, but also indirectly as a consequence of being excluded from cholesterol-rich domains. The redistribution of components is dictated by the thermodynamics of the system. The formation of domains in a biological membrane is a consequence of all of the intermolecular interactions including those among lipid molecules as well as between lipids and proteins.

  13. Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region

    PubMed Central

    Ruedas, John B.

    2014-01-01

    ABSTRACT The multidomain polymerase protein (L) of nonsegmented negative-strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure, while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild-type L in the context of a recombinant virus. Exchange of the downstream C-terminal nonconserved region abolished activity, but coexchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain or the adjacent nonconserved region thought to function as an “unstructured” linker also abrogated polymerase activity even when either domain was coexchanged with other appendage domains. Further probing of the putative linker segment using in-frame enhanced green fluorescent protein (EGFP) insertions similarly abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function. IMPORTANCE NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in

  14. Interaction between Functional Domains of Bacillus thuringiensis Insecticidal Crystal Proteins

    PubMed Central

    Rang, Cécile; Vachon, Vincent; de Maagd, Ruud A.; Villalon, Mario; Schwartz, Jean-Louis; Bosch, Dirk; Frutos, Roger; Laprade, Raynald

    1999-01-01

    Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C. PMID:10388684

  15. Staphylococcus aureus proteins differentially recognized by the ovine immune response in mastitis or nasal carriage.

    PubMed

    Seyffert, Nubia; Le Maréchal, Caroline; Jardin, Julien; McCulloch, John A; Rosado, Fabio R; Miyoshi, Anderson; Even, Sergine; Jan, Gwenaël; Berkova, Nadia; Vautor, Eric; Thiéry, Richard; Azevedo, Vasco; Le Loir, Yves

    2012-06-15

    Staphylococcus aureus is an opportunistic pathogen in dairy ruminants where it is found in healthy carriage and can be a major cause of mastitis. A better knowledge of the host-pathogen interactions is needed to tackle this serious animal health problem. This study aimed at identifying S. aureus proteins differentially expressed by S. aureus in nasal colonization versus mastitis. Serological proteome analysis (SERPA) was used to examine protein samples prepared from culture supernatants of S. aureus strains originally isolated from gangrenous mastitis and nasal carriage (O11) or subclinical mastitis (O46) and to compare patterns of immune-reactive proteins. These staphylococcal proteins were revealed by sera obtained from ewes suffering from S. aureus mastitis and by sera obtained from healthy nulliparous ewes (i.e. no lactation and no mastitis or other symptoms) that were nasally colonized by S. aureus. Altogether 49 staphylococcal immune-reactive proteins were identified in this study. Patterns of proteins revealed by sera from infected- or healthy carrier- animals were comparable and analysis singled out one immune-reactive protein, N-acetylmuramyl-L-alanine amidase, which was recognized by each of the 6 sera from infected animals, when tested individually, and not by the sera of healthy carriers. This is the first study that compares the S. aureus seroproteome in colonization versus mastitis context in ruminants. These results open avenues for studies aiming at a better understanding of the balance between infection and commensal lifestyle in this opportunistic pathogen and at new prevention strategies. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation

    DOE PAGES

    Lee, Jungsoon; Sung, Nuri; Mercado, Jonathan M.; ...

    2017-09-11

    Hsp104 is a ring-forming protein disaggregase that rescues stress-damaged proteins from an aggregated state. To facilitate protein disaggregation, Hsp104 cooperates with Hsp70 and Hsp40 chaperones (Hsp70/40) to form a bi-chaperone system. How Hsp104 recognizes its substrates, particularly the importance of the N domain, remains poorly understood and multiple, seemingly conficting mechanisms have been proposed. Although the N domain is dispensable for protein disaggregation, it is sensitive to point mutations that abolish the function of the bacterial Hsp104 homolog in vitro, and is essential for curing yeast prions by Hsp104 overexpression in vivo. Here, we present the crystal structure of anmore » N-terminal fragment of Saccharomyces cerevisiae Hsp104 with the N domain of one molecule bound to the C-terminal helix of the neighboring D1 domain. Consistent with mimicking substrate interaction, mutating the putative substrate-binding site in a constitutively active Hsp104 variant impairs the recovery of functional protein from aggregates. We fnd that the observed substrate-binding defect can be rescued by Hsp70/40 chaperones, providing a molecular explanation as to why the N domain is dispensable for protein disaggregation when Hsp70/40 is present, yet essential for the dissolution of Hsp104-specifc substrates, such as yeast prions, which likely depends on a direct N domain interaction.« less

  17. Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently.

    PubMed

    Wu, Qiang; Zhang, Wensheng; Pwee, Keng-Hock; Kumar, Prakash P

    2003-03-01

    We analyzed the DNA-binding and DNA-bending properties of recombinant HMGB1 proteins based on a rice HMGB1 cDNA. Electrophoretic mobility shift assay demonstrated that rice HMGB1 can bind synthetic four-way junction (4H) DNA and DNA minicircles efficiently but the binding to 4H can be completed out by HMGA and histone H1. Conformational changes were detected by circular dichroism analysis with 4H DNA bound to various concentrations of HMGB1 or its truncated forms. T4 ligase-mediated circularization assays with short DNA fragments of 123 bp showed that the protein is capable of increasing DNA flexibility. The 123-bp DNA formed closed circular monomers efficiently in its presence, similar to that in an earlier study on maize HMG. Additionally, our results show for the first time that the basic N-terminal domain enhances the affinity of the plant HMGB1 protein for 4H DNA, while the acidic C-terminal domain has the converse effects.

  18. The floral homeotic protein APETALA2 recognizes and acts through an AT-rich sequence element.

    PubMed

    Dinh, Thanh Theresa; Girke, Thomas; Liu, Xigang; Yant, Levi; Schmid, Markus; Chen, Xuemei

    2012-06-01

    Cell fate specification in development requires transcription factors for proper regulation of gene expression. In Arabidopsis, transcription factors encoded by four classes of homeotic genes, A, B, C and E, act in a combinatorial manner to control proper floral organ identity. The A-class gene APETALA2 (AP2) promotes sepal and petal identities in whorls 1 and 2 and restricts the expression of the C-class gene AGAMOUS (AG) from whorls 1 and 2. However, it is unknown how AP2 performs these functions. Unlike the other highly characterized floral homeotic proteins containing MADS domains, AP2 has two DNA-binding domains referred to as the AP2 domains and its DNA recognition sequence is still unknown. Here, we show that the second AP2 domain in AP2 binds a non-canonical AT-rich target sequence, and, using a GUS reporter system, we demonstrate that the presence of this sequence in the AG second intron is important for the restriction of AG expression in vivo. Furthermore, we show that AP2 binds the AG second intron and directly regulates AG expression through this sequence element. Computational analysis reveals that the binding site is highly conserved in the second intron of AG orthologs throughout Brassicaceae. By uncovering a biologically relevant AT-rich target sequence, this work shows that AP2 domains have wide-ranging target specificities and provides a missing link in the mechanisms that underlie flower development. It also sets the foundation for understanding the basis of the broad biological functions of AP2 in Arabidopsis, as well as the divergent biological functions of AP2 orthologs in dicotyledonous plants.

  19. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains.

    PubMed

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J; Eggeling, Christian; Hell, Stefan W; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-30

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

  20. Continuous and discontinuous domains: an algorithm for the automatic generation of reliable protein domain definitions.

    PubMed Central

    Siddiqui, A. S.; Barton, G. J.

    1995-01-01

    An algorithm is presented for the fast and accurate definition of protein structural domains from coordinate data without prior knowledge of the number or type of domains. The algorithm explicitly locates domains that comprise one or two continuous segments of protein chain. Domains that include more than two segments are also located. The algorithm was applied to a nonredundant database of 230 protein structures and the results compared to domain definitions obtained from the literature, or by inspection of the coordinates on molecular graphics. For 70% of the proteins, the derived domains agree with the reference definitions, 18% show minor differences and only 12% (28 proteins) show very different definitions. Three screens were applied to identify the derived domains least likely to agree with the subjective definition set. These screens revealed a set of 173 proteins, 97% of which agree well with the subjective definitions. The algorithm represents a practical domain identification tool that can be run routinely on the entire structural database. Adjustment of parameters also allows smaller compact units to be identified in proteins. PMID:7663343

  1. Domain structure and organisation in extracellular matrix proteins.

    PubMed

    Hohenester, Erhard; Engel, Jürgen

    2002-03-01

    Extracellular matrix (ECM) proteins are large modular molecules built up from a limited set of modules, or domains. The basic folds of many domains have now been determined by crystallography or NMR spectroscopy. Recent structures of domain pairs and larger tandem arrays, as well as of oligomerisation domains, have begun to reveal the principles underlying the higher order architecture of ECM proteins. Structural information, coupled with site-directed mutagenesis, has been instrumental in showing how adjacent domains can co-operate in ligand binding. Very recently, the first heterotypic ECM protein complexes have become available. Here, we review the advances of the last 5 years in understanding ECM protein structure, with special emphasis on those structures that have given insight into the biological functions of ECM proteins.

  2. Identification of the ubiquitin-protein ligase that recognizes oxidized IRP2.

    PubMed

    Yamanaka, Koji; Ishikawa, Haruto; Megumi, Yuzuru; Tokunaga, Fuminori; Kanie, Masato; Rouault, Tracey A; Morishima, Isao; Minato, Nagahiro; Ishimori, Koichiro; Iwai, Kazuhiro

    2003-04-01

    The ubiquitin system is involved in several basic cellular functions. Ubiquitination is carried out by a cascade of three reactions catalysed by the E1, E2 and E3 enzymes. Among these, the E3 ubiquitin-protein ligases have a pivotal role in determining the specificity of the system by recognizing the target substrates through defined targeting motifs. Although RING finger proteins constitute an important family of E3 ligases, only a few post-transcriptional modifications, including phosphorylation, proline hydroxylation and glycosylation, are known to function as recognition signals for E3. Iron regulatory protein 2 (IRP2), a modulator of iron metabolism, is regulated by iron-induced ubiquitination and degradation. Here we show that the RING finger protein HOIL-1 functions as an E3 ligase for oxidized IRP2, suggesting that oxidation is a specific recognition signal for ubiquitination. The oxidation of IRP2 is generated by haem, which binds to IRP2 in iron-rich cells, and by oxygen, indicating that the iron sensing of IRP2 depends on the synthesis and availability of haem.

  3. The nature of protein domain evolution: shaping the interaction network.

    PubMed

    Bagowski, Christoph P; Bruins, Wouter; Te Velthuis, Aartjan J W

    2010-08-01

    The proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. These protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. An important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. Changes in this information may bring about new folds, functions and protein architectures. With the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. Such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving inter- and intra-molecular interactions. In turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. Additionally, these studies can be used for the design of new and optimized protein domains for therapy. In this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks.

  4. The Nature of Protein Domain Evolution: Shaping the Interaction Network

    PubMed Central

    Bagowski, Christoph P; Bruins, Wouter; te Velthuis, Aartjan J.W

    2010-01-01

    The proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. These protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. An important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. Changes in this information may bring about new folds, functions and protein architectures. With the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. Such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving inter- and intra-molecular interactions. In turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. Additionally, these studies can be used for the design of new and optimized protein domains for therapy. In this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks. PMID:21286315

  5. ELISA: a unified, multidimensional view of the protein domain universe.

    PubMed

    Shakhnovich, Boris E; Harvey, John Max; Delisi, Charles

    2004-01-01

    ELISA (http://romi.bu.edu/elisa/) is a database that was designed for flexibility in defining interesting queries about protein domain evolution. We have defined and included both the inherent characteristics of the domains such as structure and function and comparisons of these characteristics between domains. Thus, the database is useful in defining structural and functional links between related protein domains and by extension sequences that encode them. In this database we introduce and employ a novel method of functional annotation and comparison. For each protein domain we create a probabilistic functional annotation tree using GO. We have designed an algorithm that accurately compares these trees and thus provides a measure of "functional distance" between two protein domains. Along with functional annotation, we have also included structural comparison between protein domains and best sequence comparisons to all known genomes. The latter enables researchers to dynamically do searches for domains sharing similar phylogenetic profiles. This combination of data and tools enables the researcher to design complex queries to carry out research in the areas of protein domain evolution, structure prediction and functional annotation of novel sequences.

  6. Delineation of modular proteins: domain boundary prediction from sequence information.

    PubMed

    Kong, Lesheng; Ranganathan, Shoba

    2004-06-01

    The delineation of domain boundaries of a given sequence in the absence of known 3D structures or detectable sequence homology to known domains benefits many areas in protein science, such as protein engineering, protein 3D structure determination and protein structure prediction. With the exponential growth of newly determined sequences, our ability to predict domain boundaries rapidly and accurately from sequence information alone is both essential and critical from the viewpoint of gene function annotation. Anyone attempting to predict domain boundaries for a single protein sequence is invariably confronted with a plethora of databases that contain boundary information available from the internet and a variety of methods for domain boundary prediction. How are these derived and how well do they work? What definition of 'domain' do they use? We will first clarify the different definitions of protein domains, and then describe the available public databases with domain boundary information. Finally, we will review existing domain boundary prediction methods and discuss their strengths and weaknesses.

  7. Protein universe containing a PUA RNA-binding domain.

    PubMed

    Cerrudo, Carolina S; Ghiringhelli, Pablo D; Gomez, Daniel E

    2014-01-01

    Here, we review current knowledge about pseudouridine synthase and archaeosine transglycosylase (PUA)-domain-containing proteins to illustrate progress in this field. A methodological analysis of the literature about the topic was carried out, together with a 'qualitative comparative analysis' to give a more comprehensive review. Bioinformatics methods for whole-protein or protein-domain identification are commonly based on pairwise protein sequence comparisons; we added comparison of structures to detect the whole universe of proteins containing the PUA domain. We present an update of proteins having this domain, focusing on the specific proteins present in Homo sapiens (dyskerin, MCT1, Nip7, eIF2D and Nsun6), and explore the existence of these in other species. We also analyze the phylogenetic distribution of the PUA domain in different species and proteins. Finally, we performed a structural comparison of the PUA domain through data mining of structural databases, determining a conserved structural motif, despite the differences in the sequence, even among eukaryotes, archaea and bacteria. All data discussed in this review, both bibliographic and analytical, corroborate the functional importance of the PUA domain in RNA-binding proteins.

  8. Measles virus recognizes its receptor, CD46, via two distinct binding domains within SCR1-2.

    PubMed

    Manchester, M; Gairin, J E; Patterson, J B; Alvarez, J; Liszewski, M K; Eto, D S; Atkinson, J P; Oldstone, M B

    1997-06-23

    Measles virus (MV) enters cells by attachment of the viral hemagglutinin to the major cell surface receptor CD46 (membrane cofactor protein). CD46 is a transmembrane glycoprotein whose ectodomain is largely composed of four conserved modules called short consensus repeats (SCRs). We have previously shown that MV interacts with SCR1 and SCR2 of CD46. (M. Manchester et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2303-2307) Here we report mapping the MV interaction with SCR1 and SCR2 of CD46 using a combination of peptide inhibition and mutagenesis studies. By testing a series of overlapping peptides corresponding to the 126 amino acid SCR1-2 region for inhibition of MV infection, two domains were identified that interacted with MV. One domain was found within SCR1 (amino acids 37-56) and another within SCR2 (amino acids 85-104). These results were confirmed by constructing chimeras with complementary regions from structurally similar, but non-MV-binding, SCRs of decay accelerating factor (DAF; CD55). These results indicate that MV contacts at least two distinct sites within SCR1-2.

  9. Conformational Epitopes Recognized by Protective Anti-Neisserial Surface Protein A Antibodies

    PubMed Central

    Hou, Victor C.; Moe, Gregory R.; Raad, Zyde; Wuorimaa, Tomi; Granoff, Dan M.

    2003-01-01

    NspA is a conserved membrane protein that elicits protective antibody responses in mice against Neisseria meningitidis. A recent crystallographic study showed that NspA adopts an eight-stranded β-barrel structure when reconstituted in detergent. In order to define the segments of NspA-containing epitopes recognized by protective murine anti-NspA antibodies, we studied the binding of two bactericidal and protective anti-NspA monoclonal antibodies (MAbs), AL12 and 14C7. Neither MAb binds to overlapping synthetic peptides (10-mers, 12-mers, and cyclic 12-mers) corresponding to the entire mature sequence of NspA, or to denatured recombinant NspA (rNspA), although binding to the protein can be restored by refolding in liposomes. Based on the ability of the two MAbs to bind to Escherichia coli microvesicles prepared from a set of rNspA variants created by site-specific mutagenesis, the most important contacts between the MAbs and NspA appear to be located within the LGG segment of loop 3. The conformation of loop 2 also appears to be an important determinant, as particular combinations of residues in this segment resulted in loss of antibody binding. Thus, the two anti-NspA MAbs recognize discontinuous conformational epitopes that result from the close proximity of loops 2 and 3 in the three-dimensional structure of NspA. The data suggest that optimally immunogenic vaccines using rNspA will require formulations that permit proper folding of the protein. PMID:14638771

  10. Protein domain decomposition using a graph-theoretic approach.

    PubMed

    Xu, Y; Xu, D; Gabow, H N; Gabow, H

    2000-12-01

    Automatic decomposition of a multi-domain protein into individual domains represents a highly interesting and unsolved problem. As the number of protein structures in PDB is growing at an exponential rate, there is clearly a need for more reliable and efficient methods for protein domain decomposition simply to keep the domain databases up-to-date. We present a new algorithm for solving the domain decomposition problem, using a graph-theoretic approach. We have formulated the problem as a network flow problem, in which each residue of a protein is represented as a node of the network and each residue--residue contact is represented as an edge with a particular capacity, depending on the type of the contact. A two-domain decomposition problem is solved by finding a bottleneck (or a minimum cut) of the network, which minimizes the total cross-edge capacity, using the classical Ford--Fulkerson algorithm. A multi-domain decomposition problem is solved through repeatedly solving a series of two-domain problems. The algorithm has been implemented as a computer program, called DomainParser. We have tested the program on a commonly used test set consisting of 55 proteins. The decomposition results are 78.2% in agreement with the literature on both the number of decomposed domains and the assignments of residues to each domain, which compares favorably to existing programs. On the subset of two-domain proteins (20 in number), the program assigned 96.7% of the residues correctly when we require that the number of decomposed domains is two.

  11. Reconstituting Protein Interaction Networks Using Parameter-Dependent Domain-Domain Interactions

    DTIC Science & Technology

    2013-05-07

    for yeast ( Saccharomyces cerevisiae ) using sequences of 5,884 proteins,a downloaded from the Saccharomyces Genome Database (SGD) [31] and yeast...Riles L, Mortimer RK, et al: Genetic and physical maps of Saccharomyces cerevisiae . Nature 1997, 387(6632 Suppl):67–73. 32. Finn RD, Mistry J, Tate J...downloaded from the Saccharomyces Genome Database [31]. NP, proteins with at least one domain annotation. NS, protein-domain amino acid sequence

  12. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    PubMed Central

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-01

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes. PMID:25635869

  13. Protein domain definition should allow for conditional disorder.

    PubMed

    Yegambaram, Kavestri; Bulloch, Esther M M; Kingston, Richard L

    2013-11-01

    Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.

  14. CBS domains: structure, function, and pathology in human proteins.

    PubMed

    Ignoul, Sofie; Eggermont, Jan

    2005-12-01

    The cystathionine-beta-synthase (CBS) domain is an evolutionarily conserved protein domain that is present in the proteome of archaebacteria, prokaryotes, and eukaryotes. CBS domains usually come in tandem repeats and are found in cytosolic and membrane proteins performing different functions (metabolic enzymes, kinases, and channels). Crystallographic studies of bacterial CBS domains have shown that two CBS domains form an intramolecular dimeric structure (CBS pair). Several human hereditary diseases (homocystinuria, retinitis pigmentosa, hypertrophic cardiomyopathy, myotonia congenital, etc.) can be caused by mutations in CBS domains of, respectively, cystathionine-beta-synthase, inosine 5'-monophosphate dehydrogenase, AMP kinase, and chloride channels. Despite their clinical relevance, it remains to be established what the precise function of CBS domains is and how they affect the structural and/or functional properties of an enzyme, kinase, or channel. Depending on the protein in which they occur, CBS domains have been proposed to affect multimerization and sorting of proteins, channel gating, and ligand binding. However, recent experiments revealing that CBS domains can bind adenosine-containing ligands such ATP, AMP, or S-adenosylmethionine have led to the hypothesis that CBS domains function as sensors of intracellular metabolites.

  15. Methods for comprehensive identification of membrane proteins recognized by a large number of monoclonal antibodies.

    PubMed

    Kurosawa, Gene; Sumitomo, Mariko; Akahori, Yasushi; Matsuda, Kazuki; Muramatsu, Chiho; Takasaki, Akihiko; Iba, Yoshitaka; Eguchi, Keiko; Tanaka, Miho; Suzuki, Kazuhiro; Morita, Miwa; Sato, Noriko; Sugiura, Mototaka; Sugioka, Atsushi; Hayashi, Nobuhiro; Kurosawa, Yoshikazu

    2009-12-31

    In order to isolate monoclonal antibodies (mAbs) that bind to tumor-associated antigens (Ags) we developed the following strategy. Using the phage-display Ab library we isolated a large number of mAbs that bind to the surface of human tumor cells. The mAbs were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. Thereafter, the Ags recognized by the mAbs were identified. For identification of the Ags by MS candidate molecules had to be purified either by immunoprecipitation or by affinity chromatography. We isolated several hundred mAbs that showed cancer-specific staining patterns. In order to identify the Ags that were recognized by the numerous mAbs within a short time we developed two methods. Using the GFC [grouping of clones by flow cytometry (FCM)] method many Abs could be grouped by comparing the staining patterns of FCM. Members in each group turned out to bind to the same molecule in many cases. After a candidate Ag was revealed, the polypeptide corresponding to its extracellular portion was prepared and used for identification of clones that bound to the Ag among all the mAbs by SITE (simultaneous identification of clones through three dimensional ELISA) method. Both methods can be generally applicable to various kinds of membrane proteins and the mAbs against them.

  16. Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR)

    PubMed Central

    Fukui, Kenji; Bessho, Yoshitaka; Shimada, Atsuhiro; Yokoyama, Shigeyuki; Kuramitsu, Seiki

    2013-01-01

    Polymerase chain reaction (PCR)-related technologies are hampered mainly by two types of error: nonspecific amplification and DNA polymerase-generated mutations. Here, we report that both errors can be suppressed by the addition of a DNA mismatch-recognizing protein, MutS, from a thermophilic bacterium. Although it had been expected that MutS has a potential to suppress polymerase-generated mutations, we unexpectedly found that it also reduced nonspecific amplification. On the basis of this finding, we propose that MutS binds a mismatched primer-template complex, thereby preventing the approach of DNA polymerase to the 3′ end of the primer. Our simple methodology improves the efficiency and accuracy of DNA amplification and should therefore benefit various PCR-based applications, ranging from basic biological research to applied medical science. PMID:23519109

  17. Discrete structure of van der Waals domains in globular proteins.

    PubMed

    Berezovsky, Igor N

    2003-03-01

    Most globular proteins are divisible by domains, distinct substructures of the globule. The notion of hierarchy of the domains was introduced earlier via van der Waals energy profiles that allow one to subdivide the proteins into domains (subdomains). The question remains open as to what is the possible structural connection of the energy profiles. The recent discovery of the loop-n-lock elements in the globular proteins suggests such a structural connection. A direct comparison of the segmentation by van der Waals energy criteria with the maps of the locked loops of nearly standard size reveals a striking correlation: domains in general appear to consist of one to several such loops. In addition, it was demonstrated that a variety of subdivisions of the same protein into domains is just a regrouping of the loop-n-lock elements.

  18. Allosteric properties of PH domains in Arf regulatory proteins.

    PubMed

    Roy, Neeladri Sekhar; Yohe, Marielle E; Randazzo, Paul A; Gruschus, James M

    2016-01-01

    Pleckstrin Homology (PH) domains bind phospholipids and proteins. They are critical regulatory elements of a number enzymes including guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) for Ras-superfamily guanine nucleotide binding proteins such as ADP-ribosylation factors (Arfs). Recent studies have indicated that many PH domains may bind more than one ligand cooperatively. Here we discuss the molecular basis of PH domain-dependent allosteric behavior of 2 ADP-ribosylation factor exchange factors, Grp1 and Brag2, cooperative binding of ligands to the PH domains of Grp1 and the Arf GTPase-activating protein, ASAP1, and the consequences for activity of the associated catalytic domains.

  19. The application of modular protein domains in proteomics

    PubMed Central

    Jadwin, Joshua A.; Ogiue-Ikeda, Mari; Machida, Kazuya

    2012-01-01

    The ability of modular protein domains to independently fold and bind short peptide ligands both in vivo and in vitro has allowed a significant number of protein-protein interaction studies to take advantage of them as affinity and detection reagents. Here, we refer to modular domain based proteomics as “domainomics” to draw attention to the potential of using domains and their motifs as tools in proteomics. In this review we describe core concepts of domainomics, established and emerging technologies, and recent studies by functional category. Accumulation of domain-motif binding data should ultimately provide the foundation for domain-specific interactomes, which will likely reveal the underlying substructure of protein networks as well as the selectivity and plasticity of signal transduction. PMID:22710164

  20. Protective activity of the CnaBE3 domain conserved among Staphylococcus aureus Sdr proteins.

    PubMed

    Becherelli, Marco; Prachi, Prachi; Viciani, Elisa; Biagini, Massimiliano; Fiaschi, Luigi; Chiarot, Emiliano; Nosari, Sarah; Brettoni, Cecilia; Marchi, Sara; Biancucci, Marco; Fontana, Maria Rita; Montagnani, Francesca; Bagnoli, Fabio; Barocchi, Michèle A; Manetti, Andrea G O

    2013-01-01

    Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.

  1. Inference of domain-disease associations from domain-protein, protein-disease and disease-disease relationships.

    PubMed

    Zhang, Wangshu; Coba, Marcelo P; Sun, Fengzhu

    2016-01-11

    Protein domains can be viewed as portable units of biological function that defines the functional properties of proteins. Therefore, if a protein is associated with a disease, protein domains might also be associated and define disease endophenotypes. However, knowledge about such domain-disease relationships is rarely available. Thus, identification of domains associated with human diseases would greatly improve our understanding of the mechanism of human complex diseases and further improve the prevention, diagnosis and treatment of these diseases. Based on phenotypic similarities among diseases, we first group diseases into overlapping modules. We then develop a framework to infer associations between domains and diseases through known relationships between diseases and modules, domains and proteins, as well as proteins and disease modules. Different methods including Association, Maximum likelihood estimation (MLE), Domain-disease pair exclusion analysis (DPEA), Bayesian, and Parsimonious explanation (PE) approaches are developed to predict domain-disease associations. We demonstrate the effectiveness of all the five approaches via a series of validation experiments, and show the robustness of the MLE, Bayesian and PE approaches to the involved parameters. We also study the effects of disease modularization in inferring novel domain-disease associations. Through validation, the AUC (Area Under the operating characteristic Curve) scores for Bayesian, MLE, DPEA, PE, and Association approaches are 0.86, 0.84, 0.83, 0.83 and 0.79, respectively, indicating the usefulness of these approaches for predicting domain-disease relationships. Finally, we choose the Bayesian approach to infer domains associated with two common diseases, Crohn's disease and type 2 diabetes. The Bayesian approach has the best performance for the inference of domain-disease relationships. The predicted landscape between domains and diseases provides a more detailed view about the disease

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

    PubMed Central

    Lenaerts, Tom; Ferkinghoff-Borg, Jesper; Stricher, Francois; Serrano, Luis; Schymkowitz, Joost WH; Rousseau, Frederic

    2008-01-01

    Background Efficient communication between distant sites within a protein is essential for cooperative biological response. Although often associated with large allosteric movements, more subtle changes in protein dynamics can also induce long-range correlations. However, an appropriate formalism that directly relates protein structural dynamics to information exchange between functional sites is still lacking. Results Here we introduce a method to analyze protein dynamics within the framework of information theory and show that signal transduction within proteins can be considered as a particular instance of communication over a noisy channel. In particular, we analyze the conformational correlations between protein residues and apply the concept of mutual information to quantify information exchange. Mapping out changes of mutual information on the protein structure then allows visualizing how distal communication is achieved. We illustrate the approach by analyzing information transfer by the SH2 domain of Fyn tyrosine kinase, obtained from Monte Carlo dynamics simulations. Our analysis reveals that the Fyn SH2 domain forms a noisy communication channel that couples residues located in the phosphopeptide and specificity binding sites and a number of residues at the other side of the domain near the linkers that connect the SH2 domain to the SH3 and kinase domains. We find that for this particular domain, communication is affected by a series of contiguous residues that connect distal sites by crossing the core of the SH2 domain. Conclusion As a result, our method provides a means to directly map the exchange of biological information on the structure of protein domains, making it clear how binding triggers conformational changes in the protein structure. As such it provides a structural road, next to the existing attempts at sequence level, to predict long-range interactions within protein structures. PMID:18842137

  3. Automatic domain decomposition of proteins by a Gaussian Network Model.

    PubMed

    Kundu, Sibsankar; Sorensen, Dan C; Phillips, George N

    2004-12-01

    Proteins are often comprised of domains of apparently independent folding units. These domains can be defined in various ways, but one useful definition divides the protein into substructures that seem to move more or less independently. The same methods that allow fairly accurate calculation of motion can be used to help classify these substructures. We show how the Gaussian Network Model (GNM), commonly used for determining motion, can also be adapted to automatically classify domains in proteins. Parallels between this physical network model and graph theory implementation are apparent. The method is applied to a nonredundant set of 55 proteins, and the results are compared to the visual assignments by crystallographers. Apart from decomposing proteins into structural domains, the algorithm can generally be applied to any large macromolecular system to decompose it into motionally decoupled sub-systems. Copyright 2004 Wiley-Liss, Inc.

  4. Structure of synaptophysin: a hexameric MARVEL-domain channel protein.

    PubMed

    Arthur, Christopher P; Stowell, Michael H B

    2007-06-01

    Synaptophysin I (SypI) is an archetypal member of the MARVEL-domain family of integral membrane proteins and one of the first synaptic vesicle proteins to be identified and cloned. Most all MARVEL-domain proteins are involved in membrane apposition and vesicle-trafficking events, but their precise role in these processes is unclear. We have purified mammalian SypI and determined its three-dimensional (3D) structure by using electron microscopy and single-particle 3D reconstruction. The hexameric structure resembles an open basket with a large pore and tenuous interactions within the cytosolic domain. The structure suggests a model for Synaptophysin's role in fusion and recycling that is regulated by known interactions with the SNARE machinery. This 3D structure of a MARVEL-domain protein provides a structural foundation for understanding the role of these important proteins in a variety of biological processes.

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

    PubMed

    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.

  6. Domain tree-based analysis of protein architecture evolution.

    PubMed

    Forslund, Kristoffer; Henricson, Anna; Hollich, Volker; Sonnhammer, Erik L L

    2008-02-01

    Understanding the dynamics behind domain architecture evolution is of great importance to unravel the functions of proteins. Complex architectures have been created throughout evolution by rearrangement and duplication events. An interesting question is how many times a particular architecture has been created, a form of convergent evolution or domain architecture reinvention. Previous studies have approached this issue by comparing architectures found in different species. We wanted to achieve a finer-grained analysis by reconstructing protein architectures on complete domain trees. The prevalence of domain architecture reinvention in 96 genomes was investigated with a novel domain tree-based method that uses maximum parsimony for inferring ancestral protein architectures. Domain architectures were taken from Pfam. To ensure robustness, we applied the method to bootstrap trees and only considered results with strong statistical support. We detected multiple origins for 12.4% of the scored architectures. In a much smaller data set, the subset of completely domain-assigned proteins, the figure was 5.6%. These results indicate that domain architecture reinvention is a much more common phenomenon than previously thought. We also determined which domains are most frequent in multiply created architectures and assessed whether specific functions could be attributed to them. However, no strong functional bias was found in architectures with multiple origins.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26512702

  8. The evolution of filamin-a protein domain repeat perspective.

    PubMed

    Light, Sara; Sagit, Rauan; Ithychanda, Sujay S; Qin, Jun; Elofsson, Arne

    2012-09-01

    Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. The evolution of filamin – A protein domain repeat perspective

    PubMed Central

    Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

    2013-01-01

    Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. PMID:22414427

  10. Classification of Domain Movements in Proteins Using Dynamic Contact Graphs

    PubMed Central

    Taylor, Daniel; Cawley, Gavin; Hayward, Steven

    2013-01-01

    A new method for the classification of domain movements in proteins is described and applied to 1822 pairs of structures from the Protein Data Bank that represent a domain movement in two-domain proteins. The method is based on changes in contacts between residues from the two domains in moving from one conformation to the other. We argue that there are five types of elemental contact changes and that these relate to five model domain movements called: “free”, “open-closed”, “anchored”, “sliding-twist”, and “see-saw.” A directed graph is introduced called the “Dynamic Contact Graph” which represents the contact changes in a domain movement. In many cases a graph, or part of a graph, provides a clear visual metaphor for the movement it represents and is a motif that can be easily recognised. The Dynamic Contact Graphs are often comprised of disconnected subgraphs indicating independent regions which may play different roles in the domain movement. The Dynamic Contact Graph for each domain movement is decomposed into elemental Dynamic Contact Graphs, those that represent elemental contact changes, allowing us to count the number of instances of each type of elemental contact change in the domain movement. This naturally leads to sixteen classes into which the 1822 domain movements are classified. PMID:24260562

  11. Classification of domain movements in proteins using dynamic contact graphs.

    PubMed

    Taylor, Daniel; Cawley, Gavin; Hayward, Steven

    2013-01-01

    A new method for the classification of domain movements in proteins is described and applied to 1822 pairs of structures from the Protein Data Bank that represent a domain movement in two-domain proteins. The method is based on changes in contacts between residues from the two domains in moving from one conformation to the other. We argue that there are five types of elemental contact changes and that these relate to five model domain movements called: "free", "open-closed", "anchored", "sliding-twist", and "see-saw." A directed graph is introduced called the "Dynamic Contact Graph" which represents the contact changes in a domain movement. In many cases a graph, or part of a graph, provides a clear visual metaphor for the movement it represents and is a motif that can be easily recognised. The Dynamic Contact Graphs are often comprised of disconnected subgraphs indicating independent regions which may play different roles in the domain movement. The Dynamic Contact Graph for each domain movement is decomposed into elemental Dynamic Contact Graphs, those that represent elemental contact changes, allowing us to count the number of instances of each type of elemental contact change in the domain movement. This naturally leads to sixteen classes into which the 1822 domain movements are classified.

  12. Selection of soluble protein expression constructs: the experimental determination of protein domain boundaries.

    PubMed

    Dyson, Michael R

    2010-08-01

    Proteins can contain multiple domains each of which is capable of possessing a separate independent function and three-dimensional structure. It is often useful to clone and express individual protein domains to study their biochemical properties and for structure determination. However, the annotated domain boundaries in databases such as Pfam or SMART are not always accurate. The present review summarizes various strategies for the experimental determination of protein domain boundaries.

  13. Exogenous agents that target transmembrane domains of proteins.

    PubMed

    Yin, Hang

    2008-01-01

    Although membrane proteins account for approximately one third of all proteins encoded in the human genome, the functions and structures of their transmembrane domains are much less understood than the water-soluble regions. A major hurdle in studying these transmembrane domains is the lack of appropriate exogenous agents that can be used as specific probes. Despite the daunting challenges, major strides have recently been made in targeting the transmembrane domains of a variety of membrane proteins. High affinity and selectivity have been achieved in model biophysical systems, membranes of bacteria, and mammalian cells.

  14. Cryptic protein-protein interaction motifs in the cytoplasmic domain of MHCI proteins.

    PubMed

    Frietze, Karla K; Pappy, Adlai L; Melson, Jack W; O'Driscoll, Emily E; Tyler, Carolyn M; Perlman, David H; Boulanger, Lisa M

    2016-07-19

    Major histocompatibility complex class I (MHCI) proteins present antigenic peptides for immune surveillance and play critical roles in nervous system development and plasticity. Most MHCI are transmembrane proteins. The extracellular domain of MHCI interacts with immunoreceptors, peptides, and co-receptors to mediate immune signaling. While the cytoplasmic domain also plays important roles in endocytic trafficking, cross-presentation of extracellularly derived antigens, and CTL priming, the molecular mediators of cytoplasmic signaling by MHCI remain largely unknown. Here we show that the cytoplasmic domain of MHCI contains putative protein-protein interaction domains known as PDZ (PSD95/disc large/zonula occludens-1) ligands. PDZ ligands are motifs that bind to PDZ domains to organize and mediate signaling at cell-cell contacts. PDZ ligands are short, degenerate motifs, and are therefore difficult to identify via sequence homology alone, but several lines of evidence suggest that putative PDZ ligand motifs in MHCI are under positive selective pressure. Putative PDZ ligands are found in all of the 99 MHCI proteins examined from diverse species, and are enriched in the cytoplasmic domain, where PDZ interactions occur. Both the position of the PDZ ligand and the class of ligand motif are conserved across species, as well as among genes within a species. Non-synonymous substitutions, when they occur, frequently preserve the motif. Of the many specific possible PDZ ligand motifs, a handful are strikingly and selectively overrepresented in MHCI's cytoplasmic domain, but not elsewhere in the same proteins. Putative PDZ ligands in MHCI encompass conserved serine and tyrosine residues that are targets of phosphorylation, a post-translational modification that can regulate PDZ interactions. Finally, proof-of-principle in vitro interaction assays demonstrate that the cytoplasmic domains of particular MHCI proteins can bind directly and specifically to PDZ1 and PDZ4&5 of MAGI

  15. An ambiguity principle for assigning protein structural domains.

    PubMed

    Postic, Guillaume; Ghouzam, Yassine; Chebrek, Romain; Gelly, Jean-Christophe

    2017-01-01

    Ambiguity is the quality of being open to several interpretations. For an image, it arises when the contained elements can be delimited in two or more distinct ways, which may cause confusion. We postulate that it also applies to the analysis of protein three-dimensional structure, which consists in dividing the molecule into subunits called domains. Because different definitions of what constitutes a domain can be used to partition a given structure, the same protein may have different but equally valid domain annotations. However, knowledge and experience generally displace our ability to accept more than one way to decompose the structure of an object-in this case, a protein. This human bias in structure analysis is particularly harmful because it leads to ignoring potential avenues of research. We present an automated method capable of producing multiple alternative decompositions of protein structure (web server and source code available at www.dsimb.inserm.fr/sword/). Our innovative algorithm assigns structural domains through the hierarchical merging of protein units, which are evolutionarily preserved substructures that describe protein architecture at an intermediate level, between domain and secondary structure. To validate the use of these protein units for decomposing protein structures into domains, we set up an extensive benchmark made of expert annotations of structural domains and including state-of-the-art domain parsing algorithms. The relevance of our "multipartitioning" approach is shown through numerous examples of applications covering protein function, evolution, folding, and structure prediction. Finally, we introduce a measure for the structural ambiguity of protein molecules.

  16. An ambiguity principle for assigning protein structural domains

    PubMed Central

    Postic, Guillaume; Ghouzam, Yassine; Chebrek, Romain; Gelly, Jean-Christophe

    2017-01-01

    Ambiguity is the quality of being open to several interpretations. For an image, it arises when the contained elements can be delimited in two or more distinct ways, which may cause confusion. We postulate that it also applies to the analysis of protein three-dimensional structure, which consists in dividing the molecule into subunits called domains. Because different definitions of what constitutes a domain can be used to partition a given structure, the same protein may have different but equally valid domain annotations. However, knowledge and experience generally displace our ability to accept more than one way to decompose the structure of an object—in this case, a protein. This human bias in structure analysis is particularly harmful because it leads to ignoring potential avenues of research. We present an automated method capable of producing multiple alternative decompositions of protein structure (web server and source code available at www.dsimb.inserm.fr/sword/). Our innovative algorithm assigns structural domains through the hierarchical merging of protein units, which are evolutionarily preserved substructures that describe protein architecture at an intermediate level, between domain and secondary structure. To validate the use of these protein units for decomposing protein structures into domains, we set up an extensive benchmark made of expert annotations of structural domains and including state-of-the-art domain parsing algorithms. The relevance of our “multipartitioning” approach is shown through numerous examples of applications covering protein function, evolution, folding, and structure prediction. Finally, we introduce a measure for the structural ambiguity of protein molecules. PMID:28097215

  17. Domain view: a web tool for protein domain visualization and analysis.

    PubMed

    Pan, Xiaokang; Bingman, Craig A; Wesenberg, Gary E; Sun, Zhaohui; Phillips, George N

    2010-12-01

    The identification of sequence-based protein domains and their boundaries is often a prelude to structure determination. An accurate prediction of disordered regions, secondary structures and low complexity segments of target protein sequences can improve the efficiency of selection in structural genomics and also aid in design of constructs for directed structural biology studies. At the Center for Eukaryotic Structural Genomics (CESG) we have developed DomainView, a web tool to visualize and analyze predicted protein domains, disordered regions, secondary structures and low complexity segments of target protein sequences for selection of experimental protein structure attempts. DomainView consists of a relational database and a web graphical-user interface. The database was developed based on MySQL, which stores data from target protein sequences and their domains, disordered regions, secondary structures and low complexity segments. The program of the web user interface is a Perl CGI script. When a user searches for a target protein sequence, the script displays the combinational information about the domains and other features of that target sequence graphically on a web page by querying the database. The graphical representation for each feature is linked to a web page showing more detailed annotation information or to a new window directly running the corresponding prediction program to show further information about that feature.

  18. 14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response.

    PubMed

    Mohammad, Duaa H; Yaffe, Michael B

    2009-09-02

    The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phosphorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G(2)/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.

  19. Defining subdomains of the K domain important for protein-protein interactions of plant MADS proteins.

    PubMed

    Yang, Yingzhen; Jack, Thomas

    2004-05-01

    The MADS proteins APETALA3 (AP3), PISTILLATA (PI), SEPALLATAI (SEPI), SEP2, SEP3, AGAMOUS, and APETALA are required for proper floral organ identity in Arabidopsis flowers. All of these floral MADS proteins conserve two domains: the MADS domain that mediates DNA binding and dimerization, and the K domain that mediates protein protein interaction. The K domain is postulated to form a several amphipathic c-helices referred to as K1, K2, and K3. The K1 and K2 helicies are located entirely within the K domain while the K3 helix spans the K domain-C domain boundary. Here we report on our studies on the interactions of the B class MADS proteins AP3 and PI with the E class MADS proteins SEP1, SEP2, and SEP3. A comparative analysis of mutants in the K domain reveals that the subdomains mediating the PI/AP3 interaction are different from the subdomains mediating the PI/SEP3 (or PI/SEP1) interaction. The strong PI/SEP3 (or PI/SEP1) interaction requires K2, part of K3, and the interhelical region between K1 and K2. By contrast, K1, K2 and the region between K1 and K2 are important for strong AP3/PI interaction. Most of the K3 helix does not appear to be important for either the PI/AP3 or the PI/SEP3 (or PI/SEP1) interaction. Conserved hydrophobic positions are most important for the strength of both PI/AP3 and PI/SEP3 dimerization, though ionic and/or polar interactions appear to play a secondary role.

  20. Proteasomes and protein conjugation across domains of life.

    PubMed

    Maupin-Furlow, Julie

    2011-12-19

    Like other energy-dependent proteases, proteasomes, which are found across the three domains of life, are self-compartmentalized and important in the early steps of proteolysis. Proteasomes degrade improperly synthesized, damaged or misfolded proteins and hydrolyse regulatory proteins that must be specifically removed or cleaved for cell signalling. In eukaryotes, proteins are typically targeted for proteasome-mediated destruction through polyubiquitylation, although ubiquitin-independent pathways also exist. Interestingly, actinobacteria and archaea also covalently attach small proteins (prokaryotic ubiquitin-like protein (Pup) and small archaeal modifier proteins (Samps), respectively) to certain proteins, and this may serve to target the modified proteins for degradation by proteasomes.

  1. Identification of structural domains in proteins by a graph heuristic.

    PubMed

    Wernisch, L; Hunting, M; Wodak, S J

    1999-05-15

    A novel automatic procedure for identifying domains from protein atomic coordinates is presented. The procedure, termed STRUDL (STRUctural Domain Limits), does not take into account information on secondary structures and handles any number of domains made up of contiguous or non-contiguous chain segments. The core algorithm uses the Kernighan-Lin graph heuristic to partition the protein into residue sets which display minimum interactions between them. These interactions are deduced from the weighted Voronoi diagram. The generated partitions are accepted or rejected on the basis of optimized criteria, representing basic expected physical properties of structural domains. The graph heuristic approach is shown to be very effective, it approximates closely the exact solution provided by a branch and bound algorithm for a number of test proteins. In addition, the overall performance of STRUDL is assessed on a set of 787 representative proteins from the Protein Data Bank by comparison to domain definitions in the CATH protein classification. The domains assigned by STRUDL agree with the CATH assignments in at least 81% of the tested proteins. This result is comparable to that obtained previously using PUU (Holm and Sander, Proteins 1994;9:256-268), the only other available algorithm designed to identify domains with any number of non-contiguous chain segments. A detailed discussion of the structures for which our assignments differ from those in CATH brings to light some clear inconsistencies between the concept of structural domains based on minimizing inter-domain interactions and that of delimiting structural motifs that represent acceptable folding topologies or architectures. Considering both concepts as complementary and combining them in a layered approach might be the way forward.

  2. Distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins.

    PubMed

    Brändle, Simone M; Obermeier, Birgit; Senel, Makbule; Bruder, Jessica; Mentele, Reinhard; Khademi, Mohsen; Olsson, Tomas; Tumani, Hayrettin; Kristoferitsch, Wolfgang; Lottspeich, Friedrich; Wekerle, Hartmut; Hohlfeld, Reinhard; Dornmair, Klaus

    2016-07-12

    Oligoclonal Ig bands (OCBs) of the cerebrospinal fluid are a hallmark of multiple sclerosis (MS), a disabling inflammatory disease of the central nervous system (CNS). OCBs are locally produced by clonally expanded antigen-experienced B cells and therefore are believed to hold an important clue to the pathogenesis. However, their target antigens have remained unknown, mainly because it was thus far not possible to isolate distinct OCBs against a background of polyclonal antibodies. To overcome this obstacle, we copurified disulfide-linked Ig heavy and light chains from distinct OCBs for concurrent analysis by mass spectrometry and aligned patient-specific peptides to corresponding transcriptome databases. This method revealed the full-length sequences of matching chains from distinct OCBs, allowing for antigen searches using recombinant OCB antibodies. As validation, we demonstrate that an OCB antibody from a patient with an infectious CNS disorder, neuroborreliosis, recognized a Borrelia protein. Next, we produced six recombinant antibodies from four MS patients and identified three different autoantigens. All of them are conformational epitopes of ubiquitous intracellular proteins not specific to brain tissue. Our findings indicate that the B-cell response in MS is heterogeneous and partly directed against intracellular autoantigens released during tissue destruction. In addition to helping elucidate the role of B cells in MS, our approach allows the identification of target antigens of OCB antibodies in other neuroinflammatory diseases and the production of therapeutic antibodies in infectious CNS diseases.

  3. Autism-specific maternal autoantibodies recognize critical proteins in developing brain

    PubMed Central

    Braunschweig, D; Krakowiak, P; Duncanson, P; Boyce, R; Hansen, R L; Ashwood, P; Hertz-Picciotto, I; Pessah, I N; Van de Water, J

    2013-01-01

    Autism spectrum disorders (ASDs) are neurodevelopmental in origin, affecting an estimated 1 in 88 children in the United States. We previously described ASD-specific maternal autoantibodies that recognize fetal brain antigens. Herein, we demonstrate that lactate dehydrogenase A and B (LDH), cypin, stress-induced phosphoprotein 1 (STIP1), collapsin response mediator proteins 1 and 2 (CRMP1, CRMP2) and Y-box-binding protein to comprise the seven primary antigens of maternal autoantibody-related (MAR) autism. Exclusive reactivity to specific antigen combinations was noted in 23% of mothers of ASD children and only 1% of controls. ASD children from mothers with specific reactivity to LDH, STIP1 and CRMP1 and/or cypin (7% vs 0% in controls; P<0.0002; odds ratios of 24.2 (95% confidence interval: 1.45–405)) had elevated stereotypical behaviors compared with ASD children from mothers lacking these antibodies. We describe the first panel of clinically significant biomarkers with over 99% specificity for autism risk thereby advancing our understanding of the etiologic mechanisms and therapeutic possibilities for MAR autism. PMID:23838888

  4. Activation of Holliday junction recognizing protein involved in the chromosomal stability and immortality of cancer cells.

    PubMed

    Kato, Tatsuya; Sato, Nagato; Hayama, Satoshi; Yamabuki, Takumi; Ito, Tomoo; Miyamoto, Masaki; Kondo, Satoshi; Nakamura, Yusuke; Daigo, Yataro

    2007-09-15

    We identified a novel gene HJURP (Holliday junction-recognizing protein) whose activation seemed to play a pivotal role in the immortality of cancer cells. HJURP was considered a possible downstream target for ataxia telangiectasia mutated signaling, and its expression was increased by DNA double-strand breaks (DSB). HJURP was involved in the homologous recombination pathway in the DSB repair process through interaction with hMSH5 and NBS1, which is a part of the MRN protein complex. HJURP formed nuclear foci in cells at S phase and those subjected to DNA damage. In vitro assays implied that HJURP bound directly to the Holliday junction and rDNA arrays. Treatment of cancer cells with small interfering RNA (siRNA) against HJURP caused abnormal chromosomal fusions and led to genomic instability and senescence. In addition, HJURP overexpression was observed in a majority of lung cancers and was associated with poor prognosis as well. We suggest that HJURP is an indispensable factor for chromosomal stability in immortalized cancer cells and is a potential novel therapeutic target for the development of anticancer drugs.

  5. Distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins

    PubMed Central

    Brändle, Simone M.; Obermeier, Birgit; Senel, Makbule; Bruder, Jessica; Mentele, Reinhard; Khademi, Mohsen; Olsson, Tomas; Tumani, Hayrettin; Kristoferitsch, Wolfgang; Lottspeich, Friedrich; Wekerle, Hartmut; Hohlfeld, Reinhard; Dornmair, Klaus

    2016-01-01

    Oligoclonal Ig bands (OCBs) of the cerebrospinal fluid are a hallmark of multiple sclerosis (MS), a disabling inflammatory disease of the central nervous system (CNS). OCBs are locally produced by clonally expanded antigen-experienced B cells and therefore are believed to hold an important clue to the pathogenesis. However, their target antigens have remained unknown, mainly because it was thus far not possible to isolate distinct OCBs against a background of polyclonal antibodies. To overcome this obstacle, we copurified disulfide-linked Ig heavy and light chains from distinct OCBs for concurrent analysis by mass spectrometry and aligned patient-specific peptides to corresponding transcriptome databases. This method revealed the full-length sequences of matching chains from distinct OCBs, allowing for antigen searches using recombinant OCB antibodies. As validation, we demonstrate that an OCB antibody from a patient with an infectious CNS disorder, neuroborreliosis, recognized a Borrelia protein. Next, we produced six recombinant antibodies from four MS patients and identified three different autoantigens. All of them are conformational epitopes of ubiquitous intracellular proteins not specific to brain tissue. Our findings indicate that the B-cell response in MS is heterogeneous and partly directed against intracellular autoantigens released during tissue destruction. In addition to helping elucidate the role of B cells in MS, our approach allows the identification of target antigens of OCB antibodies in other neuroinflammatory diseases and the production of therapeutic antibodies in infectious CNS diseases. PMID:27325759

  6. Autoantibodies in canine masticatory muscle myositis recognize a novel myosin binding protein-C family member.

    PubMed

    Wu, Xiaohua; Li, Zhi-fang; Brooks, Randolph; Komives, Elizabeth A; Torpey, Justin W; Engvall, Eva; Gonias, Steven L; Shelton, G Diane

    2007-10-01

    Inflammatory myopathies are a group of autoimmune diseases that affect muscles. In humans, the most common inflammatory myopathies are polymyositis, dermatomyositis, and inclusion body myositis. Autoantibodies may be found in humans with inflammatory myopathies, and these play an important role in diagnosis and disease classification. However, these Abs are typically not muscle specific. Spontaneously occurring canine inflammatory myopathies may be good parallel disorders and provide insights into human myositis. In dogs with inflammatory myopathy, muscle-specific autoantibodies have been found, especially in masticatory muscle myositis. We have identified the major Ag recognized by the autoantibodies in canine masticatory muscle myositis. This Ag is a novel member of the myosin binding protein-C family, which we call masticatory myosin binding protein-C (mMyBP-C). mMyBP-C is localized not only within the masticatory muscle fibers, but also at or near their cell surface, perhaps making it accessible as an immunogen. The gene for mMyBP-C also exists in humans, and mMyBP-C could potentially play a role in certain human inflammatory myopathies. Understanding the role of mMyBP-C in this canine inflammatory myopathy may advance our knowledge of mechanisms of autoimmune inflammatory muscle diseases, not only in dogs, but also in humans.

  7. Domain fusion analysis by applying relational algebra to protein sequence and domain databases

    PubMed Central

    Truong, Kevin; Ikura, Mitsuhiko

    2003-01-01

    Background Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. Results This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at . Conclusion As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time. PMID:12734020

  8. Domain fusion analysis by applying relational algebra to protein sequence and domain databases.

    PubMed

    Truong, Kevin; Ikura, Mitsuhiko

    2003-05-06

    Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at http://calcium.uhnres.utoronto.ca/pi. As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time.

  9. RNA polymerase II conserved protein domains as platforms for protein-protein interactions

    PubMed Central

    García-López, M Carmen

    2011-01-01

    RNA polymerase II establishes many protein-protein interactions with transcriptional regulators to coordinate gene expression, but little is known about protein domains involved in the contact with them. We use a new approach to look for conserved regions of the RNA pol II of S. cerevisiae located at the surface of the structure of the complex, hypothesizing that they might be involved in the interaction with transcriptional regulators. We defined five different conserved domains and demonstrate that all of them make contact with transcriptional regulators. PMID:21922063

  10. EVEREST: a collection of evolutionary conserved protein domains

    PubMed Central

    Portugaly, Elon; Linial, Nathan; Linial, Michal

    2007-01-01

    Protein domains are subunits of proteins that recur throughout the protein world. There are many definitions attempting to capture the essence of a protein domain, and several systems that identify protein domains and classify them into families. EVEREST, recently described in Portugaly et al. (2006) BMC Bioinformatics, 7, 277, is one such system that performs the task automatically, using protein sequence alone. Herein we describe EVEREST release 2.0, consisting of 20 029 families, each defined by one or more HMMs. The current EVEREST database was constructed by scanning UniProt 8.1 and all PDB sequences (total over 3 000 000 sequences) with each of the EVEREST families. EVEREST annotates 64% of all sequences, and covers 59% of all residues. EVEREST is available at . The website provides annotations given by SCOP, CATH, Pfam A and EVEREST. It allows for browsing through the families of each of those sources, graphically visualizing the domain organization of the proteins in the family. The website also provides access to analyzes of relationships between domain families, within and across domain definition systems. Users can upload sequences for analysis by the set of EVEREST families. Finally an advanced search form allows querying for families matching criteria regarding novelty, phylogenetic composition and more. PMID:17099230

  11. EVEREST: a collection of evolutionary conserved protein domains.

    PubMed

    Portugaly, Elon; Linial, Nathan; Linial, Michal

    2007-01-01

    Protein domains are subunits of proteins that recur throughout the protein world. There are many definitions attempting to capture the essence of a protein domain, and several systems that identify protein domains and classify them into families. EVEREST, recently described in Portugaly et al. (2006) BMC Bioinformatics, 7, 277, is one such system that performs the task automatically, using protein sequence alone. Herein we describe EVEREST release 2.0, consisting of 20,029 families, each defined by one or more HMMs. The current EVEREST database was constructed by scanning UniProt 8.1 and all PDB sequences (total over 3,000,000 sequences) with each of the EVEREST families. EVEREST annotates 64% of all sequences, and covers 59% of all residues. EVEREST is available at http://www.everest.cs.huji.ac.il/. The website provides annotations given by SCOP, CATH, Pfam A and EVEREST. It allows for browsing through the families of each of those sources, graphically visualizing the domain organization of the proteins in the family. The website also provides access to analyzes of relationships between domain families, within and across domain definition systems. Users can upload sequences for analysis by the set of EVEREST families. Finally an advanced search form allows querying for families matching criteria regarding novelty, phylogenetic composition and more.

  12. CDD: a Conserved Domain Database for protein classification.

    PubMed

    Marchler-Bauer, Aron; Anderson, John B; Cherukuri, Praveen F; DeWeese-Scott, Carol; Geer, Lewis Y; Gwadz, Marc; He, Siqian; Hurwitz, David I; Jackson, John D; Ke, Zhaoxi; Lanczycki, Christopher J; Liebert, Cynthia A; Liu, Chunlei; Lu, Fu; Marchler, Gabriele H; Mullokandov, Mikhail; Shoemaker, Benjamin A; Simonyan, Vahan; Song, James S; Thiessen, Paul A; Yamashita, Roxanne A; Yin, Jodie J; Zhang, Dachuan; Bryant, Stephen H

    2005-01-01

    The Conserved Domain Database (CDD) is the protein classification component of NCBI's Entrez query and retrieval system. CDD is linked to other Entrez databases such as Proteins, Taxonomy and PubMed, and can be accessed at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. CD-Search, which is available at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, is a fast, interactive tool to identify conserved domains in new protein sequences. CD-Search results for protein sequences in Entrez are pre-computed to provide links between proteins and domain models, and computational annotation visible upon request. Protein-protein queries submitted to NCBI's BLAST search service at http://www.ncbi.nlm.nih.gov/BLAST are scanned for the presence of conserved domains by default. While CDD started out as essentially a mirror of publicly available domain alignment collections, such as SMART, Pfam and COG, we have continued an effort to update, and in some cases replace these models with domain hierarchies curated at the NCBI. Here, we report on the progress of the curation effort and associated improvements in the functionality of the CDD information retrieval system.

  13. Targeting of passenger protein domains to multiple intracellular membranes.

    PubMed Central

    Janiak, F; Glover, J R; Leber, B; Rachubinski, R A; Andrews, D W

    1994-01-01

    The role of passenger domains in protein targeting was examined by fusing previously characterized targeting motifs to different protein sequences. To compare the targeting requirements for a variety of subcellular compartments, targeting of the fusion proteins was examined for endoplasmic reticulum, mitochondria and peroxisomes in vitro and in yeast. Although most passenger domains were only partially passive to translocation, motif-dependent targeting via motifs positioned at either end of one passenger domain (gPA) was demonstrated for all of the subcellular compartments tested. The data presented extend earlier suggestions that translocation competence is an intrinsic property of the passenger protein. However, the properties that determine protein targeting are not mutually exclusive for the compartments tested. Therefore, although the primary determinant of specificity is the targeting motif, our results suggest that translocation competence of the targeted protein augments the fidelity of transport. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8198533

  14. Isolation of a monoclonal antibody that recognizes the origin binding domain of JCV, but not SV40, large T-antigen.

    PubMed

    Grubman, Shelley A; Shin, Jong; Phelan, Paul J; Gong, Aaron; Can, Hande; Dilworth, Ryan; Kini, Sandeep Kuntadi; Gagnon, David; Archambault, Jacques; Meinke, Gretchen; Bohm, Andrew; Jefferson, Douglas M; Bullock, Peter A

    2016-10-01

    Within immunocompromised populations, the JC polyomavirus is the cause of the often-fatal disease Progressive Multifocal Leukoencephalopathy (PML). JC virus encodes a protein, termed T-antigen (T-ag), which is essential for its replication and pathogenicity. Previous studies of JCV T-ag have, in general, used antibodies raised against SV40 T-ag. Unfortunately, SV40 T-ag is also detected in humans and therefore there have been concerns about cross-reactivity. To address this issue, we have isolated a monoclonal antibody that binds to the JCV, but not the SV40, T-ag origin-binding domain (OBD). Furthermore, the region on the surface of the JCV T-ag OBD that is recognized by the "anti-JCV OBD mAb" has been mapped. We also demonstrate that the "anti-JCV OBD mAb" will be a useful reagent for standard techniques (e.g., Westerns blots and ELISAs). Finally, we note that additional monoclonal Abs that are specific for the T-ags encoded by the other human polyomaviruses could be generated by adopting the approach described herein.

  15. Assembly of cell regulatory systems through protein interaction domains.

    PubMed

    Pawson, Tony; Nash, Piers

    2003-04-18

    The sequencing of complete genomes provides a list that includes the proteins responsible for cellular regulation. However, this does not immediately reveal what these proteins do, nor how they are assembled into the molecular machines and functional networks that control cellular behavior. The regulation of many different cellular processes requires the use of protein interaction domains to direct the association of polypeptides with one another and with phospholipids, small molecules, or nucleic acids. The modular nature of these domains, and the flexibility of their binding properties, have likely facilitated the evolution of cellular pathways. Conversely, aberrant interactions can induce abnormal cellular behavior and disease. The fundamental properties of protein interaction domains are discussed in this review and in detailed reviews on individual domains at Science's STKE at http://www.sciencemag.org/cgi/content/full/300/5618/445/DC1.

  16. Evolution of a protein domain interaction network

    NASA Astrophysics Data System (ADS)

    Gao, Li-Feng; Shi, Jian-Jun; Guan, Shan

    2010-01-01

    In this paper, we attempt to understand complex network evolution from the underlying evolutionary relationship between biological organisms. Firstly, we construct a Pfam domain interaction network for each of the 470 completely sequenced organisms, and therefore each organism is correlated with a specific Pfam domain interaction network; secondly, we infer the evolutionary relationship of these organisms with the nearest neighbour joining method; thirdly, we use the evolutionary relationship between organisms constructed in the second step as the evolutionary course of the Pfam domain interaction network constructed in the first step. This analysis of the evolutionary course shows: (i) there is a conserved sub-network structure in network evolution; in this sub-network, nodes with lower degree prefer to maintain their connectivity invariant, and hubs tend to maintain their role as a hub is attached preferentially to new added nodes; (ii) few nodes are conserved as hubs; most of the other nodes are conserved as one with very low degree; (iii) in the course of network evolution, new nodes are added to the network either individually in most cases or as clusters with relative high clustering coefficients in a very few cases.

  17. Tandem-repeat protein domains across the tree of life

    PubMed Central

    Jernigan, Kristin K.

    2015-01-01

    Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20–40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny. In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins. We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life. A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species. PMID:25653910

  18. de Gennes Narrowing Describes the Relative Motion of Protein Domains

    NASA Astrophysics Data System (ADS)

    Hong, Liang; Smolin, Nikolai; Smith, Jeremy C.

    2014-04-01

    The relative motion of structural domains is essential for the biological function of many proteins. Here, by analyzing neutron scattering data and performing molecular dynamics simulations, we find that interdomain motion in several proteins obeys the principle of de Gennes narrowing, in which the wave vector dependence of the interdomain diffusion coefficient is inversely proportional to the interdomain structure factor. Thus, the rate of interdomain motion is inversely proportional to the probability distribution of the spatial configurations of domains.

  19. Identification of immunoreactive proteins of Dirofilaria immitis and D. repens recognized by sera from patients with pulmonary and subcutaneous dirofilariosis.

    PubMed

    González-Miguel, Javier; Rosario, Luis; Rota-Nodari, Elena; Morchón, Rodrigo; Simón, Fernando

    2010-06-01

    Human pulmonary and subcutaneous dirofilariosis caused by Dirofilaria immitis and Dirofilaria repens are worldwide diagnosed with increasing frequency. These species are responsible for the development of benign pulmonary and subcutaneous nodules, respectively, that can be confused with lung or cutaneous cancer. The aim of the present work was to identify D. immitis and D. repens proteins differentially recognized by serum samples from individuals with human pulmonary and subcutaneous dirofilariosis, using two-dimensional electrophoresis and mass spectrometry. Twenty-three immunoreactive proteins of D. immitis and 15 of D. repens were identified. The results point to the existence of differential antigenic recognition in each species, both in the number and type of proteins recognized. Individuals with pulmonary dirofilariosis recognized, on the proteome of D. immitis, among others, different isoforms of 6 enzymes involved in glycolysis, 3 redox-related proteins with antioxidant capacity and 3 heat shock proteins. Individuals with subcutaneous dirofilariosis recognized on the proteome of D. repens only 3 glycolytic enzymes, one protein involved in redox processes and one heat shock protein. These data suggest that in cases of pulmonary dirofilariosis there exists a wider recognition of immunoreactive D. immitis proteins related to key survival processes, such as energy generation, the struggle against oxidative stress and molecular repair, than in cases of human subcutaneous dirofilariosis against D. repens. This could contribute to explain the differences described in the capacity of D. immitis and D. repens development and in the frequency of occurrence of pulmonary and subcutaneous dirofilariosis in the human host.

  20. Polypeptide Modulators of Caspase Recruitment Domain (CARD)-CARD-mediated Protein-Protein Interactions*

    PubMed Central

    Palacios-Rodríguez, Yadira; García-Laínez, Guillermo; Sancho, Mónica; Gortat, Anna; Orzáez, Mar; Pérez-Payá, Enrique

    2011-01-01

    The caspase recruitment domain (CARD) is present in a large number of proteins. Initially, the CARD was recognized as part of the caspase activation machinery. CARD-CARD interactions play a role in apoptosis and are responsible for the Apaf-1-mediated activation of procaspase-9 in the apoptosome. CARD-containing proteins mediate the inflammasome-dependent activation of proinflammatory caspase-1. More recently, new roles for CARD-containing proteins have been reported in signaling pathways associated with immune responses. The functional role of CARD-containing proteins and CARDs in coordinating apoptosis and inflammatory and immune responses is not completely understood. We have explored the putative cross-talk between apoptosis and inflammation by analyzing the modulatory activity on both the Apaf-1/procaspase-9 interaction and the inflammasome-mediated procaspase-1 activation of CARD-derived polypeptides. To this end, we analyzed the activity of individual recombinant CARDs, rationally designed CARD-derived peptides, and peptides derived from phage display. PMID:22065589

  1. Stochastic lattice model of synaptic membrane protein domains

    NASA Astrophysics Data System (ADS)

    Li, Yiwei; Kahraman, Osman; Haselwandter, Christoph A.

    2017-05-01

    Neurotransmitter receptor molecules, concentrated in synaptic membrane domains along with scaffolds and other kinds of proteins, are crucial for signal transmission across chemical synapses. In common with other membrane protein domains, synaptic domains are characterized by low protein copy numbers and protein crowding, with rapid stochastic turnover of individual molecules. We study here in detail a stochastic lattice model of the receptor-scaffold reaction-diffusion dynamics at synaptic domains that was found previously to capture, at the mean-field level, the self-assembly, stability, and characteristic size of synaptic domains observed in experiments. We show that our stochastic lattice model yields quantitative agreement with mean-field models of nonlinear diffusion in crowded membranes. Through a combination of analytic and numerical solutions of the master equation governing the reaction dynamics at synaptic domains, together with kinetic Monte Carlo simulations, we find substantial discrepancies between mean-field and stochastic models for the reaction dynamics at synaptic domains. Based on the reaction and diffusion properties of synaptic receptors and scaffolds suggested by previous experiments and mean-field calculations, we show that the stochastic reaction-diffusion dynamics of synaptic receptors and scaffolds provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the observed single-molecule trajectories, and spatial heterogeneity in the effective rates at which receptors and scaffolds are recycled at the cell membrane. Our work sheds light on the physical mechanisms and principles linking the collective properties of membrane protein domains to the stochastic dynamics that rule their molecular components.

  2. Singlet CH domain containing human multidomain proteins: an inventory.

    PubMed

    Friedberg, Felix

    2010-03-01

    The actin cytoskeleton presents the basic force in processes such as cytokinesis, endocytosis, vesicular trafficking and cell migration. Here, we list 30 human singlet CH (calpononin homology/actin binding) containing multidomain molecules, each encoded by one gene. We show the domain distributions as given by the SMART program. These mosaic proteins organize geographically the placement of selected proteins in proximity within the cell. In most instances, their precise location, their actin binding capacity by way of the singlet CH (or by other domains?) and their physiological functions need further elucidation. A dendrogram based solely on the relationship for the human singlet CH domains (in terms of AA sequences) for the various molecules that possess the domain, implies that the singlet descended from a common ancestor which in turn sprouted three main branches of protein products. Each branch bifurcated multiple times thus accounting for a cornucopia of products. Wherever, additional (unassigned), highly homologous regions exist in related proteins (e.g., in LIM and LMO7 or in Tangerin and EH/BP1), these unrecognized domain regions await assignment as specific functional domains. Frequently genes coding multidomain proteins duplicated. The varying modular nature within multidomain proteins should have accelerated evolutionary changes to a degree not feasible to achieve by means of mere post-duplication mutational changes.

  3. FANCI protein binds to DNA and interacts with FANCD2 to recognize branched structures.

    PubMed

    Yuan, Fenghua; El Hokayem, Jimmy; Zhou, Wen; Zhang, Yanbin

    2009-09-04

    In this study, we report that the purified wild-type FANCI (Fanconi anemia complementation group I) protein directly binds to a variety of DNA substrates. The DNA binding domain roughly encompasses residues 200-1000, as suggested by the truncation study. When co-expressed in insect cells, a small fraction of FANCI forms a stable complex with FANCD2 (Fanconi anemia complementation group D2). Intriguingly, the purified FANCI-FANCD2 complex preferentially binds to the branched DNA structures when compared with either FANCI or FANCD2 alone. Co-immunoprecipitation with purified proteins indicates that FANCI interacts with FANCD2 through its C-terminal amino acid 1001-1328 fragment. Although the C terminus of FANCI is dispensable for direct DNA binding, it seems to be involved in the regulation of DNA binding activity. This notion is further enhanced by two C-terminal point mutations, R1285Q and D1301A, which showed differentiated DNA binding activity. We also demonstrate that FANCI forms discrete nuclear foci in HeLa cells in the absence or presence of exogenous DNA damage. The FANCI foci are colocalized perfectly with FANCD2 and partially with proliferating cell nuclear antigen irrespective of mitomycin C treatment. An increased number of FANCI foci form and become resistant to Triton X extraction in response to mitomycin C treatment. Our data suggest that the FANCI-FANCD2 complex may participate in repair of damaged replication forks through its preferential recognition of branched structures.

  4. Conditional random field approach to prediction of protein-protein interactions using domain information.

    PubMed

    Hayashida, Morihiro; Kamada, Mayumi; Song, Jiangning; Akutsu, Tatsuya

    2011-06-20

    For understanding cellular systems and biological networks, it is important to analyze functions and interactions of proteins and domains. Many methods for predicting protein-protein interactions have been developed. It is known that mutual information between residues at interacting sites can be higher than that at non-interacting sites. It is based on the thought that amino acid residues at interacting sites have coevolved with those at the corresponding residues in the partner proteins. Several studies have shown that such mutual information is useful for identifying contact residues in interacting proteins. We propose novel methods using conditional random fields for predicting protein-protein interactions. We focus on the mutual information between residues, and combine it with conditional random fields. In the methods, protein-protein interactions are modeled using domain-domain interactions. We perform computational experiments using protein-protein interaction datasets for several organisms, and calculate AUC (Area Under ROC Curve) score. The results suggest that our proposed methods with and without mutual information outperform EM (Expectation Maximization) method proposed by Deng et al., which is one of the best predictors based on domain-domain interactions. We propose novel methods using conditional random fields with and without mutual information between domains. Our methods based on domain-domain interactions are useful for predicting protein-protein interactions.

  5. Anchors aweigh: protein localization and transport mediated by transmembrane domains.

    PubMed

    Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S

    2013-10-01

    The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in cytosolic domains, TMD sorting determinants are not conserved amino acid sequences but physical properties such as the length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized, but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport.

  6. Anchors Aweigh: Protein Traffic Mediated by Transmembrane Domains

    PubMed Central

    Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S.

    2013-01-01

    The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in the cytosolic domains, TMD sorting determinants are not conserved amino-acid sequences but physical properties such as length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport. PMID:23806646

  7. Crystal Structure of the Human, FIC-Domain Containing Protein HYPE and Implications for Its Functions

    PubMed Central

    Bunney, Tom D.; Cole, Ambrose R.; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W.; Katan, Matilda

    2014-01-01

    Summary Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein, HYPE, which has remained poorly characterized. Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of autoAMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition. PMID:25435325

  8. Crystal structure of the human, FIC-domain containing protein HYPE and implications for its functions.

    PubMed

    Bunney, Tom D; Cole, Ambrose R; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W; Katan, Matilda

    2014-12-02

    Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein,HYPE, which has remained poorly characterized.Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of auto AMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition.

  9. Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

    PubMed

    Merabet, Samir; Litim-Mecheri, Isma; Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

    2011-10-01

    Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.

  10. Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains

    PubMed Central

    Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K.; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

    2011-01-01

    Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences. PMID:22046139

  11. Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein.

    PubMed

    Park, Chin-Ju; Ko, Junsang; Ryu, Kyoung-Seok; Choi, Byong-Seok

    2014-02-01

    RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1-α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.

  12. Domain organizations of modular extracellular matrix proteins and their evolution.

    PubMed

    Engel, J

    1996-11-01

    Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

  13. Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins.

    PubMed Central

    Gillooly, D J; Simonsen, A; Stenmark, H

    2001-01-01

    PtdIns3P is a phosphoinositide 3-kinase product that has been strongly implicated in regulating membrane trafficking in both mammalian and yeast cells. PtdIns3P has been shown to be specifically located on membranes associated with the endocytic pathway. Proteins that contain FYVE zinc-finger domains are recruited to PtdIns3P-containing membranes. Structural information is now available concerning the interaction between FYVE domains and PtdIns3P. A number of proteins have been identified which contain a FYVE domain, and in this review we discuss the functions of PtdIns3P and its FYVE-domain-containing effector proteins in membrane trafficking, cytoskeletal regulation and receptor signalling. PMID:11284710

  14. Recombinant human IgG antibodies recognizing distinct extracellular domains of EGF receptor exhibit different degrees of growth inhibitory effects on human A431 cancer cells.

    PubMed

    Chang, Chialun; Takayanagi, Atsushi; Yoshida, Tetsuhiko; Shimizu, Nobuyoshi

    2013-05-01

    Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.

  15. CD-Search: protein domain annotations on the fly.

    PubMed

    Marchler-Bauer, Aron; Bryant, Stephen H

    2004-07-01

    We describe the Conserved Domain Search service (CD-Search), a web-based tool for the detection of structural and functional domains in protein sequences. CD-Search uses BLAST(R) heuristics to provide a fast, interactive service, and searches a comprehensive collection of domain models. Search results are displayed as domain architecture cartoons and pairwise alignments between the query and domain-model consensus sequences. Search results may be visualized in further detail by embedding the query sequence into multiple alignment displays and by mapping onto three-dimensional molecular graphic displays of known structures within the domain family. CD-Search can be accessed at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi.

  16. Domain conservation in several volvocalean cell wall proteins.

    PubMed

    Woessner, J P; Molendijk, A J; van Egmond, P; Klis, F M; Goodenough, U W; Haring, M A

    1994-11-01

    Based on our previous work demonstrating that (SerPro)x epitopes are common to extensin-like cell wall proteins in Chlamydomonas' reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)10 oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.

  17. Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

    SciTech Connect

    Wang, Chengliang; Zhang, Qiongdi; Hang, Tianrong; Tao, Yue; Ma, Xukai; Wu, Minhao; Zhang, Xuan Zang, Jianye

    2015-08-28

    The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66. The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66{sup 176–C} complexed with Rpl8{sup 204–224} in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

  18. NMR structure of the single QALGGH zinc finger domain from the Arabidopsis thaliana SUPERMAN protein.

    PubMed

    Isernia, Carla; Bucci, Enrico; Leone, Marilisa; Zaccaro, Laura; Di Lello, Paola; Digilio, Giuseppe; Esposito, Sabrina; Saviano, Michele; Di Blasio, Benedetto; Pedone, Carlo; Pedone, Paolo V; Fattorusso, Roberto

    2003-03-03

    Zinc finger domains of the classical type represent the most abundant DNA binding domains in eukaryotic transcription factors. Plant proteins contain from one to four zinc finger domains, which are characterized by high conservation of the sequence QALGGH, shown to be critical for DNA-binding activity. The Arabidopsis thaliana SUPERMAN protein, which contains a single QALGGH zinc finger, is necessary for proper spatial development of reproductive floral tissues and has been shown to specifically bind to DNA. Here, we report the synthesis and UV and NMR spectroscopic structural characterization of a 37 amino acid SUPERMAN region complexed to a Zn(2+) ion (Zn-SUP37) and present the first high-resolution structure of a classical zinc finger domain from a plant protein. The NMR structure of the SUPERMAN zinc finger domain consists of a very well-defined betabetaalpha motif, typical of all other Cys(2)-His(2) zinc fingers structurally characterized. As a consequence, the highly conserved QALGGH sequence is located at the N terminus of the alpha helix. This region of the domain of animal zinc finger proteins consists of hypervariable residues that are responsible for recognizing the DNA bases. Therefore, we propose a peculiar DNA recognition code for the QALGGH zinc finger domain that includes all or some of the amino acid residues at positions -1, 2, and 3 (numbered relative to the N terminus of the helix) and possibly others at the C-terminal end of the recognition helix. This study further confirms that the zinc finger domain, though very simple, is an extremely versatile DNA binding motif.

  19. The PUB domain: a putative protein-protein interaction domain implicated in the ubiquitin-proteasome pathway.

    PubMed

    Suzuki, T; Park, H; Till, E A; Lennarz, W J

    2001-10-12

    Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al. J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism. Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified. Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway. We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions.

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

    PubMed

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

    2015-12-01

    This article presents a historical review of the protein structure classification database CATH. Together with the SCOP database, CATH remains comprehensive and reasonably up-to-date with the now more than 100,000 protein structures in the PDB. We review the expansion of the CATH and SCOP resources to capture predicted domain structures in the genome sequence data and to provide information on the likely functions of proteins mediated by their constituent domains. The establishment of comprehensive function annotation resources has also meant that domain families can be functionally annotated allowing insights into functional divergence and evolution within protein families. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Viral Macro Domains Reverse Protein ADP-Ribosylation.

    PubMed

    Li, Changqing; Debing, Yannick; Jankevicius, Gytis; Neyts, Johan; Ahel, Ivan; Coutard, Bruno; Canard, Bruno

    2016-10-01

    ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD(+) to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular integrity are demodified

  2. Viral Macro Domains Reverse Protein ADP-Ribosylation

    PubMed Central

    Li, Changqing; Debing, Yannick; Jankevicius, Gytis; Neyts, Johan; Ahel, Ivan

    2016-01-01

    ABSTRACT ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD+ to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. IMPORTANCE Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular

  3. On the detection of functionally coherent groups of protein domains with an extension to protein annotation

    PubMed Central

    McLaughlin, William A; Chen, Ken; Hou, Tingjun; Wang, Wei

    2007-01-01

    Background Protein domains coordinate to perform multifaceted cellular functions, and domain combinations serve as the functional building blocks of the cell. The available methods to identify functional domain combinations are limited in their scope, e.g. to the identification of combinations falling within individual proteins or within specific regions in a translated genome. Further effort is needed to identify groups of domains that span across two or more proteins and are linked by a cooperative function. Such functional domain combinations can be useful for protein annotation. Results Using a new computational method, we have identified 114 groups of domains, referred to as domain assembly units (DASSEM units), in the proteome of budding yeast Saccharomyces cerevisiae. The units participate in many important cellular processes such as transcription regulation, translation initiation, and mRNA splicing. Within the units the domains were found to function in a cooperative manner; and each domain contributed to a different aspect of the unit's overall function. The member domains of DASSEM units were found to be significantly enriched among proteins contained in transcription modules, defined as genes sharing similar expression profiles and presumably similar functions. The observation further confirmed the functional coherence of DASSEM units. The functional linkages of units were found in both functionally characterized and uncharacterized proteins, which enabled the assessment of protein function based on domain composition. Conclusion A new computational method was developed to identify groups of domains that are linked by a common function in the proteome of Saccharomyces cerevisiae. These groups can either lie within individual proteins or span across different proteins. We propose that the functional linkages among the domains within the DASSEM units can be used as a non-homology based tool to annotate uncharacterized proteins. PMID:17937820

  4. Trichomonas vaginalis initiator binding protein (IBP39) and RNA polymerase II large subunit carboxy terminal domain interaction.

    PubMed

    Lau, Audrey O T; Smith, Alias J; Brown, Mark T; Johnson, Patricia J

    2006-11-01

    The core promoter that directs RNA polymerase to the start of transcription in the protist Trichomonas vaginalis is an initiator (Inr) element recognized by the Inr Binding Protein, IBP39. This nuclear protein is composed of two domains: a 14.5 kDa amino (N-terminal) and a 25 kDa carboxy terminal domain (C-domain). Here we describe the identification of an IBP39-interacting protein by screening a T. vaginalis expression library using a two-hybrid system with the IBP39 C-domain as bait. The CTD of the large subunit of RNAP II was found to specifically interact with the C-domain. The specificity and nature of the interaction between the CTD of RNAP II and the C-domain of IBP39 was validated by three independent biochemical methods: co-immunoprecipitation with epitope-tagged proteins, affinity chromatography and enzyme linked ligand sorbent (ELLSA) assays. Binding was shown to involve hydrophobic bonds and to have a disassociation constant (K(d)) of 690 nM (+/-55). These results confirm and extend our previous binding studies using a peptide composed of the last nine amino acids of RNAP II CTD [Schumacher MA, Lau AOT, Johnson PJ. Structural basis of core promoter recognition in a primitive eukaryote. Cell 2003;115:413-24] that predicted an interaction between the CTD and IBP39. These data further demonstrate that IBP39 minimally possesses two functional domains: a N-terminal DNA binding domain (that recognizes the Inr) [Liston DR, Johnson PJ. Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element. Mol Cell Biol 1999;19:2380-8] and a C-terminal protein binding domain that recognizes the RNAP II CTD, an interaction that may be critical for recruiting RNAP II for initiation of transcription.

  5. Protein domain repetition is enriched in Streptococcal cell-surface proteins.

    PubMed

    Lin, I-Hsuan; Hsu, Ming-Ta; Chang, Chuan-Hsiung

    2012-12-01

    Tandem repetition of domain in protein sequence occurs in all three domains of life. It creates protein diversity and adds functional complexity in organisms. In this work, we analyzed 52 streptococcal genomes and found 3748 proteins contained domain repeats. Proteins not harboring domain repeats are significantly enriched in cytoplasm, whereas proteins with domain repeats are significantly enriched in cytoplasmic membrane, cell wall and extracellular locations. Domain repetition occurs most frequently in S. pneumoniae and least in S. thermophilus and S. pyogenes. DUF1542 is the highest repeated domain in a single protein, followed by Rib, CW_binding_1, G5 and HemolysinCabind. 3D structures of 24 repeat-containing proteins were predicted to investigate the structural and functional effect of domain repetition. Several repeat-containing streptococcal cell surface proteins are known to be virulence-associated. Surface-associated tandem domain-containing proteins without experimental functional characterization may be potentially involved in the pathogenesis of streptococci and deserve further investigation. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Insights into jumonji c-domain containing protein 6 (JMJD6): a multifactorial role in FMDV replication in cells

    USDA-ARS?s Scientific Manuscript database

    The Jumonji C-domain containing protein 6 (JMJD6) has had a convoluted history. It was first identified as the phosphatidylserine receptor (PSR) on the cell surface responsible for recognizing phosphatidylserine on the surface of apoptotic cells resulting in their engulfment by phagocytic cells. Sub...

  7. Quantifying protein–protein interactions in high throughput using protein domain microarrays

    PubMed Central

    Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

    2011-01-01

    Protein microarrays provide an efficient way to identify and quantify protein–protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain–peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (KDs) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein–ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein–protein interaction networks. PMID:20360771

  8. Understanding protein domain-swapping using structure-based models of protein folding.

    PubMed

    Mascarenhas, Nahren Manuel; Gosavi, Shachi

    2017-09-01

    In domain-swapping, two or more identical protein monomers exchange structural elements and fold into dimers or multimers whose units are structurally similar to the original monomer. Domain-swapping is of biotechnological interest because inhibiting domain-swapping can reduce disease-causing fibrillar protein aggregation. To achieve such inhibition, it is important to understand both the energetics that stabilize the domain-swapped structure and the protein dynamics that enable the swapping. Structure-based models (SBMs) encode the folded structure of the protein in their potential energy functions. SBMs have been successfully used to understand diverse aspects of monomer folding. Symmetrized SBMs model interactions between two identical protein chains using only intra-monomer interactions. Molecular dynamics simulations of such symmetrized SBMs have been used to correctly predict the domain-swapped structure and to understand the mechanism of domain-swapping. Here, we review such models and illustrate that monomer topology determines key aspects of domain-swapping. However, in some proteins, specifics of local energetic interactions modulate domain-swapping and these need to be added to the symmetrized SBMs. We then summarize some general principles of the mechanism of domain-swapping that emerge from the symmetrized SBM simulations. Finally, using our own results, we explore how symmetrized SBMs could be used to design domain-swapping in proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Comprehensive Mapping of Common Immunodominant Epitopes in the Eastern Equine Encephalitis Virus E2 Protein Recognized by Avian Antibody Responses

    PubMed Central

    Sun, EnCheng; Zhao, Jing; Sun, Liang; Xu, QingYuan; Yang, Tao; Qin, YongLi; Wang, WenShi; Wei, Peng; Sun, Jing; Wu, DongLai

    2013-01-01

    Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb) response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211–226 and 331–352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11–26, 30–45 and 151–166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV) and Duck Plague Virus (DPV). The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein. PMID:23922704

  10. Comprehensive mapping of common immunodominant epitopes in the eastern equine encephalitis virus E2 protein recognized by avian antibody responses.

    PubMed

    Sun, Encheng; Zhao, Jing; Sun, Liang; Xu, Qingyuan; Yang, Tao; Qin, Yongli; Wang, Wenshi; Wei, Peng; Sun, Jing; Wu, Donglai

    2013-01-01

    Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb) response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211-226 and 331-352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11-26, 30-45 and 151-166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV) and Duck Plague Virus (DPV). The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein.

  11. Partitioning protein structures into domains: why is it so difficult?

    PubMed

    Holland, Timothy A; Veretnik, Stella; Shindyalov, Ilya N; Bourne, Philip E

    2006-08-18

    This analysis takes an in-depth look into the difficulties encountered by automatic methods for domain decomposition from three-dimensional structure. The analysis involves a multi-faceted set of criteria including the integrity of secondary structure elements, the tendency toward fragmentation of domains, domain boundary consistency and topology. The strength of the analysis comes from the use of a new comprehensive benchmark dataset, which is based on consensus among experts (CATH, SCOP and AUTHORS of the 3D structures) and covers 30 distinct architectures and 211 distinct topologies as defined by CATH. Furthermore, over 66% of the structures are multi-domain proteins; each domain combination occurring once per dataset. The performance of four automatic domain assignment methods, DomainParser, NCBI, PDP and PUU, is carefully analyzed using this broad spectrum of topology combinations and knowledge of rules and assumptions built into each algorithm. We conclude that it is practically impossible for an automatic method to achieve the level of performance of human experts. However, we propose specific improvements to automatic methods as well as broadening the concept of a structural domain. Such work is prerequisite for establishing improved approaches to domain recognition. (The benchmark dataset is available from http://pdomains.sdsc.edu).

  12. Biochemical and functional characterization of the ROC domain of DAPK establishes a new paradigm of GTP regulation in ROCO proteins.

    PubMed

    Bialik, Shani; Kimchi, Adi

    2012-10-01

    DAPK (death-associated protein kinase) is a newly recognized member of the mammalian family of ROCO proteins, characterized by common ROC (Ras of complex proteins) and COR (C-terminal of ROC) domains. In the present paper, we review our recent work showing that DAPK is functionally a ROCO protein; its ROC domain binds and hydrolyses GTP. Furthermore, GTP binding regulates DAPK catalytic activity in a novel manner by enhancing autophosphorylation on inhibitory Ser308, thereby promoting the kinase 'off' state. This is a novel mechanism for in cis regulation of kinase activity by the distal ROC domain. The functional similarities between DAPK and the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat protein kinase 2), another member of the ROCO family, are also discussed.

  13. Characterization of Two Dinoflagellate Cold Shock Domain Proteins

    PubMed Central

    Beauchemin, Mathieu; Roy, Sougata; Pelletier, Sarah; Averback, Alexandra; Lanthier, Frederic

    2016-01-01

    ABSTRACT Roughly two-thirds of the proteins annotated as transcription factors in dinoflagellate transcriptomes are cold shock domain-containing proteins (CSPs), an uncommon condition in eukaryotic organisms. However, no functional analysis has ever been reported for a dinoflagellate CSP, and so it is not known if they do in fact act as transcription factors. We describe here some of the properties of two CSPs from the dinoflagellate Lingulodinium polyedrum, LpCSP1 and LpCSP2, which contain a glycine-rich C-terminal domain and an N-terminal cold shock domain phylogenetically related to those in bacteria. However, neither of the two LpCSPs act like the bacterial CSP, since they do not functionally complement the Escherichia coli quadruple cold shock domain protein mutant BX04, and cold shock does not induce LpCSP1 and LpCSP2 to detectable levels, based on two-dimensional gel electrophoresis. Both CSPs bind to RNA and single-stranded DNA in a nonspecific manner in electrophoretic mobility shift assays, and both proteins also bind double-stranded DNA nonspecifically, albeit more weakly. These CSPs are thus unlikely to act alone as sequence-specific transcription factors. IMPORTANCE Dinoflagellate transcriptomes contain cold shock domain proteins as the major component of the proteins annotated as transcription factors. We show here that the major family of cold shock domain proteins in the dinoflagellate Lingulodinium do not bind specific sequences, suggesting that transcriptional control is not a predominant mechanism for regulating gene expression in this group of protists. PMID:27303711

  14. Negative Cooperativity and High Affinity in Chitooligosaccharide Binding by a Mycobacterium smegmatis Protein Containing LysM and Lectin Domains.

    PubMed

    Patra, Dhabaleswar; Mishra, Padmanabh; Vijayan, Mamannamana; Surolia, Avadhesha

    2016-01-12

    LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides has been underexplored. Binding of a Mycobacterium smegmatis protein containing a lectin (MSL) and one LysM domain to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration that demonstrate the presence of two binding sites of nonidentical affinities per dimeric MSL-LysM molecule. The affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in the MSL-LysM molecule, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the case presented here, two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of the MSL-LysM molecule for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far.

  15. Rapid similarity search of proteins using alignments of domain arrangements.

    PubMed

    Terrapon, Nicolas; Weiner, January; Grath, Sonja; Moore, Andrew D; Bornberg-Bauer, Erich

    2014-01-15

    Homology search methods are dominated by the central paradigm that sequence similarity is a proxy for common ancestry and, by extension, functional similarity. For determining sequence similarity in proteins, most widely used methods use models of sequence evolution and compare amino-acid strings in search for conserved linear stretches. Probabilistic models or sequence profiles capture the position-specific variation in an alignment of homologous sequences and can identify conserved motifs or domains. While profile-based search methods are generally more accurate than simple sequence comparison methods, they tend to be computationally more demanding. In recent years, several methods have emerged that perform protein similarity searches based on domain composition. However, few methods have considered the linear arrangements of domains when conducting similarity searches, despite strong evidence that domain order can harbour considerable functional and evolutionary signal. Here, we introduce an alignment scheme that uses a classical dynamic programming approach to the global alignment of domains. We illustrate that representing proteins as strings of domains (domain arrangements) and comparing these strings globally allows for a both fast and sensitive homology search. Further, we demonstrate that the presented methods complement existing methods by finding similar proteins missed by popular amino-acid-based comparison methods. An implementation of the presented algorithms, a web-based interface as well as a command-line program for batch searching against the UniProt database can be found at http://rads.uni-muenster.de. Furthermore, we provide a JAVA API for programmatic access to domain-string–based search methods.

  16. RapA2 Is a Calcium-binding Lectin Composed of Two Highly Conserved Cadherin-like Domains That Specifically Recognize Rhizobium leguminosarum Acidic Exopolysaccharides*

    PubMed Central

    Abdian, Patricia L.; Caramelo, Julio J.; Ausmees, Nora; Zorreguieta, Angeles

    2013-01-01

    In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like β-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins. PMID:23235153

  17. Dynamics of domain coverage of the protein sequence universe.

    PubMed

    Rekapalli, Bhanu; Wuichet, Kristin; Peterson, Gregory D; Zhulin, Igor B

    2012-11-16

    The currently known protein sequence space consists of millions of sequences in public databases and is rapidly expanding. Assigning sequences to families leads to a better understanding of protein function and the nature of the protein universe. However, a large portion of the current protein space remains unassigned and is referred to as its "dark matter". Here we suggest that true size of "dark matter" is much larger than stated by current definitions. We propose an approach to reducing the size of "dark matter" by identifying and subtracting regions in protein sequences that are not likely to contain any domain. Recent improvements in computational domain modeling result in a decrease, albeit slowly, in the relative size of "dark matter"; however, its absolute size increases substantially with the growth of sequence data.

  18. Dynamics of domain coverage of the protein sequence universe

    PubMed Central

    2012-01-01

    Background The currently known protein sequence space consists of millions of sequences in public databases and is rapidly expanding. Assigning sequences to families leads to a better understanding of protein function and the nature of the protein universe. However, a large portion of the current protein space remains unassigned and is referred to as its “dark matter”. Results Here we suggest that true size of “dark matter” is much larger than stated by current definitions. We propose an approach to reducing the size of “dark matter” by identifying and subtracting regions in protein sequences that are not likely to contain any domain. Conclusions Recent improvements in computational domain modeling result in a decrease, albeit slowly, in the relative size of “dark matter”; however, its absolute size increases substantially with the growth of sequence data. PMID:23157439

  19. Repeat proteins challenge the concept of structural domains.

    PubMed

    Espada, Rocío; Parra, R Gonzalo; Sippl, Manfred J; Mora, Thierry; Walczak, Aleksandra M; Ferreiro, Diego U

    2015-10-01

    Structural domains are believed to be modules within proteins that can fold and function independently. Some proteins show tandem repetitions of apparent modular structure that do not fold independently, but rather co-operate in stabilizing structural forms that comprise several repeat-units. For many natural repeat-proteins, it has been shown that weak energetic links between repeats lead to the breakdown of co-operativity and the appearance of folding sub-domains within an apparently regular repeat array. The quasi-1D architecture of repeat-proteins is crucial in detailing how the local energetic balances can modulate the folding dynamics of these proteins, which can be related to the physiological behaviour of these ubiquitous biological systems.

  20. An Algebro-Topological Description of Protein Domain Structure

    PubMed Central

    Penner, Robert Clark; Knudsen, Michael; Wiuf, Carsten; Andersen, Jørgen Ellegaard

    2011-01-01

    The space of possible protein structures appears vast and continuous, and the relationship between primary, secondary and tertiary structure levels is complex. Protein structure comparison and classification is therefore a difficult but important task since structure is a determinant for molecular interaction and function. We introduce a novel mathematical abstraction based on geometric topology to describe protein domain structure. Using the locations of the backbone atoms and the hydrogen bonds, we build a combinatorial object – a so-called fatgraph. The description is discrete yet gives rise to a 2-dimensional mathematical surface. Thus, each protein domain corresponds to a particular mathematical surface with characteristic topological invariants, such as the genus (number of holes) and the number of boundary components. Both invariants are global fatgraph features reflecting the interconnectivity of the domain by hydrogen bonds. We introduce the notion of robust variables, that is variables that are robust towards minor changes in the structure/fatgraph, and show that the genus and the number of boundary components are robust. Further, we invesigate the distribution of different fatgraph variables and show how only four variables are capable of distinguishing different folds. We use local (secondary) and global (tertiary) fatgraph features to describe domain structures and illustrate that they are useful for classification of domains in CATH. In addition, we combine our method with two other methods thereby using primary, secondary, and tertiary structure information, and show that we can identify a large percentage of new and unclassified structures in CATH. PMID:21629687

  1. Bpur, the Lyme Disease Spirochete's PUR Domain Protein

    PubMed Central

    Jutras, Brandon L.; Chenail, Alicia M.; Carroll, Dustin W.; Miller, M. Clarke; Zhu, Haining; Bowman, Amy; Stevenson, Brian

    2013-01-01

    The PUR domain is a nucleic acid-binding motif found in critical regulatory proteins of higher eukaryotes and in certain species of bacteria. During investigations into mechanisms by which the Lyme disease spirochete controls synthesis of its Erp surface proteins, it was discovered that the borrelial PUR domain protein, Bpur, binds with high affinity to double-stranded DNA adjacent to the erp transcriptional promoter. Bpur was found to enhance the effects of the erp repressor protein, BpaB. Bpur also bound single-stranded DNA and RNA, with relative affinities RNA > double-stranded DNA > single-stranded DNA. Rational site-directed mutagenesis of Bpur identified amino acid residues and domains critical for interactions with nucleic acids, and it revealed that the PUR domain has a distinct mechanism of interaction with each type of nucleic acid ligand. These data shed light on both gene regulation in the Lyme spirochete and functional mechanisms of the widely distributed PUR domain. PMID:23846702

  2. Formation and organization of protein domains in the immunological synapse

    NASA Astrophysics Data System (ADS)

    Carlson, Andreas; Mahadevan, L.

    2014-11-01

    The cellular basis for the adaptive immune response during antigen recognition relies on a specialized protein interface known as the immunological synapse. Here, we propose a minimal mathematical model for the dynamics of the IS that encompass membrane mechanics, hydrodynamics and protein kinetics. Simple scaling laws describe the dynamics of protein clusters as a function of membrane stiffness, rigidity of the adhesive proteins, and fluid flow in the synaptic cleft. Numerical simulations complement the scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment suggests that passive dynamics suffices to describe the short-time formation and organization of protein clusters, while the stabilization and long time dynamics of the synapse is likely determined by active cytoskeleton processes triggered by receptor binding. Our study reveals that the fluid flow generated by the interplay between membrane deformation and protein binding kinetics can assist immune cells in regulating protein sorting.

  3. Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein.

    PubMed

    Wong, Jaslyn E M M; Alsarraf, Husam M A B; Kaspersen, Jørn Døvling; Pedersen, Jan Skov; Stougaard, Jens; Thirup, Søren; Blaise, Mickaël

    2014-02-01

    Cellulose, chitin and peptidoglycan are major long-chain carbohydrates in living organisms, and constitute a substantial fraction of the biomass. Characterization of the biochemical basis of dynamic changes and degradation of these β,1-4-linked carbohydrates is therefore important for both functional studies of biological polymers and biotechnology. Here, we investigated the functional role of multiplicity of the carbohydrate-binding lysin motif (LysM) domain that is found in proteins involved in bacterial peptidoglycan synthesis and remodelling. The Bacillus subtilis peptidoglycan-hydrolysing NlpC/P60 D,L-endopeptidase, cell wall-lytic enzyme associated with cell separation, possesses four LysM domains. The contribution of each LysM domain was determined by direct carbohydrate-binding studies in aqueous solution with microscale thermophoresis. We found that bacterial LysM domains have affinity for N-acetylglucosamine (GlcNac) polymers in the lower-micromolar range. Moreover, we demonstrated that a single LysM domain is able to bind carbohydrate ligands, and that LysM domains act additively to increase the binding affinity. Our study reveals that affinity for GlcNAc polymers correlates with the chain length of the carbohydrate, and suggests that binding of long carbohydrates is mediated by LysM domain cooperativity. We also show that bacterial LysM domains, in contrast to plant LysM domains, do not discriminate between GlcNAc polymers, and recognize both peptidoglycan fragments and chitin polymers with similar affinity. Finally, an Ala replacement study suggested that the carbohydrate-binding site in LysM-containing proteins is conserved across phyla. © 2013 FEBS.

  4. CD8+ T cells recognize an inclusion membrane-associated protein from the vacuolar pathogen Chlamydia trachomatis

    PubMed Central

    Fling, Steven P.; Sutherland, R. Alec; Steele, Lisa N.; Hess, Bruce; D'Orazio, Sarah E. F.; Maisonneuve, Jean-François; Lampe, Mary F.; Probst, Peter; Starnbach, Michael N.

    2001-01-01

    During infection with Chlamydia trachomatis, CD8+ T cells are primed, even though the bacteria remain confined to a host cell vacuole throughout their developmental cycle. Because CD8+ T cells recognize antigens processed from cytosolic proteins, the Chlamydia antigens recognized by these CD8+ T cells very likely have access to the host cell cytoplasm during infection. The identity of these C. trachomatis proteins has remained elusive, even though their localization suggests they may play important roles in the biology of the organism. Here we use a retroviral expression system to identify Cap1, a 31-kDa protein from C. trachomatis recognized by protective CD8+ T cells. Cap1 contains no strong homology to any known protein. Immunofluorescence microscopy by using Cap1-specific antibody demonstrates that this protein is localized to the vacuolar membrane. Cap1 is virtually identical among the human C. trachomatis serovars, suggesting that a vaccine incorporating Cap1 might enable the vaccine to protect against all C. trachomatis serovars. The identification of proteins such as Cap1 that associate with the inclusion membrane will be required to fully understand the interaction of C. trachomatis with its host cell. PMID:11158611

  5. Effective Moment Feature Vectors for Protein Domain Structures

    PubMed Central

    Shi, Jian-Yu; Yiu, Siu-Ming; Zhang, Yan-Ning; Chin, Francis Yuk-Lun

    2013-01-01

    Imaging processing techniques have been shown to be useful in studying protein domain structures. The idea is to represent the pairwise distances of any two residues of the structure in a 2D distance matrix (DM). Features and/or submatrices are extracted from this DM to represent a domain. Existing approaches, however, may involve a large number of features (100–400) or complicated mathematical operations. Finding fewer but more effective features is always desirable. In this paper, based on some key observations on DMs, we are able to decompose a DM image into four basic binary images, each representing the structural characteristics of a fundamental secondary structure element (SSE) or a motif in the domain. Using the concept of moments in image processing, we further derive 45 structural features based on the four binary images. Together with 4 features extracted from the basic images, we represent the structure of a domain using 49 features. We show that our feature vectors can represent domain structures effectively in terms of the following. (1) We show a higher accuracy for domain classification. (2) We show a clear and consistent distribution of domains using our proposed structural vector space. (3) We are able to cluster the domains according to our moment features and demonstrate a relationship between structural variation and functional diversity. PMID:24391828

  6. ELMO Domains, Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases*

    PubMed Central

    East, Michael P.; Bowzard, J. Bradford; Dacks, Joel B.; Kahn, Richard A.

    2012-01-01

    The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases. PMID:23014990

  7. Protein domain recurrence and order can enhance prediction of protein functions.

    PubMed

    Messih, Mario Abdel; Chitale, Meghana; Bajic, Vladimir B; Kihara, Daisuke; Gao, Xin

    2012-09-15

    Burgeoning sequencing technologies have generated massive amounts of genomic and proteomic data. Annotating the functions of proteins identified in this data has become a big and crucial problem. Various computational methods have been developed to infer the protein functions based on either the sequences or domains of proteins. The existing methods, however, ignore the recurrence and the order of the protein domains in this function inference. We developed two new methods to infer protein functions based on protein domain recurrence and domain order. Our first method, DRDO, calculates the posterior probability of the Gene Ontology terms based on domain recurrence and domain order information, whereas our second method, DRDO-NB, relies on the naïve Bayes methodology using the same domain architecture information. Our large-scale benchmark comparisons show strong improvements in the accuracy of the protein function inference achieved by our new methods, demonstrating that domain recurrence and order can provide important information for inference of protein functions. The new models are provided as open source programs at http://sfb.kaust.edu.sa/Pages/Software.aspx. dkihara@cs.purdue.edu, xin.gao@kaust.edu.sa Supplementary data are available at Bioinformatics Online.

  8. Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

    PubMed Central

    Smith, Everett Clinton; Smith, Stacy E.; Carter, James R.; Webb, Stacy R.; Gibson, Kathleen M.; Hellman, Lance M.; Fried, Michael G.; Dutch, Rebecca Ellis

    2013-01-01

    Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

  9. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the TIR domain from the Brucella melitensis TIR-domain-containing protein TcpB.

    PubMed

    Alaidarous, Mohammed; Ve, Thomas; Ullah, M Obayed; Valkov, Eugene; Mansell, Ashley; Schembri, Mark A; Sweet, Matthew J; Kobe, Bostjan

    2013-10-01

    In mammals, Toll-like receptors (TLRs) recognize conserved microbial molecular signatures and induce an early innate immune response in the host. TLR signalling is mediated by interactions between the cytosolic TIR (Toll/interleukin-1 receptor) domains of the receptor and the adaptor proteins. Increasingly, it is apparent that pathogens target this interaction via pathogen-expressed TIR-domain-containing proteins to modulate immune responses. A TIR-domain-containing protein TcpB has been reported in the pathogenic bacterium Brucella melitensis. Studies have shown that TcpB interferes with the TLR2 and TLR4 signalling pathways to inhibit TLR-mediated inflammatory responses. Such interference may involve TIR-TIR-domain interactions between bacterial and mammalian proteins, but there is a lack of information about these interactions at the molecular level. In this study, the cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the protein construct corresponding to the TIR domain of TcpB (residues 120-250) are reported. The crystals diffracted to 2.6 Å resolution, have the symmetry of the monoclinic space group P2₁ and are most likely to contain four molecules in the asymmetric unit. The structure should help in understanding the molecular basis of how TcpB affects the innate immunity of the host.

  10. SNP@Domain: a web resource of single nucleotide polymorphisms (SNPs) within protein domain structures and sequences

    PubMed Central

    Han, Areum; Kang, Hyo Jin; Cho, Yoobok; Lee, Sunghoon; Kim, Young Joo; Gong, Sungsam

    2006-01-01

    The single nucleotide polymorphisms (SNPs) in conserved protein regions have been thought to be strong candidates that alter protein functions. Thus, we have developed SNP@Domain, a web resource, to identify SNPs within human protein domains. We annotated SNPs from dbSNP with protein structure-based as well as sequence-based domains: (i) structure-based using SCOP and (ii) sequence-based using Pfam to avoid conflicts from two domain assignment methodologies. Users can investigate SNPs within protein domains with 2D and 3D maps. We expect this visual annotation of SNPs within protein domains will help scientists select and interpret SNPs associated with diseases. A web interface for the SNP@Domain is freely available at and from . PMID:16845090

  11. From keys to bulldozers: expanding roles for winged helix domains in nucleic-acid-binding proteins.

    PubMed

    Harami, Gábor M; Gyimesi, Máté; Kovács, Mihály

    2013-07-01

    The winged helix domain (WHD) is a widespread nucleic-acid-binding protein structural element found in all kingdoms of life. Although the overall structure of the WHD is conserved, its functional properties and interaction profiles are extremely versatile. WHD-containing proteins can exploit nearly the full spectrum of nucleic acid structural features for recognition and even covalent modification or noncovalent rearrangement of target molecules. WHD functions range from sequence-recognizing keys in transcription factors and bulldozer-like strand-separating wedges in helicases to mediators of protein-protein interactions (PPIs). Further investigations are needed to understand the contribution of WHD structural dynamics to nucleic-acid-modifying enzymatic functions.

  12. Cysteine-rich domains related to Frizzled receptors and Hedgehog-interacting proteins

    PubMed Central

    Pei, Jimin; Grishin, Nick V

    2012-01-01

    Frizzled and Smoothened are homologous seven-transmembrane proteins functioning in the Wnt and Hedgehog signaling pathways, respectively. They harbor an extracellular cysteine-rich domain (FZ-CRD), a mobile evolutionary unit that has been found in a number of other metazoan proteins and Frizzled-like proteins in Dictyostelium. Domains distantly related to FZ-CRDs, in Hedgehog-interacting proteins (HHIPs), folate receptors and riboflavin-binding proteins (FRBPs), and Niemann-Pick Type C1 proteins (NPC1s), referred to as HFN-CRDs, exhibit similar structures and disulfide connectivity patterns compared with FZ-CRDs. We used computational analyses to expand the homologous set of FZ-CRDs and HFN-CRDs, providing a better understanding of their evolution and classification. First, FZ-CRD-containing proteins with various domain compositions were identified in several major eukaryotic lineages including plants and Chromalveolata, revealing a wider phylogenetic distribution of FZ-CRDs than previously recognized. Second, two new and distinct groups of highly divergent FZ-CRDs were found by sensitive similarity searches. One of them is present in the calcium channel component Mid1 in fungi and the uncharacterized FAM155 proteins in metazoans. Members of the other new FZ-CRD group occur in the metazoan-specific RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) proteins that are putative tumor suppressors acting as inhibitors of matrix metalloproteases. Finally, sequence and three-dimensional structural comparisons helped us uncover a divergent HFN-CRD in glypicans, which are important morphogen-binding heparan sulfate proteoglycans. Such a finding reinforces the evolutionary ties between the Wnt and Hedgehog signaling pathways and underscores the importance of gene duplications in creating essential signaling components in metazoan evolution. PMID:22693159

  13. Pleiotropic roles of cold shock domain proteins in plants.

    PubMed

    Sasaki, Kentaro; Imai, Ryozo

    2011-01-01

    The cold shock domain (CSD) is a nucleic acid binding domain that is widely conserved from bacteria to higher plants and animals. In Escherichia coli, cold shock proteins (CSPs) are composed solely of a CSD and function as RNA chaperones that destabilize RNA secondary structures. Cellular RNAs tend to be folded into unfavorable structures under low temperature conditions, and RNA chaperones resolve these structures, recovering functionality of the RNAs. CSP functions are associated mainly with cold adaptation, but they are also involved in other biological processes under normal growth conditions. Eukaryotic CSD proteins contain auxiliary domains in addition to the CSD and regulate many biological processes such as development and stress tolerance. In plants, it has been demonstrated that CSD proteins play essential roles in acquiring freezing tolerance. In addition, it has been suggested that some plant CSD proteins regulate embryo development, flowering time, and fruit development. In this review, we summarize the pleiotropic biological functions of CSP proteins in plants and discuss possible mechanisms by which plant CSD proteins regulate the functions of RNA molecules.

  14. BRCT-domain protein BRIT1 influences class switch recombination.

    PubMed

    Yen, Wei-Feng; Chaudhry, Ashutosh; Vaidyanathan, Bharat; Yewdell, William T; Pucella, Joseph N; Sharma, Rahul; Liang, Yulong; Li, Kaiyi; Rudensky, Alexander Y; Chaudhuri, Jayanta

    2017-07-19

    DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs.

  15. Methods of use of cellulose binding domain proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1997-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  16. Methods of use of cellulose binding domain proteins

    SciTech Connect

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1997-09-23

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  17. Methods of use of cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1997-09-23

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  18. Inferring Selection on Amino Acid Preference in Protein Domains

    PubMed Central

    Durbin, Richard

    2009-01-01

    Models that explicitly account for the effect of selection on new mutations have been proposed to account for “codon bias” or the excess of “preferred” codons that results from selection for translational efficiency and/or accuracy. In principle, such models can be applied to any mutation that results in a preferred allele, but in most cases, the fitness effect of a specific mutation cannot be predicted. Here we show that it is possible to assign preferred and unpreferred states to amino acid changing mutations that occur in protein domains. We propose that mutations that lead to more common amino acids (at a given position in a domain) can be considered “preferred alleles” just as are synonymous mutations leading to codons for more abundant tRNAs. We use genome-scale polymorphism data to show that alleles for preferred amino acids in protein domains occur at higher frequencies in the population, as has been shown for preferred codons. We show that this effect is quantitative, such that there is a correlation between the shift in frequency of preferred alleles and the predicted fitness effect. As expected, we also observe a reduction in the numbers of polymorphisms and substitutions at more important positions in domains, consistent with stronger selection at those positions. We examine the derived allele frequency distribution and polymorphism to divergence ratios of preferred and unpreferred differences and find evidence for both negative and positive selections acting to maintain protein domains in the human population. Finally, we analyze a model for selection on amino acid preferences in protein domains and find that it is consistent with the quantitative effects that we observe. PMID:19095755

  19. Structure of the Noncatalytic Domains and Global Fold of the Protein Disulfide Isomerase ERp72

    SciTech Connect

    Kozlov, G.; Määttänen, P; Schrag, J; Hura, G; Gabrielli, L; Cygler, M; Thomas, D; Gehring, K

    2009-01-01

    Protein disulfide isomerases are a family of proteins that catalyze the oxidation and isomerization of disulfide bonds in newly synthesized proteins in the endoplasmic reticulum. The family includes general enzymes such as PDI that recognize unfolded proteins, and others that are selective for specific classes of proteins. Here, we report the X-ray crystal structure of central non-catalytic domains of a specific isomerase, ERp72 (also called CaBP2 and protein disulfide-isomerase A4) from Rattus norvegicus. The structure reveals strong similarity to ERp57, a PDI-family member that interacts with the lectin-like chaperones calnexin and calreticulin but, unexpectedly, ERp72 does not interact with calnexin as shown by isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. Small-angle X-ray scattering (SAXS) of ERp72 was used to develop models of the full-length protein using both rigid body refinement and ab initio simulated annealing of dummy atoms. The two methods show excellent agreement and define the relative positions of the five thioredoxin-like domains of ERp72 and potential substrate or chaperone binding sites.

  20. C-terminal Domain of Leucyl-tRNA Synthetase from Pathogenic Candida albicans Recognizes both tRNASer and tRNALeu*

    PubMed Central

    Ji, Quan-Quan; Fang, Zhi-Peng; Ye, Qing; Ruan, Zhi-Rong; Zhou, Xiao-Long; Wang, En-Duo

    2016-01-01

    Leucyl-tRNA synthetase (LeuRS) is a multidomain enzyme that catalyzes Leu-tRNALeu formation and is classified into bacterial and archaeal/eukaryotic types with significant diversity in the C-terminal domain (CTD). CTDs of both bacterial and archaeal LeuRSs have been reported to recognize tRNALeu through different modes of interaction. In the human pathogen Candida albicans, the cytoplasmic LeuRS (CaLeuRS) is distinguished by its capacity to recognize a uniquely evolved chimeric tRNASer (CatRNASer(CAG)) in addition to its cognate CatRNALeu, leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNASer, suggesting the significance of their highly divergent CTDs in tRNASer recognition. The results of this study provided the first evidence of the indispensable function of the CTD of eukaryotic LeuRS in recognizing non-cognate CatRNASer and cognate CatRNALeu. Three lysine residues were identified as involved in mediating enzyme-tRNA interaction in the leucylation process: mutation of all three sites totally ablated the leucylation activity. The importance of the three lysine residues was further verified by gel mobility shift assays and complementation of a yeast leuS gene knock-out strain. PMID:26677220

  1. Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

    PubMed

    Sawatsky, Bevan; Bente, Dennis A; Czub, Markus; von Messling, Veronika

    2016-05-01

    The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

  2. The modular organization of domain structures: insights into protein-protein binding.

    PubMed

    del Sol, Antonio; Carbonell, Pablo

    2007-12-01

    Domains are the building blocks of proteins and play a crucial role in protein-protein interactions. Here, we propose a new approach for the analysis and prediction of domain-domain interfaces. Our method, which relies on the representation of domains as residue-interacting networks, finds an optimal decomposition of domain structures into modules. The resulting modules comprise highly cooperative residues, which exhibit few connections with other modules. We found that non-overlapping binding sites in a domain, involved in different domain-domain interactions, are generally contained in different modules. This observation indicates that our modular decomposition is able to separate protein domains into regions with specialized functions. Our results show that modules with high modularity values identify binding site regions, demonstrating the predictive character of modularity. Furthermore, the combination of modularity with other characteristics, such as sequence conservation or surface patches, was found to improve our predictions. In an attempt to give a physical interpretation to the modular architecture of domains, we analyzed in detail six examples of protein domains with available experimental binding data. The modular configuration of the TEM1-beta-lactamase binding site illustrates the energetic independence of hotspots located in different modules and the cooperativity of those sited within the same modules. The energetic and structural cooperativity between intramodular residues is also clearly shown in the example of the chymotrypsin inhibitor, where non-binding site residues have a synergistic effect on binding. Interestingly, the binding site of the T cell receptor beta chain variable domain 2.1 is contained in one module, which includes structurally distant hot regions displaying positive cooperativity. These findings support the idea that modules possess certain functional and energetic independence. A modular organization of binding sites confers

  3. Comparison of antigenic proteins from Lactococcus garvieae KG- and KG+ strains that are recognized by olive flounder (Paralichthys olivaceus) antibodies.

    PubMed

    Shin, Gee-Wook; Nho, Seong-Won; Park, Seong-Bin; Jang, Ho-Bin; Cha, In-Seok; Ha, Mi-Ae; Kim, Young-Rim; Dalvi, Rishikesh S; Joh, Seong-Joon; Jung, Tae-Sung

    2009-10-20

    Lactococcus garvieae is an important etiological agent of lactococcosis in various fish species including olive flounder (Paralichthys olivaceus). In this study, proteomic and immunoproteomic analyses were employed to compare the antigenic profiles of strains KG9408, MS93003, and NSS9310 strains of L. garvieae. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) revealed differences in five protein spots among the different L. garvieae strains. In immunoproteomic analysis, there was a significant difference in the 2-DE immunoblot profiles of the L. garvieae strains using sera collected from fish surviving infection with either L. garvieae strains KG9408 or NSS9310. These sera reacted with 8 and 7 unique antigenic protein spots, respectively. Heat shock protein (HSP) 70 and DNA-directed RNA polymerase were among the specific antigens recognized by the anti-NSS9310 serum. In addition, the anti-NSS9310 and anti-KG9408 olive flounder sera reacted with 25 common antigenic protein spots of all the L. garvieae strains, which included elongation factor (EF)-Tu, arginine deiminase (AD), inosine-5'-monophosphate dehydrogenase (IMPD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphomannomutase (PMM), L-lactate dehydrogenase (L-LDH), 6-phosphofructokinase and UDP-galactose 4-epimerase (UDP-galactose). Based on the present results, the 8 antigens recognized by the anti-KG9408 serum and the 25 common antigens recognized by both sera may serve as potential markers for developing an effective vaccine against this bacterium.

  4. Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins

    PubMed Central

    Xu, Zhixiong; Meng, Xianzhang; Cai, Ying; Liang, Hong; Nagarajan, Lalitha; Brandt, Stephen J.

    2007-01-01

    The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and β-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins. PMID:17437998

  5. Inter-Domain Dynamics in a Two-Domain Protein Studied by NMR

    NASA Astrophysics Data System (ADS)

    Ryabov, Yaroslav; Fushman, David

    2006-03-01

    Domain orientation and dynamics often play an important role in regulation of multidomain proteins function. Here we consider a two-domain system, Lys48-linked di-ubiquitin (Ub2), which is the simplest model of the polyubiquitin chain involved in the ubiquitin-proteasome pathway. Under physiological conditions Ub2 adopts a compact conformation, in which the functionally important hydrophobic residues are sequestered at the interface between the two Ub2 domains. Here we present a dynamic model that combines the anisotropic overall rotational diffusion with intra- and interdomain dynamics. This model describes the interdomain motion as a transition between two distinct conformational states. The model is applied to experimental ^15N relaxation data for Lys48-linked Ub2 acquired at neutral (pH 6.8) and acidic (pH 4.5) conditions. The model provides complete picture of Ub2 domain mobility including domain orientations, time scales of domain motions, and occupation probabilities for both states of Ub2. The obtained results are consistent with independent data on chemical shift perturbation mapping and spin labeling.

  6. TOPDOM: database of conservatively located domains and motifs in proteins.

    PubMed

    Varga, Julia; Dobson, László; Tusnády, Gábor E

    2016-09-01

    The TOPDOM database-originally created as a collection of domains and motifs located consistently on the same side of the membranes in α-helical transmembrane proteins-has been updated and extended by taking into consideration consistently localized domains and motifs in globular proteins, too. By taking advantage of the recently developed CCTOP algorithm to determine the type of a protein and predict topology in case of transmembrane proteins, and by applying a thorough search for domains and motifs as well as utilizing the most up-to-date version of all source databases, we managed to reach a 6-fold increase in the size of the whole database and a 2-fold increase in the number of transmembrane proteins. TOPDOM database is available at http://topdom.enzim.hu The webpage utilizes the common Apache, PHP5 and MySQL software to provide the user interface for accessing and searching the database. The database itself is generated on a high performance computer. tusnady.gabor@ttk.mta.hu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

  7. Targeting proteins to liquid-ordered domains in lipid membranes.

    PubMed

    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. Defining the boundaries: structure and function of LOB domain proteins.

    PubMed

    Majer, Christine; Hochholdinger, Frank

    2011-01-01

    The plant-specific LBD (Lateral Organ Boundaries Domain) gene family is essential in the regulation of plant lateral organ development and is involved in the regulation of anthocyanin and nitrogen metabolism. LBD proteins contain a characteristic LOB domain composed of a C-motif required for DNA-binding, a conserved glycine residue, and a leucine-zipper-like sequence required for protein-protein interactions. Recently, several LBD genes associated with mutant phenotypes related to almost all aspects of plant development, including embryo, root, leaf, and inflorescence development have been functionally characterized. These novel insights contribute to a better understanding of the molecular definition of boundaries between organs or boundaries between organs and meristems and the regulation of these processes by environmental cues and phytohormones.

  9. A Bayesian Sampler for Optimization of Protein Domain Hierarchies

    PubMed Central

    2014-01-01

    Abstract The process of identifying and modeling functionally divergent subgroups for a specific protein domain class and arranging these subgroups hierarchically has, thus far, largely been done via manual curation. How to accomplish this automatically and optimally is an unsolved statistical and algorithmic problem that is addressed here via Markov chain Monte Carlo sampling. Taking as input a (typically very large) multiple-sequence alignment, the sampler creates and optimizes a hierarchy by adding and deleting leaf nodes, by moving nodes and subtrees up and down the hierarchy, by inserting or deleting internal nodes, and by redefining the sequences and conserved patterns associated with each node. All such operations are based on a probability distribution that models the conserved and divergent patterns defining each subgroup. When we view these patterns as sequence determinants of protein function, each node or subtree in such a hierarchy corresponds to a subgroup of sequences with similar biological properties. The sampler can be applied either de novo or to an existing hierarchy. When applied to 60 protein domains from multiple starting points in this way, it converged on similar solutions with nearly identical log-likelihood ratio scores, suggesting that it typically finds the optimal peak in the posterior probability distribution. Similarities and differences between independently generated, nearly optimal hierarchies for a given domain help distinguish robust from statistically uncertain features. Thus, a future application of the sampler is to provide confidence measures for various features of a domain hierarchy. PMID:24494927

  10. Membrane shape instabilities induced by BAR domain proteins

    NASA Astrophysics Data System (ADS)

    Baumgart, Tobias

    2014-03-01

    Membrane curvature has developed into a forefront of membrane biophysics. Numerous proteins involved in membrane curvature sensing and membrane curvature generation have recently been discovered, including proteins containing the crescent-shaped BAR domain as membrane binding and shaping module. Accordingly, the structure determination of these proteins and their multimeric complexes is increasingly well-understood. Substantially less understood, however, are thermodynamic and kinetic aspects and the detailed mechanisms of how these proteins interact with membranes in a curvature-dependent manner. New experimental approaches need to be combined with established techniques to be able to fill in these missing details. Here we use model membrane systems in combination with a variety of biophysical techniques to characterize mechanistic aspects of BAR domain protein function. This includes a characterization of membrane curvature sensing and membrane generation. We also establish kinetic and thermodynamic aspects of BAR protein dimerization in solution, and investigate kinetic aspects of membrane binding. We present two new approaches to investigate membrane shape instabilities and demonstrate that membrane shape instabilities can be controlled by protein binding and lateral membrane tension. This work is supported through NIH grant GM-097552 and NSF grant CBET-1053857.

  11. Identification of an inflammation-inducible serum protein recognized by anti-disialic acid antibodies as carbonic anhydrase II.

    PubMed

    Yasukawa, Zenta; Sato, Chihiro; Kitajima, Ken

    2007-03-01

    Acute-phase proteins are an important marker of inflammation and sometimes have a role in the general defense response towards tissue injury. In the present study, we identified a 32-kDa protein that was immunoreactive with monoclonal antibody 2-4B (mAb.2-4B), which is specific to di/oligoNeu5Gc structures, and that behaved as an acute-phase protein following stimulation with either turpentine oil or lipopolysaccharides. The 32-kDa protein was identified as carbonic anhydrase II (CA-II), based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses of the purified protein. Mouse and human CA-II was immunoreactive and immunoprecipitated with mAb.2-4B, but contained no sialic acid. In addition to mAb.2-4B, the mAb. S2-566 an antibody specific for diNeu5Ac-containing glycans, recognized the CA-II, whereas an anti-oligo/polysialic acid antibody did not. These results indicate that a part of the CA-II protein structure mimics the disialic acid structure recognized by the monoclonal antibodies. This is the first report that CA-II circulates in the serum following inflammation.

  12. EVEREST: automatic identification and classification of protein domains in all protein sequences

    PubMed Central

    Portugaly, Elon; Harel, Amir; Linial, Nathan; Linial, Michal

    2006-01-01

    Background Proteins are comprised of one or several building blocks, known as domains. Such domains can be classified into families according to their evolutionary origin. Whereas sequencing technologies have advanced immensely in recent years, there are no matching computational methodologies for large-scale determination of protein domains and their boundaries. We provide and rigorously evaluate a novel set of domain families that is automatically generated from sequence data. Our domain family identification process, called EVEREST (EVolutionary Ensembles of REcurrent SegmenTs), begins by constructing a library of protein segments that emerge in an all vs. all pairwise sequence comparison. It then proceeds to cluster these segments into putative domain families. The selection of the best putative families is done using machine learning techniques. A statistical model is then created for each of the chosen families. This procedure is then iterated: the aforementioned statistical models are used to scan all protein sequences, to recreate a library of segments and to cluster them again. Results Processing the Swiss-Prot section of the UniProt Knoledgebase, release 7.2, EVEREST defines 20,230 domains, covering 85% of the amino acids of the Swiss-Prot database. EVEREST annotates 11,852 proteins (6% of the database) that are not annotated by Pfam A. In addition, in 43,086 proteins (20% of the database), EVEREST annotates a part of the protein that is not annotated by Pfam A. Performance tests show that EVEREST recovers 56% of Pfam A families and 63% of SCOP families with high accuracy, and suggests previously unknown domain families with at least 51% fidelity. EVEREST domains are often a combination of domains as defined by Pfam or SCOP and are frequently sub-domains of such domains. Conclusion The EVEREST process and its output domain families provide an exhaustive and validated view of the protein domain world that is automatically generated from sequence data. The

  13. EVEREST: automatic identification and classification of protein domains in all protein sequences.

    PubMed

    Portugaly, Elon; Harel, Amir; Linial, Nathan; Linial, Michal

    2006-06-02

    Proteins are comprised of one or several building blocks, known as domains. Such domains can be classified into families according to their evolutionary origin. Whereas sequencing technologies have advanced immensely in recent years, there are no matching computational methodologies for large-scale determination of protein domains and their boundaries. We provide and rigorously evaluate a novel set of domain families that is automatically generated from sequence data. Our domain family identification process, called EVEREST (EVolutionary Ensembles of REcurrent SegmenTs), begins by constructing a library of protein segments that emerge in an all vs. all pairwise sequence comparison. It then proceeds to cluster these segments into putative domain families. The selection of the best putative families is done using machine learning techniques. A statistical model is then created for each of the chosen families. This procedure is then iterated: the aforementioned statistical models are used to scan all protein sequences, to recreate a library of segments and to cluster them again. Processing the Swiss-Prot section of the UniProt Knoledgebase, release 7.2, EVEREST defines 20,230 domains, covering 85% of the amino acids of the Swiss-Prot database. EVEREST annotates 11,852 proteins (6% of the database) that are not annotated by Pfam A. In addition, in 43,086 proteins (20% of the database), EVEREST annotates a part of the protein that is not annotated by Pfam A. Performance tests show that EVEREST recovers 56% of Pfam A families and 63% of SCOP families with high accuracy, and suggests previously unknown domain families with at least 51% fidelity. EVEREST domains are often a combination of domains as defined by Pfam or SCOP and are frequently sub-domains of such domains. The EVEREST process and its output domain families provide an exhaustive and validated view of the protein domain world that is automatically generated from sequence data. The EVEREST library of domain

  14. BC-box protein domain-related mechanism for VHL protein degradation

    PubMed Central

    Pozzebon, Maria Elena; Varadaraj, Archana; Mattoscio, Domenico; Jaffray, Ellis G.; Miccolo, Claudia; Galimberti, Viviana; Tommasino, Massimo; Hay, Ronald T.; Chiocca, Susanna

    2013-01-01

    The tumor suppressor VHL (von Hippel–Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasome-dependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins. PMID:24145437

  15. Monoclonal Antibodies that Recognize Proteins Unique to Somatic Embryos of Daucus carota.

    DTIC Science & Technology

    1985-01-01

    For enzyme immunoassay (EIA), 6 M urea extracts of the lyophilized tissues were prepared (-., personal communication), their protein content was...of hybridoma supernates on EIA plates coated with 6 M urea extracts (1 mg protein/well) of lyophilized callus cells (A), somatic embryo cells (B), and...101Ali reacts with denatured proteins that were identified as LHCP by their apparent molecular weight and reaction with rabbit antisera to maize LHCP

  16. Functional characterization of rice CW-domain containing zinc finger proteins involved in histone recognition.

    PubMed

    Zhang, Zhe; Zhang, Feng; Cheng, Zhi-Jun; Liu, Ling-Long; Lin, Qi-Bing; Wu, Fu-Qing; Zhang, Huan; Wang, Jiu-Lin; Wang, Jie; Guo, Xiu-Ping; Zhang, Xin; Lei, Cai-Lin; Zhao, Zhi-Chao; Zhu, Shan-Shan; Wan, Jian-Min

    2017-10-01

    Histone recognition is important for understanding the mechanisms of histone modification, which play a pivotal role in transcriptional regulation during plant development. Here, we identified three cysteine-tryptophan (CW)-domain containing zinc finger (ZF) proteins involved in histone recognition, namely OsCW-ZF3, OsCW-ZF5 and OsCW-ZF7. Protein sequence analysis showed that they have two unknown motifs in addition to the CW domain. All three OsCW-ZFs were expressed in aerial tissues, with relatively high levels in developing panicles. Subcellular localization revealed that the OsCW-ZFs target the cell nucleus and CW domains are not necessary for their nuclear localization. In contrast to OsCW-ZF3 and OsCW-ZF5 where the CW domains bind histone H3 lysine 4 with different methylated forms (H3K4me), the CW domain from OsCW-ZF7 recognizes only trimethylated histone H3 lysine 4 (H3K4me3). Analysis of mutant suggested that three conserved tryptophan residues in the CW domain are essential for binding to H3K4me. Further study found that OsCW-ZF7 interacts with TAFII20, a transcription initiation factor TFIID 20kDa subunit. Knockout of OsCW-ZF7 caused defective development of awns. This study provides new insights into our understanding of the CW domain and lays a foundation for further investigation of its roles in rice. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Structures of YAP protein domains reveal promising targets for development of new cancer drugs

    PubMed Central

    Sudol, Marius; Shields, Denis C.; Farooq, Amjad

    2012-01-01

    YAP (Yes-associated protein) is a potent oncogene and a major effector of the mammalian Hippo tumor suppressor pathway. In this review, our emphasis is on the structural basis of how YAP recognizes its various cellular partners. In particular, we discuss the role of LATS kinase and AMOTL1 junction protein, two key cellular partners of YAP that bind to its WW domain, in mediating cytoplasmic localization of YAP and thereby playing a key role in the regulation of its transcriptional activity. Importantly, the crystal structure of an amino-terminal domain of YAP in complex with the carboxy-terminal domain of TEAD transcription factor was only recently solved at atomic resolution, while the structure of WW domain of YAP in complex with a peptide containing the PPxY motif has been available for more than a decade. We discuss how such structural information may be exploited for the rational development of novel anti-cancer therapeutics harboring greater efficacy coupled with low toxicity. We also embark on a brief discussion of how recent in silico studies led to identification of the cardiac glycoside digitoxin as a potential modulator of WW domain-ligand interactions. Conversely, dobutamine was identified in a screen of known drugs as a compound that promotes cytoplasmic localization of YAP, thereby resulting in growth suppressing activity. Finally, we discuss how a recent study on the dynamics of WW domain folding on a biologically critical time scale may provide a tool to generate repertoires of WW domain variants for regulation of the Hippo pathway toward desired, non-oncogenic outputs. PMID:22609812

  18. Domain formation in membranes caused by lipid wetting of protein.

    PubMed

    Akimov, Sergey A; Frolov, Vladimir A J; Kuzmin, Peter I; Zimmerberg, Joshua; Chizmadzhev, Yuri A; Cohen, Fredric S

    2008-05-01

    Formation of rafts and other domains in cell membranes is considered as wetting of proteins by lipids. The membrane is modeled as a continuous elastic medium. Thermodynamic functions of the lipid films that wet proteins are calculated using a mean-field theory of liquid crystals as adapted to biomembranes. This approach yields the conditions necessary for a macroscopic wetting film to form; its thickness could also be determined. It is shown that films of macroscopic thicknesses form around large (tens nanometers in diameter) lipid-protein aggregates; only thin adsorption films form around single proteins or small complexes. The means by which wetting films can facilitate the merger of these aggregates is considered. It is shown that a wetting film prevents a protein from leaving an aggregate. Using experimentally derived values of elastic moduli and spontaneous curvatures as well as height mismatch between aggregates and bulk membrane, we obtained numerical results, which can be compared with the experimental data.

  19. DNA topoisomerase II alpha is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2.

    PubMed Central

    Taagepera, S; Rao, P N; Drake, F H; Gorbsky, G J

    1993-01-01

    We have determined that the major mitotic phosphoprotein in chromosomes recognized by the antiphosphoprotein antibody MPM-2 is the 170-kDa isoform of topoisomerase II (topo II), the isoform predominant in proliferating cells. As a prerequisite to making this discovery, it was necessary to develop protocols to protect chromosomal proteins from dephosphorylation during cell extraction and chromosome isolation procedures. Immunofluorescence analysis of the large chromosomes prepared from Indian Muntjac cells revealed colocalization of MPM-2 and anti-topo II antibodies to the chromosomal centromeres and to the axial regions of the chromosomal arms. For biochemical fractionation studies, large quantities of chromosomes from the P388D1 mouse lymphocyte cell line were isolated and treated to remove DNA and histone proteins. Immunoblot and immunoprecipitation experiments with this chromosome scaffold fraction identified the major MPM-2-reactive phosphoprotein to be DNA topo II. Using a panel of anti-peptide antibodies specific to the isoforms of topo II, we determined that the major phosphoprotein recognized by MPM-2 is the 170-kDa isoform of topo II, topo II alpha. The 180-kDa isoform, topo II beta, present in the isolated chromosomes in much smaller quantities, is also recognized by MPM-2. The mitotic phosphorylation of the topo II proteins may be critical for proper chromosome condensation and segregation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7690961

  20. Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

    PubMed Central

    Rossey, Iebe; Gilman, Morgan S. A.; Kabeche, Stephanie C.; Sedeyn, Koen; Wrapp, Daniel; Kanekiyo, Masaru; Chen, Man; Mas, Vicente; Spitaels, Jan; Melero, José A.; Graham, Barney S.; Schepens, Bert; McLellan, Jason S.; Saelens, Xavier

    2017-01-01

    Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV. PMID:28194013

  1. Prediction of Cancer Proteins by Integrating Protein Interaction, Domain Frequency, and Domain Interaction Data Using Machine Learning Algorithms

    PubMed Central

    2015-01-01

    Many proteins are known to be associated with cancer diseases. It is quite often that their precise functional role in disease pathogenesis remains unclear. A strategy to gain a better understanding of the function of these proteins is to make use of a combination of different aspects of proteomics data types. In this study, we extended Aragues's method by employing the protein-protein interaction (PPI) data, domain-domain interaction (DDI) data, weighted domain frequency score (DFS), and cancer linker degree (CLD) data to predict cancer proteins. Performances were benchmarked based on three kinds of experiments as follows: (I) using individual algorithm, (II) combining algorithms, and (III) combining the same classification types of algorithms. When compared with Aragues's method, our proposed methods, that is, machine learning algorithm and voting with the majority, are significantly superior in all seven performance measures. We demonstrated the accuracy of the proposed method on two independent datasets. The best algorithm can achieve a hit ratio of 89.4% and 72.8% for lung cancer dataset and lung cancer microarray study, respectively. It is anticipated that the current research could help understand disease mechanisms and diagnosis. PMID:25866773

  2. N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells.

    PubMed Central

    Puddington, L; Bevan, M J; Rose, J K; Lefrançois, L

    1986-01-01

    The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti-VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed. Images PMID:3022003

  3. Domain Hierarchy and closed Loops (DHcL): a server for exploring hierarchy of protein domain structure

    PubMed Central

    Koczyk, Grzegorz; Berezovsky, Igor N.

    2008-01-01

    Domain hierarchy and closed loops (DHcL) (http://sitron.bccs.uib.no/dhcl/) is a web server that delineates energy hierarchy of protein domain structure and detects domains at different levels of this hierarchy. The server also identifies closed loops and van der Waals locks, which constitute a structural basis for the protein domain hierarchy. The DHcL can be a useful tool for an express analysis of protein structures and their alternative domain decompositions. The user submits a PDB identifier(s) or uploads a 3D protein structure in a PDB format. The results of the analysis are the location of domains at different levels of hierarchy, closed loops, van der Waals locks and their interactive visualization. The server maintains a regularly updated database of domains, closed loop and van der Waals locks for all X-ray structures in PDB. DHcL server is available at: http://sitron.bccs.uib.no/dhcl. PMID:18502776

  4. Measuring interactions of FERM domain-containing sorting Nexin proteins with endosomal lipids and cargo molecules.

    PubMed

    Ghai, Rajesh; Mobli, Mehdi; Collins, Brett M

    2014-01-01

    Endosomal recycling pathways regulate cellular homeostasis via the transport of internalized material back to the plasma membrane. Phox homology (PX) and band 4.1/ezrin/radixin/moesin (FERM) domain-containing proteins are a recently identified subfamily of PX proteins that are critical for the recycling of numerous transmembrane cargo molecules. The PX-FERM subfamily includes three endosome-associated proteins called sorting nexin (SNX) 17, SNX27, and SNX31. These are modular peripheral membrane proteins that act as central scaffolds mediating protein-lipid interactions, cargo binding, and regulatory protein recruitment. This chapter outlines the methodology employed to classify the PX-FERM family using combined bioinformatics and structure prediction tools. It further details the application of isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to understand the mechanisms that underpin their endosomal membrane recruitment and subsequent recognition of NPxY/NxxY peptide sorting motifs, present in many cargo receptors and required for their trafficking. It is now increasingly recognized that the formation of a stable trafficking complex is dictated by a multitude of coordinated protein-protein and protein-lipid interactions, and the approaches highlighted here will be useful for future studies aimed at understanding these biomolecular interactions in greater detail.

  5. ADDA: a domain database with global coverage of the protein universe.

    PubMed

    Heger, Andreas; Wilton, Christopher Andrew; Sivakumar, Ashwin; Holm, Liisa

    2005-01-01

    We used the Automatic Domain Decomposition Algorithm (ADDA) to generate a database of protein domain families with complete coverage of all protein sequences. Sequences are split into domains and domains are grouped into protein domain families in a completely automated process. The current database contains domains for more than 1.5 million sequences in more than 40,000 domain families. In particular, there are 3828 novel domain families that do not overlap with the curated domain databases Pfam, SCOP and InterPro. The data are freely available for downloading and querying via a web interface (http://ekhidna.biocenter.helsinki.fi:9801/sqgraph/pairsdb).

  6. Glucosidase II and MRH-domain containing proteins in the secretory pathway

    PubMed Central

    D’Alessio, Cecilia; Dahms, Nancy M.

    2015-01-01

    N -glycosylation in the endoplasmic reticulum (ER) consists of the transfer of a pre-assembled glycan conserved among species (Glc3Man9GlcNAc2) from a lipid donor to a consensus sequence within a nascent protein that is entering the ER. The protein-linked glycans are then processed by glycosidases and glycosyltransferases in the ER producing specific structures that serve as signalling molecules for the fate of the folding glycoprotein: to stay in the ER during the folding process, to be retrotranslocated to the cytosol for proteasomal degradation if irreversibly misfolded, or to pursue transit through the secretory pathway as a mature glycoprotein. In the ER, each glycan signalling structure is recognized by a specific lectin. A domain similar to that of the mannose 6-phosphate receptors (MPRs) has been identified in several proteins of the secretory pathway. These include the beta subunit of glucosidase II (GII), a key enzyme in the early processing of the transferred glycan that removes middle and innermost glucoses and is involved in quality control of glycoprotein folding in the ER (QC), the lectins OS-9 and XTP3-B, proteins involved in the delivery of ER misfolded proteins to degradation (ERAD), the gamma subunit of the Golgi GlcNAc-1-phosphotransferase, an enzyme involved in generating the mannose 6-phosphate (M6P) signal for sorting acidic hydrolases to lysosomes, and finally the MPRs that deliver those hydrolytic enzymes to the lysosome. Each of the MRH-containing proteins recognizes a different signalling N-glycan structure. Three-dimensional structures of some of the MRH domains have been solved, providing the basis to understand recognition mechanisms. PMID:25692846

  7. Evolutionary history and genome organization of DUF1220 protein domains.

    PubMed

    O'Bleness, Majesta S; Dickens, C Michael; Dumas, Laura J; Kehrer-Sawatzki, Hildegard; Wyckoff, Gerald J; Sikela, James M

    2012-09-01

    DUF1220 protein domains exhibit the most extreme human lineage-specific (HLS) copy number increase of any protein coding region in the human genome and have recently been linked to evolutionary and pathological changes in brain size (e.g., 1q21-associated microcephaly). These findings lend support to the view that DUF1220 domain dosage is a key factor in the determination of primate (and human) brain size. Here we analyze 41 animal genomes and present the most complete account to date of the evolutionary history and genome organization of DUF1220 domains and the gene family that encodes them (NBPF). Included among the novel features identified by this analysis is a DUF1220 domain precursor in nonmammalian vertebrates, a unique predicted promoter common to all mammalian NBPF genes, six distinct clades into which DUF1220 sequences can be subdivided, and a previously unknown member of the NBPF gene family (NBPF25). Most importantly, we show that the exceptional HLS increase in DUF1220 copy number (from 102 in our last common ancestor with chimp to 272 in human; an average HLS increase of ~28 copies every million years since the Homo/Pan split) was driven by intragenic domain hyperamplification. This increase primarily involved a 4.7 kb, tandemly repeated three DUF1220 domain unit we have named the HLS DUF1220 triplet, a motif that is a likely candidate to underlie key properties unique to the Homo sapiens brain. Interestingly, all copies of the HLS DUF1220 triplet lie within a human-specific pericentric inversion that also includes the 1q12 C-band, a polymorphic heterochromatin expansion that is unique to the human genome. Both cytogenetic features likely played key roles in the rapid HLS DUF1220 triplet hyperamplification, which is among the most striking genomic changes specific to the human lineage.

  8. Intra-domain phage display (ID-PhD) of peptides and protein mini-domains censored from canonical pIII phage display.

    PubMed

    Tjhung, Katrina F; Deiss, Frédérique; Tran, Jessica; Chou, Ying; Derda, Ratmir

    2015-01-01

    In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD) allows for simple quality control of the display and the natural variations in the displayed sequences. Additionally, direct linkage to phage propagation allows efficient monitoring of sequence cleavage, providing a convenient system for selection and evolution of protease-susceptible or protease-resistant sequences. As an example of the benefits of such an ID-PhD system, we displayed a negatively charged FLAG sequence, which is known to be post-translationally excised from pIII when displayed on the N-terminus, as well as positively charged sequences which suppress production of phage when displayed on the N-terminus. ID-PhD of FLAG exhibited sub-nanomolar apparent Kd suggesting multivalent nature of the display. A TEV-protease recognition sequence (TEVrs) co-expressed in tandem with FLAG, allowed us to demonstrate that 99.9997% of the phage displayed the FLAG-TEVrs tandem and can be recognized and cleaved by TEV-protease. The residual 0.0003% consisted of phage clones that have excised the insert from their genome. ID-PhD is also amenable to display of protein mini-domains, such as the 33-residue minimized Z-domain of protein A. We show that it is thus possible to use ID-PhD for multivalent display and selection of mini-domain proteins (Affibodies, scFv, etc.).

  9. Functions of the C-terminal domains of apoptosis-related proteins of the Bcl-2 family.

    PubMed

    Gómez-Fernández, Juan C

    2014-10-01

    Bcl-2 family proteins are involved in cell homeostasis, where they regulate cell death. Some of these proteins are pro-apoptotic and others pro-survival. Moreover, many of them share a similar domain composition with several of the so-called BH domains, although some only have a BH3 domain. A C-terminal domain is present in all the multi-BH domain proteins and in some of the BH3-only ones. This C-terminal domain is hydrophobic or amphipathic, for which reason it was thought when they were discovered that they were membrane anchors. Although this is indeed one of their functions, it has since been observed that they may also serve as regulators of the function of some members of this family, such as Bax. They may also serve to recognize the target membrane of some of these proteins, which only after an apoptotic signal, are incorporated into a membrane. It has been shown that peptides that imitate the sequence of C-terminal domains can form pores and may serve as a model to design cytotoxic molecules.

  10. Control of domain swapping in bovine odorant-binding protein.

    PubMed Central

    Ramoni, Roberto; Vincent, Florence; Ashcroft, Alison E; Accornero, Paolo; Grolli, Stefano; Valencia, Christel; Tegoni, Mariella; Cambillau, Christian

    2002-01-01

    As revealed by the X-ray structure, bovine odorant-binding protein (OBPb) is a domain swapped dimer [Tegoni, Ramoni, Bignetti, Spinelli and Cambillau (1996) Nat. Struct. Biol. 3, 863-867; Bianchet, Bains, Petosi, Pevsner, Snyder, Monaco and Amzel (1996) Nat. Struct. Biol. 3, 934-939]. This contrasts with all known mammalian OBPs, which are monomers, and in particular with porcine OBP (OBPp), sharing 42.3% identity with OBPb. By the mechanism of domain swapping, monomers are proposed to evolve into dimers and oligomers, as observed in human prion. Comparison of bovine and porcine OBP sequences pointed at OBPp glycine 121, in the hinge linking the beta-barrel to the alpha-helix. The absence of this residue in OBPb might explain why the normal lipocalin beta-turn is not formed. In order to decipher the domain swapping determinants we have produced a mutant of OBPb in which a glycine residue was inserted after position 121, and a mutant of OBPp in which glycine 121 was deleted. The latter mutation did not result in dimerization, while OBPb-121Gly+ became monomeric, suggesting that domain swapping was reversed. Careful structural analysis revealed that besides the presence of a glycine in the hinge, the dimer interface formed by the C-termini and by the presence of the lipocalins conserved disulphide bridge may also control domain swapping. PMID:11931632

  11. Properties of natural and artificial proteins displaying multiple ubiquitin-binding domains.

    PubMed

    Lopitz-Otsoa, Fernando; Rodríguez, Manuel S; Aillet, Fabienne

    2010-02-01

    Ubiquitylation provides a rapid alternative to control the activity of crucial cellular factors through the remodelling of a target protein. Diverse ubiquitin chains are recognized by domains with affinity for UBDs (ubiquitin-binding domains) present in receptor/effector proteins. Interestingly, some proteins contain more than one UBD and the preservation of this structure in many species suggests an evolutionary advantage for this topology. Here, we review some typical proteins that naturally contain more than one UBD and emphasize how such structures contribute to the mechanism they mediate. Characteristics such as higher affinities for polyubiquitin chains and chain-linkage preferences can be replicated by the TUBEs (tandem ubiquitin-binding entities). Furthermore, TUBEs show two additional properties: protection of ubiquitylated substrates from deubiquitylating enzymes and interference with the action of the proteasome. Consequently, TUBEs behave as 'ubiquitin traps' that efficiently capture endogenous ubiquitylated proteins. Interpretations and hypothetical models proposed by different groups to understand the synchronous action of multiple UBDs are discussed herein.

  12. The functional domain grouping of microtubule associated proteins

    PubMed Central

    Deane, Charlotte M; Wakefield, James G

    2008-01-01

    Microtubules (MTs), which play crucial roles in normal cell function, are regulated by MT associated proteins (MAPs). Using a combinatorial approach that includes biochemistry, proteomics and bioinformatics, we have recently identified 270 putative MAPs from Drosophila embryos and characterized some of those required for correct progression through mitosis. Here we identify functional groups of these MAPs using a reciprocal hits sequence alignment technique and assign InterPro functional domains to 28 previously uncharacterized proteins. This approach gives insight into the potential functions of MAPs and how their roles may affect MTs. PMID:19704789

  13. Roles for the coat protein telokin-like domain and the scaffolding protein amino-terminus

    PubMed Central

    Suhanovsky, Margaret M.; Teschke, Carolyn M.

    2011-01-01

    Assembly of icosahedral capsids of proper size and symmetry is not understood. Residue F170 in bacteriophage P22 coat protein is critical for conformational switching during assembly. Substitutions at this site cause assembly of tubes of hexamerically arranged coat protein. Intragenic suppressors of the ts phenotype of F170A and F170K coat protein mutants were isolated. Suppressors were repeatedly found in the coat protein telokin-like domain at position 285, which caused coat protein to assemble into petite procapsids and capsids. Petite capsid assembly strongly correlated to the side chain volume of the substituted amino acid. We hypothesize that larger side chains at position 285 torque the telokin-like domain, changing flexibility of the subunit and intercapsomer contacts. Thus, a single amino acid substitution in coat protein is sufficient to change capsid size. In addition, the products of assembly of the variant coat proteins were affected by the size of the internal scaffolding protein. PMID:21784500

  14. Single-domain protein folding: a multi-faceted problem

    NASA Astrophysics Data System (ADS)

    Junier, Ivan; Ritort, Felix

    2006-08-01

    We review theoretical approaches, experiments and numerical simulations that have been recently proposed to investigate the folding problem in single-domain proteins. From a theoretical point of view, we emphasize the energy landscape approach. As far as experiments are concerned, we focus on the recent development of single-molecule techniques. In particular, we compare the results obtained with two main techniques: single protein force measurements with optical tweezers and single-molecule fluorescence in studies on the same protein (RNase H). This allows us to point out some controversial issues such as the nature of the denatured and intermediate states and possible folding pathways. After reviewing the various numerical simulation techniques, we show that on-lattice protein-like models can help to understand many controversial issues.

  15. Single Molecular Film for Recognizing Biological Molecular Interaction: DNA-Protein Interaction and Enzyme Reaction

    NASA Astrophysics Data System (ADS)

    Kurihara, Kazue

    Protein-protein and protein-substrate interactions play essential roles in biological functions. Surface forces measurement and atomic force microscopy, which directly measure the interaction forces as a function of the surface separation, enable us to quantitatively evaluate these interactions [1-3]. We have employed the surface forces measurement [4] and colloidal probe atomic force microscopy [5] to study interactions involved in specific molecular recognition of DNA-protein and enzyme-substrate reaction. Studied are interactions between nucleic acid bases (adenine and thymine) [6], Spo0A-DB (the DNA-binding site of a transcription factor Spo0A), and DNA [7,8], those between subunits I and II of heptaprenyl diphosphate (HepPP) synthase in the presence of a substrate ((E,E)-farnesyl diphosphate, FPP) and a cofactor (Mg2+) [9-11], and the selectivity of the substrates in this enzymatic reaction [12]. Keys of our approach are the preparation of well-defined samples and the appropriate analysis. We have modified he substrate surfaces with these proteins using the Langmuir-Blodgett (LB) method. This chapter reviews the LB modification method and subsequent demonstrations of biological specific interactions employing this approach.

  16. Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2.

    PubMed

    Sasai, Kazumi; Fetterer, Raymond H; Lillehoj, Hyun; Matusra, Satomi; Constantinoiu, Constantin C; Matsubayashi, Makoto; Tani, Hiroyuki; Baba, Eiichiroh

    2008-12-01

    The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.

  17. The Anaplasma marginale msp5 gene encodes a 19-kilodalton protein conserved in all recognized Anaplasma species.

    PubMed Central

    Visser, E S; McGuire, T C; Palmer, G H; Davis, W C; Shkap, V; Pipano, E; Knowles, D P

    1992-01-01

    Immunization with Anaplasma marginale outer membranes induced immunity against clinical disease which correlated with antibody titer to outer membrane proteins, including a 19-kDa protein (N. Tebele, T. C. McGuire, and G. H. Palmer, Infect. Immun. 59:3199-3204, 1991). This 19-kDa protein, designated major surface protein 5 (MSP-5), was encoded by a single-copy 633-bp gene. The molecular mass of MSP-5, defined in immunoblots by binding to monoclonal antibody ANAF16C1, was conserved among all recognized species of Anaplasma: A. marginale, A. centrale, and A. ovis. Recombinant MSP-5, which absorbed the antibody reactivity of bovine immune serum to native MSP-5, was recognized by anti-A. marginale and anti-A. centrale immune sera in a competitive inhibition assay with monoclonal antibody ANAF16C1. The presence of antibody to the epitope defined by monoclonal antibody ANAF16C1 in all postinfection sera tested indicates that this epitope is a potential diagnostic antigen for use in identifying persistently infected cattle. Images PMID:1280624

  18. Identification and phylogenetic analyses of VASt, an uncharacterized protein domain associated with lipid-binding domains in Eukaryotes.

    PubMed

    Khafif, Mehdi; Cottret, Ludovic; Balagué, Claudine; Raffaele, Sylvain

    2014-06-26

    Several regulators of programmed cell death (PCD) in plants encode proteins with putative lipid-binding domains. Among them, VAD1 is a regulator of PCD propagation harboring a GRAM putative lipid-binding domain. However the function of VAD1 at the subcellular level is unknown and the domain architecture of VAD1 has not been analyzed in details. We analyzed sequence conservation across the plant kingdom in the VAD1 protein and identified an uncharacterized VASt (VAD1 Analog of StAR-related lipid transfer) domain. Using profile hidden Markov models (profile HMMs) and phylogenetic analysis we found that this domain is conserved among eukaryotes and generally associates with various lipid-binding domains. Proteins containing both a GRAM and a VASt domain include notably the yeast Ysp2 cell death regulator and numerous uncharacterized proteins. Using structure-based phylogeny, we found that the VASt domain is structurally related to Bet v1-like domains. We identified a novel protein domain ubiquitous in Eukaryotic genomes and belonging to the Bet v1-like superfamily. Our findings open perspectives for the functional analysis of VASt-containing proteins and the characterization of novel mechanisms regulating PCD.

  19. Peptidic degron in EID1 is recognized by an SCF E3 ligase complex containing the orphan F-box protein FBXO21

    PubMed Central

    Zhang, Cuiyan; Li, Xiaotong; Adelmant, Guillaume; Dobbins, Jessica; Geisen, Christoph; Oser, Matthew G.; Wucherpfenning, Kai W.; Marto, Jarrod A.; Kaelin, William G.

    2015-01-01

    EP300-interacting inhibitor of differentiation 1 (EID1) belongs to a protein family implicated in the control of transcription, differentiation, DNA repair, and chromosomal maintenance. EID1 has a very short half-life, especially in G0 cells. We discovered that EID1 contains a peptidic, modular degron that is necessary and sufficient for its polyubiquitylation and proteasomal degradation. We found that this degron is recognized by an Skp1, Cullin, and F-box (SCF)-containing ubiquitin ligase complex that uses the F-box Only Protein 21 (FBXO21) as its substrate recognition subunit. SCFFBXO21 polyubiquitylates EID1 both in vitro and in vivo and is required for the efficient degradation of EID1 in both cycling and quiescent cells. The EID1 degron partially overlaps with its retinoblastoma tumor suppressor protein-binding domain and is congruent with a previously defined melanoma-associated antigen-binding motif shared by EID family members, suggesting that binding to retinoblastoma tumor suppressor and melanoma-associated antigen family proteins could affect the polyubiquitylation and turnover of EID family members in cells. PMID:26631746

  20. Recognition of 2'-O-methylated 3'-end of piRNA by the PAZ domain of a Piwi protein.

    PubMed

    Simon, Bernd; Kirkpatrick, John P; Eckhardt, Stephanie; Reuter, Michael; Rocha, Elsa A; Andrade-Navarro, Miguel A; Sehr, Peter; Pillai, Ramesh S; Carlomagno, Teresa

    2011-02-09

    Piwi proteins are germline-specific Argonautes that associate with small RNAs called Piwi-interacting RNAs (piRNAs), and together with these RNAs are implicated in transposon silencing. The PAZ domain of Argonaute proteins recognizes the 3'-end of the RNA, which in the case of piRNAs is invariably modified with a 2'-O-methyl group. Here, we present the solution structure of the PAZ domain from the mouse Piwi protein, MIWI, in complex with an 8-mer piRNA mimic. The methyl group is positioned in a hydrophobic cavity made of conserved amino acids from strand β7 and helix α3, where it is contacted by the side chain of methionine-382. Our structure is similar to that of Ago-PAZ, but subtle differences illustrate how the PAZ domain has evolved to accommodate distinct 3' ends from a variety of RNA substrates.

  1. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  2. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  3. Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology.

    PubMed

    Cano-Garrido, Olivia; Sánchez-Chardi, Alejandro; Parés, Sílvia; Giró, Irene; Tatkiewicz, Witold I; Ferrer-Miralles, Neus; Ratera, Imma; Natalello, Antonino; Cubarsi, Rafael; Veciana, Jaume; Bach, Àlex; Villaverde, Antonio; Arís, Anna; Garcia-Fruitós, Elena

    2016-10-01

    Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest. The development of both natural and synthetic biomaterials for biomedical applications is a field in constant development. In this context, E. coli is a bacteria that has been widely studied for its ability to naturally produce functional biomaterials with broad biomedical uses. Despite being effective, products derived from this species contain membrane

  4. The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant.

    PubMed Central

    Schultze, B; Gross, H J; Brossmer, R; Herrler, G

    1991-01-01

    The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface. S protein is proposed to be responsible for the primary attachement of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated. Images PMID:1920630

  5. Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins.

    PubMed

    Martin-Serrano, Juan; Yarovoy, Anton; Perez-Caballero, David; Bieniasz, Paul D; Yaravoy, Anton

    2003-10-14

    The release of enveloped viruses from infected cells often requires a virally encoded activity, termed a late-budding domain (L domain), encoded by essential PTAP, PPXY, or YPDL sequence motifs. PTAP-type L domains recruit one of three endosomal sorting complexes required for transport (ESCRT-I). However, subsequent events in viral budding are poorly defined, and neither YPDL nor PPXY-type L domains require ESCRT-I. Here, we show that ESCRT-I and other class E vacuolar protein sorting (VPS) factors are linked by a complex series of protein-protein interactions. In particular, interactions between ESCRT-I and ESCRT-III are bridged by AIP-1/ALIX, a mammalian orthologue of the yeast class E VPS factor, Bro1. Expression of certain ESCRT-III components as fusion proteins induces a late budding defect that afflicts all three L-domain types, suggesting that ESCRT-III integrity is required in a general manner. Notably, the prototype YPDL-type L domain encoded by equine infectious anemia virus (EIAV) acts by recruiting AIP-1/ALIX and expression of a truncated form of AIP-1/ALIX or small interfering RNA-induced AIP-1/ALIX depletion specifically inhibits EIAV YPDL-type L-domain function. Overall, these findings indicate that L domains subvert a subset of class E VPS factors to mediate viral budding, some of which are required for each of the L-domain types, whereas others apparently act as adaptors to physically link specific L-domain types to the class E VPS machinery.

  6. Proteins on the catwalk: modelling the structural domains of the CCN family of proteins.

    PubMed

    Holbourn, Kenneth P; Perbal, Bernard; Ravi Acharya, K

    2009-03-01

    The CCN family of proteins (CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6) are multifunctional mosaic proteins that play keys roles in crucial areas of physiology such as angiogenesis, skeletal development tumourigenesis, cell proliferation, adhesion and survival. This expansive repertoire of functions comes through a modular structure of 4 discrete domains that act both independently and in concert. How these interactions with ligands and with neighbouring domains lead to the biological effects is still to be explored but the molecular structure of the domains is likely to play an important role in this. In this review we have highlighted some of the key features of the individual domains of CCN family of proteins based on their biological effects using a homology modelling approach.

  7. A DNA-binding protein containing two widely separated zinc finger motifs that recognize the same DNA sequence.

    PubMed

    Fan, C M; Maniatis, T

    1990-01-01

    We have isolated a full-length cDNA clone encoding a protein (PRDII-BF1) that binds specifically to a positive regulatory domain (PRDII) of the human IFN-beta gene promoter, and to a similar sequence present in a number of other promoters and enhancers. The sequence of this protein reveals two novel structural features. First, it is the largest sequence-specific DNA-binding protein reported to date (298 kD). Second, it contains two widely separated sets of C2-H2-type zinc fingers. Remarkably, each set of zinc fingers binds to the same DNA sequence motif with similar affinities and methylation interference patterns. Thus, this protein may act by binding simultaneously to reiterated copies of the same recognition sequence. Although the function of PRDII-BF1 is not known, the level of its mRNA is inducible by serum and virus, albeit with different kinetics.

  8. PDZ domain protein GIPC interacts with the cytoplasmic tail of melanosomal membrane protein gp75 (tyrosinase-related protein-1).

    PubMed

    Liu, T F; Kandala, G; Setaluri, V

    2001-09-21

    Tyrosinase and tyrosinase-related proteins (TRPs) are a family of melanosomal membrane proteins involved in mammalian pigmentation. Whereas the melanogenic functions of TRPs are localized in their amino-terminal domains that reside within the lumen of melanosomes, the sorting and targeting of these proteins to melanosomes is mediated by signals in their cytoplasmic domains. To identify proteins that interact with the cytoplasmic tail of gp75 (TRP-1), the most abundant melanosomal membrane protein, we performed yeast two-hybrid screening of a melanocyte cDNA library. Here, we show that the cytoplasmic domain of gp75 interacts with a PDZ domain-containing protein. The gp75-interacting protein is identical to GIPC, an RGS (regulator of G protein signaling)/GAIP-interacting protein, and to SEMCAP-1, a transmembrane semaphorin-binding protein. Carboxyl-terminal amino acid residues, Ser-Val-Val, of gp75 are necessary and sufficient for interaction of gp75 with the single PDZ domain in GIPC. Although endogenous and transfected GIPCs bind efficiently to transiently expressed gp75, only a small amount of GIPC is found associated with gp75 at steady state. Using a strategy to selectively synchronize the biosynthesis of endogenous gp75, we demonstrate that only newly synthesized gp75 associates with GIPC, primarily in the juxtanuclear Golgi region. Our data suggest that GIPC/SEMCAP-1 plays a role in biosynthetic sorting of proteins, specifically gp75, to melanosomes.

  9. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera

    SciTech Connect

    Aris, J.P.; Blobel, G. )

    1991-02-01

    The authors have isolated a 1.1-kilobase cDNA clone that encodes human fibrillarin by screening a hepatoma library in parallel with DNA probes derived from the fibrillarin genes of Saccharomyces cerevisiae (NOP1) and Xenopus laevis. RNA blot analysis indicates that the corresponding mRNA is {approximately}1,300 nucleotides in length. Human fibrillarin expressed in vitro migrates on SDS gels as a 36-kDa protein that is specifically immunoprecipitated by antisera from humans with scleroderma autoimmune disease. Human fibrillarin contains an amino-terminal repetitive domain {approximately}75-80 amino acids in length that is rich in glycine and arginine residues and is similar to amino-terminal domains in the yeast and Xenopus fibrillarins. The occurrence of a putative RNA-binding domain and an RNP consensus sequence within the protein is consistent with the association of fibrillarin with small nucleolar RNAs. Protein sequence alignments show that 67% of amino acids from human fibrillarin are identical to those in yeast fibrillarin and that 81% are identical to those in Xenopus fibrillarin. This identity suggests the evolutionary conservation of an important function early in the pathway for ribosome biosynthesis.

  10. The β1 domain of protein G can replace the chorismate mutase domain of the T-protein.

    PubMed

    Osuna, Joel; Flores, Humberto; Saab-Rincón, Gloria

    2012-02-17

    T-protein is composed of chorismate mutase (AroQ(T)) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQ(T) with the β1-domain of protein G (Gβ1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gβ1-TyrA folds into a dimeric conformation. Amino acid substitutions in the Gβ1 domain of Gβ1-TyrA identified residues involved in stabilizing the TyrA dimeric conformation. Gβ1 substitutions in the N-terminal β-hairpin eliminated Gβ1-TyrA expression, whereas Gβ1-TyrA tolerated Gβ1 substitutions in the C-terminal β-hairpin and in the α-helix. All of the characterized variants folded into a dimeric conformation. The importance of the β2-strand in forming a Gβ1 homo-dimerization interface explains the relevance of the first-β-hairpin in stabilizing the dimeric TyrA protein.

  11. Recognizing Faces

    ERIC Educational Resources Information Center

    Ellis, Hadyn D.

    1975-01-01

    The proposition that the mechanisms underlying facial recognition are different from those involved in recognizing other classes of pictorial material was assessed following a general review of the literature concerned with recognizing faces. (Author/RK)

  12. To con protection: TIR-domain containing proteins (Tcp) and innate immune evasion.

    PubMed

    Patterson, Nicholas J; Werling, Dirk

    2013-09-15

    The innate immune system provides the host's first line of defence against invading pathogens. Key to the stimulation of the innate immune response is pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs), which recognize microbial-associated molecular patterns (MAMPs). Binding of MAMPs to TLRs triggers a signalling cascade resulting in the production of pro-inflammatory mediators. Central to this TLR signalling pathway are heterotypic protein-protein interactions mediated through Toll/interleukin-1 receptor (TIR) domains found in both the cytoplasmic regions of TLRs and several key adaptor proteins. Interestingly, TIR-domain containing proteins (Tcps) do not seem to be unique to the mammalian TLR system, but occurs in abundance in many biological forms. Recent evidence suggests that pathogenic bacteria have developed a range of ingenuous strategies to evade the host immune mechanisms involving Tcps. There is increasing evidence to suggest that these pathogen-encoded Tcps interfere directly with the TLR signalling pathway and thus inhibit the activation of NF-κB, with different modes of action and roles in virulence. Here, we review the current state of knowledge on the possible roles and mechanisms of action of bacterial encoded Tcp. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Normalized Cut Algorithm for Automated Assignment of Protein Domains

    NASA Technical Reports Server (NTRS)

    Samanta, M. P.; Liang, S.; Zha, H.; Biegel, Bryan A. (Technical Monitor)

    2002-01-01

    We present a novel computational method for automatic assignment of protein domains from structural data. At the core of our algorithm lies a recently proposed clustering technique that has been very successful for image-partitioning applications. This grap.,l-theory based clustering method uses the notion of a normalized cut to partition. an undirected graph into its strongly-connected components. Computer implementation of our method tested on the standard comparison set of proteins from the literature shows a high success rate (84%), better than most existing alternative In addition, several other features of our algorithm, such as reliance on few adjustable parameters, linear run-time with respect to the size of the protein and reduced complexity compared to other graph-theory based algorithms, would make it an attractive tool for structural biologists.

  14. Using support vector machine for improving protein-protein interaction prediction utilizing domain interactions

    SciTech Connect

    Singhal, Mudita; Shah, Anuj R.; Brown, Roslyn N.; Adkins, Joshua N.

    2010-10-02

    Understanding protein interactions is essential to gain insights into the biological processes at the whole cell level. The high-throughput experimental techniques for determining protein-protein interactions (PPI) are error prone and expensive with low overlap amongst them. Although several computational methods have been proposed for predicting protein interactions there is definite room for improvement. Here we present DomainSVM, a predictive method for PPI that uses computationally inferred domain-domain interaction values in a Support Vector Machine framework to predict protein interactions. DomainSVM method utilizes evidence of multiple interacting domains to predict a protein interaction. It outperforms existing methods of PPI prediction by achieving very high explanation ratios, precision, specificity, sensitivity and F-measure values in a 10 fold cross-validation study conducted on the positive and negative PPIs in yeast. A Functional comparison study using GO annotations on the positive and the negative test sets is presented in addition to discussing novel PPI predictions in Salmonella Typhimurium.

  15. Molecular analysis of the binding mode of Toll/interleukin-1 receptor (TIR) domain proteins during TLR2 signaling.

    PubMed

    Nada, Masatoshi; Ohnishi, Hidenori; Tochio, Hidehito; Kato, Zenichiro; Kimura, Takeshi; Kubota, Kazuo; Yamamoto, Takahiro; Kamatari, Yuji O; Tsutsumi, Naotaka; Shirakawa, Masahiro; Kondo, Naomi

    2012-10-01

    Toll-like receptor (TLR) signaling is initiated by the binding of various adaptor proteins through ligand-induced oligomerization of the Toll/interleukin-1 receptor (TIR) domains of the TLRs. TLR2, which recognizes peptidoglycans, lipoproteins or lipopeptides derived from Gram-positive bacteria, is known to use the TIR domain-containing adaptor proteins myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal). Molecular analyses of the binding specificity of MyD88, Mal, and TLR2 are important for understanding the initial defenses mounted against Gram-positive bacterial infections such as Streptococcus pneumoniae. However, the detailed molecular mechanisms involved in the multiple interactions of these TIR domains remain unclear. Our study demonstrates that the TIR domain proteins MyD88, Mal, TLR1, and TLR2 directly bind to each other in vitro. We have also identified two binding interfaces of the MyD88 TIR domain for the TLR2 TIR domain. A residue at these interfaces has recently been found to be mutated in innate immune deficiency patients. These novel insights into the binding mode of TIR proteins will contribute to elucidation of the mechanisms underlying innate immune deficiency diseases, and to future structural studies of hetero-oligomeric TIR-TIR complexes. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. The Rapamycin-Binding Domain of the Protein Kinase mTOR is a Destabilizing Domain*

    PubMed Central

    Edwards, Sarah R.; Wandless, Thomas J.

    2013-01-01

    Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding domain (FRB) of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to ten-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retain the ability to inhibit mTOR, albeit with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wildtype FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems. PMID:17350953

  17. An Epigenetic Regulator: Methyl-CpG-Binding Domain Protein 1 (MBD1)

    PubMed Central

    Li, Lu; Chen, Bi-Feng; Chan, Wai-Yee

    2015-01-01

    DNA methylation is an important form of epigenetic regulation in both normal development and cancer. Methyl-CpG-binding domain protein 1 (MBD1) is highly related to DNA methylation. Its MBD domain recognizes and binds to methylated CpGs. This binding allows it to trigger methylation of H3K9 and results in transcriptional repression. The CXXC3 domain of MBD1 makes it a unique member of the MBD family due to its affinity to unmethylated DNA. MBD1 acts as an epigenetic regulator via different mechanisms, such as the formation of the MCAF1/MBD1/SETDB1 complex or the MBD1-HDAC3 complex. As methylation status always changes along with carcinogenesis or neurogenesis, MBD1 with its interacting partners, including proteins and non-coding RNAs, participates in normal or pathological processes and functions in different regulatory systems. Because of the important role of MBD1 in epigenetic regulation, it is a good candidate as a therapeutic target for diseases. PMID:25751725

  18. Method for identification of rigid domains and hinge residues in proteins based on exhaustive enumeration.

    PubMed

    Sim, Jaehyun; Sim, Jun; Park, Eunsung; Lee, Julian

    2015-06-01

    Many proteins undergo large-scale motions where relatively rigid domains move against each other. The identification of rigid domains, as well as the hinge residues important for their relative movements, is important for various applications including flexible docking simulations. In this work, we develop a method for protein rigid domain identification based on an exhaustive enumeration of maximal rigid domains, the rigid domains not fully contained within other domains. The computation is performed by mapping the problem to that of finding maximal cliques in a graph. A minimal set of rigid domains are then selected, which cover most of the protein with minimal overlap. In contrast to the results of existing methods that partition a protein into non-overlapping domains using approximate algorithms, the rigid domains obtained from exact enumeration naturally contain overlapping regions, which correspond to the hinges of the inter-domain bending motion. The performance of the algorithm is demonstrated on several proteins.

  19. Anti-HmuY Antibodies Specifically Recognize Porphyromonas gingivalis HmuY Protein but Not Homologous Proteins in Other Periodontopathogens

    PubMed Central

    Śmiga, Michał; Bielecki, Marcin; Olczak, Mariusz; Smalley, John W.; Olczak, Teresa

    2015-01-01

    Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium. PMID:25658942

  20. Novel leucine rich repeat domains in proteins from unicellular eukaryotes and bacteria.

    PubMed

    Miyashita, Hiroki; Kuroki, Yoshio; Matsushima, Norio

    2014-03-01

    Leucine rich repeats (LRRs) are present in over 20,000 proteins from viruses to eukaryotes. Two to sixty-two LRRs occur in tandem. Each repeat is typically 20-30 residues long and can be divided into an HCS (Highly conserved segment) and a VS (Variable segment). The HCS part consists of an eleven or a twelve residue stretch, LxxLxLxxNx(x/-)L, in which "L" is Leu, Ile, Val, or Phe, "N" is Asn, Thr, Ser, or Cys, "x" is a non-conserved residue, and "-" is a possible deletion site. Eight classes have been recognized. However, there are many unclassified or unrecognized LRRs. Here we performed to search novel LRRs using protein sequence database. The novel LRR domains are present over three hundred proteins, which include fungal ECM33 protein and Monosiga brevicollis LRR receptor kinase, from unicellular eukaryotes and bacteria. The HCS part is clearly different from that of the known LRRs and consists of a twelve or a thirteen residue stretch, VxGx(L/F)x(L/C)xxNx(x/-)L, that is characterized by the addition of Gly between the first conserved Val and the second conserved Leu. The novel LRRs identified here form a new family. The novel LRR domains were classified into four classes. The VS parts of the two classes are consistent with those of known, normal "SDS22-like" and "IRREKO" classes, while the other two classes have unique VS parts. The structures, functions, and evolution of the novel LRR domains and their proteins are described. The present results should stimulate various experimental studies.

  1. A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts.

    PubMed

    Asakura, Yukari; Barkan, Alice

    2007-12-01

    The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.

  2. Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase

    PubMed Central

    Vance, Steven; Tkachenko, Olga; Thomas, Ben; Bassuni, Mona; Hong, Hui; Nietlispach, Daniel; Broadhurst, William

    2016-01-01

    Type I modular polyketide synthases (PKSs) produce polyketide natural products by passing a growing acyl substrate chain between a series of enzyme domains housed within a gigantic multifunctional polypeptide assembly. Throughout each round of chain extension and modification reactions, the substrate stays covalently linked to an acyl carrier protein (ACP) domain. In the present study we report on the solution structure and dynamics of an ACP domain excised from MLSA2, module 9 of the PKS system that constructs the macrolactone ring of the toxin mycolactone, cause of the tropical disease Buruli ulcer. After modification of apo ACP with 4′-phosphopantetheine (Ppant) to create the holo form, 15N nuclear spin relaxation and paramagnetic relaxation enhancement (PRE) experiments suggest that the prosthetic group swings freely. The minimal chemical shift perturbations displayed by Ppant-attached C3 and C4 acyl chains imply that these substrate-mimics remain exposed to solvent at the end of a flexible Ppant arm. By contrast, hexanoyl and octanoyl chains yield much larger chemical shift perturbations, indicating that they interact with the surface of the domain. The solution structure of octanoyl-ACP shows the Ppant arm bending to allow the acyl chain to nestle into a nonpolar pocket, whereas the prosthetic group itself remains largely solvent exposed. Although the highly reduced octanoyl group is not a natural substrate for the ACP from MLSA2, similar presentation modes would permit partner enzyme domains to recognize an acyl group while it is bound to the surface of its carrier protein, allowing simultaneous interactions with both the substrate and the ACP. PMID:26920023

  3. Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase.

    PubMed

    Vance, Steven; Tkachenko, Olga; Thomas, Ben; Bassuni, Mona; Hong, Hui; Nietlispach, Daniel; Broadhurst, William

    2016-04-15

    Type I modular polyketide synthases (PKSs) produce polyketide natural products by passing a growing acyl substrate chain between a series of enzyme domains housed within a gigantic multifunctional polypeptide assembly. Throughout each round of chain extension and modification reactions, the substrate stays covalently linked to an acyl carrier protein (ACP) domain. In the present study we report on the solution structure and dynamics of an ACP domain excised from MLSA2, module 9 of the PKS system that constructs the macrolactone ring of the toxin mycolactone, cause of the tropical disease Buruli ulcer. After modification of apo ACP with 4'-phosphopantetheine (Ppant) to create the holo form, (15)N nuclear spin relaxation and paramagnetic relaxation enhancement (PRE) experiments suggest that the prosthetic group swings freely. The minimal chemical shift perturbations displayed by Ppant-attached C3 and C4 acyl chains imply that these substrate-mimics remain exposed to solvent at the end of a flexible Ppant arm. By contrast, hexanoyl and octanoyl chains yield much larger chemical shift perturbations, indicating that they interact with the surface of the domain. The solution structure of octanoyl-ACP shows the Ppant arm bending to allow the acyl chain to nestle into a nonpolar pocket, whereas the prosthetic group itself remains largely solvent exposed. Although the highly reduced octanoyl group is not a natural substrate for the ACP from MLSA2, similar presentation modes would permit partner enzyme domains to recognize an acyl group while it is bound to the surface of its carrier protein, allowing simultaneous interactions with both the substrate and the ACP. © 2016 The Author(s).

  4. Sugar-binding proteins from fish: selection of high affinity "lambodies" that recognize biomedically relevant glycans.

    PubMed

    Hong, Xia; Ma, Mark Z; Gildersleeve, Jeffrey C; Chowdhury, Sudipa; Barchi, Joseph J; Mariuzza, Roy A; Murphy, Michael B; Mao, Li; Pancer, Zeev

    2013-01-18

    Glycan-binding proteins are important for a wide variety of basic research and clinical applications, but proteins with high affinity and selectivity for carbohydrates are difficult to obtain. Here we describe a facile and cost-effective strategy to generate monoclonal lamprey antibodies, called lambodies, that target glycan determinants. We screened a library of yeast surface-displayed (YSD) lamprey variable lymphocyte receptors (VLR) for clones that can selectively bind various biomedically important glycotopes. These glycoconjugates included tumor-associated carbohydrate antigens (Tn and TFα), Lewis antigens (LeA and LeX), N-glycolylneuraminic acid, targets of broadly neutralizing HIV antibodies (poly-Man9 and the HIV gp120), and the glycoproteins asialo-ovine submaxillary mucin (aOSM) and asialo-human glycophorin A (aGPA). We isolated clones that bind each of these targets in a glycan-dependent manner and with very strong binding constants, for example, 6.2 nM for Man9 and 44.7 nM for gp120, determined by surface plasmon resonance (SPR). One particular lambody, VLRB.aGPA.23, was shown by glycan array analysis to be selective for the blood group H type 3 trisaccharide (BG-H3, Fucα1-2Galβ1-3GalNAcα), aGPA, and TFα (Galβ1-3GalNAcα), with affinity constants of 0.2, 1, and 8 nM, respectively. In human tissue microarrays this lambody selectively detected cancer-associated carbohydrate antigens in 14 different types of cancers. It stained 27% of non-small cell lung cancer (NSCLC) samples in a pattern that correlated with poor patient survival. Lambodies with exquisite affinity and selectivity for glycans may find myriad uses in glycobiology and biomedical research.

  5. Identifying the hierarchy of dynamic domains in proteins using the data of molecular dynamics simulations.

    PubMed

    Yesylevskyy, Semen O

    2010-04-01

    The Hierarchical Domain-Wise Alignment (HDWA) technique of domain identification in proteins is presented. HDWA is designed to identify hierarchically organized dynamic domains in proteins using the MD trajectories by eliminating systematic motions from MD trajectories recursively in a model-free manner. The method is tested on the proteins from different structural classes.

  6. M.(phi)BssHII, a novel cytosine-C5-DNA-methyltransferase with target-recognizing domains at separated locations of the enzyme.

    PubMed Central

    Sethmann, S; Ceglowski, P; Willert, J; Iwanicka-Nowicka, R; Trautner, T A; Walter, J

    1999-01-01

    In all cytosine-C5-DNA-methyltransferases (MTases) from prokaryotes and eukaryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order. In addition, one variable region, which includes the target-recognizing domain(s) (TRDs) characteristic for each enzyme, has been localized in one region between the same blocks of these conserved elements. This conservation in the order of conserved and variable sequences suggests stringent structural constraints in the primary structure to obtain the correct folding of the enzymes. Here we report the characterization of a new type of a multispecific MTase, M.(phiphi)BssHII, which is expressed as two isoforms. Isoform I is an entirely novel type of MTase which has, in addition to the TRDs at the conventional location, one TRD located at a non-canonical position at its N-terminus. Isoform II is represented by the same MTase, but without the N-terminal TRD. The N-terminal TRD provides HaeII methylation specificity to isoform I. The TRD is fully functional when engineered into either the conventional variable region of M.(phiphi)BssHII or the related monospecific M.phi3TII MTase. The implications of this structural plasticity with respect to the evolution of MTases are discussed. PMID:10369689

  7. Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

    PubMed

    Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

    2017-04-07

    Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

    PubMed

    Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

    2010-10-01

    The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion.

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

    PubMed Central

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

    2016-01-01

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

  10. Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

    PubMed

    Brewer, Matthew T; Agbedanu, Prince N; Zamanian, Mostafa; Day, Tim A; Carlson, Steve A

    2013-11-01

    Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

  11. The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins

    PubMed Central

    Dickey, Chad A.; Kamal, Adeela; Lundgren, Karen; Klosak, Natalia; Bailey, Rachel M.; Dunmore, Judith; Ash, Peter; Shoraka, Sareh; Zlatkovic, Jelena; Eckman, Christopher B.; Patterson, Cam; Dickson, Dennis W.; Nahman, N. Stanley; Hutton, Michael; Burrows, Francis; Petrucelli, Leonard

    2007-01-01

    A primary pathologic component of Alzheimer’s disease (AD) is the formation of neurofibrillary tangles composed of hyperphosphorylated tau (p-tau). Expediting the removal of these p-tau species may be a relevant therapeutic strategy. Here we report that inhibition of Hsp90 led to decreases in p-tau levels independent of heat shock factor 1 (HSF1) activation. A critical mediator of this mechanism was carboxy terminus of Hsp70–interacting protein (CHIP), a tau ubiquitin ligase. Cochaperones were also involved in Hsp90-mediated removal of p-tau, while those of the mature Hsp90 refolding complex prevented this effect. This is the first demonstration to our knowledge that blockade of the refolding pathway promotes p-tau turnover through degradation. We also show that peripheral administration of a novel Hsp90 inhibitor promoted selective decreases in p-tau species in a mouse model of tauopathy, further suggesting a central role for the Hsp90 complex in the pathogenesis of tauopathies. When taken in the context of known high-affinity Hsp90 complexes in affected regions of the AD brain, these data implicate a central role for Hsp90 in the development of AD and other tauopathies and may provide a rationale for the development of novel Hsp90-based therapeutic strategies. PMID:17304350

  12. Characterization of carbohydrate structural features recognized by anti-arabinogalactan-protein monoclonal antibodies.

    PubMed

    Yates, E A; Valdor, J F; Haslam, S M; Morris, H R; Dell, A; Mackie, W; Knox, J P

    1996-03-01

    Arabinogalactan-proteins (AGPs) are a diverse class of plant cell surface proteoglycans implicated in a range of fundamental processes associated with plant cell development. Anti-AGP monoclonal antibodies have been used extensively for the investigation of the developmental regulation of AGPs although virtually nothing is known about the structure of the carbohydrate epitopes recognised by these antibodies. In this report, a series of methyl glycosides of monosaccharides and a range of oligosaccharides that are elements of the carbohydrate component of AGPs have been investigated for recognition by previously derived anti-AGP monoclonal antibodies. No clear evidence was obtained for the involvement of terminal arabinofuranosides, nor of the galactan backbone, in the recognition of the glycan structure of AGPs by any of the antibodies used in this study. Interestingly, the most effective inhibitor of the binding of the monoclonal antibodies MAC207, JIM4 and JIM13 to exudate gum antigens was an acidic trisaccharide, isolated from a partial acid hydrolysate of gum karaya which has the structure: GlcA beta(1-->3) GalA alpha(1-->2)Rha, determined by a combination of FAB-MS, GC-MS and NMR spectroscopy.

  13. T Cells that Recognize HPV Protein Can Target Virus-Infected Cells | Center for Cancer Research

    Cancer.gov

    Adoptive T-cell transfer (ACT) is a promising form of cancer immunotherapy. Treating patients with T cells isolated from a tumor and subsequently expanded in the lab can cause the complete regression of some melanomas and cervical cancers, but the treatment is currently restricted to a few cancer types. An approach that may be applied to a wider array of cancers involves modifying peripheral blood T cells with chimeric antigen receptors or T-cell receptors (TCR) that target specific tumor antigens. Unfortunately, epithelial cancers, which are the vast majority of cancers diagnosed, have proven difficult to treat this way because most identified antigens are shared with healthy tissues and targeting them leads to toxic side effects. However, cancers caused by persistent human papillomavirus (HPV) infection, including cervical, head and neck, anal, vaginal, vulvar, and penile cancers, may be particularly amenable to the latter form of ACT since the E6 and E7 viral proteins are essential for cancer formation but are not produced in normal tissues. To test this idea, Christian Hinrichs, M.D., and his colleagues examined tumor infiltrating lymphocytes (TILs) from a patient who experienced a prolonged disease-free period after her second surgical removal of metastatic anal cancer in the hopes of identifying a TCR against one of the HPV oncoproteins.

  14. Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions

    PubMed Central

    Delaforge, Elise; Milles, Sigrid; Huang, Jie-rong; Bouvier, Denis; Jensen, Malene Ringkjøbing; Sattler, Michael; Hart, Darren J.; Blackledge, Martin

    2016-01-01

    Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales. PMID:27679800

  15. Hydrophobic-cluster analysis of plant protein sequences. A domain homology between storage and lipid-transfer proteins.

    PubMed Central

    Henrissat, B; Popineau, Y; Kader, J C

    1988-01-01

    Hydrophobic-cluster analysis was used to characterize a conserved domain located near the C-terminal amino acid sequence of wheat (Triticum aestivum) storage proteins. This domain was transformed into a linear template for a global search for similarities in over 5200 protein sequences. In addition to proteins that had already been found to exhibit homology to wheat storage proteins, a previously unreported homology was found with non-specific lipid-transfer proteins from castor bean (Ricinus communis) and from spinach (Spinacia oleracea) leaf. Hydrophobic-cluster analysis of various members of the present protein group clearly shows a typical domain structure where (i) variable and conserved domains are located along the sequence at precise positions, (ii) the conserved domains probably reflect a common ancestor, and (iii) the unique properties of a given protein (chain cut into subunits, repetitive domains, trypsin-inhibitor active site) are associated with the variable domains. PMID:3214430

  16. Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT).

    PubMed Central

    Reisz-Porszasz, S; Probst, M R; Fukunaga, B N; Hankinson, O

    1994-01-01

    The activated aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) bind DNA as a heterodimer. Both proteins represent a novel class of basic helix-loop-helix (bHLH)-containing transcription factors in that (i) activation of AHR requires the binding of ligand (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]), (ii) the xenobiotic responsive element (XRE) recognized by the AHR/ARNT heterodimer differs from the recognition sequence for nearly all other bHLH proteins, and (iii) both proteins contain a PAS homology region, which in the Drosophila PER and SIM proteins functions as a dimerization domain. A cDNA for mouse ARNT has been cloned, and potential functional domains of ARNT were investigated by deletion analysis. A mutant lacking all regions of ARNT other than the bHLH and PAS regions is unimpaired in TCDD-dependent dimerization and subsequent XRE binding and only modestly reduced in ability to complement an ARNT-deficient mutant cell line, c4, in vivo. Both the first and second alpha helices of the bHLH region are required for dimerization. The basic region is required for XRE binding but not for dimerization. Deletion of either the A or B segments of the PAS region slightly affects TCDD-induced heterodimerization, while deletion of the complete PAS region severely affects (but does not eliminate) dimerization. Thus, ARNT possesses multiple domains required for maximal heterodimerization. Mutants deleted for PAS A, PAS B, and the complete PAS region all retain some degree of XRE binding, yet none can rescue the c4 mutant. Therefore, both the PAS A and PAS B segments, besides contributing to dimerization, apparently fulfill additional, unknown functions required for biological activity of ARNT. Images PMID:8065341

  17. Experimental mapping of soluble protein domains using a hierarchical approach.

    PubMed

    Pedelacq, Jean-Denis; Nguyen, Hau B; Cabantous, Stephanie; Mark, Brian L; Listwan, Pawel; Bell, Carolyn; Friedland, Natasha; Lockard, Meghan; Faille, Alexandre; Mourey, Lionel; Terwilliger, Thomas C; Waldo, Geoffrey S

    2011-10-01

    Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.

  18. Experimental mapping of soluble protein domains using a hierarchical approach

    PubMed Central

    Pedelacq, Jean-Denis; Nguyen, Hau B.; Cabantous, Stephanie; Mark, Brian L.; Listwan, Pawel; Bell, Carolyn; Friedland, Natasha; Lockard, Meghan; Faille, Alexandre; Mourey, Lionel; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2011-01-01

    Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained. PMID:21771856

  19. The binding domain structure of retinoblastoma-binding proteins.

    PubMed Central

    Figge, J.; Breese, K.; Vajda, S.; Zhu, Q. L.; Eisele, L.; Andersen, T. T.; MacColl, R.; Friedrich, T.; Smith, T. F.

    1993-01-01

    The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding. PMID:8382993

  20. AGL15, a MADS domain protein expressed in developing embryos.

    PubMed Central

    Heck, G R; Perry, S E; Nichols, K W; Fernandez, D E

    1995-01-01

    To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo. PMID:7549483

  1. Genome-wide analysis of the WW domain-containing protein genes in silkworm and their expansion in eukaryotes.

    PubMed

    Meng, Gang; Dai, Fangyin; Tong, Xiaoling; Li, Niannian; Ding, Xin; Song, Jiangbo; Lu, Cheng

    2015-06-01

    WW domains are protein modules that mediate protein-protein interactions through recognition of proline-rich peptide motifs and phosphorylated serine/threonine-proline sites. WW domains are found in many different structural and signaling proteins that are involved in a variety of cellular processes. WW domain-containing proteins (WWCPs) and complexes have been implicated in major human diseases including cancer as well as in major signaling cascades such as the Hippo tumor suppressor pathway, making them targets for new diagnostics and therapeutics. There are a number of reports about the WWCPs in different species, but systematic analysis of the WWCP genes and its ligands is still lacking in silkworm and the other organisms. In this study, WWCP genes and PY motif-containing proteins have been identified and analyzed in 56 species including silkworm. Whole-genome screening of B. mori identified thirty-three proteins with thirty-nine WW domains located on thirteen chromosomes. In the 39 silkworm WW domains, 15 domains belong to the Group I WW domain; 14 domains were in Group II/III, 9 domains derived from 8 silkworm WWCPs could not be classified into any group, and Group IV contains only one WW domain. Based on gene annotation, silkworm WWCP genes have functions in multi-biology processes. A detailed list of WWCPs from the other 55 species was sorted in this work. In 14,623 silkworm predicted proteins, nearly 18 % contained PY motif, nearly 30 % contained various motifs totally that could be recognized by WW domains. Gene Ontology and KEGG analysis revealed that dozens of WW domain-binding proteins are involved in Wnt, Hedgehog, Notch, mTOR, EGF and Jak-STAT signaling pathway. Tissue expression patterns of WWCP genes and potential WWCP-binding protein genes on the third day of the fifth instar (L5D3) were examined by microarray analysis. Tissue expression profile analysis found that several WWCP genes and poly-proline or PY motif-containing protein genes took

  2. A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins.

    PubMed

    Bhatia, Vikram Kjøller; Hatzakis, Nikos S; Stamou, Dimitrios

    2010-06-01

    The discovery of proteins that recognize membrane curvature created a paradigm shift by suggesting that membrane shape may act as a cue for protein localization that is independent of lipid or protein composition. Here we review recent data on membrane curvature sensing by three structurally unrelated motifs: BAR domains, amphipathic helices and membrane-anchored proteins. We discuss the conclusion that the curvature of the BAR dimer is not responsible for sensing and that the sensing properties of all three motifs can be rationalized by the physicochemical properties of the curved membrane itself. We thus anticipate that membrane curvature will promote the redistribution of proteins that are anchored in membranes through any type of hydrophobic moiety, a thesis that broadens tremendously the implications of membrane curvature for protein sorting, trafficking and signaling in cell biology.

  3. SCAN domain-containing 2 gene (SCAND2) is a novel nuclear protein derived from the zinc finger family by exon shuffling.

    PubMed

    Dupuy, Denis; Dupérat, Véronique Guyonnet; Arveiler, Benoît

    2002-05-01

    The SCAN domain is a recently recognized protein domain that characterizes a subfamily of the Krüppel-like zinc finger proteins. We have previously described a novel SCAN domain-containing 2 gene (SCAND2) that does not belong to the zinc finger family. We report structural and sequence analyzes of all known members of the SCAN family and use these data to illustrate a model of gene family evolution. Most of the SCAN containing genes share common gene organization features that support the proposed origin for SCAND2 by disruption of an ancestral SCAN-zinc finger gene by a retroposition event and subsequent exon shuffling.

  4. Multi-megabase silencing in nucleolar dominance results from siRNA-directed de novo DNA methylation recognized by specific methylcytosine binding proteins

    PubMed Central

    Preuss, Sasha B.; Costa-Nunes, Pedro; Tucker, Sarah; Pontes, Olga; Lawrence, Richard J.; Mosher, Rebecca; Kasschau, Kristin D.; Carrington, James C.; Baulcombe, David C.; Viegas, Wanda; Pikaard, Craig S.

    2008-01-01

    In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2 and the methylcytosine binding domain proteins, MBD6 and MBD10 as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes, and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2 and the methylcytosine binding domain proteins, MBD6 and MBD10 as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes, and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. Collectively, these results indicate that in addition to directing the silencing of retrotransposons and noncoding repeats, siRNAs specify de novo cytosine methylation patterns that are recognized by MBD6 and MBD10 in the large-scale silencing of rRNA gene loci. PMID:19061642

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

    SciTech Connect

    Lechner, Mark S. . E-mail: msl27@drexel.edu; Schultz, David C.; Negorev, Dmitri; Maul, Gerd G.; Rauscher, Frank J.

    2005-06-17

    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.

  6. Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors

    PubMed Central

    Szomolay, Barbara; Liu, Jie; Brown, Paul E; Miles, John J; Clement, Mathew; Llewellyn-Lacey, Sian; Dolton, Garry; Ekeruche-Makinde, Julia; Lissina, Anya; Schauenburg, Andrea J; Sewell, Andrew K; Burrows, Scott R; Roederer, Mario; Price, David A; Wooldridge, Linda; van den Berg, Hugo A

    2016-01-01

    Evidence indicates that autoimmunity can be triggered by virus-specific CD8+ T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8+ T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8+ T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8+ T-cell clones are highly focused on their index peptide sequence and that ‘CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8+ T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases. PMID:26846725

  7. Protein Domain Analysis of Genomic Sequence Data Reveals Regulation of LRR Related Domains in Plant Transpiration in Ficus

    PubMed Central

    Lang, Tiange; Yin, Kangquan; Liu, Jinyu; Cao, Kunfang; Cannon, Charles H.; Du, Fang K.

    2014-01-01

    Predicting protein domains is essential for understanding a protein’s function at the molecular level. However, up till now, there has been no direct and straightforward method for predicting protein domains in species without a reference genome sequence. In this study, we developed a functionality with a set of programs that can predict protein domains directly from genomic sequence data without a reference genome. Using whole genome sequence data, the programming functionality mainly comprised DNA assembly in combination with next-generation sequencing (NGS) assembly methods and traditional methods, peptide prediction and protein domain prediction. The proposed new functionality avoids problems associated with de novo assembly due to micro reads and small single repeats. Furthermore, we applied our functionality for the prediction of leucine rich repeat (LRR) domains in four species of Ficus with no reference genome, based on NGS genomic data. We found that the LRRNT_2 and LRR_8 domains are related to plant transpiration efficiency, as indicated by the stomata index, in the four species of Ficus. The programming functionality established in this study provides new insights for protein domain prediction, which is particularly timely in the current age of NGS data expansion. PMID:25269070

  8. The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import.

    PubMed Central

    Weis, K; Ryder, U; Lamond, A I

    1996-01-01

    Nuclear proteins are targeted through the nuclear pore complex (NPC) in an energy-dependent reaction. The import reaction is mediated by nuclear localization sequences (NLS) in the substrate which are recognized by heterodimeric cytoplasmic receptors. hSRP1 alpha is an NLS-binding subunit of the human NLS receptor complex and is complexed in vivo with a second subunit of 97 kDa (p97). We show here that a short amino-terminal domain in hSRP1 alpha is necessary and sufficient for its interaction with p97. This domain is conserved in other SRP1-like proteins and its fusion to a cytoplasmic reporter protein is sufficient to promote complete nuclear import, circumventing the usual requirement for an NLS receptor interaction. The same amino-terminal domain inhibits import of NLS-containing proteins when added to an in vitro nuclear transport assay. While full-length hSRP alpha is able to leave the nucleus, the amino-terminal domain alone is not sufficient to promote exit. We conclude that hSRP1 alpha functions as an adaptor to tether NLS-containing substrates to the protein import machinery. Images PMID:8617227

  9. Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions.

    PubMed

    Wood, Kathleen H; Johnson, Brian S; Welsh, Sarah A; Lee, Jun Y; Cui, Yue; Krizman, Elizabeth; Brodkin, Edward S; Blendy, Julie A; Robinson, Michael B; Bartolomei, Marisa S; Zhou, Zhaolan

    2016-04-01

    DNA methylation is recognized by methyl-CpG-binding domain (MBD) proteins. Multiple MBDs are linked to neurodevelopmental disorders in humans and mice. However, the functions of MBD2 are poorly understood. We characterized Mbd2 knockout mice and determined spatiotemporal expression of MBDs and MBD2-NuRD (nucleosome remodeling deacetylase) interactions. We analyzed behavioral phenotypes, generated biotin-tagged MBD1 and MBD2 knockin mice, and performed biochemical studies of MBD2-NuRD. Most behavioral measures are minimally affected in Mbd2 knockout mice. In contrast to other MBDs, MBD2 shows distinct expression patterns. Unlike most MBDs, MBD2 is ubiquitously expressed in all tissues examined and appears dispensable for brain functions measured in this study. We provide novel genetic tools and reveal new directions to investigate MBD2 functions in vivo.

  10. Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions

    PubMed Central

    Wood, Kathleen H; Johnson, Brian S; Welsh, Sarah A; Lee, Jun Y; Cui, Yue; Krizman, Elizabeth; Brodkin, Edward S; Blendy, Julie A; Robinson, Michael B; Bartolomei, Marisa S; Zhou, Zhaolan

    2016-01-01

    Aim: DNA methylation is recognized by methyl-CpG-binding domain (MBD) proteins. Multiple MBDs are linked to neurodevelopmental disorders in humans and mice. However, the functions of MBD2 are poorly understood. We characterized Mbd2 knockout mice and determined spatiotemporal expression of MBDs and MBD2–NuRD (nucleosome remodeling deacetylase) interactions. Experimental procedures: We analyzed behavioral phenotypes, generated biotin-tagged MBD1 and MBD2 knockin mice, and performed biochemical studies of MBD2–NuRD. Results: Most behavioral measures are minimally affected in Mbd2 knockout mice. In contrast to other MBDs, MBD2 shows distinct expression patterns. Conclusion: Unlike most MBDs, MBD2 is ubiquitously expressed in all tissues examined and appears dispensable for brain functions measured in this study. We provide novel genetic tools and reveal new directions to investigate MBD2 functions in vivo. PMID:27066839

  11. A potent anti-dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface

    PubMed Central

    Fibriansah, Guntur; Tan, Joanne L; Smith, Scott A; Alwis, Adamberage R; Ng, Thiam-Seng; Kostyuchenko, Victor A; Ibarra, Kristie D; Wang, Jiaqi; Harris, Eva; Silva, Aravinda; Crowe, James E; Lok, Shee-Mei

    2014-01-01

    Dengue virus (DENV), which consists of four serotypes (DENV1-4), infects over 400 million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross-reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype-specific and bind to quaternary structure-dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6 Å resolution by cryo-EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI-DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection. Subject Categories Microbiology, Virology & Host Pathogen Interaction; Immunology PMID:24421336

  12. Functionalization of OEP-based benzochlorins to develop carbohydrate-conjugated photosensitizers. Attempt to target beta-galactoside-recognized proteins.

    PubMed

    Li, Guolin; Pandey, Suresh K; Graham, Andrew; Dobhal, Mahabeer P; Mehta, Ricky; Chen, Yihui; Gryshuk, Amy; Rittenhouse-Olson, Kate; Oseroff, Allan; Pandey, Ravindra K

    2004-01-09

    meso-(2-Formylvinyl)octaethylporphyrin on reaction with cyanotrimethylsilane in the presence of various catalysts [copper triflate [Cu(OTf)(2)], indium triflate [In(OTf)(3)], or magnesium bromide diethyl etherate (MgBr(2).Et(2)O)] produced a mixture of the intermediate 3-hydroxy-3-cyanopropenoporphyrin, the corresponding trimethylsilyl ether derivative, and the unexpected propenochlorins. The yields of the reaction products were found to depend on the reaction conditions and the catalysts used. The intermediate porphyrins on treatment with concentrated sulfuric acid yielded the free-base cyanobenzochlorins in major quantity along with several other novel benzochlorins as minor products. Reduction of ethyl-3-hydroxy-1-pentenoate-porphyrin with DIBAL-H/NaBH(4) and subsequent acid treatment provided the corresponding free-base 10(3)-(2-hydroxyethyl)benzochlorin, which upon a sequence of reactions gave a free-base benzochlorin bearing a carboxylic acid functionality in good yield. It was then condensed with a variety of carbohydrates (glucosamine, galactosamine, and lactosamine), and the related conjugates were screened using the galectin-binding-ability assay. Among the carbohydrate conjugates investigated, the lactose and galactose analogues displayed the galectin-binding ability with an enhancement of about 300-400-fold compared to lactose. In preliminary studies, all photosensitizers (with or without carbohydrate moieties) were found to be active in vitro [radiation-induced fibrosarcoma (RIF) tumor cells]. However, the cells incubated with lactose (known to bind to beta-galactoside-recognized proteins) prior to the addition of the photosensitizers containing the beta-galactose moiety (e.g., galactose and lactose) produced a 100% decrease in their photosensitizing efficacy. Under similar experimental conditions, benzochlorin without a beta-galactoside moiety or the related glucose conjugate did not show any inhibition in its photosensitizing efficacy. These results

  13. Conserved epitopes on HIV-1, FIV and SIV p24 proteins are recognized by HIV-1 infected subjects.

    PubMed

    Roff, Shannon R; Sanou, Missa P; Rathore, Mobeen H; Levy, Jay A; Yamamoto, Janet K

    2015-01-01

    Cross-reactive peptides on HIV-1 and FIV p24 protein sequences were studied using peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected long-term survivors (LTS; >10 y of infection without antiretroviral therapy, ART), short-term HIV-1 infected subjects not on ART, and ART-treated HIV-1 infected subjects. IFNγ-ELISpot and CFSE-proliferation analyses were performed with PBMC using overlapping HIV-1 and FIV p24 peptides. Over half of the HIV-1 infected subjects tested (22/31 or 71%) responded to one or more FIV p24 peptide pools by either IFNγ or T-cell proliferation analysis. PBMC and T cells from infected subjects in all 3 HIV(+) groups predominantly recognized one FIV p24 peptide pool (Fp14) by IFNγ production and one additional FIV p24 peptide pool (Fp9) by T-cell proliferation analysis. Furthermore, evaluation of overlapping SIV p24 peptide sequences identified conserved epitope(s) on the Fp14/Hp15-counterpart of SIV, Sp14, but none on Fp9-counterpart of SIV, Sp9. The responses to these FIV peptide pools were highly reproducible and persisted throughout 2-4 y of monitoring. Intracellular staining analysis for cytotoxins and phenotyping for CD107a determined that peptide epitopes from Fp9 and Fp14 pools induced cytotoxic T lymphocyte-associated molecules including perforin, granzyme B, granzyme A, and/or expression of CD107a. Selected FIV and corresponding SIV epitopes recognized by HIV-1 infected patients indicate that these protein sequences are evolutionarily conserved on both SIV and HIV-1 (e.g., Hp15:Fp14:Sp14). These studies demonstrate that comparative immunogenicity analysis of HIV-1, FIV, and SIV can identify evolutionarily-conserved T cell-associated lentiviral epitopes, which could be used as a vaccine for prophylaxis or immunotherapy.

  14. Electroporation of the photosynthetic membrane: structural changes in protein and lipid-protein domains.

    PubMed Central

    Rosemberg, Y; Rotenberg, M; Korenstein, R

    1994-01-01

    A biological membrane undergoes a reversible permeability increase through structural changes in the lipid domain when exposed to high external electric fields. The present study shows the occurrence of electric field-induced changes in the conductance of the proton channel of the H(+)-ATPase as well as electric field-induced structural changes in the lipid-protein domain of photosystem (PS) II in the photosynthetic membrane. The study was carried out by analyzing the electric field-stimulated delayed luminescence (EPL), which originates from charge recombination in the protein complexes of PS I and II of photosynthetic vesicles. We established that a small fraction of the total electric field-induced conductance change was abolished by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the H(+)-ATPase. This reversible electric field-induced conductance change has characteristics of a small channel and possesses a lifetime < or = 1 ms. To detect electric field-induced changes in the lipid-protein domains of PS II, we examined the effects of phospholipase A2 (PLA2) on EPL. Higher values of EPL were observed from vesicles that were exposed in the presence of PLA2 to an electroporating electric field than to a nonelectroporating electric field. The effect of the electroporating field was a long-lived one, lasting for a period > or = 2 min. This effect was attributed to long-lived electric field-induced structural changes in the lipid-protein domains of PS II. PMID:7811916

  15. A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

    PubMed

    Peterson, A J; Kyba, M; Bornemann, D; Morgan, K; Brock, H W; Simon, J

    1997-11-01

    The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

  16. Prediction of scaffold proteins based on protein interaction and domain architectures.

    PubMed

    Oh, Kimin; Yi, Gwan-Su

    2016-07-28

    Scaffold proteins are known for being crucial regulators of various cellular functions by assembling multiple proteins involved in signaling and metabolic pathways. Identification of scaffold proteins and the study of their molecular mechanisms can open a new aspect of cellular systemic regulation and the results can be applied in the field of medicine and engineering. Despite being highlighted as the regulatory roles of dozens of scaffold proteins, there was only one known computational approach carried out so far to find scaffold proteins from interactomes. However, there were limitations in finding diverse types of scaffold proteins because their criteria were restricted to the classical scaffold proteins. In this paper, we will suggest a systematic approach to predict massive scaffold proteins from interactomes and to characterize the roles of scaffold proteins comprehensively. From a total of 10,419 basic scaffold protein candidates in protein interactomes, we classified them into three classes according to the structural evidences for scaffolding, such as domain architectures, domain interactions and protein complexes. Finally, we could define 2716 highly reliable scaffold protein candidates and their characterized functional features. To assess the accuracy of our prediction, the gold standard positive and negative data sets were constructed. We prepared 158 gold standard positive data and 844 gold standard negative data based on the functional information from Gene Ontology consortium. The precision, sensitivity and specificity of our testing was 80.3, 51.0, and 98.5 % respectively. Through the function enrichment analysis of highly reliable scaffold proteins, we could confirm the significantly enriched functions that are related to scaffold protein binding. We also identified functional association between scaffold proteins and their recruited proteins. Furthermore, we checked that the disease association of scaffold proteins is higher than kinases. In

  17. Beauty is in the eye of the beholder: proteins can recognize binding sites of homologous proteins in more than one way.

    PubMed

    Martin, Juliette

    2010-06-17

    Understanding the mechanisms of protein-protein interaction is a fundamental problem with many practical applications. The fact that different proteins can bind similar partners suggests that convergently evolved binding interfaces are reused in different complexes. A set of protein complexes composed of non-homologous domains interacting with homologous partners at equivalent binding sites was collected in 2006, offering an opportunity to investigate this point. We considered 433 pairs of protein-protein complexes from the ABAC database (AB and AC binary protein complexes sharing a homologous partner A) and analyzed the extent of physico-chemical similarity at the atomic and residue level at the protein-protein interface. Homologous partners of the complexes were superimposed using Multiprot, and similar atoms at the interface were quantified using a five class grouping scheme and a distance cut-off. We found that the number of interfacial atoms with similar properties is systematically lower in the non-homologous proteins than in the homologous ones. We assessed the significance of the similarity by bootstrapping the atomic properties at the interfaces. We found that the similarity of binding sites is very significant between homologous proteins, as expected, but generally insignificant between the non-homologous proteins that bind to homologous partners. Furthermore, evolutionarily conserved residues are not colocalized within the binding sites of non-homologous proteins. We could only identify a limited number of cases of structural mimicry at the interface, suggesting that this property is less generic than previously thought. Our results support the hypothesis that different proteins can interact with similar partners using alternate strategies, but do not support convergent evolution.

  18. VISUALIZIATION OF CELLULAR PHOSPHOINOSITIDE POOLS WITH GFP-FUSED PROTEIN-DOMAINS

    PubMed Central

    Balla, Tamas; Várnai, Péter

    2011-01-01

    This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present only in tiny amounts compared to structural lipids but are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane-recruitment and activity of many protein signaling-complexes in specific membrane compartments and have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single cell level. The only available technique in live cell application is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules when fused to fluorescent proteins can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular resolution and rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. Here, we summarize the design and practical use of these constructs and also review important considerations for the interpretation of the data obtained by this technique. PMID:19283730

  19. Identification of immunogenic proteins from ovarian tissue and recognized in larval extracts of Rhipicephalus (Boophilus) microplus, through an immunoproteomic approach.

    PubMed

    Ramírez Rodríguez, Patricia Berenice; Rosario Cruz, Rodrigo; Domínguez García, Delia Inés; Hernández Gutiérrez, Rodolfo; Lagunes Quintanilla, Rodolfo Esteban; Ortuño Sahagún, Daniel; González Castillo, Celia; Gutiérrez Ortega, Abel; Herrera Rodríguez, Sara Elisa; Vallejo Cardona, Adriana; Martínez Velázquez, Moisés

    2016-11-01

    Rhipicephalus (Boophilus) microplus ticks are obligatory hematophagous ectoparasites of cattle and act as vectors for disease-causing microorganisms. Conventional tick control is based on the use of chemical acaricides; however, their uncontrolled use has increased tSresistant tick populations, as well as food and environmental contamination. Alternative immunological tick control has shown to be partially effective. The only anti-tick vaccine commercially available at present in the world is based on intestinal Bm86 protein, and shows a variable effectiveness depending on tick strains or geographic isolates. Therefore, there is a need to characterize new antigens in order to improve immunological protection. The aim of this work was to identify immunogenic proteins from ovarian tissue extracts of R. microplus, after cattle immunization. Results showed that ovarian proteins complexed with the adjuvant Montanide ISA 50 V generated a strong humoral response on vaccinated cattle. IgG levels peaked at fourth post-immunization week and remained high until the end of the experiment. 1D and 2D SDS-PAGE-Western blot assays with sera from immunized cattle recognized several ovarian proteins. Reactive bands were cut and analyzed by LC-MS/MS. They were identified as Vitellogenin, Vitellogenin-2 precursor and Yolk Cathepsin. Our findings along with bioinformatic analysis indicate that R. microplus has several Vitellogenin members, which are proteolytically processed to generate multiple polypeptide fragments. This apparent complexity of vitellogenic tick molecular targets gives the opportunity to explore their potential usefulness as vaccine candidates but, at the same time, imposes a challenge on the selection of the appropriate set of antigens. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. A functional protein pore with a "retro" transmembrane domain.

    PubMed Central

    Cheley, S.; Braha, O.; Lu, X.; Conlan, S.; Bayley, H.

    1999-01-01

    Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif. PMID:10386875

  1. Improving protein-protein interaction article classification using biological domain knowledge.

    PubMed

    Chen, Yifei; Guo, Hongjian; Liu, Feng; Manderick, Bernard

    2015-01-01

    Interaction Article Classification (IAC) is a specific text classification application in biological domain that tries to find out which articles describe Protein-Protein Interactions (PPIs) to help extract PPIs from biological literature more efficiently. However, the existing text representation and feature weighting schemes commonly used for text classification are not well suited for IAC. We capture and utilise biological domain knowledge, i.e. gene mentions also known as protein or gene names in the articles, to address the problem. We put forward a new gene mention order-based approach that highlights the important role of gene mentions to represent the texts. Furthermore, we also incorporate the information concerning gene mentions into a novel feature weighting scheme called Gene Mention-based Term Frequency (GMTF). By conducting experiments, we show that using the proposed representation and weighting schemes, our Interaction Article Classifier (IACer) performs better than other leading systems for the moment.

  2. Spike Protein VP8* of Human Rotavirus Recognizes Histo-Blood Group Antigens in a Type-Specific Manner

    PubMed Central

    Huang, Pengwei; Xia, Ming; Zhong, Weiming; Wei, Chao; Wang, Leyi; Morrow, Ardythe

    2012-01-01

    Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Leb) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Leb and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells. PMID:22345472

  3. Identification of two novel mouse nuclear proteins that bind selectively to a methylated c-Myc recognizing sequence.

    PubMed Central

    Suetake, I; Tajima, S; Asano, A

    1993-01-01

    The c-Myc recognizes the sequence CACGTG (Blackwell, T. K., Kretzner, L., Blackwood, E.M., Eisenman, R. N., and Weintraub, H. (1990) Science 250, 1149-1151), and its binding is inhibited by methylation of the core CpG (Prendergast, G. C. and Ziff, E. B. (1991) Science 251, 186-189). We identified two novel nuclear proteins, MMBP-1 and MMBP-2, that bound specifically and under physiological salt condition to the c-Myc binding motif of which cytidine in the CpG sequence was methylated. MMBP-1 was about 42 kD and MMBP-2 was about 63 kD. MMBP-1 was found in specific cells, while MMBP-2 was found in all the cell lines tested, suggesting that MMBP-1 may modulate the role of MMBP-2 in tissue specific manner. We propose that the two proteins play a role in the regulation of c-Myc function through stabilizing or destabilizing the methylation state of the c-Myc binding motif. Images PMID:8502552

  4. Four C1q domain-containing proteins involved in the innate immune response in Hyriopsis cumingii.

    PubMed

    Zhao, Ling-Ling; Jin, Min; Li, Xin-Cang; Ren, Qian; Lan, Jiang-Feng

    2016-08-01

    C1q is a key subcomponent of the complement C1 complex. This subcomponent contains a globular C1q (gC1q) domain with remarkable ligand binding properties. C1q domain-containing (C1qDC) proteins are composed of all proteins with a gC1q domain. C1qDC proteins exist in many invertebrates and recognize non-self-ligands. In our study, four C1qDC genes, namely, HcC1qDC1-HcC1qDC4, were identified from Hyriopsis cumingii. HcC1qDC1-HcC1qDC4 encode a protein of 224, 204, 305, and 332 amino acids, respectively. All C1qDC proteins consist of a gC1q domain at the C terminal. In addition to the gC1q domain, a coiled-coil region is found in HcC1qDC4. Multiple alignments and phylogenetic tree analysis revealed that the C1qDC proteins highly differ from one another. Tissue distribution analysis demonstrated that HcC1qDC1-HcC1qDC4 are widely distributed in hemocytes, hepatopancreas, gills, mantle, and foot. These C1qDC genes are regulated by bacteria to varying degrees. These recombinant HcC1qDC proteins exhibit a binding activity against different bacterial species. Our results may suggest the roles of HcC1qDC genes in anti-bacterial immune defense. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

    PubMed

    Ogawara, Hiroshi

    2016-09-01

    PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

  6. The Epitope Recognized by Monoclonal Antibody 2B6 in the B/C Domains of Classical Swine Fever Virus Glycoprotein E2 Affects Viral Binding to Hyperimmune Sera and Replication.

    PubMed

    Tong, Chao; Chen, Ning; Liao, Xun; Xie, Wenqi; Li, Dejiang; Li, Xiaoliang; Fang, Weihuan

    2015-04-01

    Classical swine fever (CSF) is a highly contagious disease of pigs caused by CSF virus (CSFV). E2 is the major viral envelope protein of immune dominance that induces neutralizing antibodies and confers protection against CSFV infection. The B/C domains of E2 are variable among CSFV isolates, which could affect immunogenicity and binding to antibodies. We attempted to characterize the epitope recognized by a monoclonal antibody 2B6 (mAb-2B6) raised against the E2 B/C domains of the vaccine C-strain and to examine if mutations in the epitope region would affect antibody binding and viral neutralization. The epitope specific for mAb-2B6 recognition is linear, spanning five residues (774)DGXNP(778) in the B/C domains. The residue N777 is indispensable for the specificity. The epitope exists only in group 1 strains, but not in those of group 2. The recombinant viruses containing individual mutations on the epitope region lost the reactivity to mAb-2B6. The mutant virus RecC-N777S had low replication potential, about 10-fold decrease in the yield of progeny virus particles, whereas the mutant virus RecC-P778A reverted to proline upon continuous passaging. The mutations on the mAb-2B6 epitope region did not affect neutralization by anti-C-strain polyclonal sera from pigs. Deletion from aa774 covering the mAb-2B6 epitope, but not that from aa781, also affected binding with the polyclonal antibodies from vaccinated pigs, although the major binding region for the vaccinated antibodies is aa690-773.

  7. Structural organization and interactions of transmembrane domains in tetraspanin proteins

    PubMed Central

    Kovalenko, Oleg V; Metcalf, Douglas G; DeGrado, William F; Hemler, Martin E

    2005-01-01

    Background Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed. Results Among 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)n. In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80. Conclusion Our results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that

  8. TRALI ASSOCIATED HNA-3a ANTIBODIES RECOGNIZE COMPLEX DETERMINANTS ON CHOLINE TRANSPORTER-LIKE PROTEIN 2 (CTL2)

    PubMed Central

    Bougie, Daniel W; Peterson, Julie A; Kanack, Adam J; Curtis, Brian R; Aster, Richard H

    2014-01-01

    Background HNA-3a specific antibodies can cause severe, sometimes fatal, transfusion related acute lung injury (TRALI) when present in transfused blood. The HNA3-a/b antigens are determined by an R154Q polymorphism in the first of five extracellular loops of the 10-membrane spanning choline transporter-like protein 2 (CTL2) expressed on neutrophils, lymphocytes and other tissues. About 50% of HNA-3a antibodies (Type 1) can be detected using CTL2 Loop 1 peptides containing R154; the remaining 50% (Type 2) fail to recognize this target. Understanding the basis for this difference could guide efforts to develop practical assays to screen blood donors for HNA-3 antibodies. Study design and methods Reactions of HNA-3a antibodies against recombinant versions of human, mouse, and human/mouse (chimeric) CTL2 were characterized using flow cytometry and various solid phase assays. Results Findings made show that, for binding to CTL2, Type 2 HNA-3a antibodies require non-polymorphic amino acid residues in the third, and possibly the second, extracellular loops of CTL2 to be in a configuration comparable to that found naturally in the cell membrane. In contrast, Type 1 antibodies require only peptides from the first extracellular loop that contain R154 for recognition. Conclusion Although Type 1 HNA-3a antibodies can readily be detected in solid phase assays that use a CTL2 peptide containing R154 as a target, development of a practical test to screen blood donors for Type 2 antibodies will pose a serious technical challenge because of the complex nature of the epitope(s) recognized by this antibody sub-group. PMID:24846273

  9. Gag Protein Epitopes Recognized by CD4+ T-Helper Lymphocytes from Equine Infectious Anemia Virus-Infected Carrier Horses

    PubMed Central

    Lonning, S. M.; Zhang, W.; McGuire, T. C.

    1999-01-01

    Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV. PMID:10196322

  10. Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

    SciTech Connect

    Asojo, Oluwatoyin A.

    2011-05-01

    The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structure was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.

  11. Mutual effects of disorder and order in fusion proteins between intrinsically disordered domains and fluorescent proteins.

    PubMed

    Lotti, Marina; Longhi, Sonia

    2012-01-01

    Intrinsically disordered proteins are being paid an increasing amount of interest due to the understanding of the crucial role that flexible regions play in molecular recognition and in signaling. Accordingly, reports focusing on the structural and functional characterization of intrinsically disordered proteins or regions are growing exponentially. Relatively few studies have however been reported on the mutual effects of ordered and disordered moieties in artificial fusion proteins. In this review, we focus on the few available experimental data based on the use of chimeras in which fluorescent proteins were fused to disordered domains of different lengths, compactness and propensity to form secondary structures. The impact of the artificial fusion on the conformational and functional properties of the resulting proteins is discussed.

  12. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    PubMed

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-02-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

  13. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    PubMed Central

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-01-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin. PMID:8552082

  14. A conserved domain targets exported PHISTb family proteins to the periphery of Plasmodium infected erythrocytes

    PubMed Central

    Tarr, Sarah J.; Moon, Robert W.; Hardege, Iris; Osborne, Andrew R.

    2014-01-01

    During blood-stage infection, malaria parasites export numerous proteins to the host erythrocyte. The Poly-Helical Interspersed Sub-Telomeric (PHIST) proteins are an exported family that share a common ‘PRESAN’ domain, and include numerous members in Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi. In P. falciparum, PHIST proteins have been implicated in protein trafficking and intercellular communication. A number of PHIST proteins are essential for parasite survival. Here, we identify nine members of the PHISTb sub-class of PHIST proteins, including one protein known to be essential for parasite survival, that localise to the erythrocyte periphery. These proteins have solubility characteristics consistent with their association with the erythrocyte cytoskeleton. Together, an extended PRESAN domain, comprising the PRESAN domain and preceding sequence, form a novel targeting-domain that is sufficient to localise a protein to the erythrocyte periphery. We validate the role of this domain in RESA, thus identifying a cytoskeleton-binding domain in RESA that functions independently of its known spectrin-binding domain. Our data suggest that some PHISTb proteins may act as cross-linkers of the erythrocyte cytoskeleton. We also show for the first time that peripherally-localised PHISTb proteins are encoded in genomes of P. knowlesi and vivax indicating a conserved role for the extended PRESAN domain of these proteins in targeting to the erythrocyte periphery. PMID:25106850

  15. Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein.

    PubMed

    Buck, Teresa M; Jordahl, Alexa S; Yates, Megan E; Preston, G Michael; Cook, Emily; Kleyman, Thomas R; Brodsky, Jeffrey L

    2017-02-01

    In the kidney, the epithelial sodium channel (ENaC) regulates blood pressure through control of sodium and volume homeostasis, and in the lung, ENaC regulates the volume of airway and alveolar fluids. ENaC is a heterotrimer of homologous α-, β- and γ-subunits, and assembles in the endoplasmic reticulum (ER) before it traffics to and functions at the plasma membrane. Improperly folded or orphaned ENaC subunits are subject to ER quality control and targeted for ER-associated degradation (ERAD). We previously established that a conserved, ER lumenal, molecular chaperone, Lhs1/GRP170, selects αENaC, but not β- or γ-ENaC, for degradation when the ENaC subunits were individually expressed. We now find that when all three subunits are co-expressed, Lhs1-facilitated ERAD was blocked. To determine which domain-domain interactions between the ENaC subunits are critical for chaperone-dependent quality control, we employed a yeast model and expressed chimeric α/βENaC constructs in the context of the ENaC heterotrimer. We discovered that the βENaC transmembrane domain was sufficient to prevent the Lhs1-dependent degradation of the α-subunit in the context of the ENaC heterotrimer. Our work also found that Lhs1 delivers αENaC for proteasome-mediated degradation after the protein has become polyubiquitinated. These data indicate that the Lhs1 chaperone selectively recognizes an immature form of αENaC, one which has failed to correctly assemble with the other channel subunits via its transmembrane domain. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  16. Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

    PubMed Central

    McAuley, Alexander J.; Torres, Maricela; Plante, Jessica A.; Huang, Claire Y.-H.; Bente, Dennis A.

    2016-01-01

    ABSTRACT Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, with in vivo pathogenesis often not being correlated with in vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence. IMPORTANCE The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric

  17. Glucocorticoids repress transcription from a negative glucocorticoid response element recognized by two homeodomain-containing proteins, Pbx and Oct-1.

    PubMed

    Subramaniam, N; Cairns, W; Okret, S

    1998-09-04

    Several studies have established that the prolactin (PRL) gene is expressed not only in lactotrophs and somatotrophs of the anterior pituitary but, albeit to a lesser extent, in non-pituitary cells like human thymocytes, decidualized endometrium, mammary glands during lactation, and some human non-pituitary cell lines. Despite the requirement in the pituitary for the pituitary-specific transcription factor Pit-1/GHF-1 for PRL expression, the expression in non-pituitary cells occurs in the absence of Pit-1/GHF-1 and can be repressed by glucocorticoids. This prompted us to investigate the transcription factors in non-pituitary cells which are involved in controlling expression and glucocorticoid repression of a previously characterized negative glucocorticoid response element from the bovine prolactin gene (PRL3 nGRE). Here we have demonstrated that non-pituitary cells (COS-7 and mouse hepatoma Hepa1c1c7 cells) conferred increased expression via the PRL3 nGRE mainly because of the binding of the ubiquitously expressed POU-homeodomain-containing octamer transcription factor-1 (Oct-1) to an AT-rich sequence present in the PRL3 sequence. However, full transcriptional activity required the binding of a second ubiquitously expressed homeodomain-containing protein, Pbx, previously shown to bind cooperatively with several homeotic selector proteins. The Pbx binding site in the PRL3 nGRE, located just upstream of the Oct-1 binding site, showed a strong sequence similarity with known Pbx binding sites and bound Pbx with an affinity similar to that of other established Pbx target sequences. Interestingly, both Oct-1 and Pbx binding to the PRL3 nGRE were found to be required for glucocorticoid repression. Addition of in vitro translated glucocorticoid receptor DNA binding domain to the nuclear extract prevented Oct-1 and Pbx from binding to the PRL element. The involvement of the homeobox protein Pbx in glucocorticoid repression via an nGRE identifies a new role for this

  18. Molecular characterization and ligand binding specificity of the PDZ domain-containing protein GIPC3 from Schistosoma japonicum

    PubMed Central

    2012-01-01

    Background Schistosomiasis is a serious global health problem that afflicts more than 230 million people in 77 countries. Long-term mass treatments with the only available drug, praziquantel, have caused growing concerns about drug resistance. PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins are recognized as potential targets for the next generation of drug development. However, the PDZ domain-containing protein family in parasites has largely been unexplored. Methods We present the molecular characteristics of a PDZ domain-containing protein, GIPC3, from Schistosoma japonicum (SjGIPC3) according to bioinformatics analysis and experimental approaches. The ligand binding specificity of the PDZ domain of SjGIPC3 was confirmed by screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays. The native ligand candidates were predicted by Tailfit software based on the C-terminal binding specificity, and further validated by Y2H assays. Results SjGIPC3 is a single PDZ domain-containing protein comprised of 328 amino acid residues. Structural prediction revealed that a conserved PDZ domain was presented in the middle region of the protein. Phylogenetic analysis revealed that SjGIPC3 and other trematode orthologues clustered into a well-defined cluster but were distinguishable from those of other phyla. Transcriptional analysis by quantitative RT-PCR revealed that the SjGIPC3 gene was relatively highly expressed in the stages within the host, especially in male adult worms. By using Y2H assays to screen an arbitrary peptide library, we confirmed the C-terminal binding specificity of the SjGIPC3-PDZ domain, which could be deduced as a consensus sequence, -[SDEC]-[STIL]-[HSNQDE]-[VIL]*. Furthermore, six proteins were predicted to be native ligand candidates of SjGIPC3 based on the C-terminal binding properties and other biological information; four of these were confirmed to be potential ligands using the Y2H system. Conclusions In this study, we first

  19. The SBASE protein domain library, release 2.0: a collection of annotated protein sequence segments.

    PubMed Central

    Pongor, S; Skerl, V; Cserzö, M; Hátsági, Z; Simon, G; Bevilacqua, V

    1993-01-01

    SBASE 2.0 is the second release of SBASE, a collection of annotated protein domain sequences. SBASE entries represent various structural, functional, ligand-binding and topogenic segments of proteins [Pongor, S. et al. (1993) Prot. Eng., in press]. This release contains 34,518 entries provided with standardized names and it is cross-referenced to the major protein and nucleic acid databanks as well as to the PROSITE catalog of protein sequence patterns [Bairoch, A. (1992) Nucl. Acids Res., 20 suppl, 2013-2018]. SBASE can be used for establishing domain homologies using different database-search tools such as FASTA [Lipman and Pearson (1985) Science, 227, 1436-1441], FASTDB [Brutlag et al. (1990) Comp. Appl. Biosci., 6, 237-245] or BLAST3 [Altschul and Lipman (1990) Proc. Natl. Acad. Sci. USA, 87, 5509-5513] which is especially useful in the case of loosely defined domain types for which efficient consensus patterns can not be established. SBASE 2.0 and a set of search and retrieval tools are freely available on request to the authors or by anonymous 'ftp' file transfer from mean value of ftp.icgeb.trieste.it. PMID:8332532

  20. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures.

    PubMed

    Marchler-Bauer, Aron; Bo, Yu; Han, Lianyi; He, Jane; Lanczycki, Christopher J; Lu, Shennan; Chitsaz, Farideh; Derbyshire, Myra K; Geer, Renata C; Gonzales, Noreen R; Gwadz, Marc; Hurwitz, David I; Lu, Fu; Marchler, Gabriele H; Song, James S; Thanki, Narmada; Wang, Zhouxi; Yamashita, Roxanne A; Zhang, Dachuan; Zheng, Chanjuan; Geer, Lewis Y; Bryant, Stephen H

    2017-01-04

    NCBI's Conserved Domain Database (CDD) aims at annotating biomolecular sequences with the location of evolutionarily conserved protein domain footprints, and functional sites inferred from such footprints. An archive of pre-computed domain annotation is maintained for proteins tracked by NCBI's Entrez database, and live search services are offered as well. CDD curation staff supplements a comprehensive collection of protein domain and protein family models, which have been imported from external providers, with representations of selected domain families that are curated in-house and organized into hierarchical classifications of functionally distinct families and sub-families. CDD also supports comparative analyses of protein families via conserved domain architectures, and a recent curation effort focuses on providing functional characterizations of distinct subfamily architectures using SPARCLE: Subfamily Protein Architecture Labeling Engine. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.

  1. The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

    PubMed

    Swiderski, Michal R; Birker, Doris; Jones, Jonathan D G

    2009-02-01

    In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

  2. Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions.

    PubMed Central

    Meyers, S; Downing, J R; Hiebert, S W

    1993-01-01

    The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) translocation associated with acute myelogenous leukemia and encodes a protein with a central 118-amino-acid domain with 69% homology to the Drosophila pair-rule gene, runt. We demonstrate that AML-1 is a DNA-binding protein which specifically interacts with a sequence belonging to the group of enhancer core motifs, TGT/cGGT. Electrophoretic mobility shift analysis of cell extracts identified two AML-1-containing protein-DNA complexes whose electrophoretic mobilities were slower than those of complexes formed with AML-1 produced in vitro. Mixing of in vitro-produced AML-1 with cell extracts prior to gel mobility shift analysis resulted in the formation of higher-order complexes. Deletion mutagenesis of AML-1 revealed that the runt homology domain mediates both sequence-specific DNA binding and protein-protein interactions. The hybrid product, AML-1/ETO, which results from the (8;21) translocation and retains the runt homology domain, both recognizes the AML-1 consensus sequence and interacts with other cellular proteins. Images PMID:8413232

  3. Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta

    PubMed Central

    2009-01-01

    Background Cloning of the Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases. Results Comprehensive in silico analyses indicate that the recently identified Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed. Conclusion The identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family. PMID:19930663

  4. ThreaDom: extracting protein domain boundary information from multiple threading alignments

    PubMed Central

    Xue, Zhidong; Xu, Dong; Wang, Yan; Zhang, Yang

    2013-01-01

    Motivation: Protein domains are subunits that can fold and evolve independently. Identification of domain boundary locations is often the first step in protein folding and function annotations. Most of the current methods deduce domain boundaries by sequence-based analysis, which has low accuracy. There is no efficient method for predicting discontinuous domains that consist of segments from separated sequence regions. As template-based methods are most efficient for protein 3D structure modeling, combining multiple threading alignment information should increase the accuracy and reliability of computational domain predictions. Result: We developed a new protein domain predictor, ThreaDom, which deduces domain boundary locations based on multiple threading alignments. The core of the method development is the derivation of a domain conservation score that combines information from template domain structures and terminal and internal alignment gaps. Tested on 630 non-redundant sequences, without using homologous templates, ThreaDom generates correct single- and multi-domain classifications in 81% of cases, where 78% have the domain linker assigned within ±20 residues. In a second test on 486 proteins with discontinuous domains, ThreaDom achieves an average precision 84% and recall 65% in domain boundary prediction. Finally, ThreaDom was examined on 56 targets from CASP8 and had a domain overlap rate 73, 87 and 85% with the target for Free Modeling, Hard multiple-domain and discontinuous domain proteins, respectively, which are significantly higher than most domain predictors in the CASP8. Similar results were achieved on the targets from the most recently CASP9 and CASP10 experiments. Availability: http://zhanglab.ccmb.med.umich.edu/ThreaDom/. Contact: zhng@umich.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23812990

  5. Structural Basis for the Discriminative Recognition of N6-Methyladenosine RNA by the Human YT521-B Homology Domain Family of Proteins.

    PubMed

    Xu, Chao; Liu, Ke; Ahmed, Hazem; Loppnau, Peter; Schapira, Matthieu; Min, Jinrong

    2015-10-09

    N(6)-Methyladenosine (m(6)A) is the most abundant internal modification in RNA and is specifically recognized by YT521-B homology (YTH) domain-containing proteins. Recently we reported that YTHDC1 prefers guanosine and disfavors adenosine at the position preceding the m(6)A nucleotide in RNA and preferentially binds to the GG(m(6)A)C sequence. Now we systematically characterized the binding affinities of the YTH domains of three other human proteins and yeast YTH domain protein Pho92 and determined the crystal structures of the YTH domains of human YTHDF1 and yeast Pho92 in complex with a 5-mer m(6)A RNA, respectively. Our binding and structural data revealed that the YTH domain used a conserved aromatic cage to recognize m(6)A. Nevertheless, none of these YTH domains, except YTHDC1, display sequence selectivity at the position preceding the m(6)A modification. Structural comparison of these different YTH domains revealed that among those, only YTHDC1 harbors a distinctly selective binding pocket for the nucleotide preceding the m(6)A nucleotide. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Structural Basis for the Discriminative Recognition of N6-Methyladenosine RNA by the Human YT521-B Homology Domain Family of Proteins*

    PubMed Central

    Xu, Chao; Liu, Ke; Ahmed, Hazem; Loppnau, Peter; Schapira, Matthieu; Min, Jinrong

    2015-01-01

    N6-Methyladenosine (m6A) is the most abundant internal modification in RNA and is specifically recognized by YT521-B homology (YTH) domain-containing proteins. Recently we reported that YTHDC1 prefers guanosine and disfavors adenosine at the position preceding the m6A nucleotide in RNA and preferentially binds to the GG(m6A)C sequence. Now we systematically characterized the binding affinities of the YTH domains of three other human proteins and yeast YTH domain protein Pho92 and determined the crystal structures of the YTH domains of human YTHDF1 and yeast Pho92 in complex with a 5-mer m6A RNA, respectively. Our binding and structural data revealed that the YTH domain used a conserved aromatic cage to recognize m6A. Nevertheless, none of these YTH domains, except YTHDC1, display sequence selectivity at the position preceding the m6A modification. Structural comparison of these different YTH domains revealed that among those, only YTHDC1 harbors a distinctly selective binding pocket for the nucleotide preceding the m6A nucleotide. PMID:26318451

  7. The PDZ Domain of the LIM Protein Enigma Binds to β-Tropomyosin

    PubMed Central

    Guy, Pamela M.; Kenny, Daryn A.; Gill, Gordon N.

    1999-01-01

    PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein α-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal β-TM). The interaction between Enigma and skeletal β-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal β-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal β-TM in transfected cells. The association of Enigma with skeletal β-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells. PMID:10359609

  8. Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

    PubMed Central

    2012-01-01

    Background Proteins convey the majority of biochemical and cellular activities in organisms. Over the course of evolution, proteins undergo normal sequence mutations as well as large scale mutations involving domain duplication and/or domain shuffling. These events result in the generation of new proteins and protein families. Processes that affect proteome evolution drive species diversity and adaptation. Herein, change over the course of metazoan evolution, as defined by birth/death and duplication/deletion events within protein families and domains, was examined using the proteomes of 9 metazoan and two outgroup species. Results In studying members of the three major metazoan groups, the vertebrates, arthropods, and nematodes, we found that the number of protein families increased at the majority of lineages over the course of metazoan evolution where the magnitude of these increases was greatest at the lineages leading to mammals. In contrast, the number of protein domains decreased at most lineages and at all terminal lineages. This resulted in a weak correlation between protein family birth and domain birth; however, the correlation between domain birth and domain member duplication was quite strong. These data suggest that domain birth and protein family birth occur via different mechanisms, and that domain shuffling plays a role in the formation of protein families. The ratio of protein family birth to protein domain birth (domain shuffling index) suggests that shuffling had a more demonstrable effect on protein families in nematodes and arthropods than in vertebrates. Through the contrast of high and low domain shuffling indices at the lineages of Trichinella spiralis and Gallus gallus, we propose a link between protein redundancy and evolutionary changes controlled by domain shuffling; however, the speed of adaptation among the different lineages was relatively invariant. Evaluating the functions of protein families that appeared or disappeared at the

  9. Co-evolutionary Analysis of Domains in Interacting Proteins Reveals Insights into Domain–Domain Interactions Mediating Protein–Protein Interactions

    PubMed Central

    Jothi, Raja; Cherukuri, Praveen F.; Tasneem, Asba; Przytycka, Teresa M.

    2006-01-01

    Recent advances in functional genomics have helped generate large-scale high-throughput protein interaction data. Such networks, though extremely valuable towards molecular level understanding of cells, do not provide any direct information about the regions (domains) in the proteins that mediate the interaction. Here, we performed co-evolutionary analysis of domains in interacting proteins in order to understand the degree of co-evolution of interacting and non-interacting domains. Using a combination of sequence and structural analysis, we analyzed protein–protein interactions in F1-ATPase, Sec23p/Sec24p, DNA-directed RNA polymerase and nuclear pore complexes, and found that interacting domain pair(s) for a given interaction exhibits higher level of co-evolution than the noninteracting domain pairs. Motivated by this finding, we developed a computational method to test the generality of the observed trend, and to predict large-scale domain–domain interactions. Given a protein–protein interaction, the proposed method predicts the domain pair(s) that is most likely to mediate the protein interaction. We applied this method on the yeast interactome to predict domain–domain interactions, and used known domain–domain interactions found in PDB crystal structures to validate our predictions. Our results show that the prediction accuracy of the proposed method is statistically significant. Comparison of our prediction results with those from two other methods reveals that only a fraction of predictions are shared by all the three methods, indicating that the proposed method can detect known interactions missed by other methods. We believe that the proposed method can be used with other methods to help identify previously unrecognized domain–domain interactions on a genome scale, and could potentially help reduce the search space for identifying interaction sites. PMID:16949097

  10. CDvist: A webserver for identification and visualization of conserved domains in protein sequences

    SciTech Connect

    Adebali, Ogun; Ortega, Davi R.; Zhulin, Igor B.

    2014-12-18

    Identification of domains in protein sequences allows their assigning to biological functions. Several webservers exist for identification of protein domains using similarity searches against various databases of protein domain models. However, none of them provides comprehensive domain coverage while allowing bulk querying and their visualization schemes can be improved. To address these issues, we developed CDvist (a comprehensive domain visualization tool), which combines the best available search algorithms and databases into a user-friendly framework. First, a given protein sequence is matched to domain models using high-specificity tools and only then unmatched segments are subjected to more sensitive algorithms resulting in a best possible comprehensive coverage. In conclusion, bulk querying and rich visualization and download options provide improved functionality to domain architecture analysis.

  11. CDvist: A webserver for identification and visualization of conserved domains in protein sequences

    DOE PAGES

    Adebali, Ogun; Ortega, Davi R.; Zhulin, Igor B.

    2014-12-18

    Identification of domains in protein sequences allows their assigning to biological functions. Several webservers exist for identification of protein domains using similarity searches against various databases of protein domain models. However, none of them provides comprehensive domain coverage while allowing bulk querying and their visualization schemes can be improved. To address these issues, we developed CDvist (a comprehensive domain visualization tool), which combines the best available search algorithms and databases into a user-friendly framework. First, a given protein sequence is matched to domain models using high-specificity tools and only then unmatched segments are subjected to more sensitive algorithms resulting inmore » a best possible comprehensive coverage. In conclusion, bulk querying and rich visualization and download options provide improved functionality to domain architecture analysis.« less

  12. NovelFam3000 – Uncharacterized human protein domains conserved across model organisms

    PubMed Central

    Kemmer, Danielle; Podowski, Raf M; Arenillas, David; Lim, Jonathan; Hodges, Emily; Roth, Peggy; Sonnhammer, Erik LL; Höög, Christer; Wasserman, Wyeth W

    2006-01-01

    Background Despite significant efforts from the research community, an extensive portion of the proteins encoded by human genes lack an assigned cellular function. Most metazoan proteins are composed of structural and/or functional domains, of which many appear in multiple proteins. Once a domain is characterized in one protein, the presence of a similar sequence in an uncharacterized protein serves as a basis for inference of function. Thus knowledge of a domain's function, or the protein within which it arises, can facilitate the analysis of an entire set of proteins. Description From the Pfam domain database, we extracted uncharacterized protein domains represented in proteins from humans, worms, and flies. A data centre was created to facilitate the analysis of the uncharacterized domain-containing proteins. The centre both provides researchers with links to dispersed internet resources containing gene-specific experimental data and enables them to post relevant experimental results or comments. For each human gene in the system, a characterization score is posted, allowing users to track the progress of characterization over time or to identify for study uncharacterized domains in well-characterized genes. As a test of the system, a subset of 39 domains was selected for analysis and the experimental results posted to the NovelFam3000 system. For 25 human protein members of these 39 domain families, detailed sub-cellular localizations were determined. Specific observations are presented based on the analysis of the integrated information provided through the online NovelFam3000 system. Conclusion Consistent experimental results between multiple members of a domain family allow for inferences of the domain's functional role. We unite bioinformatics resources and experimental data in order to accelerate the functional characterization of scarcely annotated domain families. PMID:16533400

  13. Glycan Reader is improved to recognize most sugar types and chemical modifications in the Protein Data Bank.

    PubMed

    Park, Sang-Jun; Lee, Jumin; Patel, Dhilon S; Ma, Hongjing; Lee, Hui Sun; Jo, Sunhwan; Im, Wonpil

    2017-10-01

    Glycans play a central role in many essential biological processes. Glycan Reader was originally developed to simplify the reading of Protein Data Bank (PDB) files containing glycans through the automatic detection and annotation of sugars and glycosidic linkages between sugar units and to proteins, all based on atomic coordinates and connectivity information. Carbohydrates can have various chemical modifications at different positions, making their chemical space much diverse. Unfortunately, current PDB files do not provide exact annotations for most carbohydrate derivatives and more than 50% of PDB glycan chains have at least one carbohydrate derivative that could not be correctly recognized by the original Glycan Reader. Glycan Reader has been improved and now identifies most sugar types and chemical modifications (including various glycolipids) in the PDB, and both PDB and PDBx/mmCIF formats are supported. CHARMM-GUI Glycan Reader is updated to generate the simulation system and input of various glycoconjugates with most sugar types and chemical modifications. It also offers a new functionality to edit the glycan structures through addition/deletion/modification of glycosylation types, sugar types, chemical modifications, glycosidic linkages, and anomeric states. The simulation system and input files can be used for CHARMM, NAMD, GROMACS, AMBER, GENESIS, LAMMPS, Desmond, OpenMM, and CHARMM/OpenMM. Glycan Fragment Database in GlycanStructure.Org is also updated to provide an intuitive glycan sequence search tool for complex glycan structures with various chemical modifications in the PDB. http://www.charmm-gui.org/input/glycan and http://www.glycanstructure.org. wonpil@lehigh.edu. Supplementary data are available at Bioinformatics online.

  14. Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

    NASA Technical Reports Server (NTRS)

    Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

    1997-01-01

    A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

  15. Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

    NASA Technical Reports Server (NTRS)

    Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

    1997-01-01

    A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

  16. Archaeal surface layer proteins contain beta propeller, PKD, and beta helix domains and are related to metazoan cell surface proteins.

    PubMed

    Jing, Hua; Takagi, Junichi; Liu, Jin-huan; Lindgren, Sara; Zhang, Rong-guang; Joachimiak, A; Wang, Jia-huai; Springer, Timothy A

    2002-10-01

    The surface layer of archaeobacteria protects cells from extreme environments and, in Methanosarcina, may regulate cell adhesion. We identify three domain types that account for the complete architecture of numerous Methanosarcina surface layer proteins (SLPs). We solve the crystal structure for two of these domains, which correspond to the two N-terminal domains of an M. mazei SLP. One domain displays a unique, highly symmetrical, seven-bladed beta propeller fold, and the other belongs to the polycystic kidney disease (PKD) superfamily fold. The third domain is predicted to adopt a beta helix fold. These domains have homologs in metazoan cell surface proteins, suggesting remarkable relationships between domains in archaeal SLPs and metazoan cell surface proteins.

  17. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    SciTech Connect

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  18. Impact of protein domains on PE_PGRS30 polar localization in Mycobacteria.

    PubMed

    De Maio, Flavio; Maulucci, Giuseppe; Minerva, Mariachiara; Anoosheh, Saber; Palucci, Ivana; Iantomasi, Raffaella; Palmieri, Valentina; Camassa, Serena; Sali, Michela; Sanguinetti, Maurizio; Bitter, Wilbert; Manganelli, Riccardo; De Spirito, Marco; Delogu, Giovanni

    2014-01-01

    PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.

  19. Multimodal Recognition of Diverse Peptides by the C-Terminal SH2 Domain of Phospholipase C-γ1 Protein.

    PubMed

    McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

    2017-04-11

    SH2 domains recognize phosphotyrosine (pY)-containing peptide ligands and play key roles in the regulation of receptor tyrosine kinase pathways. Each SH2 domain has individualized specificity, encoded in the amino acids neighboring the pY, for defined targets that convey their distinct functions. The C-terminal SH2 domain (PLCC) of the phospholipase C-γ1 full-length protein (PLCγ1) typically binds peptides containing small and hydrophobic amino acids adjacent to the pY, including a peptide derived from platelet-derived growth factor receptor B (PDGFRB) and an intraprotein recognition site (Y783 of PLCγ1) involved in the regulation of the protein's lipase activity. Remarkably, PLCC also recognizes unexpected peptides containing amino acids with polar or bulky side chains that deviate from this pattern. This versatility in recognition specificity may allow PLCγ1 to participate in diverse, previously unrecognized, signaling pathways in response to binding chemically dissimilar partners. We have used structural approaches, including nuclear magnetic resonance and X-ray crystallography, to elucidate the mechanisms of noncognate peptide binding to PLCC by ligands derived from receptor tyrosine kinase ErbB2 and from the insulin receptor. The high-resolution peptide-bound structures reveal that PLCC has a relatively static backbone but contains a chemically rich protein surface comprised of a combination of hydrophobic pockets and amino acids with charged side chains. We demonstrate that this expansive and chemically diverse PLCC interface, in addition to peptide conformational plasticity, permits PLCC to recognize specific noncognate peptide ligands with multimodal specificity.

  20. Comparative analysis of SET domain proteins in maize and Arabidopsis reveals multiple duplications preceding the divergence of monocots and dicots.

    PubMed

    Springer, Nathan M; Napoli, Carolyn A; Selinger, David A; Pandey, Ritu; Cone, Karen C; Chandler, Vicki L; Kaeppler, Heidi F; Kaeppler, Shawn M

    2003-06-01

    Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.

  1. SET and MYND domain containing protein 3 in cancer

    PubMed Central

    Huang, Lei; Xu, A-Man

    2017-01-01

    Lysine methylation plays a vital role in histone modification. Deregulations of lysine methyltransferases and demethylases have been frequently observed in human cancers. The SET and MYND domain containing protein 3 (SMYD3) is a novel histone lysine methyltransferase and it functions by regulating chromatin during the development of myocardial and skeletal muscle. It has been recently unveiled to play significant roles in human cancer genesis and progression via regulating various key cancer-associated genes and pathways and promoting cell proliferation and migration. Upregulation of SMYD3 expression is present in multiple cancer types, suggesting it as a potential prognostic marker. Herein the structure, substrates and targets of SMYD3, and its effects on initiation, invasion and metastasis of diverse tumors (e.g., esophageal squamous cell carcinoma, gastric cancer, hepatocellular carcinoma, cholangiocarcinoma, breast cancer, prostate cancer, and leukemia) are systematically reviewed, providing clues for the development of novel SMYD3-specific personalized anti-cancer therapy. SMYD3 inhibitors (e.g., BCI-121 and novobiocin) could hopefully fight against tumors with efficacy. PMID:28123630

  2. The role of internal duplication in the evolution of multi-domain proteins.

    PubMed

    Nacher, J C; Hayashida, M; Akutsu, T

    2010-08-01

    Many proteins consist of several structural domains. These multi-domain proteins have likely been generated by selective genome growth dynamics during evolution to perform new functions as well as to create structures that fold on a biologically feasible time scale. Domain units frequently evolved through a variety of genetic shuffling mechanisms. Here we examine the protein domain statistics of more than 1000 organisms including eukaryotic, archaeal and bacterial species. The analysis extends earlier findings on asymmetric statistical laws for proteome to a wider variety of species. While proteins are composed of a wide range of domains, displaying a power-law decay, the computation of domain families for each protein reveals an exponential distribution, characterizing a protein universe composed of a thin number of unique families. Structural studies in proteomics have shown that domain repeats, or internal duplicated domains, represent a small but significant fraction of genome. In spite of its importance, this observation has been largely overlooked until recently. We model the evolutionary dynamics of proteome and demonstrate that these distinct distributions are in fact rooted in an internal duplication mechanism. This process generates the contemporary protein structural domain universe, determines its reduced thickness, and tames its growth. These findings have important implications, ranging from protein interaction network modeling to evolutionary studies based on fundamental mechanisms governing genome expansion.

  3. Characterization of domain-peptide interaction interface: prediction of SH3 domain-mediated protein-protein interaction network in yeast by generic structure-based models.

    PubMed

    Hou, Tingjun; Li, Nan; Li, Youyong; Wang, Wei

    2012-05-04

    Determination of the binding specificity of SH3 domain, a peptide recognition module (PRM), is important to understand their biological functions and reconstruct the SH3-mediated protein-protein interaction network. In the present study, the SH3-peptide interactions for both class I and II SH3 domains were characterized by the intermolecular residue-residue interaction network. We developed generic MIEC-SVM models to infer SH3 domain-peptide recognition specificity that achieved satisfactory prediction accuracy. By investigating the domain-peptide recognition mechanisms at the residue level, we found that the class-I and class-II binding peptides have different binding modes even though they occupy the same binding site of SH3. Furthermore, we predicted the potential binding partners of SH3 domains in the yeast proteome and constructed the SH3-mediated protein-protein interaction network. Comparison with the experimentally determined interactions confirmed the effectiveness of our approach. This study showed that our sophisticated computational approach not only provides a powerful platform to decipher protein recognition code at the molecular level but also allows identification of peptide-mediated protein interactions at a proteomic scale. We believe that such an approach is general to be applicable to other domain-peptide interactions.

  4. Botulinum Neurotoxin Serotype A Recognizes Its Protein Receptor SV2 by a Different Mechanism than Botulinum Neurotoxin B Synaptotagmin

    PubMed Central

    Weisemann, Jasmin; Stern, Daniel; Mahrhold, Stefan; Dorner, Brigitte G.; Rummel, Andreas

    2016-01-01

    Botulinum neurotoxins (BoNTs) exhibit extraordinary potency due to their exquisite neurospecificity, which is achieved by dual binding to complex polysialo-gangliosides and synaptic vesicle proteins. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 isoforms, SV2A‐C, identified as protein receptors for the most relevant serotype BoNT/A, binds within the 50 kDa cell binding domain HC of BoNT/A. Here, we deciphered the BoNT/A‐SV2 interactions in more detail. In pull down assays, the binding of HCA to SV2-LD4 isoforms decreases from SV2C >> SV2A > SV2B. A binding constant of 200 nM was determined for BoNT/A to rat SV2C-LD4 in GST pull down assay. A similar binding constant was determined by surface plasmon resonance for HCA to rat SV2C and to human SV2C, the latter being slightly lower due to the substitution L563F in LD4. At pH 5, as measured in acidic synaptic vesicles, the binding constant of HCA to hSV2C is increased more than 10-fold. Circular dichroism spectroscopy reveals that the quadrilateral helix of SV2C-LD4 already exists in solution prior to BoNT/A binding. Hence, the BoNT/A‐SV2C interaction is of different nature compared to BoNT/B‐Syt-II. In particular, the preexistence of the quadrilateral β-sheet helix of SV2 and its pH-dependent binding to BoNT/A via backbone–backbone interactions constitute major differences. Knowledge of the molecular details of BoNT/A‐SV2 interactions drives the development of high affinity peptides to counteract BoNT/A intoxications or to capture functional BoNT/A variants in innovative detection systems for botulism diagnostic. PMID:27196927

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

    SciTech Connect

    Dahms, Sven O.; Mayer, Magnus C.; Roeser, Dirk; Multhaup, Gerd; Than, Manuel E.

    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.

  6. Small Molecule-Induced Domain Swapping as a Mechanism for Controlling Protein Function and Assembly

    PubMed Central

    Karchin, Joshua M.; Ha, Jeung-Hoi; Namitz, Kevin E.; Cosgrove, Michael S.; Loh, Stewart N.

    2017-01-01

    Domain swapping is the process by which identical proteins exchange reciprocal segments to generate dimers. Here we introduce induced domain swapping (INDOS) as a mechanism for regulating protein function. INDOS employs a modular design consisting of the fusion of two proteins: a recognition protein that binds a triggering molecule, and a target protein that undergoes a domain swap in response to binding of the triggering ligand. The recognition protein (FK506 binding protein) is inserted into functionally-inactivated point mutants of two target proteins (staphylococcal nuclease and ribose binding protein). Binding of FK506 to the FKBP domain causes the target domain to first unfold, then refold via domain swap. The inactivating mutations become ‘swapped out’ in the dimer, increasing nuclease and ribose binding activities by 100-fold and 15-fold, respectively, restoring them to near wild-type values. INDOS is intended to convert an arbitrary protein into a functional switch, and is the first example of rational design in which a small molecule is used to trigger protein domain swapping and subsequent activation of biological function. PMID:28287617

  7. Expansion of divergent SEA domains in cell surface proteins and nucleoporin 54.

    PubMed

    Pei, Jimin; Grishin, Nick V

    2017-03-01

    SEA (sea urchin sperm protein, enterokinase, agrin) domains, many of which possess autoproteolysis activity, have been found in a number of cell surface and secreted proteins. Despite high sequence divergence, SEA domains were also proposed to be present in dystroglycan based on a conserved autoproteolysis motif and receptor-type protein phosphatase IA-2 based on structural similarity. The presence of a SEA domain adjacent to the transmembrane segment appears to be a recurring theme in quite a number of type I transmembrane proteins on the cell surface, such as MUC1, dystroglycan, IA-2, and Notch receptors. By comparative sequence and structural analyses, we identified dystroglycan-like proteins with SEA domains in Capsaspora owczarzaki of the Filasterea group, one of the closest single-cell relatives of metazoans. We also detected novel and divergent SEA domains in a variety of cell surface proteins such as EpCAM, α/ε-sarcoglycan, PTPRR, collectrin/Tmem27, amnionless, CD34, KIAA0319, fibrocystin-like protein, and a number of cadherins. While these proteins are mostly from metazoans or their single cell relatives such as choanoflagellates and Filasterea, fibrocystin-like proteins with SEA domains were found in several other eukaryotic lineages including green algae, Alveolata, Euglenozoa, and Haptophyta, suggesting an ancient evolutionary origin. In addition, the intracellular protein Nucleoporin 54 (Nup54) acquired a divergent SEA domain in choanoflagellates and metazoans. © 2016 The Protein Society.

  8. Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction

    PubMed Central

    2012-01-01

    Background Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ). We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD)1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1−/− NMJs. Methods Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1−/− mouse skeletal muscle. Results Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1−/− mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1–4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1−/− mouse skeletal muscle than in wild-type skeletal muscle. Conclusion EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1−/− muscle may be due to functional compensation by other EHD paralogs. PMID:22974368

  9. Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

    SciTech Connect

    Choi, Yoo Seong; Pack, Seung Pil; Yoo, Young Je . E-mail: yjyoo@snu.ac.kr

    2005-04-22

    A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.

  10. Genetic, structural, and molecular insights into the function of ras of complex proteins domains.

    PubMed

    Civiero, Laura; Dihanich, Sybille; Lewis, Patrick A; Greggio, Elisa

    2014-07-17

    Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson's disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural information available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored.

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

    PubMed

    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.

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

    SciTech Connect

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

    2012-03-15

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

  13. The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins.

    PubMed

    Casey, Lachlan W; Lavrencic, Peter; Bentham, Adam R; Cesari, Stella; Ericsson, Daniel J; Croll, Tristan; Turk, Dušan; Anderson, Peter A; Mark, Alan E; Dodds, Peter N; Mobli, Mehdi; Kobe, Bostjan; Williams, Simon J

    2016-10-17

    Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world's most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.

  14. Definition of an epitope on Japanese encephalitis virus (JEV) envelope protein recognized by JEV-specific murine CD8+ cytotoxic T lymphocytes.

    PubMed

    Takada, K; Masaki, H; Konishi, E; Takahashi, M; Kurane, I

    2000-01-01

    We defined an epitope on the Japanese encephalitis virus (JEV) envelope (E) protein recognized by CD8+ cytotoxic T lymphocytes (CTLs). CTLs induced in JEV-infected BALB/c (H-2d) mice recognized E and/or premembrane (PrM) proteins, while CTLs in C57BL/6J (H-2b) and C3H/HeJ (H-2k) mice did not. JEV-specific CTLs had a phenotype of CD3+ CD4- CD8+. Twenty-four 9-amino acid (a.a.) peptides, which had binding motifs for H-2Kd, H-2Ld or H-2Dd, were synthesized according to the amino acid sequences of PrM and E proteins. CTLs induced by JEV infection recognized only the peptide K-3. Immunization of BALB/c mice with only a group of peptides including K-3 induced CTLs which recognized the homologous K-3 peptide, while immunization with other peptides did not. The peptide K-3 had a binding motif for H-2Kd. This is consistent with the finding that JEV-specific CTLs in BALB/c mice was H-2Kd-restricted. These results indicate that the epitope recognized by CTLs in BALB/c mice is located between a.a. 60 and 68 on the E protein, corresponding to an a.a. sequence of CYHASVTDI.

  15. Defining the Domain Arrangement of the Mammalian Target of Rapamycin Complex Component Rictor Protein

    PubMed Central

    Zhou, Ping; Zhang, Ning; Nussinov, Ruth

    2015-01-01

    Abstract Mammalian target of rapamycin (mTOR) complexes play a pivotal role in the cell. Raptor and Rictor proteins interact with mTOR to form two distinct complexes, mTORC1 and mTORC2, respectively. While the domain structure of Raptor is known, curre