Science.gov

Sample records for recognizing protein domains

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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…

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  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. 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.

  15. 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

  16. 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.

  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. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. Protein function prediction using domain families

    PubMed Central

    2013-01-01

    Here we assessed the use of domain families for predicting the functions of whole proteins. These 'functional families' (FunFams) were derived using a protocol that combines sequence clustering with supervised cluster evaluation, relying on available high-quality Gene Ontology (GO) annotation data in the latter step. In essence, the protocol groups domain sequences belonging to the same superfamily into families based on the GO annotations of their parent proteins. An initial test based on enzyme sequences confirmed that the FunFams resemble enzyme (domain) families much better than do families produced by sequence clustering alone. For the CAFA 2011 experiment, we further associated the FunFams with GO terms probabilistically. All target proteins were first submitted to domain superfamily assignment, followed by FunFam assignment and, eventually, function assignment. The latter included an integration step for multi-domain target proteins. The CAFA results put our domain-based approach among the top ten of 31 competing groups and 56 prediction methods, confirming that it outperforms simple pairwise whole-protein sequence comparisons. PMID:23514456

  5. 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.

  6. 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.

  7. 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…

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD

    PubMed Central

    Suh, Man-Hee; Ye, Ping; Zhang, Mincheng; Hausmann, Stéphane; Shuman, Stewart; Gnatt, Averell L.; Fu, Jianhua

    2005-01-01

    Fcp1 is an essential protein phosphatase that hydrolyzes phosphoserines within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). Fcp1 plays a major role in the regulation of CTD phosphorylation and, hence, critically influences the function of Pol II throughout the transcription cycle. The basic understanding of Fcp1–CTD interaction has remained ambiguous because two different modes have been proposed: the “dockingsite” model versus the “distributive” mechanism. Here we demonstrate biochemically that Fcp1 recognizes and dephosphorylates the CTD directly, independent of the globular non-CTD part of the Pol II structure. We point out that the recognition of CTD by the phosphatase is based on random access and is not driven by Pol II conformation. Results from three different types of experiments reveal that the overall interaction between Fcp1 and Pol II is not stable but dynamic. In addition, we show that Fcp1 also interacts with a region on the polymerase distinct from the CTD. We emphasize that this non-CTD site is functionally distinct from the docking site invoked previously as essential for the CTD phosphatase activity of Fcp1. We speculate that Fcp1 interaction with the non-CTD site may mediate its stimulatory effect on transcription elongation reported previously. PMID:16301539

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  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. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. 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

  10. 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

  11. A new and unexpected domain-domain interaction in the AraC protein.

    PubMed

    Cole, Stephanie Dirla; Schleif, Robert

    2012-05-01

    An interaction between the dimerization domains and DNA binding domains of the dimeric AraC protein has previously been shown to facilitate repression of the Escherichia coli araBAD operon by AraC in the absence of arabinose. A new interaction between the domains of AraC in the presence of arabinose is reported here, the regulatory consequences of which are unknown. Evidence for the interaction is the following: the dissociation rate of arabinose-bound AraC from half-site DNA is considerably faster than that of free DNA binding domain, and the affinity of the dimerization domains for arabinose is increased when half-site DNA is bound. In addition, an increase in the fluorescence intensity of tryptophan residues located in the arabinose-bound dimerization domain is observed upon binding of half-site DNA to the DNA binding domains. Direct physical evidence of the new domain-domain interaction is demonstrated by chemical crosslinking and NMR experiments.

  12. Global Patterns of Protein Domain Gain and Loss in Superkingdoms

    PubMed Central

    Nasir, Arshan; Kim, Kyung Mo; Caetano-Anollés, Gustavo

    2014-01-01

    Domains are modules within proteins that can fold and function independently and are evolutionarily conserved. Here we compared the usage and distribution of protein domain families in the free-living proteomes of Archaea, Bacteria and Eukarya and reconstructed species phylogenies while tracing the history of domain emergence and loss in proteomes. We show that both gains and losses of domains occurred frequently during proteome evolution. The rate of domain discovery increased approximately linearly in evolutionary time. Remarkably, gains generally outnumbered losses and the gain-to-loss ratios were much higher in akaryotes compared to eukaryotes. Functional annotations of domain families revealed that both Archaea and Bacteria gained and lost metabolic capabilities during the course of evolution while Eukarya acquired a number of diverse molecular functions including those involved in extracellular processes, immunological mechanisms, and cell regulation. Results also highlighted significant contemporary sharing of informational enzymes between Archaea and Eukarya and metabolic enzymes between Bacteria and Eukarya. Finally, the analysis provided useful insights into the evolution of species. The archaeal superkingdom appeared first in evolution by gradual loss of ancestral domains, bacterial lineages were the first to gain superkingdom-specific domains, and eukaryotes (likely) originated when an expanding proto-eukaryotic stem lineage gained organelles through endosymbiosis of already diversified bacterial lineages. The evolutionary dynamics of domain families in proteomes and the increasing number of domain gains is predicted to redefine the persistence strategies of organisms in superkingdoms, influence the make up of molecular functions, and enhance organismal complexity by the generation of new domain architectures. This dynamics highlights ongoing secondary evolutionary adaptations in akaryotic microbes, especially Archaea. PMID:24499935

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  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. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  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. 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.

  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. 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

  1. 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

  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. 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.).

  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. 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.

  6. 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.

  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. 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.

  9. 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.

  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. 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.

  12. 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.

  13. Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

    PubMed

    Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca Ellis

    2017-02-17

    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 meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements which 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 EC, et al. Trimeric transmembrane domain interactions in paramyxovirus fusion proteins. 2013. J Biol Chem. 288, 35726). 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins

    PubMed Central

    Holbrook, Lisa-Marie; Kwong, Lai-Shan; Metcalfe, Clive L.; Fenouillet, Emmanuel; Jones, Ian M.; Barclay, A. Neil

    2016-01-01

    ABSTRACT In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents. PMID:26986548

  5. OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins.

    PubMed

    Holbrook, Lisa-Marie; Kwong, Lai-Shan; Metcalfe, Clive L; Fenouillet, Emmanuel; Jones, Ian M; Barclay, A Neil

    2016-01-01

    In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.

  6. 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.

  7. 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

  8. 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.

  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. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  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. 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

  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. 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.

  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. 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

  7. 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

  8. 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

  9. 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.

  10. Computational Prediction of Protein Function Based on Weighted Mapping of Domains and GO Terms

    PubMed Central

    Teng, Zhixia; Guo, Maozu; Dai, Qiguo; Wang, Chunyu; Li, Jin; Liu, Xiaoyan

    2014-01-01

    In this paper, we propose a novel method, SeekFun, to predict protein function based on weighted mapping of domains and GO terms. Firstly, a weighted mapping of domains and GO terms is constructed according to GO annotations and domain composition of the proteins. The association strength between domain and GO term is weighted by symmetrical conditional probability. Secondly, the mapping is extended along the true paths of the terms based on GO hierarchy. Finally, the terms associated with resident domains are transferred to host protein and real annotations of the host protein are determined by association strengths. Our careful comparisons demonstrate that SeekFun outperforms the concerned methods on most occasions. SeekFun provides a flexible and effective way for protein function prediction. It benefits from the well-constructed mapping of domains and GO terms, as well as the reasonable strategy for inferring annotations of protein from those of its domains. PMID:24868539

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  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. 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.

  10. 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

  11. 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

  12. Recognizing abuse.

    PubMed

    Davidhizar, R; Newman-Giger, J

    1996-01-01

    After years as a taboo topic, abuse has come "out of the closet" and is being talked about openly in society. Yet, while abuse in the workplace is being confronted, abuse within families still often goes unrecognized by outsiders, including by nurses. Failure of nurses to recognize abuse is unfortunate since frequently they are the first point of contact with the victim of abuse in the emergency room, clinic and home. Understanding and insight into the problem of family violence by nurses is critical in addressing this problem. Knowledge is crucial in planning strategies that will have the long-lasting effect of decreasing the cycle of abuse in families.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  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. 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.

  19. Physical Characteristics of a Citrullinated Pro-Filaggrin Epitope Recognized by Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Sera

    PubMed Central

    Trier, Nicole Hartwig; Holm, Bettina Eide; Slot, Ole; Locht, Henning; Lindegaard, Hanne; Svendsen, Anders; Houen, Gunnar

    2016-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease of complex etiology. A characteristic feature of a subset of RA is the presence of anti-citrullinated protein antibodies (ACPA), which correlate with a progressive disease course. In this study, we employed streptavidin capture enzyme-linked immunosorbent assay to analyze ACPA reactivity. Using the pro-filaggrin peptide HQCHQEST-Cit-GRSRGRCGRSGS, as template, we analyzed the reactivity of RA sera and healthy donor sera to various peptides in order to determine the physical characteristics of the citrullinated pro-filaggrin epitope and to examine whether biotin labelling influence antibody recognition. The full-length cyclic pro-filaggrin peptide and a linear form with a N-terminal biotin, was recognized to the same level, whereas, a notable difference in ACPA reactivity to the linear peptides with a C-terminal biotin was found, probably due to steric hindrance. Screening of linear and cyclic truncated peptides, revealed that small cyclic peptides containing 10–12 amino acids are favored over the linear. Moreover, the charged amino acids C-terminal to citrulline were found to be essential for antibody reactivity, most important was the charged amino acid in position 4 C-terminal to citrulline. Collectively, peptide structure, length, the presence of charged amino acids and biotin labelling markedly influence antibody reactivity. In relation to the clinical diagnostics of ACPA, these findings may reflect the differences in diagnostic assays used for detection of ACPA, which relates to differences in sensitivity and specificity dependent on the assay applied. PMID:28002483

  20. 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.

  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. 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.

  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. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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, current bioinformatics tools failed to classify the domains in Rictor. Here we focus on identifying specific domains in Rictor by searching for conserved regions. We scanned the pdb structural database and constructed three protein domain datasets. Next we carried out multiple pairwise sequence alignments of the proteins in the domain dataset. By analyzing the z-scores of Rictor sequence similarity to protein sequences in the dataset, we assigned the structural and functional domains of Rictor. We found that, like Raptor, Rictor also has HEAT and WD40 domains, which could be the common motif binding to mTORC. Rictor may also have pleckstrin homology domains, which mediate cellular localization and transmit signals to downstream targets, as well as a domain that is homologous to 50S protein L17 and human 39S protein L17. This putative ribosome binding domain could mediate mTORC2–ribosome interaction. PMID:26176550

  11. 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

  12. ALP/Enigma PDZ-LIM domain proteins in the heart.

    PubMed

    Zheng, Ming; Cheng, Hongqiang; Banerjee, Indroneal; Chen, Ju

    2010-04-01

    Actinin-associated LIM protein (ALP) and Enigma are two subfamilies of Postsynaptic density 95, discs large and zonula occludens-1 (PDZ)-Lin-11, Isl1 and Mec-3 (LIM) domain containing proteins. ALP family members have one PDZ and one LIM domain, whereas Enigma proteins contain one PDZ and three LIM domains. Four ALP and three Enigma proteins have been identified in mammals, each having multiple splice variants and unique expression patterns. Functionally, these proteins bind through their PDZ domains to alpha-actinin and bind through their LIM domains or other internal protein interaction domains to other proteins, including signaling molecules. ALP and Enigma proteins have been implicated in cardiac and skeletal muscle structure, function and disease, neuronal function, bipolar disorder, tumor growth, platelet and epithelial cell motility and bone formation. This review will focus on recent advances in the biological roles of ALP/Enigma PDZ-LIM domain proteins in cardiac muscle and provide insights into mechanisms by which mutations in these proteins are related to human cardiac disease.

  13. Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4.

    PubMed

    Otani, Junji; Arita, Kyohei; Kato, Tsuyoshi; Kinoshita, Mariko; Kimura, Hironobu; Suetake, Isao; Tajima, Shoji; Ariyoshi, Mariko; Shirakawa, Masahiro

    2013-03-01

    The methyl-CpG binding domain (MBD) protein MBD4 participates in DNA repair as a glycosylase that excises mismatched thymine bases in CpG sites and also functions in transcriptional repression. Unlike other MBD proteins, MBD4 recognizes not only methylated CpG dinucleotides ((5m)CG/(5m)CG) but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine ((5m)CG/TG). The glycosylase activity of MBD4 is also implicated in active DNA demethylation initiated by the deaminase-catalyzed conversion of 5-methylcytosine to thymine. Here, we report the crystal structures of the MBD of MBD4 (MBDMBD4) complexed with (5m)CG/(5m)CG and (5m)CG/TG. The crystal structures show that the DNA interface of MBD4 has flexible structural features and harbors an extensive water network that supports its dual base specificities. Combined with the results of biochemical analyses, the crystal structure of MBD4 bound to 5-hydroxymethylcytosine further demonstrates that MBDMBD4 is able to recognize a wide range of 5-methylcytosine modifications through the unique water network. The versatile base recognition ability of MBDMBD4 implies multifunctional roles for MBD4 in the regulation of dynamic DNA methylation patterns coupled with deamination and/or oxidation of 5-methylcytosine.

  14. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

    SciTech Connect

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M.F.; Downes, C. Peter; Batty, Ian H.

    2012-10-16

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

  15. Structure and function of Toll/interleukin-1 receptor/resistance protein (TIR) domains.

    PubMed

    Ve, Thomas; Williams, Simon J; Kobe, Bostjan

    2015-02-01

    The Toll/interleukin-1 receptor/resistance protein (TIR) domain is a protein-protein interaction domain consisting of 125-200 residues, widely distributed in animals, plants and bacteria but absent from fungi, archea and viruses. In plants and animals, these domains are found in proteins with functions in innate immune pathways, while in bacteria, some TIR domain-containing proteins interfere with the innate immune pathways in the host. TIR domains function as protein scaffolds, mostly involving self-association and homotypic interactions with other TIR domains. In the last 15 years, the three-dimensional structures of TIR domains from several mammalian, plant and bacterial proteins have been reported. These structures, jointly with functional data including the identification of interacting proteins, have started to provide insight into the molecular basis of the assembly of animal and plant immune signaling complexes, and for host immunosuppression by bacterial pathogens. This review focuses on the current knowledge of the structures of the TIR domains and how the structure relates to function.

  16. Crystal Structure of the Protein Kinase Domain of Yeast AMP-Activated Protein Kinase Snf1

    SciTech Connect

    Rudolph,M.; Amodeo, G.; Bai, Y.; Tong, L.

    2005-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator, and is an important target for drug development against diabetes, obesity, and other diseases. AMPK is a hetero-trimeric enzyme, with a catalytic ({alpha}) subunit, and two regulatory ({beta} and {gamma}) subunits. Here we report the crystal structure at 2.2 Angstrom resolution of the protein kinase domain (KD) of the catalytic subunit of yeast AMPK (commonly known as SNF1). The Snf1-KD structure shares strong similarity to other protein kinases, with a small N-terminal lobe and a large C-terminal lobe. Two negative surface patches in the structure may be important for the recognition of the substrates of this kinase.

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

    DTIC Science & Technology

    2013-05-07

    Superfamily ( SF ) [33], and SMART [34,35]. PFAM domains: FH2, Drf_FH3, and two Drf_GBD domains; SF domains: Formin homology 2 domain (FH2 domain) and ARM...annotation data from six commonly used annotation databases: PFAM-A (release 25.0) [32], Superfamily ( SF ) [33], SMART [34,35], PRODOM [36], TIGRFAM [37... SF 3,651 62.1 962,602 33.0 1,355 1,307 0.79 SMART 3,023 51.4 455,523 15.6 392 379 0.66 PRODOM 146 2.5 19,760 0.7 111 111 0.02 TIGRFAM 3,019 51.3

  18. Different functional modes of BAR domain proteins in formation and plasticity of mammalian postsynapses.

    PubMed

    Kessels, Michael M; Qualmann, Britta

    2015-09-01

    A plethora of cell biological processes involve modulations of cellular membranes. By using extended lipid-binding interfaces, some proteins have the power to shape membranes by attaching to them. Among such membrane shapers, the superfamily of Bin-Amphiphysin-Rvs (BAR) domain proteins has recently taken center stage. Extensive structural work on BAR domains has revealed a common curved fold that can serve as an extended membrane-binding interface to modulate membrane topologies and has allowed the grouping of the BAR domain superfamily into subfamilies with structurally slightly distinct BAR domain subtypes (N-BAR, BAR, F-BAR and I-BAR). Most BAR superfamily members are expressed in the mammalian nervous system. Neurons are elaborately shaped and highly compartmentalized cells. Therefore, analyses of synapse formation and of postsynaptic reorganization processes (synaptic plasticity) - a basis for learning and memory formation - has unveiled important physiological functions of BAR domain superfamily members. These recent advances, furthermore, have revealed that the functions of BAR domain proteins include different aspects. These functions are influenced by the often complex domain organization of BAR domain proteins. In this Commentary, we review these recent insights and propose to classify BAR domain protein functions into (1) membrane shaping, (2) physical integration, (3) action through signaling components, and (4) suppression of other BAR domain functions.

  19. The J-domain proteins of Arabidopsis thaliana: an unexpectedly large and diverse family of chaperones.

    PubMed

    Miernyk, J A

    2001-07-01

    A total of 89 J-domain proteins were identified in the genome of the model flowering plant Arabidopsis thaliana. The deduced amino acid sequences of the J-domain proteins were analyzed for an assortment of structural features and motifs. Based on the results of sequence comparisons and structure and function predictions, 51 distinct families were identified. The families ranged in size from 1 to 6 members. Subcellular localizations of the A thaliana J-domain proteins were predicted; species were found in both the soluble and membrane compartments of all cellular organelles. Based on digital Northern analysis, the J-domain proteins could be separated into groups of low, medium, and moderate expression levels. This genomics-based analysis of the A thaliana J-domain proteins establishes a framework for detailed studies of biological function and specificity. It additionally provides a comprehensive basis for evolutionary comparisons.

  20. Implications of 3D domain swapping for protein folding, misfolding and function.

    PubMed

    Rousseau, Frederic; Schymkowitz, Joost; Itzhaki, Laura S

    2012-01-01

    Three-dimensional domain swapping is the process by which two identical protein chains exchange a part of their structure to form an intertwined dimer or higher-order oligomer. The phenomenon has been observed in the crystal structures of a range of different proteins. In this chapter we review the experiments that have been performed in order to understand the sequence and structural determinants of domain-swapping and these show how the general principles obtained can be used to engineer proteins to domain swap. We discuss the role of domain swapping in regulating protein function and as one possible mechanism of protein misfolding that can lead to aggregation and disease. We also review a number of interesting pathways of macromolecular assembly involving β-strand insertion or complementation that are related to the domain-swapping phenomenon.

  1. Dynamics and Adaptive Benefits of Protein Domain Emergence and Arrangements during Plant Genome Evolution

    PubMed Central

    Kersting, Anna R.; Bornberg-Bauer, Erich; Moore, Andrew D.; Grath, Sonja

    2012-01-01

    Plant genomes are generally very large, mostly paleopolyploid, and have numerous gene duplicates and complex genomic features such as repeats and transposable elements. Many of these features have been hypothesized to enable plants, which cannot easily escape environmental challenges, to rapidly adapt. Another mechanism, which has recently been well described as a major facilitator of rapid adaptation in bacteria, animals, and fungi but not yet for plants, is modular rearrangement of protein-coding genes. Due to the high precision of profile-based methods, rearrangements can be well captured at the protein level by characterizing the emergence, loss, and rearrangements of protein domains, their structural, functional, and evolutionary building blocks. Here, we study the dynamics of domain rearrangements and explore their adaptive benefit in 27 plant and 3 algal genomes. We use a phylogenomic approach by which we can explain the formation of 88% of all arrangements by single-step events, such as fusion, fission, and terminal loss of domains. We find many domains are lost along every lineage, but at least 500 domains are novel, that is, they are unique to green plants and emerged more or less recently. These novel domains duplicate and rearrange more readily within their genomes than ancient domains and are overproportionally involved in stress response and developmental innovations. Novel domains more often affect regulatory proteins and show a higher degree of structural disorder than ancient domains. Whereas a relatively large and well-conserved core set of single-domain proteins exists, long multi-domain arrangements tend to be species-specific. We find that duplicated genes are more often involved in rearrangements. Although fission events typically impact metabolic proteins, fusion events often create new signaling proteins essential for environmental sensing. Taken together, the high volatility of single domains and complex arrangements in plant genomes

  2. Polydom: a secreted protein with pentraxin, complement control protein, epidermal growth factor and von Willebrand factor A domains.

    PubMed Central

    Gilgès, D; Vinit, M A; Callebaut, I; Coulombel, L; Cacheux, V; Romeo, P H; Vigon, I

    2000-01-01

    To identify extracellular proteins with epidermal growth factor (EGF) domains that are potentially involved in the control of haemopoiesis, we performed degenerate reverse-transcriptase-mediated PCR on the murine bone-marrow stromal cell line MS-5 and isolated a new partial cDNA encoding EGF-like domains related to those in the Notch proteins. Cloning and sequencing of the full-length cDNA showed that it encoded a new extracellular multi-domain protein that we named polydom. This 387 kDa mosaic protein contained a signal peptide followed by a new association of eight different protein domains, including a pentraxin domain and a von Willebrand factor type A domain, ten EGF domains, and 34 complement control protein modules. The human polydom mRNA is strongly expressed in placenta, its expression in the other tissues being weak or undetectable. The particular multidomain structure of the encoded protein suggests an important biological role in cellular adhesion and/or in the immune system. PMID:11062057

  3. Cytoplasmic Ig-Domain Proteins: Cytoskeletal Regulators with a Role in Human Disease

    PubMed Central

    Otey, Carol A.; Dixon, Richard; Stack, Christianna; Goicoechea, Silvia M.

    2009-01-01

    Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin. Together with results obtained from cultured cells and mouse models, these clinical studies have yielded novel insights into the unexpected roles of Ig domain proteins in mechanotransduction and signaling to the nucleus. An emerging theme in this field is that cytoskeleton-associated Ig domain proteins are more than structural elements of the cell, and may have evolved to fill different needs in different cellular compartments. PMID:19466753

  4. Versatile TPR domains accommodate different modes of target protein recognition and function.

    PubMed

    Allan, Rudi Kenneth; Ratajczak, Thomas

    2011-07-01

    The tetratricopeptide repeat (TPR) motif is one of many repeat motifs that form structural domains in proteins that can act as interaction scaffolds in the formation of multi-protein complexes involved in numerous cellular processes such as transcription, the cell cycle, protein translocation, protein degradation and host defence against invading pathogens. The crystal structures of many TPR domain-containing proteins have been determined, showing TPR motifs as two anti-parallel α-helices packed in tandem arrays to form a structure with an amphipathic groove which can bind a target peptide. This is however not the only mode of target recognition by TPR domains, with short amino acid insertions and alternative TPR motif conformations also shown to contribute to protein interactions, highlighting diversity in TPR domains and the versatility of this structure in mediating biological events.

  5. Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes

    PubMed Central

    Andreu-Fernández, Vicente; Sancho, Mónica; Genovés, Ainhoa; Lucendo, Estefanía; Todt, Franziska; Lauterwasser, Joachim; Funk, Kathrin; Jahreis, Günther; Pérez-Payá, Enrique; Mingarro, Ismael; Edlich, Frank; Orzáez, Mar

    2017-01-01

    The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation. PMID:28028215

  6. Maximum occurrence analysis of protein conformations for different distributions of paramagnetic metal ions within flexible two-domain proteins.

    PubMed

    Luchinat, Claudio; Nagulapalli, Malini; Parigi, Giacomo; Sgheri, Luca

    2012-02-01

    Multidomain proteins are composed of rigid domains connected by (flexible) linkers. Therefore, the domains may experience a large degree of reciprocal reorientation. Pseudocontact shifts and residual dipolar couplings arising from one or more paramagnetic metals successively placed in a single metal binding site in the protein can be used as restraints to assess the degree of mobility of the different domains. They can be used to determine the maximum occurrence (MO) of each possible protein conformation, i.e. the maximum weight that such conformations can have independently of the real structural ensemble, in agreement with the provided restraints. In the case of two-domain proteins, the metal ions can be placed all in the same domain, or distributed between the two domains. It has been demonstrated that the quantity of independent information for the characterization of the system is larger when all metals are bound in the same domain. At the same time, it has been shown that there are practical advantages in placing the metals in different domains. Here, it is shown that distributing the metals between the domains provides a tool for defining a coefficient of compatibility among the restraints obtained from different metals, without a significant decrease of the capability of the MO values to discriminate among conformations with different weights.

  7. Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures

    SciTech Connect

    Pykäläinen, Anette; Boczkowska, Malgorzata; Zhao, Hongxia; Saarikangas, Juha; Rebowski, Grzegorz; Jansen, Maurice; Hakanen, Janne; Koskela, Essi V.; Peränen, Johan; Vihinen, Helena; Jokitalo, Eija; Salminen, Marjo; Ikonen, Elina; Dominguez, Roberto; Lappalainen, Pekka

    2013-05-29

    Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR- and F-BAR-domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR-domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique membrane-deforming activity.

  8. Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein.

    PubMed

    Chen, Shuqing; Vincent, Sarah; Wilson, David B; Ganem, Bruce

    2003-02-01

    The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the l-tyrosine biosynthetic pathway. The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine. Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains. Residues 1-88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback-inhibition activities were localized in residues 94-373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.

  9. Specificity Profiling of Protein-Binding Domains Using One-Bead-One-Compound Peptide Libraries

    PubMed Central

    Kunys, Andrew R.; Lian, Wenlong; Pei, Dehua

    2013-01-01

    One-bead-one-compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need of any special equipment and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains) and subsequently using the specificity information to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains and the related data analysis. PMID:23788558

  10. Gene3D: Multi-domain annotations for protein sequence and comparative genome analysis.

    PubMed

    Lees, Jonathan G; Lee, David; Studer, Romain A; Dawson, Natalie L; Sillitoe, Ian; Das, Sayoni; Yeats, Corin; Dessailly, Benoit H; Rentzsch, Robert; Orengo, Christine A

    2014-01-01

    Gene3D (http://gene3d.biochem.ucl.ac.uk) is a database of protein domain structure annotations for protein sequences. Domains are predicted using a library of profile HMMs from 2738 CATH superfamilies. Gene3D assigns domain annotations to Ensembl and UniProt sequence sets including >6000 cellular genomes and >20 million unique protein sequences. This represents an increase of 45% in the number of protein sequences since our last publication. Thanks to improvements in the underlying data and pipeline, we see large increases in the domain coverage of sequences. We have expanded this coverage by integrating Pfam and SUPERFAMILY domain annotations, and we now resolve domain overlaps to provide highly comprehensive composite multi-domain architectures. To make these data more accessible for comparative genome analyses, we have developed novel search algorithms for searching genomes to identify related multi-domain architectures. In addition to providing domain family annotations, we have now developed a pipeline for 3D homology modelling of domains in Gene3D. This has been applied to the human genome and will be rolled out to other major organisms over the next year.

  11. Occurrence of protein disulfide bonds in different domains of life: a comparison of proteins from the Protein Data Bank.

    PubMed

    Bošnjak, I; Bojović, V; Šegvić-Bubić, T; Bielen, A

    2014-03-01

    Disulfide bonds (SS bonds) are important post-translational modifications of proteins. They stabilize a three-dimensional (3D) structure (structural SS bonds) and also have the catalytic or regulatory functions (redox-active SS bonds). Although SS bonds are present in all groups of organisms, no comparative analyses of their frequency in proteins from different domains of life have been made to date. Using the Protein Data Bank, the number and subcellular locations of SS bonds in Archaea, Bacteria and Eukarya have been compared. Approximately three times higher frequency of proteins with SS bonds in eukaryotic secretory organelles (e.g. endoplasmic reticulum) than in bacterial periplasmic/secretory pathways was calculated. Protein length also affects the SS bond frequency: the average number of SS bonds is positively correlated with the length for longer proteins (>200 amino acids), while for the shorter and less stable proteins (<200 amino acids) this correlation is negative. Medium-sized proteins (250-350 amino acids) indicated a high number of SS bonds only in Archaea which could be explained by the need for additional protein stabilization in hyperthermophiles. The results emphasize higher capacity for the SS bond formation and isomerization in Eukarya when compared with Archaea and Bacteria.

  12. Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity.

    PubMed Central

    Johnson, J D; Zhang, W; Rudnick, A; Rutter, W J; German, M S

    1997-01-01

    LIM-homeodomain proteins direct cellular differentiation by activating transcription of cell-type-specific genes, but this activation requires cooperation with other nuclear factors. The LIM-homeodomain protein Lmx1 cooperates with the basic helix-loop-helix (bHLH) protein E47/Pan-1 to activate the insulin promoter in transfected fibroblasts. In this study, we show that two proteins originally called Lmx1 are the closely related products of two distinct vertebrate genes, Lmx1.1 and Lmx1.2. We have used yeast genetic systems to delineate the functional domains of the Lmx1 proteins and to characterize the physical interactions between Lmx1 proteins and E47/Pan-1 that produce synergistic transcriptional activation. The LIM domains of the Lmx1 proteins, and particularly the second LIM domain, mediate both specific physical interactions and transcriptional synergy with E47/Pan-1. The LIM domains of the LIM-homeodomain protein Isl-1, which cannot mediate transcriptional synergy with E47/Pan-1, do not interact with E47/Pan-1. In vitro studies demonstrate that the Lmx1.1 LIM2 domain interacts specifically with the bHLH domain of E47/Pan-1. These studies provide the basis for a model of the assembly of LIM-homeodomain-containing complexes on DNA elements that direct cell-type-restricted transcription in differentiated tissues. PMID:9199284

  13. Clustering amino acid contents of protein domains: biochemical functions of proteins and implications for origin of biological macromolecules.

    PubMed

    Torshin, I Y

    2001-04-01

    Structural classes of protein domains correlate with their amino acid compositions. Several successful algorithms (that use only amino acid composition) have been elaborated for the prediction of structural class or potential biochemical significance. This work deals with dynamic classification (clustering) of the domains on the basis of their amino acid composition. Amino acid contents of domains from a non-redundant PDB set were clustered in 20-dimensional space of amino acid contents. Despite the variations of an empirical parameter and non-redundancy of the set, only one large cluster (tens-hundreds of proteins) surrounded by hundreds of small clusters (1-5 proteins), was identified. The core of the largest cluster contains at least 64% DNA (nucleotide)-interacting protein domains from various sources. About 90% of the proteins of the core are intracellular proteins. 83% of the DNA/nucleotide interacting domains in the core belong to the mixed alpha-beta folds (a+b, a/b), 14% are all-alpha (mostly helices) and all-beta (mostly beta-strands) proteins. At the same time, when core domains that belong to one organism (E.coli) are considered, over 80% of them prove to be DNA/nucleotide interacting proteins. The core is compact: amino acid contents of domains from the core lie in relatively narrow and specific ranges. The core also contains several Fe-S cluster-binding domains, amino acid contents of the core overlap with ferredoxin and CO-dehydrogenase clusters, the oldest known proteins. As Fe-S clusters are thought to be the first biocatalysts, the results are discussed in relation to contemporary experiments and models dealing with the origin of biological macromolecules. The origin of most primordial proteins is considered here to be a result of co-adsorption of nucleotides and amino acids on specific clays, followed by en-block polymerization of the adsorbed mixtures of amino acids.

  14. SH2 domain proteins as high-affinity receptor tyrosine kinase substrates.

    PubMed

    Sierke, S L; Koland, J G

    1993-09-28

    Activation of a growth factor receptor tyrosine kinase (RTK) is accompanied by a rapid autophosphorylation of the receptor on tyrosine residues. Receptor activation has been shown to promote the association of signal-transducing proteins containing SH2 domains (second domain of src homology). These receptor-associated proteins can, in turn, be phosphorylated by the RTK, an event which presumably regulates their activities. It has been suggested that SH2 domains in signal-transducing proteins target these proteins as substrates of the activated RTK. To test this hypothesis, recombinant proteins were generated that contained tyrosine phosphorylation sites of the erbB3 receptor and/or the SH2 domain of c-src. Incorporation of the SH2 domain led to a decrease in KM and an increase in Vmax for the substrate. The KM determined for one chimeric SH2/erbB3 substrate was among the lowest reported for epidermal growth factor RTK substrates. Experiments with a truncated kinase lacking C-terminal autophosphorylation sites indicated that the reduction in KM for these substrates was mediated by interactions between the substrate SH2 domain and phosphotyrosine residues of the RTK. These interactions could also inhibit RTK activity. These results demonstrate that the SH2 domain can effectively target substrates to a RTK and that SH2 domain proteins can regulate RTK activity.

  15. Structure and Function of the TIR Domain from the Grape NLR Protein RPV1

    PubMed Central

    Williams, Simon J.; Yin, Ling; Foley, Gabriel; Casey, Lachlan W.; Outram, Megan A.; Ericsson, Daniel J.; Lu, Jiang; Boden, Mikael; Dry, Ian B.; Kobe, Bostjan

    2016-01-01

    The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR) domain has been shown to be both necessary and sufficient for defense signaling in the model plants flax and Arabidopsis. In examples from these organisms, TIR domain self-association is required for signaling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevine Muscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signaling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices (“AE” interface). This interface is shared with the interface discovered in the dimeric complex of the TIR domains from the Arabidopsis RPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signaling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signaling. PMID:28008335

  16. Cooperative folding of intrinsically disordered domains drives assembly of a strong elongated protein

    NASA Astrophysics Data System (ADS)

    Gruszka, Dominika T.; Whelan, Fiona; Farrance, Oliver E.; Fung, Herman K. H.; Paci, Emanuele; Jeffries, Cy M.; Svergun, Dmitri I.; Baldock, Clair; Baumann, Christoph G.; Brockwell, David J.; Potts, Jennifer R.; Clarke, Jane

    2015-06-01

    Bacteria exploit surface proteins to adhere to other bacteria, surfaces and host cells. Such proteins need to project away from the bacterial surface and resist significant mechanical forces. SasG is a protein that forms extended fibrils on the surface of Staphylococcus aureus and promotes host adherence and biofilm formation. Here we show that although monomeric and lacking covalent cross-links, SasG maintains a highly extended conformation in solution. This extension is mediated through obligate folding cooperativity of the intrinsically disordered E domains that couple non-adjacent G5 domains thermodynamically, forming interfaces that are more stable than the domains themselves. Thus, counterintuitively, the elongation of the protein appears to be dependent on the inherent instability of its domains. The remarkable mechanical strength of SasG arises from tandemly arrayed `clamp' motifs within the folded domains. Our findings reveal an elegant minimal solution for the assembly of monomeric mechano-resistant tethers of variable length.

  17. Mapping of domains on HIV envelope protein mediating association with calnexin and protein-disulfide isomerase.

    PubMed

    Papandréou, Marie-Jeanne; Barbouche, Rym; Guieu, Régis; Rivera, Santiago; Fantini, Jacques; Khrestchatisky, Michel; Jones, Ian M; Fenouillet, Emmanuel

    2010-04-30

    The cell catalysts calnexin (CNX) and protein-disulfide isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope (Env) glycoprotein. Following HIV binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX, whereas only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in vitro conditions, which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in cellulo with CNX or in vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2, and the C terminus of V3 did not bind CNX-associated Env, whereas those against C1, V1/V2, and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discrete, largely nonoverlapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents.

  18. Mapping of Domains on HIV Envelope Protein Mediating Association with Calnexin and Protein-disulfide Isomerase*

    PubMed Central

    Papandréou, Marie-Jeanne; Barbouche, Rym; Guieu, Régis; Rivera, Santiago; Fantini, Jacques; Khrestchatisky, Michel; Jones, Ian M.; Fenouillet, Emmanuel

    2010-01-01

    The cell catalysts calnexin (CNX) and protein-disulfide isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope (Env) glycoprotein. Following HIV binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX, whereas only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in vitro conditions, which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in cellulo with CNX or in vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2, and the C terminus of V3 did not bind CNX-associated Env, whereas those against C1, V1/V2, and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discrete, largely nonoverlapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents. PMID:20202930

  19. Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

    NASA Astrophysics Data System (ADS)

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

  20. Crystal Structure of the Chromodomain Helicase DNA-binding Protein 1 (Chd1) DNA-binding Domain in Complex with DNA

    SciTech Connect

    Sharma A.; Heroux A.; Jenkins K. R.; Bowman G. D.

    2011-12-09

    Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves.

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

    SciTech Connect

    Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun; Chun, ChangJu; Im, Young Jun

    2014-09-12

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

  2. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    PubMed

    Neuvonen, Maarit; Ahola, Tero

    2009-01-09

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  3. Redox-coupled structural changes of the catalytic a' domain of protein disulfide isomerase.

    PubMed

    Inagaki, Koya; Satoh, Tadashi; Yagi-Utsumi, Maho; Le Gulluche, Anne-Charlotte; Anzai, Takahiro; Uekusa, Yoshinori; Kamiya, Yukiko; Kato, Koichi

    2015-09-14

    Protein disulfide isomerase functions as a folding catalyst in the endoplasmic reticulum. Its b' and a' domains provide substrate-binding sites and undergo a redox-dependent domain rearrangement coupled to an open-closed structural change. Here we determined the first solution structure of the a' domain in its oxidized form and thereby demonstrate that oxidation of the a' domain induces significant conformational changes not only in the vicinity of the active site but also in the distal b'-interfacial segment. Based on these findings, we propose that this conformational transition triggers the domain segregation coupled with the exposure of the hydrophobic surface.

  4. Characterization of substrate binding of the WW domains in human WWP2 protein.

    PubMed

    Jiang, Jiahong; Wang, Nan; Jiang, Yafei; Tan, Hongwei; Zheng, Jimin; Chen, Guangju; Jia, Zongchao

    2015-07-08

    WW domains harbor substrates containing proline-rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human WWP2 (hWWP2). Herein we have employed multiple techniques to investigate the second WW domain (WW2) in hWWP2. Our results show that hWWP2 is a specialized E3 for PPxY motif-containing substrates only and does not recognize other amino acids and phospho-residues. The strongest binding affinity of WW2, and the incompatibility between each WW domain, imply a novel relationship, and our SPR experiment reveals a dynamic binding mode in Class-I WW domains for the first time. The results from alanine-scanning mutagenesis and modeling further point to functionally conserved residues in WW2.

  5. Role of oligomerization domains in thrombospondins and other extracellular matrix proteins.

    PubMed

    Engel, Jürgen

    2004-06-01

    Coiled coils, collagen triple helices and globular oligomerization domains mediate the subunit assembly of many proteins in vertebrates and invertebrates. Oligomerization offers functional advantages including multivalency, increased binding strength and the combined function of different domains. These features are seen in natural proteins and may be introduced by protein engineering. The special focus of this review is on oligomerization domain of extracellular matrix proteins. For thrombospondins, initial interesting results on the functional role of oligomerization have been published. Other features remain to be explored. For example, it is not clear why thrombospondin-1 and thrombospondin-2 are trimers whereas thrombospondins-3 to -5 are pentamers. To stimulate this type of research, this review makes a survey of oligomerization domains and their functional role in extracellular matrix proteins.

  6. Solid-state nanopore analysis of the PDZ2 protein domain

    NASA Astrophysics Data System (ADS)

    Freedman, Kevin; Haq, Raza; Jurgens, Maike; Mulero, Rafael; Prabhu, Anmiv; Jemth, Per; Edel, Joshua; Kim, Minjun

    2010-03-01

    The PDZ2 protein domain plays a significant role in biology; specifically as a ubiquitous binding domain for a variety of proteins found in organisms from bacteria to humans. PDZ2 and a single-point mutant were characterized using nanopores to help elucidate the structure-function relationship of this protein and provide a framework for more complex studies involving protein folding/binding. The translocation properties and unfolding of this domain was interrogated by the ionic-current blockade method using a single digit nanometer solid-state pore. By conducting these experiments under a wide variety of fluidic conditions, significantly different ionic current blockades were recorded and provided a method for sensing the folding/unfolding characteristics of the PDZ2 protein domain and its single-point mutant.

  7. LdFlabarin, a New BAR Domain Membrane Protein of Leishmania Flagellum

    PubMed Central

    Thonnus, Magali; Salin, Bénédicte; Boissier, Fanny; Blancard, Corinne; Sauvanet, Cécile; Metzler, Christelle; Espiau, Benoît; Sahin, Annelise; Merlin, Gilles

    2013-01-01

    During the Leishmania life cycle, the flagellum undergoes successive assembly and disassembly of hundreds of proteins. Understanding these processes necessitates the study of individual components. Here, we investigated LdFlabarin, an uncharacterized L. donovani flagellar protein. The gene is conserved within the Leishmania genus and orthologous genes only exist in the Trypanosoma genus. LdFlabarin associates with the flagellar plasma membrane, extending from the base to the tip of the flagellum as a helicoidal structure. Site-directed mutagenesis, deletions and chimera constructs showed that LdFlabarin flagellar addressing necessitates three determinants: an N-terminal potential acylation site and a central BAR domain for membrane targeting and the C-terminal domain for flagellar specificity. In vitro, the protein spontaneously associates with liposomes, triggering tubule formation, which suggests a structural/morphogenetic function. LdFlabarin is the first characterized Leishmania BAR domain protein, and the first flagellum-specific BAR domain protein. PMID:24086735

  8. The b' domain provides the principal peptide-binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins.

    PubMed Central

    Klappa, P; Ruddock, L W; Darby, N J; Freedman, R B

    1998-01-01

    Protein disulfide isomerase (PDI) is a very efficient catalyst of folding of many disulfide-bonded proteins. A great deal is known about the catalytic functions of PDI, while little is known about its substrate binding. We recently demonstrated by cross-linking that PDI binds peptides and misfolded proteins, with high affinity but broad specificity. To characterize the substrate-binding site of PDI, we investigated the interactions of various recombinant fragments of human PDI, expressed in Escherichia coli, with different radiolabelled model peptides. We observed that the b' domain of human PDI is essential and sufficient for the binding of small peptides. In the case of larger peptides, specifically a 28 amino acid fragment derived from bovine pancreatic trypsin inhibitor, or misfolded proteins, the b' domain is essential but not sufficient for efficient binding, indicating that contributions from additional domains are required. Hence we propose that the different domains of PDI all contribute to the binding site, with the b' domain forming the essential core. PMID:9463371

  9. The mammalian START domain protein family in lipid transport in health and disease.

    PubMed

    Clark, Barbara J

    2012-03-01

    Lipid transfer proteins of the steroidogenic acute regulatory protein-related lipid transfer (START) domain family are defined by the presence of a conserved ∼210 amino acid sequence that folds into an α/β helix-grip structure forming a hydrophobic pocket for ligand binding. The mammalian START proteins bind diverse ligands, such as cholesterol, oxysterols, phospholipids, sphingolipids, and possibly fatty acids, and have putative roles in non-vesicular lipid transport, thioesterase enzymatic activity, and tumor suppression. However, the biological functions of many members of the START domain protein family are not well established. Recent research has focused on characterizing the cell-type distribution and regulation of the START proteins, examining the specificity and directionality of lipid transport, and identifying disease states associated with dysregulation of START protein expression. This review summarizes the current concepts of the proposed physiological and pathological roles for the mammalian START domain proteins in cholesterol and lipid trafficking.

  10. Cooperative interactions between paired domain and homeodomain.

    PubMed

    Jun, S; Desplan, C

    1996-09-01

    The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs,the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

  11. The NHR domains of Neuralized and related proteins: Beyond Notch signalling.

    PubMed

    Liu, Sili; Boulianne, Gabrielle L

    2017-01-01

    Neuralized Homology Repeats (NHRs) were first identified in Neuralized, an E3-ubiquitin ligase that plays a key role in the Notch signalling pathway. Since their original discovery, NHR domains have been shown to regulate protein-protein interactions in a broad range of developmental processes and in a wide variety of species from flies to humans. The NHR family of proteins can be categorized into three groups: (1) those that contain a RING finger, (2) those that contain a SOCS box and, (3) those that only have NHR domains. Here we review the structure and function of NHR domains in various cellular and developmental processes.

  12. A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis.

    PubMed

    Percy, Melanie J; Zhao, Quan; Flores, Adrian; Harrison, Claire; Lappin, Terence R J; Maxwell, Patrick H; McMullin, Mary Frances; Lee, Frank S

    2006-01-17

    The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the alpha-subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel-Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

  13. A Protein Domain and Family Based Approach to Rare Variant Association Analysis

    PubMed Central

    Richardson, Tom G.; Shihab, Hashem A.; Rivas, Manuel A.; McCarthy, Mark I.; Campbell, Colin; Timpson, Nicholas J.; Gaunt, Tom R.

    2016-01-01

    Background It has become common practice to analyse large scale sequencing data with statistical approaches based around the aggregation of rare variants within the same gene. We applied a novel approach to rare variant analysis by collapsing variants together using protein domain and family coordinates, regarded to be a more discrete definition of a biologically functional unit. Methods Using Pfam definitions, we collapsed rare variants (Minor Allele Frequency ≤ 1%) together in three different ways 1) variants within single genomic regions which map to individual protein domains 2) variants within two individual protein domain regions which are predicted to be responsible for a protein-protein interaction 3) all variants within combined regions from multiple genes responsible for coding the same protein domain (i.e. protein families). A conventional collapsing analysis using gene coordinates was also undertaken for comparison. We used UK10K sequence data and investigated associations between regions of variants and lipid traits using the sequence kernel association test (SKAT). Results We observed no strong evidence of association between regions of variants based on Pfam domain definitions and lipid traits. Quantile-Quantile plots illustrated that the overall distributions of p-values from the protein domain analyses were comparable to that of a conventional gene-based approach. Deviations from this distribution suggested that collapsing by either protein domain or gene definitions may be favourable depending on the trait analysed. Conclusion We have collapsed rare variants together using protein domain and family coordinates to present an alternative approach over collapsing across conventionally used gene-based regions. Although no strong evidence of association was detected in these analyses, future studies may still find value in adopting these approaches to detect previously unidentified association signals. PMID:27128313

  14. Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions.

    PubMed

    O'Seaghdha, Maghnus; van Schooten, Carina J; Kerrigan, Steven W; Emsley, Jonas; Silverman, Gregg J; Cox, Dermot; Lenting, Peter J; Foster, Timothy J

    2006-11-01

    Protein A (Spa) is a surface-associated protein of Staphylococcus aureus best known for its ability to bind to the Fc region of IgG. Spa also binds strongly to the Fab region of the immunoglobulins bearing V(H)3 heavy chains and to von Willebrand factor (vWF). Previous studies have suggested that the protein A-vWF interaction is important in S. aureus adherence to platelets under conditions of shear stress. We demonstrate that Spa expression is sufficient for adherence of bacteria to immobilized vWF under low fluid shear. The full length recombinant Ig-binding region of protein A, Spa-EDABC, fused to glutathione-S-transferase (GST), bound recombinant vWF in a dose-dependent and saturable fashion with half maximal binding of about 30 nm in immunosorbent assays. Full length-Spa did not bind recombinant vWF A3 domain but displayed binding to recombinant vWF domains A1 and D'-D3 (half maximal binding at 100 nm and 250 nm, respectively). Each recombinant protein A Ig-binding domain bound to the A1 domain in a similar manner to the full length-Spa molecule (half maximal binding 100 nm). Amino acid substitutions were introduced in the GST-SpaD protein at sites known to be involved in IgG Fc or in V(H)3 Fab binding. Mutants altered in residues that recognized IgG Fc but not those that recognized V(H)3 Fab had reduced binding to vWF A1 and D'-D3. This indicated that both vWF regions recognized a region on helices I and II that overlapped the IgG Fc binding site.

  15. Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains.

    PubMed Central

    Adinolfi, S; Bagni, C; Musco, G; Gibson, T; Mazzarella, L; Pastore, A

    1999-01-01

    FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence

  16. Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco.

    PubMed

    Felix, Georg; Boller, Thomas

    2003-02-21

    To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs). Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco. Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations. Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes. Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells. Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs. Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity. Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors. Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested. Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.

  17. Activation of nanoscale allosteric protein domain motion revealed by neutron spin echo spectroscopy

    NASA Astrophysics Data System (ADS)

    Bu, Zimei; Farago, Bela; Callaway, David

    2012-02-01

    NHERF1 is a multi-domain scaffolding protein that assembles the signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by a membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 angstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length scales and on sub-microsecond time scales upon forming a complex with ezrin. We show that a much simplified coarse-grained model is sufficient to describe inter-domain motion of a multi-domain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. The results demonstrate that propagation of allosteric signals to distal sites involves the activation of long-range coupled domain motions on submicrosecond time scales, and that these coupled motions can be distinguished and characterized by NSE.

  18. Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20

    PubMed Central

    Maio, N.; Rouault, T. A.

    2017-01-01

    Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery. PMID:27714045

  19. Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis peptides are cross recognized by anti-myelin basic protein antibodies in multiple sclerosis patients.

    PubMed

    Mameli, Giuseppe; Cossu, Davide; Cocco, Eleonora; Masala, Speranza; Frau, Jessica; Marrosu, Maria Giovanna; Sechi, Leonardo A

    2014-05-15

    Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis (MAP) have been associated to multiple sclerosis (MS). We searched for antibodies against the homologous peptides Epstein-Barr virus nuclear antigen 1 (EBNA1)400-413, MAP_0106c protein (MAP)121-132, and myelin basic protein (MBP)85-98 on a MS Sardinian cohort, showing that these antibodies are highly prevalent among MS patients compared to healthy controls. Competitive assay demonstrated that antibodies recognizing EBNA1400-413 and MAP121-132 cross-react with MBP85-98, possibly through a molecular mimicry mechanism. Indeed, the fact that peptides from different pathogens can be cross-recognized by antibodies targeting self-epitopes supports the hypothesis that EBV and MAP might trigger autoimmunity through a common target.

  20. Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats

    PubMed Central

    Pelassa, Ilaria; Fiumara, Ferdinando

    2015-01-01

    Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as “junk” sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in

  1. 3DSwap: curated knowledgebase of proteins involved in 3D domain swapping.

    PubMed

    Shameer, Khader; Shingate, Prashant N; Manjunath, S C P; Karthika, M; Pugalenthi, Ganesan; Sowdhamini, Ramanathan

    2011-01-01

    Three-dimensional domain swapping is a unique protein structural phenomenon where two or more protein chains in a protein oligomer share a common structural segment between individual chains. This phenomenon is observed in an array of protein structures in oligomeric conformation. Protein structures in swapped conformations perform diverse functional roles and are also associated with deposition diseases in humans. We have performed in-depth literature curation and structural bioinformatics analyses to develop an integrated knowledgebase of proteins involved in 3D domain swapping. The hallmark of 3D domain swapping is the presence of distinct structural segments such as the hinge and swapped regions. We have curated the literature to delineate the boundaries of these regions. In addition, we have defined several new concepts like 'secondary major interface' to represent the interface properties arising as a result of 3D domain swapping, and a new quantitative measure for the 'extent of swapping' in structures. The catalog of proteins reported in 3DSwap knowledgebase has been generated using an integrated structural bioinformatics workflow of database searches, literature curation, by structure visualization and sequence-structure-function analyses. The current version of the 3DSwap knowledgebase reports 293 protein structures, the analysis of such a compendium of protein structures will further the understanding molecular factors driving 3D domain swapping.

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

    PubMed

    Jin, Tengchuan; Huang, Mo; Smith, Patrick; Jiang, Jiansheng; Xiao, T Sam

    2013-08-01

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

  3. Molecular cloning, genomic structure, and tissue distribution of EW135, a novel chicken egg white protein with group B scavenger receptor cysteine-rich domains.

    PubMed

    Yoo, Whayoung; Nakamura, Tomohiro; Asanuma, Hideki; Matsushita, Misao

    2013-11-01

    Approximately 80 proteins are reported to be present in chicken egg white. The major function of egg white proteins isolated so far is to defend the egg yolk against infections. We recently isolated a novel protein termed EW135 from chicken egg white. In this paper, we have determined the complete amino acid sequence of EW135 based on cDNA cloning. EW135 consists of 970 amino acids with a putative signal peptide of 17 amino acids. It is composed exclusively of tandem repeats of nine group B scavenger receptor cysteine-rich (SRCR) domains separated by eight seven-amino acid peptides. The features of consensus sequences found in the group B SRCR domain were well conserved in EW135. The EW135 gene consists of putative 11 exons, with each SRCR domain being encoded by a single exon. Reverse transcription PCR showed that EW135 is expressed in only the oviduct among the 11 types of tissues tested. EW135 is a second soluble protein belonging to the group B SRCR domain superfamily identified in chickens. One of the important functions of proteins belonging to the group B SRCR domain superfamily is to recognize pathogens in innate immunity. It is, therefore, conceivable that EW135 could be involved in host defense in egg white.

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

    PubMed

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

    2014-02-21

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

  5. Modulation of neurotransmitter receptors and synaptic differentiation by proteins containing complement-related domains.

    PubMed

    Nakayama, Minoru; Hama, Chihiro

    2011-02-01

    Neurotransmitter receptors play central roles in basic neurotransmission and synaptic plasticity. Recent studies have revealed that some transmembrane and extracellular proteins bind to neurotransmitter receptors, forming protein complexes that are required for proper synaptic localization or gating of core receptor molecules. Consequently, the components of these complexes contribute to long-term potentiation, a process that is critical for learning and memory. Here, we review factors that regulate neurotransmitter receptors, with a focus on proteins containing CUB (complement C1r/C1s, Uegf, Bmp1) or CCP (complement control protein) domains, which are frequently found in complement system proteins. Proteins that contain these domains are structurally distinct from TARPs (transmembrane AMPA receptor regulatory proteins), and may constitute new protein families that modulate either the localization or function of neurotransmitter receptors. In addition, other CCP domain-containing proteins participate in dendritic patterning and/or synaptic differentiation, although current evidence has not identified any direct activities on neurotransmitter receptors. Some of these proteins are involved in pathologic conditions such as epileptic seizure and mental retardation. Together, these lines of information have shown that CUB and CCP domain-containing proteins contribute to a wide variety of neuronal events that ultimately establish neural circuits.

  6. Reciprocal Influence of Protein Domains in the Cold-Adapted Acyl Aminoacyl Peptidase from Sporosarcina psychrophila

    PubMed Central

    Parravicini, Federica; Natalello, Antonino; Papaleo, Elena; De Gioia, Luca; Doglia, Silvia Maria; Lotti, Marina; Brocca, Stefania

    2013-01-01

    Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/β-hydrolase domain and by an N-terminal β-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the β-propeller. The α1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated β-propeller. A single charged residue (E10) in the α1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution. PMID:23457536

  7. Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter.

    PubMed

    Skillman, Kristen M; Barnard, Travis J; Peterson, Janine H; Ghirlando, Rodolfo; Bernstein, Harris D

    2005-11-01

    Bacterial autotransporters are proteins that contain a small C-terminal 'beta domain' that facilitates translocation of a large N-terminal 'passenger domain' across the outer membrane (OM) by an unknown mechanism. Here we used EspP, an autotransporter produced by Escherichia coli 0157:H7, as a model protein to gain insight into the transport reaction. Initially we found that the passenger domain of a truncated version of EspP (EspPDelta1-851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspPDelta1-851 behaves as a compact monomer and strongly suggest that the channel formed by the beta domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N-terminus of EspPDelta1-851 was efficiently translocated across the OM. Further analysis revealed that the passenger domain of wild-type EspP also folds at least partially in the periplasm. To reconcile these data, we propose that the EspP beta domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.

  8. Methyl-CpG-binding domain proteins: readers of the epigenome.

    PubMed

    Du, Qian; Luu, Phuc-Loi; Stirzaker, Clare; Clark, Susan J

    2015-01-01

    How DNA methylation is interpreted and influences genome regulation remains largely unknown. Proteins of the methyl-CpG-binding domain (MBD) family are primary candidates for the readout of DNA methylation as they recruit chromatin remodelers, histone deacetylases and methylases to methylated DNA associated with gene repression. MBD protein binding requires both functional MBD domains and methyl-CpGs; however, some MBD proteins also bind unmethylated DNA and active regulatory regions via alternative regulatory domains or interaction with the nucleosome remodeling deacetylase (NuRD/Mi-2) complex members. Mutations within MBD domains occur in many diseases, including neurological disorders and cancers, leading to loss of MBD binding specificity to methylated sites and gene deregulation. Here, we summarize the current state of knowledge about MBD proteins and their role as readers of the epigenome.

  9. Protein domain networks: Scale-free mixing of positive and negative exponents

    NASA Astrophysics Data System (ADS)

    Nacher, J. C.; Hayashida, M.; Akutsu, T.

    2006-07-01

    Many biological studies have been focused on the study of proteins, since proteins are essential for most cell functions. Although proteins are unique, they share certain common properties. For example, well-defined regions within a protein can fold independently from the rest of the protein and have their own function. They are called protein domains, and served as protein building blocks. In this article, we present a theoretical model for studying the protein domain networks, where one node of the network corresponds to one protein and two proteins are connected if they contain the same domain. The resulting distribution of nodes with a given degree, k, shows not only a power-law with negative exponent γ=-1, but it resembles the superposition of two power-law functions, one with a negative exponent and another with a positive exponent β=1. We call this distribution pattern “ scale-free mixing”. To explain the emergence of this superposition of power-laws, we propose a basic model with two main components: (1) mutation and (2) duplication of domains. Precisely, duplication gives rise to complete subgraphs (i.e., cliques) on the network, thus for several values of k a large number of nodes with degree k is produced, which explains the positive power-law branch of the degree distribution. In order to compare our model with experimental data, we generate protein domain networks with data from the UniProt Knowledgebase-Swissprot database for protein sequences and using InterPro, Pfam and Smart for domain databases. Our results indicate that the signal of this scale-free mixing pattern is also observed in the experimental data and it is conserved among organisms as Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Mus musculus, and Homo sapiens.

  10. Structural Studies of the Tandem Tudor Domains of Fragile X Mental Retardation Related Proteins FXR1 and FXR2

    SciTech Connect

    Adams-Cioaba, Melanie A.; Guo, Yahong; Bian, ChuanBing; Amaya, Maria F.; Lam, Robert; Wasney, Gregory A.; Vedadi, Masoud; Xu, Chao; Min, Jinrong

    2011-11-23

    Expansion of the CGG trinucleotide repeat in the 5'-untranslated region of the FMR1, fragile X mental retardation 1, gene results in suppression of protein expression for this gene and is the underlying cause of Fragile X syndrome. In unaffected individuals, the FMRP protein, together with two additional paralogues (Fragile X Mental Retardation Syndrome-related Protein 1 and 2), associates with mRNA to form a ribonucleoprotein complex in the nucleus that is transported to dendrites and spines of neuronal cells. It is thought that the fragile X family of proteins contributes to the regulation of protein synthesis at sites where mRNAs are locally translated in response to stimuli. Here, we report the X-ray crystal structures of the non-canonical nuclear localization signals of the FXR1 and FXR2 autosomal paralogues of FMRP, which were determined at 2.50 and 1.92 {angstrom}, respectively. The nuclear localization signals of the FXR1 and FXR2 comprise tandem Tudor domain architectures, closely resembling that of UHRF1, which is proposed to bind methylated histone H3K9. The FMRP, FXR1 and FXR2 proteins comprise a small family of highly conserved proteins that appear to be important in translational regulation, particularly in neuronal cells. The crystal structures of the N-terminal tandem Tudor domains of FXR1 and FXR2 revealed a conserved architecture with that of FMRP. Biochemical analysis of the tandem Tudor doamins reveals their ability to preferentially recognize trimethylated peptides in a sequence-specific manner.

  11. Histone Code Modulation by Oncogenic PWWP-domain Protein in Breast Cancers

    DTIC Science & Technology

    2013-06-01

    Hoffmann MJ. Transcription factor networks in embryonic stem cells and testicular cancer and the definition of epigenetics. Epigenetics 2007; 2(1): 37-42...PWWP-domain Protein in Breast Cancers PRINCIPAL INVESTIGATOR: Zeng-Quan Yang, Ph.D...SUBTITLE 5a. CONTRACT NUMBER Histone Code Modulation by Oncogenic PWWP-domain Protein in Breast Cancers 5b. GRANT NUMBER W81XWH-09-1-0109 5c

  12. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

    PubMed

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-12-29

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

  13. The Transactivation Domains of the p53 Protein.

    PubMed

    Raj, Nitin; Attardi, Laura D

    2017-01-03

    The p53 tumor suppressor is a transcriptional activator, with discrete domains that participate in sequence-specific DNA binding, tetramerization, and transcriptional activation. Mutagenesis and reporter studies have delineated two distinct activation domains (TADs) and specific hydrophobic residues within these TADs that are critical for their function. Knockin mice expressing p53 mutants with alterations in either or both of the two TADs have revealed that TAD1 is critical for responses to acute DNA damage, whereas both TAD1 and TAD2 participate in tumor suppression. Biochemical and structural studies have identified factors that bind either or both TADs, including general transcription factors (GTFs), chromatin modifiers, and negative regulators, helping to elaborate a model through which p53 activates transcription. Posttranslational modifications (PTMs) of the p53 TADs through phosphorylation also regulate TAD activity. Together, these studies on p53 TADs provide great insight into how p53 serves as a tumor suppressor.

  14. Travelling lipid domains in a dynamic model for protein-induced pattern formation in biomembranes

    NASA Astrophysics Data System (ADS)

    John, Karin; Bär, Markus

    2005-06-01

    Cell membranes are composed of a mixture of lipids. Many biological processes require the formation of spatial domains in the lipid distribution of the plasma membrane. We have developed a mathematical model that describes the dynamic spatial distribution of acidic lipids in response to the presence of GMC proteins and regulating enzymes. The model encompasses diffusion of lipids and GMC proteins, electrostatic attraction between acidic lipids and GMC proteins as well as the kinetics of membrane attachment/detachment of GMC proteins. If the lipid-protein interaction is strong enough, phase separation occurs in the membrane as a result of free energy minimization and protein/lipid domains are formed. The picture is changed if a constant activity of enzymes is included into the model. We chose the myristoyl-electrostatic switch as a regulatory module. It consists of a protein kinase C that phosphorylates and removes the GMC proteins from the membrane and a phosphatase that dephosphorylates the proteins and enables them to rebind to the membrane. For sufficiently high enzymatic activity, the phase separation is replaced by travelling domains of acidic lipids and proteins. The latter active process is typical for nonequilibrium systems. It allows for a faster restructuring and polarization of the membrane since it acts on a larger length scale than the passive phase separation. The travelling domains can be pinned by spatial gradients in the activity; thus the membrane is able to detect spatial clues and can adapt its polarity dynamically to changes in the environment.

  15. A nuclear localization domain in the hnRNP A1 protein

    PubMed Central

    1995-01-01

    The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta- galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2. PMID:7730395

  16. p23 and HSP20/alpha-crystallin proteins define a conserved sequence domain present in other eukaryotic protein families.

    PubMed

    Garcia-Ranea, J A; Mirey, Gladys; Camonis, Jacques; Valencia, Alfonso

    2002-10-09

    We identified families of proteins characterized by the presence of a domain similar to human p23 protein, which include proteins such as Sgt1, involved in the yeast kinetochore assembly; melusin, involved in specific interactions with the cytoplasmic integrin beta1 domain; Rar1, related to pathogenic resistance in plants, and to development in animals; B5+B5R flavo-hemo cytochrome NAD(P)H oxidoreductase type B in humans and mice; and NudC, involved in nucleus migration during mitosis. We also found that p23 and the HSP20/alpha-crystallin family of heat shock proteins, which share the same three-dimensional folding, show a pattern of conserved residues that points to a common origin in the evolution of both protein domains. The p23 and HSP20/alpha-crystallin phylogenetic relationship and their similar role in chaperone activity suggest a common function, probably involving protein-protein interaction, for those proteins containing p23-like domains.

  17. Role of Nucleotide Binding and GTPase Domain Dimerization in Dynamin-like Myxovirus Resistance Protein A for GTPase Activation and Antiviral Activity*

    PubMed Central

    Dick, Alexej; Graf, Laura; Olal, Daniel; von der Malsburg, Alexander; Gao, Song; Kochs, Georg; Daumke, Oliver

    2015-01-01

    Myxovirus resistance (Mx) GTPases are induced by interferon and inhibit multiple viruses, including influenza and human immunodeficiency viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action. PMID:25829498

  18. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    SciTech Connect

    Bielmann, Regula; Habann, Matthias; Eugster, Marcel R.; Lurz, Rudi; Calendar, Richard; Klumpp, Jochen; Loessner, Martin J.

    2015-03-15

    Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.

  19. An alternative scenario for the formation of specialized protein nano-domains (cluster phases) in biomembranes

    NASA Astrophysics Data System (ADS)

    Destainville, N.

    2010-09-01

    We discuss a realistic scenario, accounting for the existence of sub-micrometric protein domains in cell membranes. At the biological level, such membrane domains have been shown to be specialized, in order to perform a determined biological task, in the sense that they gather one or a few protein species out of the hundreds of different ones that a cell membrane may contain. By analyzing the balance between mixing entropy and protein affinities, we propose that such protein sorting in distinct domains can be explained without appealing to pre-existing lipidic micro-phase separations, as in the lipid raft scenario. We show that the proposed scenario is compatible with known physical interactions between membrane proteins, even if thousands of different species coexist.

  20. Predicting protein N-glycosylation by combining functional domain and secretion information.

    PubMed

    Li, Sujun; Liu, Boshu; Cai, Yudong; Li, Yixue

    2007-08-01

    Protein N-glycosylation plays an important role in protein function. Yet, at present, few computational methods are available for the prediction of this protein modification. This prompted our development of a support vector machine (SVM)-based method for this task, as well as a partial least squares (PLS) regression based prediction method for comparison. A functional domain feature space was used to create SVM and PLS models, which achieved accuracies of 83.91% and 79.89%, respectively, as evaluated by a leave-one-out cross-validation. Subsequently, SVM and PLS models were developed based on functional domain and protein secretion information, which yielded accuracies of 89.13% and 86%, respectively. This analysis demonstrates that the protein functional domain and secretion information are both efficient predictors of N-glycosylation.

  1. The HPr Proteins from the Thermophile Bacillus stearothermophilus Can Form Domain-swapped Dimers

    SciTech Connect

    Sridharan, Sudharsan; Razvi, Abbas; Scholtz, J. Martin; Sacchettini, James C.

    2010-07-20

    The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar to the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B. subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.

  2. Protein Domain of Unknown Function 3233 is a Translocation Domain of Autotransporter Secretory Mechanism in Gamma proteobacteria

    PubMed Central

    Prakash, Ananth; Yogeeshwari, S.; Sircar, Sanchari; Agrawal, Shipra

    2011-01-01

    Vibrio cholerae, the enteropathogenic gram negative bacteria is one of the main causative agents of waterborne diseases like cholera. About 1/3rd of the organism's genome is uncharacterised with many protein coding genes lacking structure and functional information. These proteins form significant fraction of the genome and are crucial in understanding the organism's complete functional makeup. In this study we report the general structure and function of a family of hypothetical proteins, Domain of Unknown Function 3233 (DUF3233), which are conserved across gram negative gammaproteobacteria (especially in Vibrio sp. and similar bacteria). Profile and HMM based sequence search methods were used to screen homologues of DUF3233. The I-TASSER fold recognition method was used to build a three dimensional structural model of the domain. The structure resembles the transmembrane beta-barrel with an axial N-terminal helix and twelve antiparallel beta-strands. Using a combination of amphipathy and discrimination analysis we analysed the potential transmembrane beta-barrel forming properties of DUF3233. Sequence, structure and phylogenetic analysis of DUF3233 indicates that this gram negative bacterial hypothetical protein resembles the beta-barrel translocation unit of autotransporter Va secretory mechanism with a gene organisation that differs from the conventional Va system. PMID:22073138

  3. IS-Dom: a dataset of independent structural domains automatically delineated from protein structures

    NASA Astrophysics Data System (ADS)

    Ebina, Teppei; Umezawa, Yuki; Kuroda, Yutaka

    2013-05-01

    Protein domains that can fold in isolation are significant targets in diverse area of proteomics research as they are often readily analyzed by high-throughput methods. Here, we report IS-Dom, a dataset of Independent Structural Domains (ISDs) that are most likely to fold in isolation. IS-Dom was constructed by filtering domains from SCOP, CATH, and DomainParser using quantitative structural measures, which were calculated by estimating inter-domain hydrophobic clusters and hydrogen bonds from the full length protein's atomic coordinates. The ISD detection protocol is fully automated, and all of the computed interactions are stored in the server which enables rapid update of IS-Dom. We also prepared a standard IS-Dom using parameters optimized by maximizing the Youden's index. The standard IS-Dom, contained 54,860 ISDs, of which 25.5 % had high sequence identity and termini overlap with a Protein Data Bank (PDB) cataloged sequence and are thus experimentally shown to fold in isolation [coined autonomously folded domain (AFDs)]. Furthermore, our ISD detection protocol missed less than 10 % of the AFDs, which corroborated our protocol's ability to define structural domains that are able to fold independently. IS-Dom is available through the web server (http://domserv.lab.tuat.ac.jp/IS-Dom.html), and users can either, download the standard IS-Dom dataset, construct their own IS-Dom by interactively varying the parameters, or assess the structural independence of newly defined putative domains.

  4. ERAD of proteins containing aberrant transmembrane domains requires ubiquitylation of cytoplasmic lysine residues

    PubMed Central

    Briant, Kit; Koay, Yee-Hui; Otsuka, Yuka; Swanton, Eileithyia

    2015-01-01

    ABSTRACT Clearance of misfolded proteins from the endoplasmic reticulum (ER) is mediated by the ubiquitin-proteasome system in a process known as ER-associated degradation (ERAD). The mechanisms through which proteins containing aberrant transmembrane domains are degraded by ERAD are poorly understood. To address this question, we generated model ERAD substrates based on CD8 with either a non-native transmembrane domain but a folded ER luminal domain (CD8TMD*), or the native transmembrane domain but a misfolded luminal domain (CD8LUM*). Although both chimeras were degraded by ERAD, we found that the location of the folding defect determined the initial site of ubiquitylation. Ubiquitylation of cytoplasmic lysine residues was required for the extraction of CD8TMD* from the ER membrane during ERAD, whereas CD8LUM* continued to be degraded in the absence of cytoplasmic lysine residues. Cytoplasmic lysine residues were also required for degradation of an additional ERAD substrate containing an unassembled transmembrane domain and when a non-native transmembrane domain was introduced into CD8LUM*. Our results suggest that proteins with defective transmembrane domains are removed from the ER through a specific ERAD mechanism that depends upon ubiquitylation of cytoplasmic lysine residues. PMID:26446255

  5. Identification and purification of a novel 120-kDa protein that recognizes the cAMP-responsive element

    SciTech Connect

    Andrisani, O.; Dixon, J.E. )

    1990-02-25

    The TGACGTCA (CRE) motif required for function by a number of cellular (somatostatin, enkephalin, alpha-human chorionic gonadotropin) and viral (Ad5 E1A-inducible, HTLV-1 TAX-inducible) genes is the site of interaction of multiple sequence-specific complexes. A protocol has been developed for the fractionation and purification of these activities. We report here the purification from HeLa nuclear extracts of a novel 120-kDa polypeptide which by Southwestern blots, gel retardation, and UV cross-linking assays displays CRE-specific binding. The CRE-affinity purified 120-kDa protein displays properties distinct from those of the 43-kDa CREB/ATF polypeptide. The 120-kDa protein is readily phosphorylated in vitro by protein kinase C but not by protein kinase A, suggesting that this molecule may mediate cellular signals distinct from the cAMP-responsive pathway. In vitro transcription-complementation assays utilizing the purified 120-kDa protein failed to transactivate the cAMP-responsive somatostatin promoter suggesting that the mode of action of this 120-kDa polypeptide may require an activation step distinct from the cAMP-signaling pathway.

  6. Charting the Landscape of Tandem BRCT Domain-Mediated Protein Interactions

    PubMed Central

    Woods, Nicholas T.; Mesquita, Rafael D.; Sweet, Michael; Carvalho, Marcelo A.; Li, Xueli; Liu, Yun; Nguyen, Huey; Thomas, C. Eric; Iversen, Edwin S.; Marsillac, Sylvia; Karchin, Rachel; Koomen, John; Monteiro, Alvaro N.A.

    2014-01-01

    Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, includes a large number of proteins responsible for detection of DNA damage, promotion of repair, and coordination with cell cycle progression. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The BRCT domain is a modular protein domain critical for relaying signals in the DDR. We performed a systematic analysis of protein-protein interactions involving tandem BRCT domains (tBRCT) in the DDR by combining literature curation, yeast two hybrid (Y2H) screens, and tandem affinity purification coupled to mass spectrometry (TAP-MS). We identified one previously unrecognized BRCT protein and generated human protein-protein interaction network for this type of modular domain. This study also reveals several novel components in DNA damage signaling such as COMMD1 and mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair. PMID:22990118

  7. DNA binding residues in the RQC domain of Werner protein are critical for its catalytic activities.

    PubMed

    Tadokoro, Takashi; Kulikowicz, Tomasz; Dawut, Lale; Croteau, Deborah L; Bohr, Vilhelm A

    2012-06-01

    Werner protein (WRN), member of the RecQ helicase family, is a helicase and exonuclease, and participates in multiple DNA metabolic processes including DNA replication, recombination and DNA repair. Mutations in the WRN gene cause Werner syndrome, associated with premature aging, genome instability and cancer predisposition. The RecQ C-terminal (RQC) domain of WRN, containing α2-α3 loop and β-wing motifs, is important for DNA binding and for many protein interactions. To better understand the critical functions of this domain, we generated recombinant WRN proteins (using a novel purification scheme) with mutations in Arg-993 within the α2-α3 loop of the RQC domain and in Phe-1037 of the -wing motif. We then studied the catalytic activities and DNA binding of these mutant proteins as well as some important functional protein interactions. The mutant proteins were defective in DNA binding and helicase activity, and interestingly, they had deficient exonuclease activity and strand annealing function. The RQC domain of WRN has not previously been implicated in exonuclease or annealing activities. The mutant proteins could not stimulate NEIL1 incision activity as did the wild type. Thus, the Arg-993 and Phe-1037 in the RQC domain play essential roles in catalytic activity, and in functional interactions mediated by WRN.

  8. Differential arginine methylation of the G-protein pathway suppressor GPS-2 recognized by tumor-specific T cells in melanoma.

    PubMed

    Jarmalavicius, Saulius; Trefzer, Uwe; Walden, Peter

    2010-03-01

    The aim of the study was to identify as potential therapeutic targets specific molecular alterations in tumor cells recognized by the immune system. To identify such targets, we analyzed the human leukocyte antigen (HLA) peptidomes of human melanoma cells by 2-dimensional nano-HPLC/mass spectrometry and tested the immunological significance of the peptides by ex vivo ELISpot assays with lymphocytes from melanoma patients. The peptide SQNPRFYHK was identified as derived from the regulator of the nuclear corepressor complex (NCoR) G-protein pathway suppressor 2 (GPS-2) and to be differentially unmethylated, monomethylated or asymmetrically dimethylated at the arginine. The methylation state was specifically recognized by the immune system in that only the monomethylated variant induced T-cell responses and significantly stronger responses in patients than in healthy controls. The methylations were confirmed with synthetic analogues and in vitro radiolabeling assays with recombinant GPS-2 and synthetic peptides. The immunity of the 3 variants of GPS-2 was tested in T-cell assays with T lymphocytes of melanoma patients compared with healthy donors. The results show for the first time that GPS-2 is differentially methylated at a site that lacks known methylation motifs and that the methylation state is detected by the immune system.-Jarmalavicius, S., Trefzer, U., Walden, P. Differential arginine methylation of the G-protein pathway suppressor GPS-2 recognized by tumor-specific T cells in melanoma.

  9. Fungal Endopolygalacturonases Are Recognized as Microbe-Associated Molecular Patterns by the Arabidopsis Receptor-Like Protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES11[W

    PubMed Central

    Zhang, Lisha; Kars, Ilona; Essenstam, Bert; Liebrand, Thomas W.H.; Wagemakers, Lia; Elberse, Joyce; Tagkalaki, Panagiota; Tjoitang, Devlin; van den Ackerveken, Guido; van Kan, Jan A.L.

    2014-01-01

    Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis. PMID:24259685

  10. Fungal endopolygalacturonases are recognized as microbe-associated molecular patterns by the arabidopsis receptor-like protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1.

    PubMed

    Zhang, Lisha; Kars, Ilona; Essenstam, Bert; Liebrand, Thomas W H; Wagemakers, Lia; Elberse, Joyce; Tagkalaki, Panagiota; Tjoitang, Devlin; van den Ackerveken, Guido; van Kan, Jan A L

    2014-01-01

    Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.

  11. Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis.

    PubMed

    Lee, Hongsik; Jeong, Hyeyeun; Park, Sangeun; Yoo, Wonbaek; Choi, Soyoung; Choi, Kyungmin; Lee, Min-Goo; Lee, Mihwa; Cha, DaeRyong; Kim, Young-Sik; Han, Jeeyoung; Kim, Wonkon; Park, Sun-Hwa; Oh, Junseo

    2015-06-01

    Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein-albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug.

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

    PubMed Central

    Zhang, Chongxu; Nielsen, Maria E. O.; Chiang, Yueh-Chin; Kierkegaard, Morten; Wang, Xin; Lee, Darren J.; Andersen, Jens S.; Yao, Gang

    2013-01-01

    Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1’s defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made. PMID:22836166

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

    PubMed

    Richardson, Roy; Denis, Clyde L; Zhang, Chongxu; Nielsen, Maria E O; Chiang, Yueh-Chin; Kierkegaard, Morten; Wang, Xin; Lee, Darren J; Andersen, Jens S; Yao, Gang

    2012-09-01

    Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1's defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.

  14. In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana

    PubMed Central

    Urbanus, Susan L; de Folter, Stefan; Shchennikova, Anna V; Kaufmann, Kerstin; Immink, Richard GH; Angenent, Gerco C

    2009-01-01

    Background MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). Results We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. Conclusion This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during

  15. Sequence Analysis of Scaffolding Protein CipC and ORFXp, a New Cohesin-Containing Protein in Clostridium cellulolyticum: Comparison of Various Cohesin Domains and Subcellular Localization of ORFXp

    PubMed Central

    Pagès, Sandrine; Bélaïch, Anne; Fierobe, Henri-Pierre; Tardif, Chantal; Gaudin, Christian; Bélaïch, Jean-Pierre

    1999-01-01

    The gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum, whose partial sequence was published earlier (S. Pagès, A. Bélaïch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P. Bélaïch, J. Bacteriol. 178:2279–2286, 1996; C. Reverbel-Leroy, A. Bélaïch, A. Bernadac, C. Gaudin, J. P. Bélaïch, and C. Tardif, Microbiology 142:1013–1023, 1996), was completely sequenced. The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains. In addition, a second gene located 8.89 kb downstream of cipC was found to encode a three-domain protein, called ORFXp, which includes a cohesin domain. By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane of C. cellulolyticum and is not detected in the cellulosome fraction. Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (Ka = 4.8 × 109 M−1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lower Ka for CelA (2.6 × 108 M−1). Taken together, these data suggest that ORFXp may play a role in cellulosome assembly. PMID:10074072

  16. SPECTRUS: A Dimensionality Reduction Approach for Identifying Dynamical Domains in Protein Complexes from Limited Structural Datasets.

    PubMed

    Ponzoni, Luca; Polles, Guido; Carnevale, Vincenzo; Micheletti, Cristian

    2015-08-04

    Identifying dynamical, quasi-rigid domains in proteins provides a powerful means for characterizing functionally oriented structural changes via a parsimonious set of degrees of freedom. In fact, the relative displacements of few dynamical domains usually suffice to rationalize the mechanics underpinning biological functionality in proteins and can even be exploited for structure determination or refinement purposes. Here we present SPECTRUS, a general scheme that, by solely using amino acid distance fluctuations, can pinpoint the innate quasi-rigid domains of single proteins or large complexes in a robust way. Consistent domains are usually obtained by using either a pair of representative structures or thousands of conformers. The functional insights offered by the approach are illustrated for biomolecular systems of very different size and complexity such as kinases, ion channels, and viral capsids. The decomposition tool is available as a software package and web server at spectrus.sissa.it.

  17. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.

    PubMed

    Shi, Junwei; Wang, Eric; Milazzo, Joseph P; Wang, Zihua; Kinney, Justin B; Vakoc, Christopher R

    2015-06-01

    CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-Cas9-induced mutations to the 5' exons of candidate genes, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR-Cas9 mutagenesis to exons encoding functional protein domains. This generates a higher proportion of null mutations and substantially increases the potency of negative selection. We also show that the magnitude of negative selection can be used to infer the functional importance of individual protein domains of interest. A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identifies six known drug targets and 19 additional dependencies. A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting.

  18. Biochemical and functional significance of F-BAR domain proteins interaction with WASP/N-WASP.

    PubMed

    Chen, Yolande; Aardema, Jorie; Corey, Seth J

    2013-04-01

    The Bin-Amphiphysin-Rvs (BAR) domain family of proteins includes groups which promote positive (classical BAR, N-BAR, and F-BAR) and negative (I-BAR) membrane deformation. Of these groups, the F-BAR subfamily is the most diverse in its biochemical properties. F-BAR domain proteins dimerize to form a tight scaffold about the membrane. The F-BAR domain provides a banana-shaped, alpha-helical structure that senses membrane curvature. Different types of F-BAR domain proteins contain tyrosine kinase or GTPase activities; some interact with phosphatases and RhoGTPases. Most possess an SH3 domain that facilitates the recruitment and activation of WASP/N-WASP. Thus, F-BAR domain proteins affect remodeling of both membrane and the actin cytoskeleton. The purpose of this review is to highlight the role of F-BAR proteins in coupling WASP/N-WASP to cytoskeletal remodeling. A role for F-BAR/WASP interaction in human diseases affecting nervous, blood, and neoplastic tissues is discussed.

  19. Effect of interdomain linker length on an antagonistic folding-unfolding equilibrium between two protein domains.

    PubMed

    Cutler, Thomas A; Mills, Brandon M; Lubin, David J; Chong, Lillian T; Loh, Stewart N

    2009-02-27

    Fusion of one protein domain with another is a common event in both evolution and protein engineering experiments. When insertion is at an internal site (e.g., a surface loop or turn), as opposed to one of the termini, conformational strain can be introduced into both domains. Strain is manifested by an antagonistic folding-unfolding equilibrium between the two domains, which we previously showed can be parameterized by a coupling free-energy term (DeltaG(X)). The extent of strain is predicted to depend primarily on the ratio of the N-to-C distance of the guest protein to the distance between ends of the surface loop in the host protein. Here, we test that hypothesis by inserting ubiquitin (Ub) into the bacterial ribonuclease barnase (Bn), using peptide linkers from zero to 10 amino acids each. DeltaG(X) values are determined by measuring the extent to which Co(2+) binding to an engineered site on the Ub domain destabilizes the Bn domain. All-atom, unforced Langevin dynamics simulations are employed to gain structural insight into the mechanism of mechanically induced unfolding. Experimental and computational results find that the two domains are structurally and energetically uncoupled when linkers are long and that DeltaG(X) increases with decreasing linker length. When the linkers are fewer than two amino acids, strain is so great that one domain unfolds the other. However, the protein is able to refold as dimers and higher-order oligomers. The likely mechanism is a three-dimensional domain swap of the Bn domain, which relieves conformational strain. The simulations suggest that an effective route to mechanical unfolding begins with disruption of the hydrophobic core of Bn near the Ub insertion site.

  20. Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains*

    PubMed Central

    Kienast, Yvonne; Jucknischke, Ute; Scheiblich, Stefan; Thier, Martina; de Wouters, Mariana; Haas, Alexander; Lehmann, Christian; Brand, Verena; Bernicke, Dirk; Honold, Konrad; Lorenz, Stefan

    2016-01-01

    By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants. PMID:26677222

  1. Network mapping among the functional domains of Chikungunya virus nonstructural proteins.

    PubMed

    Rana, Jyoti; Rajasekharan, Sreejith; Gulati, Sahil; Dudha, Namrata; Gupta, Amita; Chaudhary, Vijay Kumar; Gupta, Sanjay

    2014-10-01

    Formation of virus specific replicase complex is among the most important steps that determines the fate of viral transcription and replication during Chikungunya virus (CHIKV) infection. In the present study, the authors have computationally generated a 3D structure of CHIKV late replicase complex on the basis of the interactions identified among the domains of CHIKV nonstructural proteins (nsPs) which make up the late replicase complex. The interactions among the domains of CHIKV nsPs were identified using systems such as pull down, protein interaction ELISA, and yeast two-hybrid. The structures of nsPs were generated using I-TASSER and the biological assembly of the replicase complex was determined using ZRANK and RDOCK. A total of 36 interactions among the domains and full length proteins were tested and 12 novel interactions have been identified. These interactions included the homodimerization of nsP1 and nsP4 through their respective C-ter domains; the associations of nsP2 helicase domain and C-ter domain of nsP4 with methyltransferase and membrane binding domains of nsP1; the interaction of nsP2 protease domain with C-ter domain of nsP4; and the interaction of nsP3 macro and alphavirus unique domains with the C-ter domain of nsP1. The novel interactions identified in the current study form a network of organized associations that suggest the spatial arrangement of nsPs in the late replicase complex of CHIKV.

  2. Characterization of a cDNA encoding a 34-kDa Purkinje neuron protein recognized by sera from patients with paraneoplastic cerebellar degeneration

    SciTech Connect

    Furneaux, H.M.; Dropcho, E.J.; Barbut, D.; Chen, Yaotseng; Rosenblum, M.K.; Old, L.J.; Posner, J.B. )

    1989-04-01

    Paraneoplastic cerebellar degeneration is a neurological disorder of unknown cause occurring in patients with an identified or occult cancer. An autoimmune etiology is likely since autoantibodies directed against the Purkinje cells of the cerebellum have been found in the serum and cerebrospinal fluid of some patients. Two Purkinje cell-specific antigens are recognized by these autoantibodies, a major antigen of 62 kDa (CDR 62, cerebellar degeneration-related 62-kDa protein) and a minor antigen of 34 kDa (CDR 34). Previous studies have described the isolation and characterization of a human cerebellar cDNA that encodes an epitope recognized by sera from patients with paraneoplastic cerebellar degeneration. The authors have now established by two independent methods that this gene is uniquely expressed in Purkinje cells of the cerebellum and corresponds to the minor antigen CDR 34. This antigen is also expressed in tumor tissue from a patient with paraneoplastic cerebellar degeneration.

  3. Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family

    PubMed Central

    Haire, Robert N.; Cannon, John P.; O’Driscoll, Marci L.; Ostrov, David A.; Mueller, M. Gail; Turner, Poem M.; Litman, Ronda T.; Litman, Gary W.; Yoder, Jeffrey A.

    2012-01-01

    A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs varies between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and effect parallel patterns of ligand recognition that potentially impact species-specific advantages. PMID:22386706

  4. α/β-hydrolase domain containing protein 15 (ABHD15)--an adipogenic protein protecting from apoptosis.

    PubMed

    Walenta, Evelyn; Pessentheiner, Ariane R; Pelzmann, Helmut J; Deutsch, Alexander; Goeritzer, Madeleine; Kratky, Dagmar; Hackl, Hubert; Oh, Da Young; Prokesch, Andreas; Bogner-Strauss, Juliane G

    2013-01-01

    Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15) is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic) concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.

  5. ENH, containing PDZ and LIM domains, heart/skeletal muscle-specific protein, associates with cytoskeletal proteins through the PDZ domain.

    PubMed

    Nakagawa, N; Hoshijima, M; Oyasu, M; Saito, N; Tanizawa, K; Kuroda, S

    2000-06-07

    The Enigma homologue protein (ENH), containing an N-terminal PDZ domain and three C-terminal LIM domains, is a heart and skeletal muscle-specific protein that has been shown to preferentially interact with protein kinase C beta (PKCbeta) through the LIM domains (Kuroda et al., J. Biol. Chem. 271, 31029-31032, 1996). We here demonstrate that ENH is colocalized with a cytoskeletal protein alpha-actinin in the Z-disk region of rat neonatal cardiomyocytes. Pull-down assays using the glutathione-S-transferase-fusion system also showed the interaction of the PDZ domain of ENH with actin and alpha-actinin. Furthermore, by combined use of the in silico and conventional cDNA cloning methods, we have isolated three ENH-related clones from a mouse heart-derived cDNA library: mENH1 (591 amino acid residues) corresponding to rat ENH, mENH2 (337 residues), and mENH3 (239 residues); the latter two containing only a single PDZ domain. Deciphering their cDNA sequences, these mENH1-3 mRNAs appear to be generated from a single mENH gene by alternative splicing. Northern blot analyses using human cancer cells and mouse embryos have shown expression of each mENH mRNA to vary considerably among the cell types and during the developmental stage. Together with a recent finding that PKCbeta is markedly activated in the cardiac hypertrophic signaling, these results suggest that ENH1 plays an important role in the heart development by scaffolding PKCbeta to the Z-disk region and that ENH2 and ENH3 negatively modulate the scaffolding activity of ENH1.

  6. [First open reading frame protein (ORF1p) of the Blattella germanica R1 retroposon and phylogenetically close GAG-like proteins of insects and fungi contain RRM domains].

    PubMed

    Kapelinskaia, T V; Kagramanova, A S; Korolev, A L; Mukha, D V

    2011-02-01

    The rDNA locus of insects and other arthropods contains non-LTR retrotransposons (retroposons) that are specifically inserted into 28S rRNA genes. The most frequent retroposons are R1 and R2, but the mechanism of insertion and the functions of these mobile elements have not been studied in detail. A clone containing a full-length R1 retroposon copy was islated from the cosmid library of Blattella germanica genes and sequenced. The amino acid sequences encoded by ORF1 of the R1 retroposon were subjected to bioinformatic analysis. It was found that ORF1 of this mobile element encodes a protein (ORF1p) belonging to the superfamily of zinc finger (CCHC) retroviral nucleocapsid proteins and contains two conserved RRM domains (RNA-recognizing motifs) identified on the basis of analysis of the secondary structure of this protein. The discovery of RRM domains in ORF1p of R1 retroposons can contribute to the understanding of the mechanisms of their retrotransposition. We revealed a coiled-coil motif in the N-terminal region of R1 ORF1p, which is similar to the coiled-coil domain involved in homo- or heteromultimerization of proteins and in protein-protein interactions. The domain organization of homologous Gag-like proteins of retroposons in some insects and fungi was found to be similar to the structure established by us for R1 ORF1p of B. germanica.

  7. Purification and Structural Analysis of SUN and KASH Domain Proteins.

    PubMed

    Esra Demircioglu, F; Cruz, Victor E; Schwartz, Thomas U

    2016-01-01

    Molecular tethers span the nuclear envelope to mechanically connect the cytoskeleton and nucleoskeleton. These bridge-like tethers, termed linkers of nucleoskeleton and cytoskeleton (LINC) complexes, consist of SUN proteins at the inner nuclear membrane and KASH proteins at the outer nuclear membrane. LINC complexes are central to a variety of cell activities including nuclear positioning and mechanotransduction, and LINC-related abnormalities are associated with a spectrum of tissue-specific diseases, termed laminopathies or envelopathies. Protocols used to study the biochemical and structural characteristics of core elements of SUN-KASH complexes are described here to facilitate further studies in this new field of cell biology.

  8. Roots of angiosperm formins: The evolutionary history of plant FH2 domain-containing proteins

    PubMed Central

    2008-01-01

    Background Shuffling of modular protein domains is an important source of evolutionary innovation. Formins are a family of actin-organizing proteins that share a conserved FH2 domain but their overall domain architecture differs dramatically between opisthokonts (metazoans and fungi) and plants. We performed a phylogenomic analysis of formins in most eukaryotic kingdoms, aiming to reconstruct an evolutionary scenario that may have produced the current diversity of domain combinations with focus on the origin of the angiosperm formin architectures. Results The Rho GTPase-binding domain (GBD/FH3) reported from opisthokont and Dictyostelium formins was found in all lineages except plants, suggesting its ancestral character. Instead, mosses and vascular plants possess the two formin classes known from angiosperms: membrane-anchored Class I formins and Class II formins carrying a PTEN-like domain. PTEN-related domains were found also in stramenopile formins, where they have been probably acquired independently rather than by horizontal transfer, following a burst of domain rearrangements in the chromalveolate lineage. A novel RhoGAP-related domain was identified in some algal, moss and lycophyte (but not angiosperm) formins that define a specific branch (Class III) of the formin family. Conclusion We propose a scenario where formins underwent multiple domain rearrangements in several eukaryotic lineages, especially plants and chromalveolates. In plants this replaced GBD/FH3 by a probably inactive RhoGAP-like domain, preserving a formin-mediated association between (membrane-anchored) Rho GTPases and the actin cytoskeleton. Subsequent amplification of formin genes, possibly coincident with the expansion of plants to dry land, was followed by acquisition of alternative membrane attachment mechanisms present in extant Class I and Class II formins, allowing later loss of the RhoGAP-like domain-containing formins in angiosperms. PMID:18430232

  9. Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.

    PubMed

    Phadungsil, Wansika; Smooker, Peter M; Vichasri-Grams, Suksiri; Grams, Rudi

    2016-01-01

    Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects.

  10. Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding proteome evolution is important for deciphering processes that drive species diversity and adaptation. Herein, the dynamics of change in protein families and protein domains over the course of metazoan evolution was explored. Change, as defined by birth/death and duplication/deletion ...

  11. Insights into the evolution and domain structure of ataxin-2 proteins across eukaryotes

    PubMed Central

    2014-01-01

    Background Ataxin-2 is an evolutionarily conserved protein first identified in humans as responsible for spinocerebellar ataxia type 2 (SCA2). The molecular basis of SCA2 is the expansion of a polyglutamine tract in Ataxin-2, encoding a Lsm domain that may bind RNA and a PAM2 motif that enables interaction with the poly (A) binding protein. Although the association with SCA2 has been verified, a detailed molecular function for Ataxin-2 has not been established. Results We have undertaken a survey of Ataxin-2 proteins across all eukaryotic domains. In eukaryotes, except for vertebrates and land plants, a single ortholog was identified. Notably, with the exception of birds, two Ataxin-2 genes exist in vertebrates. Expansion was observed in land plants and a novel class lacking the LsmAD domain was identified. Large polyQ tracts appear limited to primates and insects of the orders Hymenoptera and Diptera. A common feature across Ataxin-2 orthologs is the presence of proline-rich motifs, formerly described in the human protein. Conclusion Our analysis provides valuable information on the evolution and domain structure of Ataxin-2 proteins. Proline-rich motifs that may mediate protein interactions are widespread in Ataxin-2 proteins, but expansion of polyglutamine tracts associated with spinocerebellar ataxia type 2, is present only in primates, as well as some insects. Our analysis of Ataxin-2 proteins provides also a source to examine orthologs in a number of different species. PMID:25027299

  12. Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains.

    PubMed

    Rahaman, Obaidur; Kalimeri, Maria; Melchionna, Simone; Hénin, Jérôme; Sterpone, Fabio

    2015-07-23

    In this work, we address the question of whether the enhanced stability of thermophilic proteins has a direct connection with internal hydration. Our model systems are two homologous G domains of different stability: the mesophilic G domain of the elongation factor thermal unstable protein from E. coli and the hyperthermophilic G domain of the EF-1α protein from S. solfataricus. Using molecular dynamics simulation at the microsecond time scale, we show that both proteins host water molecules in internal cavities and that these molecules exchange with the external solution in the nanosecond time scale. The hydration free energy of these sites evaluated via extensive calculations is found to be favorable for both systems, with the hyperthermophilic protein offering a slightly more favorable environment to host water molecules. We estimate that, under ambient conditions, the free energy gain due to internal hydration is about 1.3 kcal/mol in favor of the hyperthermophilic variant. However, we also find that, at the high working temperature of the hyperthermophile, the cavities are rather dehydrated, meaning that under extreme conditions other molecular factors secure the stability of the protein. Interestingly, we detect a clear correlation between the hydration of internal cavities and the protein conformational landscape. The emerging picture is that internal hydration is an effective observable to probe the conformational landscape of proteins. In the specific context of our investigation, the analysis confirms that the hyperthermophilic G domain is characterized by multiple states and it has a more flexible structure than its mesophilic homologue.

  13. A domain-centric analysis of oomycete plant pathogen genomes reveals unique protein organization.

    PubMed

    Seidl, Michael F; Van den Ackerveken, Guido; Govers, Francine; Snel, Berend

    2011-02-01

    Oomycetes comprise a diverse group of organisms that morphologically resemble fungi but belong to the stramenopile lineage within the supergroup of chromalveolates. Recent studies have shown that plant pathogenic oomycetes have expanded gene families that are possibly linked to their pathogenic lifestyle. We analyzed the protein domain organization of 67 eukaryotic species including four oomycete and five fungal plant pathogens. We detected 246 expanded domains in fungal and oomycete plant pathogens. The analysis of genes differentially expressed during infection revealed a significant enrichment of genes encoding expanded domains as well as signal peptides linking a substantial part of these genes to pathogenicity. Overrepresentation and clustering of domain abundance profiles revealed domains that might have important roles in host-pathogen interactions but, as yet, have not been linked to pathogenicity. The number of distinct domain combinations (bigrams) in oomycetes was significantly higher than in fungi. We identified 773 oomycete-specific bigrams, with the majority composed of domains common to eukaryotes. The analyses enabled us to link domain content to biological processes such as host-pathogen interaction, nutrient uptake, or suppression and elicitation of plant immune responses. Taken together, this study represents a comprehensive overview of the domain repertoire of fungal and oomycete plant pathogens and points to novel features like domain expansion and species-specific bigram types that could, at least partially, explain why oomycetes are such remarkable plant pathogens.

  14. In vitro antitumor activity of Latcripin-15 regulator of chromosome condensation 1 domain protein

    PubMed Central

    Tian, Li; Wang, Xiaoli; Li, Xingyun; Liu, Ben; Zhang, Wei; Cao, Jing; Ning, Anhong; Huang, Min; Zhong, Mintao

    2016-01-01

    Cancer is one of the most significant health problems worldwide and thus the development of novel therapeutic agents with fewer side effects is required. The present study investigated the in vitro anticancer effects of a newly isolated fungal protein. In this study, Latcripin-15 (LP-15) regulator of chromosome condensation 1 (RCC1) domain protein, which is obtained from the Lentinula edodes C91-3 fungal strain, was identified, cloned, expressed, purified and re-folded to assess the in vitro antitumor activity of the protein. LP-15 RCC1 full-length cDNA was isolated from Lentinula edodes using 3′ and 5′-rapid amplification of cDNA ends and then cloned, expressed, purified and re-folded in vitro. In addition, the effects of the isolated LP-15 RCC1 protein's functional domain on the viability and apoptosis of human lung cancer A549 cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, transmission electron microscopy, flow cytometry and Hoechst 33258 staining. The LP-15 RCC1 functional domain protein was successfully expressed, purified and re-folded in vitro. Treatment with the LP-15 RCC1 functional domain protein significantly reduced tumor cell viability and induced apoptosis in A549 cells. The results of the present study indicate that the LP-15 RCC1 functional domain requires further investigation as a novel therapeutic agent for cancer therapy. PMID:27899975

  15. Crystal structure of the homology domain of the eukaryotic DNA replication proteins Sld3/Treslin.

    PubMed

    Itou, Hiroshi; Muramatsu, Sachiko; Shirakihara, Yasuo; Araki, Hiroyuki

    2014-09-02

    The initiation of eukaryotic chromosomal DNA replication requires the formation of an active replicative helicase at the replication origins of chromosomal DNA. Yeast Sld3 and its metazoan counterpart Treslin are the hub proteins mediating protein associations critical for the helicase formation. Here, we show the crystal structure of the central domain of Sld3 that is conserved in Sld3/Treslin family of proteins. The domain consists of two segments with 12 helices and is sufficient to bind to Cdc45, the essential helicase component. The structure model of the Sld3-Cdc45 complex, which is crucial for the formation of the active helicase, is proposed.

  16. Engineered staphylococcal protein A's IgG-binding domain with cathepsin L inhibitory activity

    SciTech Connect

    Bratkovic, Tomaz . E-mail: tomaz.bratkovic@ffa.uni-lj.si; Berlec, Ales; Popovic, Tatjana; Lunder, Mojca; Kreft, Samo; Urleb, Uros; Strukelj, Borut

    2006-10-13

    Inhibitory peptide of papain-like cysteine proteases, affinity selected from a random disulfide constrained phage-displayed peptide library, was grafted to staphylococcal protein A's B domain. Scaffold protein was additionally modified in order to allow solvent exposed display of peptide loop. Correct folding of fusion proteins was confirmed by CD-spectroscopy and by the ability to bind the Fc-region of rabbit IgG, a characteristic of parent domain. The recombinant constructs inhibited cathepsin L with inhibitory constants in the low-micromolar range.

  17. Bacteriophage SP6 encodes a second tailspike protein that recognizes Salmonella enterica serogroups C2 and C3.

    PubMed

    Gebhart, Dana; Williams, Steven R; Scholl, Dean

    2017-03-10

    SP6 is a salmonella phage closely related to coliphage K1-5. K1-5 is notable in that it encodes two polysaccharide-degrading tailspike proteins, an endosialidase that allows it to infect E. coli K1, and a lyase that enables it to infect K5 strains. SP6 is similar to K1-5 except that it encodes a P22-like endorhamnosidase tailspike, gp46, allowing it to infect group B Salmonella. We show here that SP6 can also infect Salmonella serogroups C2 and C3 and that a mutation in a putative second tailspike, gp47, eliminates this specificity. Gene 47 was fused to the coding region of the N-terminal portion of the Pseudomonas aeruginosa R2 pyocin tail fiber and expressed in trans such that the fusion protein becomes incorporated into pyocin particles. These pyocins, termed AvR2-SP47, killed serogroups C2 and C3Salmonella. We conclude that SP6 encodes two tail proteins providing it a broad host range among Salmonella enterica.

  18. Molecular characterization of NDP52, a novel protein of the nuclear domain 10, which is redistributed upon virus infection and interferon treatment

    PubMed Central

    1995-01-01

    The nuclear domain (ND)10 also described as POD or Kr bodies is involved in the development of acute promyelocytic leukemia and virus- host interactions. Immunofluorescence analysis using a variety of human autoimmune sera and monoclonal antibodies showed a typical dot like nuclear staining for ND10, suggesting that this structure consists of several proteins. Two of the ND10 proteins, Sp100 and PML are genetically characterized and show homology with several transcription factors. Here we describe NDP52, an additional novel protein of the ND10. We raised a new mAb C8A2, that specifically recognizes NDP52. Immunofluorescence analysis using this mAb showed a typical nuclear dot staining as it was described for ND10. Isolation and sequencing of the corresponding cDNA revealed that NDP52 has a predicted molecular mass of 52 kD. The deduced amino acid sequence exhibits an extended central coiled coil domain containing a leucine zipper motif. The COOH terminus of NDP52 shows homology with LIM domains, that have recently been described to mediate protein interactions, which let NDP52 appear as a suitable candidate for mediating interactions between ND10 proteins. In vivo, NDP52 is transcribed in all human tissues analyzed. Furthermore, we show that NDP52 colocalizes with the ND10 protein PML and can be redistributed upon viral infection and interferon treatment. These data suggest that ND10 proteins play an important role in the viral life cycle. PMID:7540613

  19. Efficient segmental isotope labeling of multi-domain proteins using Sortase A.

    PubMed

    Freiburger, Lee; Sonntag, Miriam; Hennig, Janosch; Li, Jian; Zou, Peijian; Sattler, Michael

    2015-09-01

    NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to existing protocols are (1) the efficient removal of cleaved peptide fragments by centrifugal filtration and (2) a strategic design of cleavable and non-cleavable affinity tags for purification. Our approach enables routine production of milligram amounts of purified segmentally labeled protein for NMR and other biophysical studies.

  20. Resilience of biochemical activity in protein domains in the face of structural divergence.

    PubMed

    Zhang, Dapeng; Iyer, Lakshminarayan M; Burroughs, A Maxwell; Aravind, L

    2014-06-01

    Recent studies point to the prevalence of the evolutionary phenomenon of drastic structural transformation of protein domains while continuing to preserve their basic biochemical function. These transformations span a wide spectrum, including simple domains incorporated into larger structural scaffolds, changes in the structural core, major active site shifts, topological rewiring and extensive structural transmogrifications. Proteins from biological conflict systems, such as toxin-antitoxin, restriction-modification, CRISPR/Cas, polymorphic toxin and secondary metabolism systems commonly display such transformations. These include endoDNases, metal-independent RNases, deaminases, ADP ribosyltransferases, immunity proteins, kinases and E1-like enzymes. In eukaryotes such transformations are seen in domains involved in chromatin-related peptide recognition and protein/DNA-modification. Intense selective pressures from 'arms-race'-like situations in conflict and macromolecular modification systems could favor drastic structural divergence while preserving function.

  1. Review the role of terminal domains during storage and assembly of spider silk proteins.

    PubMed

    Eisoldt, Lukas; Thamm, Christopher; Scheibel, Thomas

    2012-06-01

    Fibrous proteins in nature fulfill a wide variety of functions in different structures ranging from cellular scaffolds to very resilient structures like tendons and even extra-corporal fibers such as silks in spider webs or silkworm cocoons. Despite their different origins and sequence varieties many of these fibrous proteins share a common building principle: they consist of a large repetitive core domain flanked by relatively small non-repetitive terminal domains. Amongst protein fibers, spider dragline silk shows prominent mechanical properties that exceed those of man-made fibers like Kevlar. Spider silk fibers assemble in a spinning process allowing the transformation from an aqueous solution into a solid fiber within milliseconds. Here, we highlight the role of the non-repetitive terminal domains of spider dragline silk proteins during storage in the gland and initiation of the fiber assembly process.

  2. A Phytophthora sojae Glycoside Hydrolase 12 Protein Is a Major Virulence Factor during Soybean Infection and Is Recognized as a PAMP[OPEN

    PubMed Central

    Ma, Zhenchuan; Song, Tianqiao; Zhu, Lin; Ye, Wenwu; Wang, Yang; Shao, Yuanyuan; Dong, Suomeng; Zhang, Zhengguang; Dou, Daolong; Zheng, Xiaobo; Tyler, Brett M.; Wang, Yuanchao

    2015-01-01

    We identified a glycoside hydrolase family 12 (GH12) protein, XEG1, produced by the soybean pathogen Phytophthora sojae that exhibits xyloglucanase and β-glucanase activity. It acts as an important virulence factor during P. sojae infection but also acts as a pathogen-associated molecular pattern (PAMP) in soybean (Glycine max) and solanaceous species, where it can trigger defense responses including cell death. GH12 proteins occur widely across microbial taxa, and many of these GH12 proteins induce cell death in Nicotiana benthamiana. The PAMP activity of XEG1 is independent of its xyloglucanase activity. XEG1 can induce plant defense responses in a BAK1-dependent manner. The perception of XEG1 occurs independently of the perception of ethylene-inducing xylanase. XEG1 is strongly induced in P. sojae within 30 min of infection of soybean and then slowly declines. Both silencing and overexpression of XEG1 in P. sojae severely reduced virulence. Many P. sojae RXLR effectors could suppress defense responses induced by XEG1, including several that are expressed within 30 min of infection. Therefore, our data suggest that PsXEG1 contributes to P. sojae virulence, but soybean recognizes PsXEG1 to induce immune responses, which in turn can be suppressed by RXLR effectors. XEG1 thus represents an apoplastic effector that is recognized via the plant’s PAMP recognition machinery. PMID:26163574

  3. Different domains of Phytophthora sojae effector Avr4/6 are recognized by soybean resistance genes Rps4 and Rps6.

    PubMed

    Dou, Daolong; Kale, Shiv D; Liu, Tingli; Tang, Qinghua; Wang, Xia; Arredondo, Felipe D; Basnayake, Shiromi; Whisson, Stephen; Drenth, Andre; Maclean, Don; Tyler, Brett M

    2010-04-01

    At least 12 avirulence genes have been genetically identified and mapped in Phytophthora sojae, an oomycete pathogen causing root and stem rot of soybean. Previously, the Avr4 and Avr6 genes of P. sojae were genetically mapped within a 24 kb interval of the genome. Here, we identify Avr4 and Avr6 and show that they are actually a single gene, Avr4/6, located near the 24-kb region. Avr4/6 encodes a secreted protein of 123 amino acids with an RXLR-dEER protein translocation motif. Transient expression of Avr4/6 in soybean leaves revealed that its gene product could trigger a hypersensitive response (HR) in the presence of either Rps4 or Rps6. Silencing Avr4/6 in P. sojae stable transformants abolished the avirulence phenotype exhibited on both Rps4 and Rps6 soybean cultivars. The N terminus of Avr4/6, including the dEER motif, is sufficient to trigger Rps4-dependent HR while its C terminus is sufficient to trigger Rps6-mediated HR. Compared with alleles from avirulent races, alleles of Avr4/6 from virulent races possess nucleotide substitutions in the 5' untranslated region of the gene but not in the protein-coding region.

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

    PubMed Central

    Radakovics, Katharina; Smith, Terry K.; Bobik, Nina; Round, Adam; Djinović-Carugo, Kristina; Usón, Isabel

    2016-01-01

    Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1–83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1–83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1–240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88. PMID:27973613

  5. Alternative splicing for members of human mosaic domain superfamilies. I. The CH and LIM domains containing group of proteins.

    PubMed

    Friedberg, Felix

    2009-05-01

    In this paper we examine (restricted to homo sapiens) the products resulting from gene duplication and the subsequent alternative splicing for the members of a multidomain group of proteins which possess the evolutionary conserved calponin homology CH domain, i.e. an "actin binding domain", as a singlet and which, in addition, contain the conserved cysteine rich double Zn finger possessing Lim domain, also as a singlet. Seven genes, resulting from gene duplications, were identified that code for seven group members for which pre-mRNAs appear to have undergone multiple alternative splicing: Mical 1, 2 and 3 are located on chromosomes 6q21, 11p15 and 22q11, respectively. The LMO7 gene is present on chromosome 13q22 and the LIMCH1 gene on chromosome 4p13. Micall1 is mapped to chromosome 22q13 and Micall2 to chromosome 7p22. Translated Gen/Bank ESTs suggest the existence of multiple products alternatively spliced from the pre-mRNAs encoded by these genes. Characteristic indicators of such splicing among the proteins derived from one gene must include containment of some common extensive 100% identical regions. In some instances only one exon might be partly or completely eliminated. Sometimes alternative splicing is also associated with an increased frequency of creation of an exon or part of an exon from an intron. Not only coding regions for the body of the protein but also for its N- or -C ends could be affected by the splicing. If created forms are merely beginning at different starting points but remain identical in sequence thereafter, their existence as products of alternate splicing must be questioned. In the splicings, described in this paper, multiple isoforms rather than a single isoform appear as products during the gene expression.

  6. Flexible DNA binding of the BTB/POZ-domain protein FBI-1.

    PubMed

    Pessler, Frank; Hernandez, Nouria

    2003-08-01

    POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus. Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. FBI-1 is a BTB/POZ-domain protein that has been shown to modulate HIV-1 Tat trans-activation and to repress transcription of some cellular genes. We have used various viral and cellular FBI-1 binding sites to characterize the interaction of a POZ-domain protein with DNA in detail. We find that FBI-1 binds to inverted sequence repeats downstream of the HIV-1 transcription start site. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible. Indeed, FBI-1 binding sites in the adenovirus 2 major late promoter, the c-fos gene, and the c-myc P1 and P2 promoters reveal variously spaced direct, inverted, and everted sequence repeats with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T) for each repeat.

  7. Activity of a Two-Domain Antifreeze Protein Is Not Dependent on Linker Sequence

    PubMed Central

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

    2007-01-01

    The reported NMR structure of RD3, a naturally occurring two-domain antifreeze protein, suggests that the two nearly identical domains are oriented to allow simultaneous binding of their active regions to the ice surface. It is implied that the nine residues linking the two domains play a role in this alignment, but this has not been established. We have designed and expressed a modified form of RD3 that replaces the nine-residue linker with a generic sequence of one serine and eight glycine residues to test the importance of the linker amino acid sequence. The modified linker is shown to have significantly different characteristics compared to the original linker. Heteronuclear nuclear Overhauser effect experiments show that the new linker residues have more mobility than the linker residues in the native protein. Further, NMR data show that the folding of the C-terminal domain is somewhat perturbed by the altered linker. Finally, distributions of residual dipolar couplings indicate that the two domains tumble and move independently of each other. Nevertheless, the thermal hysteresis activity of the modified protein is indistinguishable from that of native RD3, proving that increased activity of the two-domain antifreeze protein is not dependent on structure of the linker. PMID:17056724

  8. Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton

    PubMed Central

    1992-01-01

    Interaction with extracellular matrix can trigger a variety of responses by cells including changes in specific gene expression and cell differentiation. The mechanism by which cell surface events are coupled to the transcriptional machinery is not understood, however, proteins localized at sites of cell-substratum contact are likely to function as signal transducers. We have recently purified and characterized a low abundance adhesion plaque protein called zyxin (Crawford, A. W., and M. C. Beckerle. 1991. J. Biol. Chem. 266:5847- 5853; Crawford, A. W., J. W. Michelsen, and M. C. Beckerle. 1992. J. Cell Biol. 116:1381-1393). We have now isolated and sequenced zyxin cDNA and we report here that zyxin exhibits an unusual proline-rich NH2- terminus followed by three tandemly arrayed LIM domains. LIM domains have previously been identified in proteins that play important roles in transcriptional regulation and cellular differentiation. LIM domains have been proposed to coordinate metal ions and we have demonstrated by atomic absorption spectroscopy that purified zyxin binds zinc, a result consistent with the idea that zyxin has zinc fingers. In addition, we have discovered that zyxin interacts in vitro with a 23-kD protein that also exhibits LIM domains. Microsequence analysis has revealed that the 23-kD protein (or cCRP) is the chicken homologue of the human cysteine- rich protein (hCRP). By double-label indirect immunofluorescence, we found that zyxin and cCRP are extensively colocalized in chicken embryo fibroblasts, consistent with the idea that they interact in vivo. We conclude that LIM domains are zinc-binding sequences that may be involved in protein-protein interactions. The demonstration that two cytoskeletal proteins, zyxin and cCRP, share a sequence motif with proteins important for transcriptional regulation raises the possibility that zyxin and cCRP are components of a signal transduction pathway that mediates adhesion-stimulated changes in gene

  9. Metazoans evolved by taking domains from soluble proteins to expand intercellular communication network

    PubMed Central

    Nam, Hyun-Jun; Kim, Inhae; Bowie, James U.; Kim, Sanguk

    2015-01-01

    A central question in animal evolution is how multicellular animals evolved from unicellular ancestors. We hypothesize that membrane proteins must be key players in the development of multicellularity because they are well positioned to form the cell-cell contacts and to provide the intercellular communication required for the creation of complex organisms. Here we find that a major mechanism for the necessary increase in membrane protein complexity in the transition from non-metazoan to metazoan life was the new incorporation of domains from soluble proteins. The membrane proteins that have incorporated soluble domains in metazoans are enriched in many of the functions unique to multicellular organisms such as cell-cell adhesion, signaling, immune defense and developmental processes. They also show enhanced protein-protein interaction (PPI) network complexity and centrality, suggesting an important role in the cellular diversification found in complex organisms. Our results expose an evolutionary mechanism that contributed to the development of higher life forms. PMID:25923201

  10. Metazoans evolved by taking domains from soluble proteins to expand intercellular communication network.

    PubMed

    Nam, Hyun-Jun; Kim, Inhae; Bowie, James U; Kim, Sanguk

    2015-04-29

    A central question in animal evolution is how multicellular animals evolved from unicellular ancestors. We hypothesize that membrane proteins must be key players in the development of multicellularity because they are well positioned to form the cell-cell contacts and to provide the intercellular communication required for the creation of complex organisms. Here we find that a major mechanism for the necessary increase in membrane protein complexity in the transition from non-metazoan to metazoan life was the new incorporation of domains from soluble proteins. The membrane proteins that have incorporated soluble domains in metazoans are enriched in many of the functions unique to multicellular organisms such as cell-cell adhesion, signaling, immune defense and developmental processes. They also show enhanced protein-protein interaction (PPI) network complexity and centrality, suggesting an important role in the cellular diversification found in complex organisms. Our results expose an evolutionary mechanism that contributed to the development of higher life forms.

  11. Selection on Network Dynamics Drives Differential Rates of Protein Domain Evolution

    PubMed Central

    Mannakee, Brian K.; Gutenkunst, Ryan N.

    2016-01-01

    The long-held principle that functionally important proteins evolve slowly has recently been challenged by studies in mice and yeast showing that the severity of a protein knockout only weakly predicts that protein’s rate of evolution. However, the relevance of these studies to evolutionary changes within proteins is unknown, because amino acid substitutions, unlike knockouts, often only slightly perturb protein activity. To quantify the phenotypic effect of small biochemical perturbations, we developed an approach to use computational systems biology models to measure the influence of individual reaction rate constants on network dynamics. We show that this dynamical influence is predictive of protein domain evolutionary rate within networks in vertebrates and yeast, even after controlling for expression level and breadth, network topology, and knockout effect. Thus, our results not only demonstrate the importance of protein domain function in determining evolutionary rate, but also the power of systems biology modeling to uncover unanticipated evolutionary forces. PMID:27380265

  12. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  13. PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2014-06-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.

  14. Characterization of antigens from nontypable Haemophilus influenzae recognized by human bactericidal antibodies. Role of Haemophilus outer membrane proteins.

    PubMed Central

    Gnehm, H E; Pelton, S I; Gulati, S; Rice, P A

    1985-01-01

    Major outer membrane antigens, proteins, and lipopolysaccharides (LPSs), from nontypable Haemophilus influenzae were characterized and examined as targets for complement-dependent human bactericidal antibodies. Outer membranes from two nontypable H. influenzae isolates that caused otitis media and pneumonia (middle ear and transtracheal aspirates) were prepared by shearing organisms in EDTA. These membranes were compared with membranes prepared independently by spheroplasting and lysozyme treatment of whole cells and found to have: similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the proteins; identical densities (rho = 1.22 g/cm3); and minimal d-lactose dehydrogenase activity indicating purity from cytoplasmic membranes. Outer membranes were solubilized in an LPS-disaggregating buffer and proteins were separated from LPS by molecular sieve chromatography. The SDS-PAGE patterns of outer membrane proteins (OMPs) from the two strains differed in the major band although other prominent bands appeared similar in molecular weight. LPS prepared by hot phenol water extraction of each of the strains contained 45% (pneumonia isolate) and 60% (otitis isolate) lipid (wt/wt), 49% and 50% carbohydrate (wt/wt), respectively, and less than 1%, 3-deoxy-manno octulosonic acid. Immunoglobulin M (IgM) purified from normal human serum (NHS) plus complement was bactericidal for both strains. Purified immunoglobulin G (IgG) from NHS killed the middle ear isolate and immune convalescent IgM from the serum of the patient with pneumonia killed his isolate. NHS or convalescent serum were absorbed with OMPs and LPS (0.6-110 micrograms) from each of the strains and immune specific inhibition of bactericidal antibody activity by each antigen was determined. OMPs from the pulmonary isolate inhibited bactericidal antibody activity directed against the isolate in both NHS (1.5 microgram of antigen) and immune serum (0.75 microgram of antigen). OMPs (60

  15. Signal Activation and Inactivation by the Gα Helical Domain: A Long-Neglected Partner in G Protein Signaling

    PubMed Central

    Dohlman, Henrik G.; Jones, Janice C.

    2013-01-01

    Heterotrimeric guanine nucleotide–binding proteins (G proteins) are positioned at the top of many signal transduction pathways. The G protein α subunit is composed of two domains, one that resembles Ras and another that is composed entirely of α helices. Historically, most attention has focused on the Ras-like domain, but emerging evidence reveals that the helical domain is an active participant in G protein signaling. PMID:22649098

  16. Identification of a novel contactin-associated transmembrane receptor with multiple domains implicated in protein-protein interactions.

    PubMed Central

    Peles, E; Nativ, M; Lustig, M; Grumet, M; Schilling, J; Martinez, R; Plowman, G D; Schlessinger, J

    1997-01-01

    Receptor protein tyrosine phosphatase beta (RPTPbeta) expressed on the surface of glial cells binds to the glycosylphosphatidylinositol (GPI)-anchored recognition molecule contactin on neuronal cells leading to neurite outgrowth. We describe the cloning of a novel contactin-associated transmembrane receptor (p190/Caspr) containing a mosaic of domains implicated in protein-protein interactions. The extracellular domain of Caspr contains a neurophilin/coagulation factor homology domain, a region related to fibrinogen beta/gamma, epidermal growth factor-like repeats, neurexin motifs as well as unique PGY repeats found in a molluscan adhesive protein. The cytoplasmic domain of Caspr contains a proline-rich sequence capable of binding to a subclass of SH3 domains of signaling molecules. Caspr and contactin exist as a complex in rat brain and are bound to each other by means of lateral (cis) interactions in the plasma membrane. We propose that Caspr may function as a signaling component of contactin, enabling recruitment and activation of intracellular signaling pathways in neurons. The binding of RPTPbeta to the contactin-Caspr complex could provide a mechanism for cell-cell communication between glial cells and neurons during development. PMID:9118959

  17. Spiroplasma eriocheiris Adhesin-Like Protein (ALP) Interacts with Epidermal Growth Factor (EGF) Domain Proteins to Facilitate Infection

    PubMed Central

    Hou, Libo; Liu, Yuhan; Gao, Qi; Xu, Xuechuan; Ning, Mingxiao; Bi, Jingxiu; Liu, Hui; Liu, Min; Gu, Wei; Wang, Wen; Meng, Qingguo

    2017-01-01

    Spiroplasma eriocheiris is a novel pathogen found in recent years, causing the tremor disease (TD) of Chinese mitten crab Eriocheir sinensis. Like Spiroplasma mirum, S. eriocheiris infects the newborn mouse (adult mice are not infected) and can cause cataract. Adhesion-related protein is an important protein involved in the interaction between pathogen and host. In this study, the Adhesin-like Protein (ALP) of S. eriocheiris was detected on its outer membrane by using immune electron microscopy, and was found to be involved in the bacterium's infection of mouse embryo fibroblasts (3T6-Swiss albino). Yeast two-hybrid analysis demonstrated that ALP interacts with a diverse group of mouse proteins. The interactions between recombinant partial fibulin7 (FBLN7; including two epidermal growth factor [EGF] domains) and ALP were confirmed by Far-western blotting and colocalization. We synthetized the domains of FBLN7 [EGF domain: amino acids 136–172 and complement control protein (CCP) domain: 81–134 amino acids], and demonstrated that only EGF domain of FBLN7 can interact with ALP. Because the EGF domain has high degree of similarity to EGF, it can activate the downstream EGFR signaling pathway, in key site amino acids. The EGFR pathway in 3T6 cells was restrained after rALP stimulation resulting from competitive binding of ALP to EGF. The unborn mouse, newborn mouse, and the adult mouse with cataract have a small amount of expressed FBLN7; however, none was detected in the brain and very little expression was seen in the eye of normal adult mice. In short, ALP as a S. eriocheiris surface protein, is critical for infection and further supports the role of ALP in S. eriocheiris infection by competitive effection of the EGF/EGFR axis of the target cells. PMID:28184355

  18. Single-stranded DNA Binding by the Helix-Hairpin-Helix Domain of XPF Protein Contributes to the Substrate Specificity of the ERCC1-XPF Protein Complex.

    PubMed

    Das, Devashish; Faridounnia, Maryam; Kovacic, Lidija; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2017-02-17

    The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble.

  19. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

    PubMed Central

    Pruitt, Rory N.; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R.; Chan, Leanne Jade G.; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L.; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B.; Sonti, Ramesh V.; Petzold, Christopher J.; Liu, Chang C.; Brodbelt, Jennifer S.; Felix, Georg; Ronald, Pamela C.

    2015-01-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals. PMID:26601222

  20. Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins

    PubMed Central

    Cogoi, Susanna; Paramasivam, Manikandan; Spolaore, Barbara; Xodo, Luigi E.

    2008-01-01

    The human KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) upstream of the major transcription initiation site. In this article, we demonstrate by primer-extension experiments, PAGE, chemical footprinting, CD, UV and FRET experiments that the G-rich strand of NHE (32R) folds into intra-molecular G-quadruplex structures. Fluorescence data show that 32R in 100 mM KCl melts with a biphasic profile, showing the formation of two distinct G-quadruplexes with Tm of ∼55°C (Q1) and ∼72°C (Q2). DMS-footprinting and CD suggest that Q1 can be a parallel and Q2 a mixed parallel/antiparallel G-quadruplex. When dsNHE (32R hybridized to its complementary) is incubated with a nuclear extract from Panc-1 cells, three DNA–protein complexes are observed by EMSA. The complex of slower mobility is competed by quadruplex 32R, but not by mutant oligonucleotides, which cannot form a quadruplex structure. Using paramagnetic beads coupled with 32R, we pulled down from the Panc-1 extract proteins with affinity for quadruplex 32R. One of these is the heterogeneous nuclear ribonucleoprotein A1, which was previously reported to unfold quadruplex DNA. Our study suggests a role of quadruplex DNA in KRAS transcription and provides the basis for the rationale design of molecular strategies to inhibit the expression of KRAS. PMID:18490377

  1. The emerging importance of the SPX domain-containing proteins in phosphate homeostasis.

    PubMed

    Secco, David; Wang, Chuang; Arpat, Bulak A; Wang, Zhiye; Poirier, Yves; Tyerman, Stephen D; Wu, Ping; Shou, Huixia; Whelan, James

    2012-03-01

    Plant growth and development are strongly influenced by the availability of nutrients in the soil solution. Among them, phosphorus (P) is one of the most essential and most limiting macro-elements for plants. In the environment, plants are often confronted with P starvation as a result of extremely low concentrations of soluble inorganic phosphate (Pi) in the soil. To cope with these conditions, plants have developed a wide spectrum of mechanisms aimed at increasing P use efficiency. At the molecular level, recent studies have shown that several proteins carrying the SPX domain are essential for maintaining Pi homeostasis in plants. The SPX domain is found in numerous eukaryotic proteins, including several proteins from the yeast PHO regulon, involved in maintaining Pi homeostasis. In plants, proteins harboring the SPX domain are classified into four families based on the presence of additional domains in their structure, namely the SPX, SPX-EXS, SPX-MFS and SPX-RING families. In this review, we highlight the recent findings regarding the key roles of the proteins containing the SPX domain in phosphate signaling, as well as providing further research directions in order to improve our knowledge on P nutrition in plants, thus enabling the generation of plants with better P use efficiency.

  2. Duplex (or quadruplet) CH domain containing human multidomain proteins: an inventory.

    PubMed

    Friedberg, Felix

    2010-04-01

    In this paper, the inventory presented for singlet CH (calponin homology/actin binding) domain containing human multidomain proteins is extended to several duplex and one quadruplet CH containing forms. Invariably, the duplexes are located at the begin of the molecules. The regions connecting the two CH units suggest amino acid conservations which allows the placing of 18 duplex containing molecules into six groups wherein the gene for one member in each group created the others more recently by gene duplication. The ancient multidomain proteins, possibly, were primarily the result of an exon shuffling (transposition) mechanism that also guided the placing of the CH singlet or duplex domain at the amino end of the newly created proteins. A mechanism that creates pseudogenes could conceivably produce genes that encode multi-domain proteins. Intragenomic duplications (slippage) might have facilitated the occurrence of encoding repeats, thus allowing for the creation of multiple identical domains within one molecule. Gene duplication with subsequent modification and small domain gene recombination which formed multidomain proteins are important forces driving evolution.

  3. Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence.

    PubMed

    Bernardes, Juliana; Zaverucha, Gerson; Vaquero, Catherine; Carbone, Alessandra

    2016-07-01

    Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of ancient domain

  4. Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence

    PubMed Central

    Bernardes, Juliana; Zaverucha, Gerson; Vaquero, Catherine; Carbone, Alessandra

    2016-01-01

    Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of ancient domain

  5. The conserved DNA-binding domains encoded by the herpes simplex virus type 1 ICP4, pseudorabies virus IE180, and varicella-zoster virus ORF62 genes recognize similar sites in the corresponding promoters.

    PubMed Central

    Wu, C L; Wilcox, K W

    1991-01-01

    Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), pseudorabies virus (PRV), varicella-zoster virus (VZV), and equine herpesvirus 1 (EHV-1) are all classified as Alphaherpesvirinae. Each of these five viruses encodes an essential immediate-early (IE) regulatory protein referred to as HSV-1 ICP4, HSV-2 ICP4, PRV IE180, VZV ORF62 protein, and EHV-1 IE1, respectively. These five proteins share extensive homology with each other in domains referred to as regions 2 and 4. The HSV-1 ICP4 region 2 domain contains residues that are required for the DNA-binding capability of ICP4. In this report, we describe the expression of region 2 domains from the ICP4, IE180, and ORF62 genes as fusion proteins in Escherichia coli. DNA-binding assays revealed that each of these region 2 fusion proteins binds to a sequence that overlaps the transcription start site in the promoter for the gene encoding the corresponding protein. Each of the sites with high affinity for one or more of these fusion proteins contains the sequence 5'-ATCGT-3'. This sequence spans the mRNA cap site in the HSV-2 ICP4 gene promoter and is immediately upstream from the transcription start site in the EHV-1 IE1 gene. These results suggest that formation of a specific complex between an IE protein and its own gene promoter may be a common mechanism used by Alphaherpesvirinae to autoregulate transcription of an essential IE gene. Images PMID:1847444

  6. Lipid-specific β-sheet formation in a mussel byssus protein domain.

    PubMed

    Heim, Markus; Elsner, Martina B; Scheibel, Thomas

    2013-09-09

    Intrinsically disordered proteins (IDP) or regions (IDR) can adopt multiple conformational states, depending on the interaction partners they encounter. This enables proteins or individual domains to fulfill multiple functions. Here, we analyzed the flank sequences of preCol-NG, one of three collagenous proteins of a mussel byssus thread governing its mechanical performance. preCol-NG comprises a collagen domain and nonrepetitive termini enclosing specific flank regions characterized by tandem repeats known from silk proteins, protein elastomers, and plant cell wall-associated proteins. We recombinantly produced a protein mimicking the M. galloprovincialis preCol-NG C-terminal flank region. The protein was intrinsically unfolded in solution, even at elevated temperatures. However, upon contact with small unilamellar vesicles (SUVs) reversible β-structure formation occurred, reminiscent of partitioning-folding coupling. This behavior of preCol-NG flank domains likely impacts byssogenesis and sheds new light on a distinct mechanism of how fibrous protein materials might be achieved by lipid-induced self-assembly in nature.

  7. The RNA polymerase I transcription factor UBF is a sequence-tolerant HMG-box protein that can recognize structured nucleic acids.

    PubMed Central

    Copenhaver, G P; Putnam, C D; Denton, M L; Pikaard, C S

    1994-01-01

    Upstream Binding Factor (UBF) is important for activation of ribosomal RNA transcription and belongs to a family of proteins containing nucleic acid binding domains, termed HMG-boxes, with similarity to High Mobility Group (HMG) chromosomal proteins. Proteins in this family can be sequence-specific or highly sequence-tolerant binding proteins. We show that Xenopus UBF can be classified among the sequence-tolerant class. Methylation interference assays using enhancer DNA probes failed to reveal any critical nucleotides required for UBF binding. Selection by UBF of optimal binding sites among a population of enhancer oligonucleotides with randomized sequences also failed to reveal any consensus sequence. The minor groove specific drugs chromomycin A3, distamycin A and actinomycin D competed against UBF for enhancer binding, suggesting that UBF, like other HMG-box proteins, probably interacts with the minor groove. UBF also shares with other HMG box proteins the ability to bind synthetic cruciform DNA. However, UBF appears different from other HMG-box proteins in that it can bind both RNA (tRNA) and DNA. The sequence-tolerant nature of UBF-nucleic acid interactions may accommodate the rapid evolution of ribosomal RNA gene sequences. Images PMID:8041627

  8. delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains.

    PubMed

    Bourova, Lenka; Kostrnova, Alexandra; Hejnova, Lucie; Moravcova, Zuzana; Moon, Hyo-Eun; Novotny, Jiri; Milligan, Graeme; Svoboda, Petr

    2003-04-01

    Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor

  9. Modeling membrane shaping by proteins: focus on EHD2 and N-BAR domains.

    PubMed

    Campelo, Felix; Fabrikant, Gur; McMahon, Harvey T; Kozlov, Michael M

    2010-05-03

    Cellular membranes are highly dynamic, undergoing both persistent and dynamic shape changes driven by specialized proteins. The observed membrane shaping can be simple deformations of existing shapes or membrane remodeling involving fission or fusion. Here we describe several mechanistic principles by which membrane shaping proteins act. We especially consider models for membrane bending and fission by EHD2 proteins and membrane bending by N-BAR domains. There are major challenges ahead to understand the general principles by which diverse membrane bending proteins act and to understand how some proteins appear to span multiple modes of action from driving curvature to inducing membrane remodeling.

  10. Rational design of FRET sensor proteins based on mutually exclusive domain interactions.

    PubMed

    Merkx, Maarten; Golynskiy, Misha V; Lindenburg, Laurens H; Vinkenborg, Jan L

    2013-10-01

    Proteins that switch between distinct conformational states are ideal to monitor and control molecular processes within the complexity of biological systems. Inspired by the modular architecture of natural signalling proteins, our group explores generic design strategies for the construction of FRET-based sensor proteins and other protein switches. In the present article, I show that designing FRET sensors based on mutually exclusive domain interactions provides a robust method to engineer sensors with predictable properties and an inherently large change in emission ratio. The modularity of this approach should make it easily transferable to other applications of protein switches in fields ranging from synthetic biology, optogenetics and molecular diagnostics.

  11. Preliminary X-ray analysis of the binding domain of the soybean vacuolar sorting receptor complexed with a sorting determinant of a seed storage protein.

    PubMed

    Maruyama, Nobuyuki; Goshi, Tomohiro; Sugiyama, Shigeru; Niiyama, Mayumi; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Matsumura, Hiroyoshi; Mikami, Bunzo

    2015-02-01

    β-Conglycinin is a major seed storage protein in soybeans, which are an important source of protein. The major subunits (α, α' and β) of β-conglycinin are sorted to protein-storage vacuoles in seed cells. Vacuolar sorting receptor (VSR) is an integral membrane protein that recognizes the sorting determinant of vacuolar proteins, including β-conglycinin, and regulates their sorting process. Vacuolar sorting determinants of the α' and β subunits of β-conglycinin exist in their C-terminal peptides. Here, the preliminary X-ray diffraction analysis of the binding domain of soybean VSR crystallized with the peptide responsible for the sorting determinant in β-conglycinin is reported. X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to space group P3121, with unit-cell parameters a = b = 116.4, c = 86.1 Å.

  12. Identification of FAH Domain-containing Protein 1 (FAHD1) as Oxaloacetate Decarboxylase*

    PubMed Central

    Pircher, Haymo; von Grafenstein, Susanne; Diener, Thomas; Metzger, Christina; Albertini, Eva; Taferner, Andrea; Unterluggauer, Hermann; Kramer, Christian; Liedl, Klaus R.; Jansen-Dürr, Pidder

    2015-01-01

    Fumarylacetoacetate hydrolase (FAH) domain-containing proteins occur in both prokaryotes and eukaryotes, where they carry out diverse enzymatic reactions, probably related to structural differences in their respective FAH domains; however, the precise relationship between structure of the FAH domain and the associated enzyme function remains elusive. In mammals, three FAH domain-containing proteins, FAHD1, FAHD2A, and FAHD2B, are known; however, their enzymatic function, if any, remains to be demonstrated. In bacteria, oxaloacetate is subject to enzymatic decarboxylation; however, oxaloacetate decarboxylases (ODx) were so far not identified in eukaryotes. Based on molecular modeling and subsequent biochemical investigations, we identified FAHD1 as a eukaryotic ODx enzyme. The results presented here indicate that dedicated oxaloacetate decarboxylases exist in eukaryotes. PMID:25575590

  13. From protein domains to drug candidates-natural products as guiding principles in the design and synthesis of compound libraries.

    PubMed

    Breinbauer, Rolf; Vetter, Ingrid R; Waldmann, Herbert

    2002-08-16

    In the continuing effort to find small molecules that alter protein function and ultimately might lead to new drugs, combinatorial chemistry has emerged as a very powerful tool. Contrary to original expectations that large libraries would result in the discovery of many hit and lead structures, it has been recognized that the biological relevance, design, and diversity of the library are more important. As the universe of conceivable compounds is almost infinite, the question arises: where is a biologically validated starting point from which to build a combinatorial library? Nature itself might provide an answer: natural products have been evolved to bind to proteins. Recent results in structural biology and bioinformatics indicate that the number of distinct protein families and folds is fairly limited. Often the same structural domain is used by many proteins in a more or less modified form created by divergent evolution. Recent progress in solid-phase organic synthesis has enabled the synthesis of combinatorial libraries based on the structure of complex natural products. It can be envisioned that natural-product-based combinatorial synthesis may permit hit or lead compounds to be found with enhanced probability and quality.

  14. BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature.

    PubMed

    Madsen, K L; Bhatia, V K; Gether, U; Stamou, D

    2010-05-03

    The internal membranes of eukaryotic cells are all twists and bends characterized by high curvature. During recent years it has become clear that specific proteins sustain these curvatures while others simply recognize membrane shape and use it as "molecular information" to organize cellular processes in space and time. Here we discuss this new important recognition process termed membrane curvature sensing (MCS). First, we review a new fluorescence-based experimental method that allows characterization of MCS using measurements on single vesicles and compare it to sensing assays that use bulk/ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can be extended to include the insertion of alkyl chain in the lipid membrane and consequently palmitoylated and myristoylated proteins are predicted to display similar curvature sensitive binding. Surprisingly, in all the aforementioned cases, MCS is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity as assumed so far. Finally, we integrate these new insights into the debate about which motifs are involved in sensing versus induction of membrane curvature and what role MCS proteins may play in biology.

  15. Features of Two New Proteins with OmpA-Like Domains Identified in the Genome Sequences of Leptospira interrogans

    PubMed Central

    Teixeira, Aline F.; de Morais, Zenaide M.; Kirchgatter, Karin; Romero, Eliete C.; Vasconcellos, Silvio A.; Nascimento, Ana Lucia T. O.

    2015-01-01

    Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively). Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG)-interacting proteins, capable of generating plasmin (PLA) and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis. PMID:25849456

  16. Short LOV Proteins in Methylocystis Reveal Insight into LOV Domain Photocycle Mechanisms

    PubMed Central

    El-Arab, Kaley K.; Pudasaini, Ashutosh; Zoltowski, Brian D.

    2015-01-01

    Light Oxygen Voltage (LOV) proteins are widely used in optogenetic devices, however universal signal transduction pathways and photocycle mechanisms remain elusive. In particular, short-LOV (sLOV) proteins have been discovered in bacteria and fungi, containing only the photoresponsive LOV element without any obvious signal transduction domains. These sLOV proteins may be ideal models for LOV domain function due to their ease of study as full-length proteins. Unfortunately, characterization of such proteins remains limited to select systems. Herein, we identify a family of bacterial sLOV proteins present in Methylocystis. Sequence analysis of Methylocystis LOV proteins (McLOV) demonstrates conservation with sLOV proteins from fungal systems that employ competitive dimerization as a signaling mechanism. Cloning and characterization of McLOV proteins confirms functional dimer formation and reveal unexpected photocycle mechanisms. Specifically, some McLOV photocycles are insensitive to external bases such as imidazole, in contrast to previously characterized LOV proteins. Mutational analysis identifies a key residue that imparts insensitivity to imidazole in two McLOV homologs and affects adduct decay by two orders of magnitude. The resultant data identifies a new family of LOV proteins that indicate a universal photocycle mechanism may not be present in LOV proteins. PMID:25933162

  17. Structure of the Bro1 Domain Protein BROX and Functional Analyses of the ALIX Bro1 Domain in HIV-1 Budding

    SciTech Connect

    Zhai Q.; Robinson H.; Landesman M. B.; Sundquist W. I.; Hill C. P.

    2011-12-01

    Bro1 domains are elongated, banana-shaped domains that were first identified in the yeast ESCRT pathway protein, Bro1p. Humans express three Bro1 domain-containing proteins: ALIX, BROX, and HD-PTP, which function in association with the ESCRT pathway to help mediate intraluminal vesicle formation at multivesicular bodies, the abscission stage of cytokinesis, and/or enveloped virus budding. Human Bro1 domains share the ability to bind the CHMP4 subset of ESCRT-III proteins, associate with the HIV-1 NC{sup Gag} protein, and stimulate the budding of viral Gag proteins. The curved Bro1 domain structure has also been proposed to mediate membrane bending. To date, crystal structures have only been available for the related Bro1 domains from the Bro1p and ALIX proteins, and structures of additional family members should therefore aid in the identification of key structural and functional elements. We report the crystal structure of the human BROX protein, which comprises a single Bro1 domain. The Bro1 domains from BROX, Bro1p and ALIX adopt similar overall structures and share two common exposed hydrophobic surfaces. Surface 1 is located on the concave face and forms the CHMP4 binding site, whereas Surface 2 is located at the narrow end of the domain. The structures differ in that only ALIX has an extended loop that projects away from the convex face to expose the hydrophobic Phe105 side chain at its tip. Functional studies demonstrated that mutations in Surface 1, Surface 2, or Phe105 all impair the ability of ALIX to stimulate HIV-1 budding. Our studies reveal similarities in the overall folds and hydrophobic protein interaction sites of different Bro1 domains, and show that a unique extended loop contributes to the ability of ALIX to function in HIV-1 budding.

  18. Sugar-Binding Proteins from Fish: Selection of High Affnity “Lambodies” That Recognize Biomedically Relevant Glycans

    PubMed Central

    Hong, Xia; Ma, Mark Z.; Gildersleeve, Jeffrey C.; Chowdhury, Sudipa; Barchi, Joseph J.; Mariuzza, Roy A.; Murphy, Michael B.; Mao, Li; Pancer, Zeev

    2013-01-01

    Glycan-binding proteins are important for a wide variety of basic research and clinical applications, but proteins with high affnity and selectivity for carbohydrates are diffcult 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 affnity 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 affnity and selectivity for glycans may find myriad uses in glycobiology and biomedical research. PMID:23030719

  19. Yellow fever vaccination elicits broad functional CD4+ T cell responses that recognize structural and nonstructural proteins.

    PubMed

    James, Eddie A; LaFond, Rebecca E; Gates, Theresa J; Mai, Duy T; Malhotra, Uma; Kwok, William W

    2013-12-01

    Yellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8(+) T cell responses, less is known about YFV-specific CD4(+) T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4(+) T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4(+) T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4(+) T cell responses that contract, forming a detectable memory population.

  20. Plant homologs of mammalian MBT-domain protein-regulated KDM1 histone lysine demethylases do not interact with plant Tudor/PWWP/MBT-domain proteins

    PubMed Central

    Sadiq, Irfan; Keren, Ido; Citovsky, Vitaly

    2016-01-01

    Histone lysine demethylases of the LSD1/KDM1 family play important roles in epigenetic regulation of eukaryotic chromatin, and they are conserved between plants and animals. Mammalian LSD1 is thought to be targeted to its substrates, i.e., methylated histones, by an MBT-domain protein SFMBT1 that represents a component of the LSD1-based repressor complex and binds methylated histones. Because MBT-domain proteins are conserved between different organisms, from animals to plants, we examined whether the KDM1-type histone lysine demethylases KDM1C and FLD of Arabidopsis interact with the Arabidopsis Tudor/PWWP/MBT-domain SFMBT1-like proteins SL1, SL2, SL3, and SL4. No such interaction was detected using the bimolecular fluorescence complementation assay in living plant cells. Thus, plants most likely direct their KDM1 chromatin-modifying enzymes to methylated histones of the target chromatin by a mechanism different from that employed by the mammalian cells. PMID:26826387

  1. Domain compatibility in Ire1 kinase is critical for the unfolded protein response.

    PubMed

    Poothong, Juthakorn; Sopha, Pattarawut; Kaufman, Randal J; Tirasophon, Witoon

    2010-07-16

    The unfolded protein response is a mechanism to cope with endoplasmic reticulum stress. In Saccharomyces cerevisiae, Ire1 senses the stress and mediates a signaling cascade to upregulate responsive genes through an unusual HAC1 mRNA splicing. The splicing requires interconnected activity (kinase and endoribonuclease (RNase)) of Ire1 to cleave HAC1 mRNA at the non-canonical splice sites before translation into Hac1 transcription factor. Analysis of the truncated kinase domain from Ire1 homologs revealed that this domain is highly conserved. Characterization by domain swapping indicated that a functional ATP/ADP binding domain is minimally required. However the overall domain compatibility is critical for eliciting its full RNase function.

  2. The macro domain protein family: structure, functions, and their potential therapeutic implications.

    PubMed

    Han, Weidong; Li, Xiaolei; Fu, Xiaobing

    2011-01-01

    Macro domains are ancient, highly evolutionarily conserved domains that are widely distributed throughout all kingdoms of life. The 'macro fold' is roughly 25kDa in size and is composed of a mixed α-β fold with similarity to the P loop-containing nucleotide triphosphate hydrolases. They function as binding modules for metabolites of NAD(+), including poly(ADP-ribose) (PAR), which is synthesized by PAR polymerases (PARPs). Although there is a high degree of sequence similarity within this family, particularly for residues that might be involved in catalysis or substrates binding, it is likely that the sequence variation that does exist among macro domains is responsible for the specificity of function of individual proteins. Recent findings have indicated that macro domain proteins are functionally promiscuous and are implicated in the regulation of diverse biological functions, such as DNA repair, chromatin remodeling and transcriptional regulation. Significant advances in the field of macro domain have occurred in the past few years, including biological insights and the discovery of novel signaling pathways. To provide a framework for understanding these recent findings, this review will provide a comprehensive overview of the known and proposed biochemical, cellular and physiological roles of the macro domain family. Recent data that indicate a critical role of macro domain regulation for the proper progression of cellular differentiation programs will be discussed. In addition, the effect of dysregulated expression of macro domain proteins will be considered in the processes of tumorigenesis and bacterial pathogenesis. Finally, a series of observations will be highlighted that should be addressed in future efforts to develop macro domains as effective therapeutic targets.

  3. Alphavirus-Specific Cytotoxic T Lymphocytes Recognize a Cross-Reactive Epitope from the Capsid Protein and Can Eliminate Virus from Persistently Infected Macrophages

    PubMed Central

    Linn, May La; Mateo, L.; Gardner, J.; Suhrbier, A.

    1998-01-01

    Persistent alphavirus infections in synovial and neural tissues are believed to be associated with chronic arthritis and encephalitis, respectively, and represent likely targets for CD8+ αβ cytotoxic T lymphocytes (CTL). Here we show that the capsid protein is a dominant target for alphavirus-specific CTL in BALB/c mice and that capsid-specific CTL from these mice recognize an H-2Kd restricted epitope, QYSGGRFTI. This epitope lies in the highly conserved region of the capsid protein, and QYSGGRFTI-specific CTL were cross reactive across a range of Old World alphaviruses. In vivo the acute primary viraemia of these highly cytopathic viruses was unaffected by QYSGGRFTI-specific CTL. However, in vitro these CTL were able to completely clear virus from macrophages persistently and productively infected with the arthrogenic alphavirus Ross River virus. PMID:9573286

  4. Structure of the SCAN Domain of Human Paternally Expressed Gene 3 Protein

    PubMed Central

    Rimsa, Vadim; Eadsforth, Thomas C.; Hunter, William N.

    2013-01-01

    Human paternally expressed gene 3 protein (PEG3) is a large multi-domain entity with diverse biological functions, including acting as a transcription factor. PEG3 contains twelve Cys2-His2 type zinc finger domains, extended regions of predicted disorder and at the N-terminus a SCAN domain. PEG3 has been identified as partner of the E3 ubiquitin-protein ligase Siah1, an association we sought to investigate. An efficient bacterial recombinant expression system of the human PEG3-SCAN domain was prepared and crystals appeared spontaneously when the protein was being concentrated after purification. The structure was determined at 1.95 Å resolution and reveals a polypeptide fold of five helices in an extended configuration. An extensive dimerization interface, using almost a quarter of the solvent accessible surface, and key salt bridge interactions explain the stability of the dimer. Comparison with other SCAN domains reveals a high degree of conservation involving residues that contribute to the dimer interface. The PEG3-SCAN domain appears to constitute an assembly block, enabling PEG3 homo- or heterodimerization to control gene expression in a combinatorial fashion. PMID:23936039

  5. Bacterial SET domain proteins and their role in eukaryotic chromatin modification

    PubMed Central

    Alvarez-Venegas, Raúl

    2014-01-01

    It has been shown by many researchers that SET-domain containing proteins modify chromatin structure and, as expected, genes coding for SET-domain containing proteins have been found in all eukaryotic genomes sequenced to date. However, during the last years, a great number of bacterial genomes have been sequenced and an important number of putative genes involved in histone post-translational modifications (histone PTMs) have been identified in many bacterial genomes. Here, I aim at presenting an overview of SET domain genes that have been identified in numbers of bacterial genomes based on similarity to SET domains of eukaryotic histone methyltransferases. I will argue in favor of the hypothesis that SET domain genes found in extant bacteria are of bacterial origin. Then, I will focus on the available information on pathogen and symbiont SET-domain containing proteins and their targets in eukaryotic organisms, and how such histone methyltransferases allow a pathogen to inhibit transcriptional activation of host defense genes. PMID:24765100

  6. A mathematically related singularity and the maximum size of protein domains.

    PubMed

    Szilágyi, András

    2008-06-01

    In a paper titled "A topologically related singularity suggests a maximum preferred size for protein domains" (Zbilut et al., Proteins 2007;66:621-629), Zbilut et al. claim to have found a singularity in certain geometrical properties of protein structures, and suggest that this singularity may limit the maximum size of protein domains. They find further support for the singularity in their analysis of G-factors calculated by the PROCHECK program. Here, we show that the claimed singularity is a mathematical artifact with no physical meaning, and we reanalyze the G-factors to show that Zbilut et al.'s results are due to a single outlier in the data. Thus, the existence of an actual singularity in the topological properties of proteins is not supported by the findings of Zbilut et al.

  7. ADM-1, a protein with metalloprotease- and disintegrin-like domains, is expressed in syncytial organs, sperm, and sheath cells of sensory organs in Caenorhabditis elegans.

    PubMed Central

    Podbilewicz, B

    1996-01-01

    A search was carried out for homologues of possible fusogenic proteins to study their function in a genetically tractable animal. The isolation, molecular, and cellular characterization of the Caenorhabditis elegans adm-1 gene (a disintegrin and metalloprotease domain) are described. A glycoprotein analogous to viral fusion proteins has been identified on the surface of guinea pig sperm (PH-30/fertilin) and is implicated in sperm-egg fusion. adm-1 is the first reported invertebrate gene related to PH-30 and a family of proteins containing snake venom disintegrin- and metalloprotease-like domains. ADM-1 shows a domain organization identical to PH-30. It contains prepro, metalloprotease, disintegrin, cysteine rich with putative fusion peptide, epidermal growth factor-like repeat, transmembrane, and cytoplasmic domains. Antibodies which recognize ADM-1 protein in immunoblots were generated. Using immunofluorescence and in situ hybridization, the products of adm-1 have been detected in specific cells during different stages of development. The localization of ADM-1 to the plasma membrane of embryonic cells and to the sheath cells of sensory organs suggests a function in cell adhesion. ADM-1 expression in the hypodermis, pharynx, vulva, and mature sperm is consistent with a putative role in somatic and gamete cell fusions. Images PMID:8970152

  8. Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins.

    PubMed

    Waldman, Vincent M; Stanage, Tyler H; Mims, Alexandra; Norden, Ian S; Oakley, Martha G

    2015-06-01

    The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans.

  9. Functional domains and motifs of bacterial type III effector proteins and their roles in infection.

    PubMed

    Dean, Paul

    2011-11-01

    A key feature of the virulence of many bacterial pathogens is the ability to deliver effector proteins into eukaryotic cells via a dedicated type three secretion system (T3SS). Many bacterial pathogens, including species of Chlamydia, Xanthomonas, Pseudomonas, Ralstonia, Shigella, Salmonella, Escherichia and Yersinia, depend on the T3SS to cause disease. T3SS effectors constitute a large and diverse group of virulence proteins that mimic eukaryotic proteins in structure and function. A salient feature of bacterial effectors is their modular architecture, comprising domains or motifs that confer an array of subversive functions within the eukaryotic cell. These domains/motifs therefore represent a fascinating repertoire of molecular determinants with important roles during infection. This review provides a snapshot of our current understanding of bacterial effector domains and motifs where a defined role in infection has been demonstrated.

  10. Biological effects of individually synthesized TNF-binding domain of variola virus CrmB protein.

    PubMed

    Tsyrendorzhiev, D D; Orlovskaya, I A; Sennikov, S V; Tregubchak, T V; Gileva, I P; Tsyrendorzhieva, M D; Shchelkunov, S N

    2014-06-01

    The biological characteristics of a 17-kDa protein synthesized in bacterial cells, a TNF-binding domain (VARV-TNF-BP) of a 47-kDa variola virus CrmB protein (VARV-CrmB) consisting of TNF-binding and chemokine-binding domains, were studied. Removal of the C-terminal chemokine-binding domain from VARV-CrmB protein was inessential for the efficiency of its inhibition of TNF cytotoxicity towards L929 mouse fibroblast culture and for TNF-induced oxidative metabolic activity of mouse blood leukocytes. The results of this study could form the basis for further studies of VARV-TNF-BP mechanisms of activity for prospective use in practical medicine.

  11. Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

    PubMed

    Böldicke, Thomas

    2017-03-08

    Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, non aggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic. This article is protected by copyright. All rights reserved.

  12. Molecular insights into the binding of phosphoinositides to the TH domain region of TIPE proteins.

    PubMed

    Antony, Priya; Baby, Bincy; Vijayan, Ranjit

    2016-11-01

    Phosphatidylinositols and their phosphorylated derivatives, phosphoinositides, play a central role in regulating diverse cellular functions. These phospholipids have been shown to interact with the hydrophobic TH domain of the tumor necrosis factor (TNF)-α-induced protein 8 (TIPE) family of proteins. However, the precise mechanism of interaction of these lipids is unclear. Here we report the binding mode and interactions of these phospholipids in the TH domain, as elucidated using molecular docking and simulations. Results indicate that phosphoinositides bind to the TH domain in a similar way by inserting their lipid tails in the hydrophobic cavity. The exposed head group is stabilized by interactions with critical positively charged residues on the surface of these proteins. Further MD simulations confirmed the dynamic stability of these lipids in the TH domain. This computational analysis thus provides insight into the binding mode of phospholipids in the TH domain of the TIPE family of proteins. Graphical abstract A phosphoinositide (phosphatidylinositol 4-phosphate; PtdIns4P) docked to TIPE2.

  13. Knowledge-Guided Docking of WW Domain Proteins and Flexible Ligands

    NASA Astrophysics Data System (ADS)

    Lu, Haiyun; Li, Hao; Banu Bte Sm Rashid, Shamima; Leow, Wee Kheng; Liou, Yih-Cherng

    Studies of interactions between protein domains and ligands are important in many aspects such as cellular signaling. We present a knowledge-guided approach for docking protein domains and flexible ligands. The approach is applied to the WW domain, a small protein module mediating signaling complexes which have been implicated in diseases such as muscular dystrophy and Liddle’s syndrome. The first stage of the approach employs a substring search for two binding grooves of WW domains and possible binding motifs of peptide ligands based on known features. The second stage aligns the ligand’s peptide backbone to the two binding grooves using a quasi-Newton constrained optimization algorithm. The backbone-aligned ligands produced serve as good starting points to the third stage which uses any flexible docking algorithm to perform the docking. The experimental results demonstrate that the backbone alignment method in the second stage performs better than conventional rigid superposition given two binding constraints. It is also shown that using the backbone-aligned ligands as initial configurations improves the flexible docking in the third stage. The presented approach can also be applied to other protein domains that involve binding of flexible ligand to two or more binding sites.

  14. CG dinucleotide periodicities recognized by the Dnmt3a-Dnmt3L complex are distinctive at retroelements and imprinted domains.

    PubMed

    Glass, Jacob L; Fazzari, Melissa J; Ferguson-Smith, Anne C; Greally, John M

    2009-01-01

    The Dnmt3a and Dnmt3L genes are critical mediators of cytosine methylation during gametogenesis, with major actions noted at transposable elements and imprinted loci. The Dnmt3a-Dnmt3L complex was recently described to have preferential activity at CG dinucleotides located 8-10 bp apart. Because cytosine methylation is heterogeneously distributed in the genome, we tested whether this relative sequence preference explains the effects of mutation of the Dnmt3a and Dnmt3L genes using bioinformatic analysis. We found that the human and mouse genomes are significantly enriched in a CG dinucleotide periodicity of 2 bp, leading to an increased frequency of CGs spaced 8 bp apart that represent widespread targets for this protein complex. When we broke down the human and mouse genomes by annotation, we found that this significant 2-bp periodicity and increased 8-bp periodicity are maintained in Alu SINEs in both species. The 8-bp periodicity was mapped genome-wide, identifying enrichment at the promoters of both paternally and maternally methylated imprinted genes and at CG dinucleotide-enriched sequences. We conclude that CG dinucleotide periodicity helps to explain some but not all of the relative sequence specificity of mutations of Dnmt3a or Dnmt3L in the establishment of germline cytosine methylation patterns.

  15. Molecular dynamics simulations elucidate the mode of protein recognition by Skp1 and the F-box domain in the SCF complex.

    PubMed

    Chandra Dantu, Sarath; Nathubhai Kachariya, Nitin; Kumar, Ashutosh

    2016-01-01

    Polyubiquitination of the target protein by a ubiquitin transferring machinery is key to various cellular processes. E3 ligase Skp1-Cul1-F-box (SCF) is one such complex which plays crucial role in substrate recognition and transfer of the ubiquitin molecule. Previous computational studies have focused on S-phase kinase-associated protein 2 (Skp2), cullin, and RING-finger proteins of this complex, but the roles of the adapter protein Skp1 and F-box domain of Skp2 have not been determined. Using sub-microsecond molecular dynamics simulations of full-length Skp1, unbound Skp2, Skp2-Cks1 (Cks1: Cyclin-dependent kinases regulatory subunit 1), Skp1-Skp2, and Skp1-Skp2-Cks1 complexes, we have elucidated the function of Skp1 and the F-box domain of Skp2. We found that the L16 loop of Skp1, which was deleted in previous X-ray crystallography studies, can offer additional stability to the ternary complex via its interactions with the C-terminal tail of Skp2. Moreover, Skp1 helices H6, H7, and H8 display vivid conformational flexibility when not bound to Skp2, suggesting that these helices can recognize and lock the F-box proteins. Furthermore, we observed that the F-box domain could rotate (5°-129°), and that the binding partner determined the degree of conformational flexibility. Finally, Skp1 and Skp2 were found to execute a domain motion in Skp1-Skp2 and Skp1-Skp2-Cks1 complexes that could decrease the distance between ubiquitination site of the substrate and the ubiquitin molecule by 3 nm. Thus, we propose that both the F-box domain of Skp2 and Skp1-Skp2 domain motions displaying preferential conformational control can together facilitate polyubiquitination of a wide variety of substrates.

  16. Identification and characterization of a monoclonal antibody recognizing the linear epitope RVADVI on VP1 protein of enterovirus 71.

    PubMed

    Man-Li, Tang; Szyporta, Milene; Fang, Lim Xiao; Kwang, Jimmy

    2012-10-01

    Several large outbreaks of hand-foot-mouth disease (HFMD) have occurred in the Asian-Pacific region since 1997, with Enterovirus 71 (EV71) and/or Coxsackievirus A16 (CAV16) as the main causative agents. Despite the close genetic relationship between the two viruses, only EV71 is associated with severe clinical manifestations and deaths. Effective antiviral treatment and vaccines are not available. High-quality monoclonal antibodies (mAbs) are necessary to improve the accuracy of the diagnosis of EV71. In this study, a mAb (designated 1D9) was generated using EV71 C5 strain virus particles as immunogens. Examined by indirect immunofluorescence assay (IFA) and Western blotting, 1D9 detected successfully all 11 subgenotypes of EV71 and showed no cross-reactivity to the four selected subgenogroups of Coxsackieviruses CAV4, CAV6, CAV10, and CAV16. A linear motif, R(3) VADVI(8), which is located at the N-terminus of the EV71 VP1 protein, was identified as the minimal binding region of 1D9. Alignment and comparison of the 1D9-defined epitope sequence against the listed sequences in the NCBI EV71 database indicated that this epitope R(3) VADVI(8) was highly conserved among EV71 strains, while no significant similarity was observed when blasted against the Coxsackieviruses. This suggests that the mAb 1D9 may be useful for the development of a cost-effective and accurate method for surveillance and early differentiation of EV71 from CAV16 infection.

  17. ADAR proteins: double-stranded RNA and Z-DNA binding domains.

    PubMed

    Barraud, Pierre; Allain, Frédéric H-T

    2012-01-01

    Adenosine deaminases acting on RNA (ADAR) catalyze adenosine to inosine editing within double-stranded RNA (dsRNA) substrates. Inosine is read as a guanine by most cellular processes and therefore these changes create codons for a different amino acid, stop codons or even a new splice-site allowing protein diversity generated from a single gene. We review here the current structural and molecular knowledge on RNA editing by the ADAR family of protein. We focus especially on two types of nucleic acid binding domains present in ADARs, namely the dsRNA and Z-DNA binding domains.

  18. Crystal structure of the TLDc domain of oxidation resistance protein 2 from zebrafish.

    PubMed

    Blaise, Mickaël; Alsarraf, Husam M A B; Wong, Jaslyn E M M; Midtgaard, Søren Roi; Laroche, Fabrice; Schack, Lotte; Spaink, Herman; Stougaard, Jens; Thirup, Søren

    2012-06-01

    The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C-terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from zebrafish. The structure was determined by X-ray crystallography to atomic resolution (0.97Å) and adopts an overall globular shape. Two antiparallel β-sheets form a central β-sandwich, surrounded by two helices and two one-turn helices. The fold shares low structural similarity to known structures.

  19. Interaction of the Intermembrane Space Domain of Tim23 Protein with Mitochondrial Membranes*

    PubMed Central

    Bajaj, Rakhi; Munari, Francesca; Becker, Stefan; Zweckstetter, Markus

    2014-01-01

    Tim23 mediates protein translocation into mitochondria. Although inserted into the inner membrane, the dynamic association of its intermembrane space (IMS) domain with the outer membrane promotes protein import. However, little is known about the molecular basis of this interaction. Here, we demonstrate that the IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N terminus. The structure of membrane-bound Tim23IMS is highly dynamic, allowing recognition of both the incoming presequence and other translocase components at the translocation contact. Cardiolipin enhances Tim23 membrane attachment, suggesting that cardiolipin can influence preprotein import. PMID:25349212

  20. Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development

    PubMed Central

    Dupé, Aurélien; Dumas, Carole; Papadopoulou, Barbara

    2015-01-01

    Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in

  1. Distribution and evolution of stable single α-helices (SAH domains) in myosin motor proteins

    PubMed Central

    Simm, Dominic; Hatje, Klas

    2017-01-01

    Stable single-alpha helices (SAHs) are versatile structural elements in many prokaryotic and eukaryotic proteins acting as semi-flexible linkers and constant force springs. This way SAH-domains function as part of the lever of many different myosins. Canonical myosin levers consist of one or several IQ-motifs to which light chains such as calmodulin bind. SAH-domains provide flexibility in length and stiffness to the myosin levers, and may be particularly suited for myosins working in crowded cellular environments. Although the function of the SAH-domains in human class-6 and class-10 myosins has well been characterised, the distribution of the SAH-domain in all myosin subfamilies and across the eukaryotic tree of life remained elusive. Here, we analysed the largest available myosin sequence dataset consisting of 7919 manually annotated myosin sequences from 938 species representing all major eukaryotic branches using the SAH-prediction algorithm of Waggawagga, a recently developed tool for the identification of SAH-domains. With this approach we identified SAH-domains in more than one third of the supposed 79 myosin subfamilies. Depending on the myosin class, the presence of SAH-domains can range from a few to almost all class members indicating complex patterns of independent and taxon-specific SAH-domain gain and loss. PMID:28369123

  2. Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding.

    PubMed

    Newcomer, Rebecca L; Fraser, LaTasha C R; Teschke, Carolyn M; Alexandrescu, Andrei T

    2015-12-15

    The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining (3)JNC' couplings transmitted through H-bonds, the temperature and urea-concentration dependence of (1)HN and (15)N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and (3)JNC' H-bond couplings, are identified with an accuracy of 90% by (1)HN temperature coefficients. The accuracy is improved to 95% when (15)N temperature coefficients are also included. In contrast, the urea dependence of (1)HN and (15)N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility.

  3. Solution structure of the zinc finger HIT domain in protein FON

    PubMed Central

    He, Fahu; Umehara, Takashi; Tsuda, Kengo; Inoue, Makoto; Kigawa, Takanori; Matsuda, Takayoshi; Yabuki, Takashi; Aoki, Masaaki; Seki, Eiko; Terada, Takaho; Shirouzu, Mikako; Tanaka, Akiko; Sugano, Sumio; Muto, Yutaka; Yokoyama, Shigeyuki

    2007-01-01

    The zinc finger HIT domain is a sequence motif found in many proteins, including thyroid hormone receptor interacting protein 3 (TRIP-3), which is possibly involved in maturity-onset diabetes of the young (MODY). Novel zinc finger motifs are suggested to play important roles in gene regulation and chromatin remodeling. Here, we determined the high-resolution solution structure of the zinc finger HIT domain in ZNHIT2 (protein FON) from Homo sapiens, by an NMR method based on 567 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure yielded a backbone RMSD to the mean coordinates of 0.19 Å for the structured residues 12–48. The fold consists of two consecutive antiparallel β-sheets and two short C-terminal helices packed against the second β-sheet, and binds two zinc ions. Both zinc ions are coordinated tetrahedrally via a CCCC-CCHC motif to the ligand residues of the zf-HIT domain in an interleaved manner. The tertiary structure of the zinc finger HIT domain closely resembles the folds of the B-box, RING finger, and PHD domains with a cross-brace zinc coordination mode, but is distinct from them. The unique three-dimensional structure of the zinc finger HIT domain revealed a novel zinc-binding fold, as a new member of the treble clef domain family. On the basis of the structural data, we discuss the possible functional roles of the zinc finger HIT domain. PMID:17656577

  4. dcGOR: an R package for analysing ontologies and protein domain annotations.

    PubMed

    Fang, Hai

    2014-10-01

    I introduce an open-source R package 'dcGOR' to provide the bioinformatics community with the ease to analyse ontologies and protein domain annotations, particularly those in the dcGO database. The dcGO is a comprehensive resource for protein domain annotations using a panel of ontologies including Gene Ontology. Although increasing in popularity, this database needs statistical and graphical support to meet its full potential. Moreover, there are no bioinformatics tools specifically designed for domain ontology analysis. As an add-on package built in the R software environment, dcGOR offers a basic infrastructure with great flexibility and functionality. It implements new data structure to represent domains, ontologies, annotations, and all analytical outputs as well. For each ontology, it provides various mining facilities, including: (i) domain-based enrichment analysis and visualisation; (ii) construction of a domain (semantic similarity) network according to ontology annotations; and (iii) significance analysis for estimating a contact (statistical significance) network. To reduce runtime, most analyses support high-performance parallel computing. Taking as inputs a list of protein domains of interest, the package is able to easily carry out in-depth analyses in terms of functional, phenotypic and diseased relevance, and network-level understanding. More importantly, dcGOR is designed to allow users to import and analyse their own ontologies and annotations on domains (taken from SCOP, Pfam and InterPro) and RNAs (from Rfam) as well. The package is freely available at CRAN for easy installation, and also at GitHub for version control. The dedicated website with reproducible demos can be found at http://supfam.org/dcGOR.

  5. Phase separation and bistability in a three-dimensional model for protein domain formation at biomembranes

    NASA Astrophysics Data System (ADS)

    Alonso, Sergio; Bär, Markus

    2010-12-01

    Proteins in living cells interact with membranes. They may bind to or unbind from the membrane to the cytosol depending on the lipid composition of the membrane and their interaction with cytosolic enzymes. Moreover, proteins can accumulate at the membrane and assemble in spatial domains. Here, a simple model of protein cycling at biomembranes is studied, when the total number of proteins is conserved. Specifically, we consider the spatio-temporal dynamics of MARCKS proteins and their interactions with enzymes facilitating translocation from and rebinding to the membrane. The model exhibits two qualitatively different mechanisms of protein domain formation: phase separation related to a long-wave instability of a membrane state with homogeneous protein coverage and stable coexistence of two states with different homogeneous protein coverage in bistable media. We evaluate the impact of the cytosolic volume on the occurrence of protein pattern formation by simulations in a three-dimensional model. We show that the explicit treatment of the volume in the model leads to an effective rescaling of the reaction rates. For a simplified model of protein cycling, we can derive analytical expressions for the rescaling coefficients and verify them by direct simulations with the complete three-dimensional model.

  6. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

    SciTech Connect

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G. )

    1988-08-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

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

    PubMed

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

    2006-02-10

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

  8. Structural determinants of protein partitioning into ordered membrane domains and lipid rafts.

    PubMed

    Lorent, Joseph Helmuth; Levental, Ilya

    2015-11-01

    Increasing evidence supports the existence of lateral nanoscopic lipid domains in plasma membranes, known as lipid rafts. These domains preferentially recruit membrane proteins and lipids to facilitate their interactions and thereby regulate transmembrane signaling and cellular homeostasis. The functionality of raft domains is intrinsically dependent on their selectivity for specific membrane components; however, while the physicochemical determinants of raft association for lipids are known, very few systematic studies have focused on the structural aspects that guide raft partitioning of proteins. In this review, we describe biophysical and thermodynamic aspects of raft-mimetic liquid ordered phases, focusing on those most relevant for protein partitioning. Further, we detail the variety of experimental models used to study protein-raft interactions. Finally, we review the existing literature on mechanisms for raft targeting, including lipid post-translational modifications, lipid binding, and transmembrane domain features. We conclude that while protein palmitoylation is a clear raft-targeting signal, few other general structural determinants for raft partitioning have been revealed, suggesting that many discoveries lie ahead in this burgeoning field.

  9. New Knowledge from Old: In silico discovery of novel protein domains in Streptomyces coelicolor

    PubMed Central

    Yeats, Corin; Bentley, Stephen; Bateman, Alex

    2003-01-01

    Background Streptomyces coelicolor has long been considered a remarkable bacterium with a complex life-cycle, ubiquitous environmental distribution, linear chromosomes and plasmids, and a huge range of pharmaceutically useful secondary metabolites. Completion of the genome sequence demonstrated that this diversity carried through to the genetic level, with over 7000 genes identified. We sought to expand our understanding of this organism at the molecular level through identification and annotation of novel protein domains. Protein domains are the evolutionary conserved units from which proteins are formed. Results Two automated methods were employed to rapidly generate an optimised set of targets, which were subsequently analysed manually. A final set of 37 domains or structural repeats, represented 204 times in the genome, was developed. Using these families enabled us to correlate items of information from many different resources. Several immediately enhance our understanding both of S. coelicolor and also general bacterial molecular mechanisms, including cell wall biosynthesis regulation and streptomycete telomere maintenance. Discussion Delineation of protein domain families enables detailed analysis of protein function, as well as identification of likely regions or residues of particular interest. Hence this kind of prior approach can increase the rate of discovery in the laboratory. Furthermore we demonstrate that using this type of in silico method it is possible to fairly rapidly generate new biological information from previously uncorrelated data. PMID:12625841

  10. A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis

    PubMed Central

    Morimoto, Akihiro; Shibuya, Hiroki; Zhu, Xiaoqiang; Kim, Jihye; Ishiguro, Kei-ichiro; Han, Min

    2012-01-01

    In yeasts and worms, KASH (Klarsicht/ANC-1/Syne/homology) domain and SUN (Sad-1/UNC-84) domain nuclear envelope (NE) proteins play a crucial role in meiotic chromosome movement and homologue pairing. However, although the vertebrate SUN domain protein SUN1 is involved in these processes, its partner has remained identified. Based on subcellular localization screening in mouse spermatocytes, we identified a novel germ cell–specific protein, KASH5, that localized exclusively at telomeres from the leptotene to diplotene stages in both spermatocytes and oocytes. KASH5 possesses hitherto unknown KASH-related sequences that directly interacted with SUN1 and mediated telomere localization. Thus, KASH5 is a mammalian meiosis-specific KASH domain protein. We show that meiotic chromosome movement depended on microtubules and that KASH5 interacted with the microtubule-associated dynein–dynactin complex. These results suggest that KASH5 connects the telomere-associated SUN1 protein to the cytoplasmic force–generating mechanism involved in meiotic chromosome movement. Our study strongly suggests that the meiotic homologue-pairing mechanism mediated by the SUN–KASH NE bridge is highly conserved among eukaryotes. PMID:22826121

  11. Kelch Domain of Gigaxonin Interacts with Intermediate Filament Proteins Affected in Giant Axonal Neuropathy

    PubMed Central

    Johnson-Kerner, Bethany L.; Garcia Diaz, Alejandro; Ekins, Sean; Wichterle, Hynek

    2015-01-01

    Patients with giant axonal neuropathy (GAN) show progressive loss of motor and sensory function starting in childhood and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin (GIG), a ubiquitously-expressed BTB/Kelch cytoplasmic protein believed to be an E3 ligase substrate adaptor. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. To delineate the molecular pathway between GIG deficiency and IF pathology, we undertook a proteomic screen to identify the normal binding partners of GIG. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. We showed these interactions were dependent on the Kelch domain of GIG. Furthermore, we identified the E3 ligase MYCBP2 and the heat shock proteins HSP90AA1/AB1 as interactors with the BTB domain that may result in the ubiquitination and subsequent degradation of intermediate filaments. Our open-ended proteomic screen provides support to GIG’s role as an adaptor protein, linking IF proteins through its Kelch domain to the ubiquitin pathway proteins via its BTB domain, and points to future approaches for reversing the phenotype in human patients. PMID:26460568

  12. Tudor domain proteins in protozoan parasites and characterization of Plasmodium falciparum tudor staphylococcal nuclease.

    PubMed

    Hossain, Manzar J; Korde, Reshma; Singh, Shivani; Mohmmed, Asif; Dasaradhi, P V N; Chauhan, V S; Malhotra, Pawan

    2008-04-01

    RNA-binding proteins play key roles in post-transcriptional regulation of gene expression. In eukaryotic cells, a multitude of RNA-binding proteins with several RNA-binding domains/motifs have been described. Here, we show the existence of two Tudor domain containing proteins, a survival of motor neuron (SMN)-like protein and a Staphylococcus aureus nuclease homologue referred to as TSN, in Plasmodium and other protozoan parasites. Activity analysis shows that Plasmodium falciparum TSN (PfTSN) possesses nuclease activity and Tudor domain is the RNA-binding domain. A specific inhibitor of micrococcal nucleases, 3',5'-deoxythymidine bisphosphate (pdTp) inhibits the nuclease as well as RNA-binding activities of the protein. PfTSN shows a predominant nuclear localization. Treatment of P. falciparum with pdTp, inhibited in vitro growth of both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, while a four fold concentration of pdTp did not have any significant effect on the mammalian cell line, Huh-7D12. Altogether, these results suggest that PfTSN is an essential enzyme in the parasite's life cycle.

  13. Functional domains within the human immunodeficiency virus type 2 envelope protein required to enhance virus production.

    PubMed

    Abada, Paolo; Noble, Beth; Cannon, Paula M

    2005-03-01

    Primate lentiviruses code for a protein that stimulates virus production. In human immunodeficiency virus type 1 (HIV-1), the activity is provided by the accessory protein, Vpu, while in HIV-2 and simian immunodeficiency virus it is a property of the envelope (Env) glycoprotein. Using a group of diverse retroviruses and cell types, we have confirmed the functional equivalence of the two proteins. However, despite these similarities, the two proteins have markedly different functional domains. While the Vpu activity is associated primarily with its membrane-spanning region, we have determined that the HIV-2 Env activity requires both the cytoplasmic tail and ectodomain of the protein, with the membrane-spanning domain being less important. Within the Env cytoplasmic tail, we further defined the necessary sequence as a membrane-proximal tyrosine-based motif. Providing the two Env regions separately as distinct CD8 chimeric proteins did not increase virus release. This suggests that the two domains must be either contained within a single protein or closely associated within a multiprotein oligomer, such as the Env trimer, in order to function. Finally, we observed that wild-type levels of incorporation of the HIV-2 Env into budding viruses were not required for this activity.

  14. Functional Domains within the Human Immunodeficiency Virus Type 2 Envelope Protein Required To Enhance Virus Production

    PubMed Central

    Abada, Paolo; Noble, Beth; Cannon, Paula M.

    2005-01-01

    Primate lentiviruses code for a protein that stimulates virus production. In human immunodeficiency virus type 1 (HIV-1), the activity is provided by the accessory protein, Vpu, while in HIV-2 and simian immunodeficiency virus it is a property of the envelope (Env) glycoprotein. Using a group of diverse retroviruses and cell types, we have confirmed the functional equivalence of the two proteins. However, despite these similarities, the two proteins have markedly different functional domains. While the Vpu activity is associated primarily with its membrane-spanning region, we have determined that the HIV-2 Env activity requires both the cytoplasmic tail and ectodomain of the protein, with the membrane-spanning domain being less important. Within the Env cytoplasmic tail, we further defined the necessary sequence as a membrane-proximal tyrosine-based motif. Providing the two Env regions separately as distinct CD8 chimeric proteins did not increase virus release. This suggests that the two domains must be either contained within a single protein or closely associated within a multiprotein oligomer, such as the Env trimer, in order to function. Finally, we observed that wild-type levels of incorporation of the HIV-2 Env into budding viruses were not required for this activity. PMID:15731257

  15. A novel single WAP domain-containing protein isoform with antibacterial relevance in Litopenaeus vannamei.

    PubMed

    Du, Zhi-Qiang; Yuan, Jian-Jun; Ren, Da-Ming

    2015-06-01

    Single WAP domain (SWD)-containing protein is a small protein containing a whey acidic protein (WAP) domain at the C-terminal region. SWD-containing protein exhibits structural similarity to the family of serine proteinase inhibitors. As of this writing, some SWD domain-containing proteins have been identified in crustaceans, and their functions included antibacterial and anti-proteinase activities. We identified a SWD protein isoform gene in Litopenaeus vanname (Lv-SWDi). Very high sequence similarity was found between Lv-SWDi and Lv-SWD. Results of time-course analysis for the gene expression profile showed that Lv-SWDi could produce a rapid feedback and an obvious upregulation at 12 h after Vibrio injection. Endogenous Lv-SWDi protein was obviously upregulated, and the highest expression level was reached at 24 h after Vibrio injection. The purified rLv-SWDi could directly bind to Gram-positive and Gram-negative bacteria. Results of the proteinase inhibitory assay also showed that rLv-SWDi could inhibit secretory protease activity from Bacillus subtilis. Lv-SWDi is a part of an important immunity-relevant gene and may serve important functions in defense against bacteria.

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

    SciTech Connect

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

    2009-01-01

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

  17. Sensing Domain Dynamics in Protein Kinase A-Iα Complexes by Solution X-ray Scattering*

    PubMed Central

    Cheng, Cecilia Y.; Yang, Jie; Taylor, Susan S.; Blumenthal, Donald K.

    2009-01-01

    The catalytic (C) and regulatory (R) subunits of protein kinase A are exceptionally dynamic proteins. Interactions between the R- and C-subunits are regulated by cAMP binding to the two cyclic nucleotide-binding domains in the R-subunit. Mammalian cells express four different isoforms of the R-subunit (RIα, RIβ, RIIα, and RIIβ) that all interact with the C-subunit in different ways. Here, we investigate the dynamic behavior of protein complexes between RIα and C-subunits using small angle x-ray scattering. We show that a single point mutation in RIα, R333K (which alters the cAMP-binding properties of Domain B) results in a compact shape compared with the extended shape of the wild-type R·C complex. A double mutant complex that disrupts the interaction site between the C-subunit and Domain B in RIα, RIαABR333K·C(K285P), results in a broader P(r) curve that more closely resembles the P(r) profiles of wild-type complexes. These results together suggest that interactions between RIα Domain B and the C-subunit in the RIα·C complex involve large scale dynamics that can be disrupted by single point mutations in both proteins. In contrast to RIα·C complexes. Domain B in the RIIβ·C heterodimer is not dynamic and is critical for both inhibition and complex formation. Our study highlights the functional differences of domain dynamics between protein kinase A isoforms, providing a framework for elucidating the global organization of each holoenzyme and the cross-talk between the R- and C-subunits. PMID:19837668

  18. Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase.

    PubMed

    Clark, Daniel N; Flanagan, John M; Hu, Jianming

    2017-02-01

    Hepatitis B virus (HBV) encodes a multifunction reverse transcriptase or polymerase (P), which is composed of several domains. The terminal protein (TP) domain is unique to HBV and related hepadnaviruses and is required for specifically binding to the viral pregenomic RNA (pgRNA). Subsequently, the TP domain is necessary for pgRNA packaging into viral nucleocapsids and the initiation of viral reverse transcription for conversion of the pgRNA to viral DNA. Uniquely, the HBV P protein initiates reverse transcription via a protein priming mechanism using the TP domain as a primer. No structural homologs or high-resolution structure exists for the TP domain. Secondary structure prediction identified three disordered loops in TP with highly conserved sequences. A meta-analysis of mutagenesis studies indicated these predicted loops are almost exclusively where functionally important residues are located. Newly constructed TP mutations revealed a priming loop in TP which plays a specific role in protein-primed DNA synthesis beyond simply harboring the site of priming. Substitutions of potential sites of phosphorylation surrounding the priming site demonstrated that these residues are involved in interactions critical for priming but are unlikely to be phosphorylated during viral replication. Furthermore, the first 13 and 66 TP residues were shown to be dispensable for protein priming and pgRNA binding, respectively. Combining current and previous mutagenesis work with sequence analysis has increased our understanding of TP structure and functions by mapping specific functions to distinct predicted secondary structures and will facilitate antiviral targeting of this unique domain.

  19. Modular structure of chromosomal proteins HMG-14 and HMG-17: Definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain

    SciTech Connect

    Trieschmann, L.; Postnikov, Y.V.; Rickers, A.; Bustin, M.

    1995-12-01

    This report describes how deletion mutants and peptides were used to identify the transcriptional enhancement domain and the nucleosome binding domain of two chromosomal proteins, HMG-14 and HMG-17. The research indicates that mutations involving C-terminal amino acids significantly reduces the ability of the nucleoproteins to enhance transcription from chromatin templates. 42 refs., 6 figs., 1 tab.

  20. Effect of Multimerization on Membrane Association of Rous Sarcoma Virus and HIV-1 Matrix Domain Proteins

    PubMed Central

    Dick, Robert A.; Kamynina, Elena

    2013-01-01

    In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV. PMID:24109216