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Sample records for pilz domain proteins

  1. The role of PilZ domain proteins in the virulence of Xanthomonas campestris pv. campestris.

    PubMed

    McCarthy, Yvonne; Ryan, Robert P; O'Donovan, Karen; He, Yong-Qiang; Jiang, Bo-Le; Feng, Jia-Xun; Tang, Ji-Liang; Dow, J Maxwell

    2008-11-01

    Cyclic di-GMP [(bis-(3'-5')-cyclic di-guanosine monophosphate)] is an almost ubiquitous second messenger in bacteria that is implicated in the regulation of a range of functions that include developmental transitions, aggregative behaviour, adhesion, biofilm formation and virulence. Comparatively little is known about the mechanism(s) by which cyclic di-GMP exerts these various regulatory effects. PilZ has been identified as a cyclic di-GMP binding protein domain; proteins with this domain are involved in regulation of specific cellular processes, including the virulence of animal pathogens. Here we have examined the role of PilZ domain proteins in virulence and the regulation of virulence factor synthesis in Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot of crucifers. The Xcc genome encodes four proteins (XC0965, XC2249, XC2317 and XC3221) that have a PilZ domain. Mutation of XC0965, XC2249 and XC3221 led to a significant reduction of virulence in Chinese radish. Mutation of XC2249 and XC3221 led to a reduction in motility whereas mutation of XC2249 and XC0965 affected extracellular enzyme production. All mutant strains were unaffected in biofilm formation in vitro. The reduction of virulence following mutation of XC3221 could not be wholly attributed to an effect on motility as mutation of pilA, which abolishes motility, has a lesser effect on virulence. PMID:19019010

  2. The Xanthomonas oryzae pv. oryzae PilZ Domain Proteins Function Differentially in Cyclic di-GMP Binding and Regulation of Virulence and Motility

    PubMed Central

    Yang, Fenghuan; Tian, Fang; Chen, Huamin; Hutchins, William; Yang, Ching-Hong

    2015-01-01

    The PilZ domain proteins have been demonstrated to be one of the major types of receptors mediating cyclic di-GMP (c-di-GMP) signaling pathways in several pathogenic bacteria. However, little is known about the function of PilZ domain proteins in c-di-GMP regulation of virulence in the bacterial blight pathogen of rice Xanthomonas oryzae pv. oryzae. Here, the roles of PilZ domain proteins PXO_00049 and PXO_02374 in c-di-GMP binding, regulation of virulence and motility, and subcellular localization were characterized in comparison with PXO_02715, identified previously as an interactor with the c-di-GMP receptor Filp to regulate virulence. The c-di-GMP binding motifs in the PilZ domains were conserved in PXO_00049 and PXO_02374 but were less well conserved in PXO_02715. PXO_00049 and PXO_02374 but not PXO_02715 proteins bound to c-di-GMP with high affinity in vitro, and the R141 and R10 residues in the PilZ domains of PXO_00049 and PXO_02374, respectively, were crucial for c-di-GMP binding. Gene deletion of PXO_00049 and PXO_02374 resulted in significant increases in virulence and hrp gene transcription, indicating their negative regulation of virulence via type III secretion system expression. All mutants showed significant changes in sliding motility but not exopolysaccharide production and biofilm formation. In trans expression of the full-length open reading frame (ORF) of each gene in the relevant mutants led to restoration of the phenotype to wild-type levels. Moreover, PXO_00049 and PXO_02374 displayed mainly multisite subcellular localizations, whereas PXO_02715 showed nonpolar distributions in the X. oryzae pv. oryzae cells. Therefore, this study demonstrated the different functions of the PilZ domain proteins in mediation of c-di-GMP regulation of virulence and motility in X. oryzae pv. oryzae. PMID:25911481

  3. The Structural Basis of Cyclic Diguanylate Signal Transduction by PilZ Domains

    SciTech Connect

    Benach,J.; Swaminathan, S.; Tamayo, R.; Handelman, S.; Folta-Stogniew, E.; Ramos, J.; Forouhar, F.; Neely, H.; Seetharaman, J.; et al

    2007-01-01

    The second messenger cyclic diguanylate (c-di-GMP) controls the transition between motile and sessile growth in eubacteria, but little is known about the proteins that sense its concentration. Bioinformatics analyses suggested that PilZ domains bind c-di-GMP and allosterically modulate effector pathways. We have determined a 1.9 Angstroms crystal structure of c-di-GMP bound to VCA0042/PlzD, a PilZ domain-containing protein from Vibrio cholerae. Either this protein or another specific PilZ domain-containing protein is required for V. cholerae to efficiently infect mice. VCA0042/PlzD comprises a C-terminal PilZ domain plus an N-terminal domain with a similar beta-barrel fold. C-di-GMP contacts seven of the nine strongly conserved residues in the PilZ domain, including three in a seven-residue long N-terminal loop that undergoes a conformational switch as it wraps around c-di-GMP. This switch brings the PilZ domain into close apposition with the N-terminal domain, forming a new allosteric interaction surface that spans these domains and the c-di-GMP at their interface. The very small size of the N-terminal conformational switch is likely to explain the facile evolutionary diversification of the PilZ domain.

  4. Identification of flgZ as a Flagellar Gene Encoding a PilZ Domain Protein That Regulates Swimming Motility and Biofilm Formation in Pseudomonas

    PubMed Central

    Redondo-Nieto, Miguel; González de Heredia, Elena; Baena, Irene; Martín-Martín, Irene; Rivilla, Rafael; Martín, Marta

    2014-01-01

    Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC) and one phosphodiesterase (BifA) implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ. PMID:24504373

  5. Functional divergence of FimX in PilZ binding and type IV pilus regulation.

    PubMed

    Qi, Yaning; Xu, Linghui; Dong, Xueming; Yau, Yin Hoe; Ho, Chun Loong; Koh, Siew Lee; Shochat, Susana Geifman; Chou, Shan-Ho; Tang, Kai; Liang, Zhao-Xun

    2012-11-01

    Type IV pili (T4P) are polar surface structures that play important roles in bacterial motility, biofilm formation, and pathogenicity. The protein FimX and its orthologs are known to mediate T4P formation in the human pathogen Pseudomonas aeruginosa and some other bacterial species. It was reported recently that FimX(XAC2398) from Xanthomonas axonopodis pv. citri interacts with PilZ(XAC1133) directly through the nonenzymatic EAL domain of FimX(XAC2398). Here we present experimental data to reveal that the strong interaction between FimX(XAC2398) and PilZ(XAC1133) is not conserved in P. aeruginosa and likely other Pseudomonas species. In vitro and in vivo binding experiments showed that the interaction between FimX and PilZ in P. aeruginosa is below the measurable limit. Surface plasmon resonance assays further confirmed that the interaction between the P. aeruginosa proteins is at least more than 3 orders of magnitude weaker than that between the X. axonopodis pv. citri pair. The N-terminal lobe region of FimX(XAC2398) was identified as the binding surface for PilZ(XAC1133) by amide hydrogen-deuterium exchange and site-directed mutagenesis studies. Lack of several key residues in the N-terminal lobe region of the EAL domain of FimX is likely to account for the greatly reduced binding affinity between FimX and PilZ in P. aeruginosa. All together, the results suggest that the interaction between PilZ and FimX in Xanthomonas species is not conserved in P. aeruginosa due to the evolutionary divergence among the FimX orthologs. The precise roles of FimX and PilZ in bacterial motility and T4P biogenesis are likely to vary among bacterial species. PMID:22942245

  6. 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…

  7. 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.

  8. Cellulose binding domain proteins

    DOEpatents

    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.

  9. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    PubMed

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein. PMID:27231351

  10. 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.

  11. Cellulose binding domain fusion proteins

    DOEpatents

    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.

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

    PubMed Central

    Khor, Susan

    2014-01-01

    Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where the score of a domain-pair is the ratio between observed and expected frequencies because the protein-protein interaction network is sparse. As such, many protein-pairs will be non-interacting and domain-pairs with promiscuous domains will be penalized. This domain promiscuity challenge to the problem of inferring reliable domain-domain interactions from protein-protein interactions has been recognized, and a number of work-arounds have been proposed. This paper reports on an application of Formal Concept Analysis to this problem. It is found that the relationship between formal concepts provides a natural way for rare domains to elevate the rank of promiscuous domain-pairs and enrich highly ranked domain-pairs with reliable domain-domain interactions. This piggybacking of promiscuous domain-pairs onto less promiscuous domain-pairs is possible only with concept lattices whose attribute-labels are not reduced and is enhanced by the presence of proteins that comprise both promiscuous and rare domains. PMID:24586450

  13. Analysis of multi-domain protein dynamics

    PubMed Central

    Roy, Amitava; Hua, Duy P; Post, Carol Beth

    2016-01-01

    Proteins with a modular architecture of multiple domains connected by linkers often exhibit diversity in the relative positions of domains while the domain tertiary structure remains unchanged. The biological function of these modular proteins, or the regulation of their activity depends on the variation in domain orientation and separation. Accordingly, careful characterization of inter-domain motion and correlated fluctuations of multi-domain systems is relevant for understanding the functional behavior of modular proteins. Molecular dynamics (MD) simulations provides a powerful approach to study these motions in atomic detail. Nevertheless, the common procedure for analyzing fluctuations from MD simulations after overall rigid-body alignment fails for multi-domain proteins; it greatly overestimates correlated positional fluctuations in the presence of relative domain motion. We show here that expressing the atomic motions of a multi-domain protein as a combination of displacement within the domain reference frame and motion of the relative domains correctly separates the internal motions to allow a useful description of correlated fluctuations. We illustrate the methodology of separating the domain fluctuations and local fluctuations by application to the tandem SH2 domains of human Syk protein kinase and by characterizing an effect of phosphorylation on the dynamics. Correlated motions are assessed from a distance covariance rather than the more common vector-coordinate covariance. The approach makes it possible to calculate the proper correlations in fluctuations internal to a domain as well as between domains. PMID:26675644

  14. Protein structural domains: definition and prediction.

    PubMed

    Ezkurdia, Iakes; Tress, Michael L

    2011-11-01

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

  15. Domains mediate protein-protein interactions and nucleate protein assemblies.

    PubMed

    Costa, S; Cesareni, G

    2008-01-01

    Cell physiology is governed by an intricate mesh of physical and functional links among proteins, nucleic acids and other metabolites. The recent information flood coming from large-scale genomic and proteomic approaches allows us to foresee the possibility of compiling an exhaustive list of the molecules present within a cell, enriched with quantitative information on concentration and cellular localization. Moreover, several high-throughput experimental and computational techniques have been devised to map all the protein interactions occurring in a living cell. So far, such maps have been drawn as graphs where nodes represent proteins and edges represent interactions. However, this representation does not take into account the intrinsically modular nature of proteins and thus fails in providing an effective description of the determinants of binding. Since proteins are composed of domains that often confer on proteins their binding capabilities, a more informative description of the interaction network would detail, for each pair of interacting proteins in the network, which domains mediate the binding. Understanding how protein domains combine to mediate protein interactions would allow one to add important features to the protein interaction network, making it possible to discriminate between simultaneously occurring and mutually exclusive interactions. This objective can be achieved by experimentally characterizing domain recognition specificity or by analyzing the frequency of co-occurring domains in proteins that do interact. Such approaches allow gaining insights on the topology of complexes with unknown three-dimensional structure, thus opening the prospect of adopting a more rational strategy in developing drugs designed to selectively target specific protein interactions. PMID:18491061

  16. Enhanced protein domain discovery using taxonomy

    PubMed Central

    Coin, Lachlan; Bateman, Alex; Durbin, Richard

    2004-01-01

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

  17. Diversity in protein recognition by PTB domains.

    PubMed

    Forman-Kay, J D; Pawson, T

    1999-12-01

    Phosphotyrosine-binding (PTB) domains were originally identified as modular domains that recognize phosphorylated Asn-Pro-Xxx-p Tyr-containing proteins. Recent binding and structural studies of PTB domain complexes with target peptides have revealed a number of deviations from the previously described mode of interaction, with respect to both the sequences of possible targets and their structures within the complexes. This diversity of recognition by PTB domains extends and strengthens our general understanding of modular binding domain recognition. PMID:10607674

  18. Immunosilencing a Highly Immunogenic Protein Trimerization Domain*

    PubMed Central

    Sliepen, Kwinten; van Montfort, Thijs; Melchers, Mark; Isik, Gözde; Sanders, Rogier W.

    2015-01-01

    Many therapeutic proteins and protein subunit vaccines contain heterologous trimerization domains, such as the widely used GCN4-based isoleucine zipper (IZ) and the T4 bacteriophage fibritin foldon (Fd) trimerization domains. We found that these domains induced potent anti-IZ or anti-Fd antibody responses in animals when fused to an HIV-1 envelope glycoprotein (Env) immunogen. To dampen IZ-induced responses, we constructed an IZ domain containing four N-linked glycans (IZN4) to shield the underlying protein surface. When fused to two different vaccine antigens, HIV-1 Env and influenza hemagglutinin (HA), IZN4 strongly reduced the antibody responses against the IZ, but did not affect the antibody titers against Env or HA. Silencing of immunogenic multimerization domains with glycans might be relevant for therapeutic proteins and protein vaccines. PMID:25635058

  19. Domain swapping: entangling alliances between proteins.

    PubMed Central

    Bennett, M J; Choe, S; Eisenberg, D

    1994-01-01

    The comparison of monomeric and dimeric diphtheria toxin (DT) reveals a mode for protein association which we call domain swapping. The structure of dimeric DT has been extensively refined against data to 2.0-A resolution and a three-residue loop has been corrected as compared with our published 2.5-A-resolution structure. The monomeric DT structure has also been determined, at 2.3-A resolution. Monomeric DT is a Y-shaped molecule with three domains: catalytic (C), transmembrane (T), and receptor binding (R). Upon freezing in phosphate buffer, DT forms a long-lived, metastable dimer. The protein chain tracing discloses that upon dimerization an unprecedented conformational rearrangement occurs: the entire R domain from each molecule of the dimer is exchanged for the R domain from the other. This involves breaking the noncovalent interactions between the R domain and the C and T domains, rotating the R domain by 180 degrees with atomic movements up to 65 A, and re-forming the same noncovalent interactions between the R domain and the C and T domains of the other chain of the dimer. This conformational transition explains the long life and metastability of the DT dimer. Several other intertwined, dimeric protein structures satisfy our definition of domain swapping and suggest that domain swapping may be the molecular mechanism for evolution of these oligomers and possibly of oligomeric proteins in general. Images PMID:8159715

  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. PMID:15777630

  1. Tuning Protein Autoinhibition by Domain Destabilization

    PubMed Central

    Cho, Jae-Hyun; Muralidharan, Vasant; Vila-Perello, Miquel; Raleigh, Daniel P.; Muir, Tom W.; Palmer, Arthur G.

    2012-01-01

    Activation of many multi-domain signaling proteins requires rearrangement of autoinhibitory interdomain interactions that occlude activator binding sites. In one model for activation, the major inactive conformation exists in equilibrium with activated-like conformations that can be stabilized by ligand binding or post-translational modifications. The molecular basis for this model is established for the archetypal signaling adapter protein Crk-II by measuring the thermodynamics and kinetics of the equilibrium between autoinhibited and activated-like states using fluorescence and NMR spectroscopies, together with segmental isotopic labeling via expressed protein ligation. The results demonstrate that intramolecular domain-domain interactions both stabilize the autoinhibited state and induce the activated-like conformation. A combination of favorable interdomain interactions and unfavorable intradomain structural changes fine-tunes the population of the activated-like conformation and allows facile response to activators. This mechanism suggests a general strategy for optimization of autoinhibitory interactions of multi-domain proteins. PMID:21532593

  2. Biodiversity of voltage sensor domain proteins.

    PubMed

    Okamura, Yasushi

    2007-06-01

    The six-transmembrane type voltage-gated ion channels play an essential role in neuronal excitability, muscle contraction, and secretion. The voltage sensor domain (VSD) is the key element of voltage-gated ion channels for sensing transmembrane potential, and has been studied at the levels of both biophysics and protein structure. Two recently identified proteins containing VSD without a pore domain showed unexpected biological roles: regulation of phosphatase activity and proton permeation. These proteins not only provide novel platforms to understand mechanisms of voltage sensing and ion permeation but also highlight previously unappreciated roles of membrane potential in non-neuronal cells. PMID:17347852

  3. 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. PMID:27309309

  4. ECOD: An Evolutionary Classification of Protein Domains

    PubMed Central

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

    2014-01-01

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

  5. Protein Domain Decomposition Using a Graph-Theoretic Approach

    SciTech Connect

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

    2000-08-20

    This paper presents a new algorithm for the decomposition of a multi-domain protein into individual structural domains. The underlying principle used is that residue-residue contacts are denser within a domain than between domains.

  6. MBT domain proteins in development and disease

    PubMed Central

    Bonasio, Roberto; Lecona, Emilio; Reinberg, Danny

    2013-01-01

    The Malignant Brain Tumor (MBT) domain is a “chromatin reader”, a protein module that binds to post-translational modifications on histone tails that are thought to affect a variety of chromatin processes, including transcription. More specifically, MBT domains recognize mono- and di-methylated lysines at a number of different positions on histone H3 and H4 tails. Three Drosophila proteins, SCM, L(3)MBT and SFMBT contain multiple adjacent MBT repeats and have critical roles in development, maintenance of cell identity, and tumor suppression. Although they function in different pathways, these proteins all localize to chromatin in vivo and repress transcription by a currently unknown molecular mechanism that requires the MBT domains. The human genome contains several homologues of these MBT proteins, some of which have been linked to important gene regulatory pathways, such as E2F/Rb- and Polycomb-mediated repression, and to the insurgence of certain neurological tumors. Here, we review the genetics, biochemistry, and cell biology of MBT proteins and their role in development and disease. PMID:19778625

  7. 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. PMID:26434392

  8. EH domain proteins regulate cardiac membrane protein targeting

    PubMed Central

    Gudmundsson, Hjalti; Hund, Thomas J.; Wright, Patrick J.; Kline, Crystal F.; Snyder, Jedidiah S.; Qian, Lan; Koval, Olha M.; Cunha, Shane R.; George, Manju; Rainey, Mark A.; Kashef, Farshid E.; Dun, Wen; Boyden, Penelope A.; Anderson, Mark E.; Band, Hamid; Mohler, Peter J.

    2010-01-01

    Rationale Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules. Membrane protein dynamics in health and disease are maintained by a complex ensemble of intracellular targeting, scaffolding, recycling, and degradation pathways. Surprisingly, despite decades of research linking dysfunction in membrane protein trafficking with human cardiovascular disease, essentially nothing is known regarding the molecular identity or function of these intracellular targeting pathways in excitable cardiomyocytes. Objective We sought to discover novel pathways for membrane protein targeting in primary cardiomyocytes. Methods and Results We report the initial characterization of a large family of membrane trafficking proteins in human heart. We employed a tissue-wide screen for novel ankyrin-associated trafficking proteins and identified four members of a unique Eps15 homology (EH) domain-containing protein family (EHD1, EHD2, EHD3, EHD4) that serve critical roles in endosome-based membrane protein targeting in other cell types. We show that EHD1-4 directly associate with ankyrin, provide the first information on the expression and localization of these molecules in primary cardiomyocytes, and demonstrate that EHD1-4 are co-expressed with ankyrin-B in the myocyte perinuclear region. Notably, the expression of multiple EHD proteins is increased in animal models lacking ankyrin-B, and EHD3-deficient cardiomyocytes display aberrant ankyrin-B localization and selective loss of Na/Ca exchanger expression and function. Finally, we report significant modulation of EHD expression following myocardial infarction, suggesting that these proteins may play a key role in regulating membrane excitability in normal and diseased heart. Conclusions Our findings identify and characterize a new class of cardiac trafficking proteins, define the first group of proteins associated with the ankyrin

  9. 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

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

    PubMed Central

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

    2015-01-01

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

  11. 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. PMID:22201747

  12. 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.

  13. Length constraints of multi­domain proteins in metazoans

    PubMed Central

    Middleton, Sarah; Song, Timothy; Nayak, Sudhir

    2010-01-01

    The increasing number of annotated genome sequences in public databases has made it possible to study the length distributions and domain composition of proteins at unprecedented resolution. To identify factors that influence protein length in metazoans, we performed an analysis of all domain­annotated proteins from a total of 49 animal species from Ensembl (v.56) or EnsemblMetazoa (v.3). Our results indicate that protein length constraints are not fixed as a linear function of domain count and can vary based on domain content. The presence of repeating domains was associated with relaxation of the constraints that govern protein length. Conversely, for proteins with unique domains, length constraints were generally maintained with increased domain counts. It is clear that mean (and median) protein length and domain composition vary significantly between metazoans and other kingdoms; however, the connections between function, domain content, and length are unclear. We incorporated Gene Ontology (GO) annotation to identify biological processes, cellular components, or molecular functions that favor the incorporation of multi­domain proteins. Using this approach, we identified multiple GO terms that favor the incorporation of multi-domain proteins; interestingly, several of the GO terms with elevated domain counts were not restricted to a single gene family. The findings presented here represent an important step in resolving the complex relationship between protein length, function, and domain content. The comparison of the data presented in this work to data from other kingdoms is likely to reveal additional differences in the regulation of protein length. PMID:20975906

  14. A non-chromatographic protein purification strategy using Src 3 homology domains as generalized capture domains.

    PubMed

    Kim, Heejae; Chen, Wilfred

    2016-09-20

    Protein purification using inverse phase transition of elastin-like polypeptide (ELP) domains is a useful alternative to chromatography. Genetic fusions of ELP domains to various proteins have the ability to reversibly transition between soluble monomers and micron-sized aggregates and this has been used to selectively purify many ELP fusions. Affinity domains can enhance this technology by using specific protein binding domains to enable ELP mediated affinity capture (EMAC) of proteins of interest (POI) that have been fused to corresponding affinity ligands. In this paper, we highlight the use of Src homology 3 (SH3) domains and corresponding peptide ligands in EMAC that have differential binding affinities towards SH3 for efficient capture and elution of proteins. Furthermore, differences between capture and elution of a monomeric and a multimeric protein were also studied. PMID:27457699

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

    PubMed Central

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

    2012-01-01

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

  16. 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.

  17. Emerging Roles of JmjC Domain-Containing Proteins.

    PubMed

    Accari, Sandra L; Fisher, Paul R

    2015-01-01

    Jumonji C (JmjC) domain-containing proteins are a diverse superfamily of proteins containing a characteristic, evolutionarily conserved β-barrel structure that normally contains binding sites for Fe(II) and α-ketoglutarate. In the best studied JmjC-domain proteins, the JmjC barrel has a histone demethylase catalytic activity. Histones are evolutionarily conserved proteins intimately involved in the packaging of DNA within the nucleus of eukaryotic organisms. The N-termini ("tails") of the histone proteins are subject to a diverse array of posttranslational modifications including methylation. Unlike many of the other histone modifications which are transient, methylation was thought to be permanent, until the relatively recent identification of the first demethylases. Jumonji C domain-containing proteins were first identified with a role in the modulation of histone methylation marks. This family of proteins is broken up into seven distinct subgroups based on domain architecture and their ability to antagonize specific histone methylation marks. Their biological functions derive from their ability to regulate gene expression and include roles in cell differentiation, growth, proliferation, and stress responses. However, one subgroup remains, the largest, in which the JmjC domain has no known biochemical function. These proteins belong to the JmjC-domain-only subgroup and as their name suggests, the only bioinformatically recognizable domain they contain is the highly conserved JmjC domain. PMID:26404469

  18. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  19. Computational Methods for Domain Partitioning of Protein Structures

    NASA Astrophysics Data System (ADS)

    Veretnik, Stella; Shindyalov, Ilya

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

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  2. 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. PMID:26253692

  3. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    PubMed Central

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

    2015-01-01

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

  4. Small protein domains fold inside the ribosome exit tunnel.

    PubMed

    Marino, Jacopo; von Heijne, Gunnar; Beckmann, Roland

    2016-03-01

    Cotranslational folding of small protein domains within the ribosome exit tunnel may be an important cellular strategy to avoid protein misfolding. However, the pathway of cotranslational folding has so far been described only for a few proteins, and therefore, it is unclear whether folding in the ribosome exit tunnel is a common feature for small protein domains. Here, we have analyzed nine small protein domains and determined at which point during translation their folding generates sufficient force on the nascent chain to release translational arrest by the SecM arrest peptide, both in vitro and in live E. coli cells. We find that all nine protein domains initiate folding while still located well within the ribosome exit tunnel. PMID:26879042

  5. Interaction of ion channels and receptors with PDZ domain proteins.

    PubMed

    Kornau, H C; Seeburg, P H; Kennedy, M B

    1997-06-01

    The complex anatomy of neurons demands a high degree of functional organization. Therefore, membrane receptors and ion channels are often localized to selected subcellular sites and coupled to specific signal transduction machineries. PDZ domains have come into focus as protein interaction modules that mediate the binding of a class of submembraneous proteins to membrane receptors and ion channels and thus subserve these organizational aspects. The structures of two PDZ domains have been resolved, which has led to a structural understanding of the specificity of interactions of various PDZ domains with their respective partners. The functional implications of PDZ domain interactions are now being addressed in vitro and in vivo. PMID:9232802

  6. 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

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

    PubMed

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

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  8. Proteasomes and protein conjugation across domains of life

    PubMed Central

    Maupin-Furlow, Julie

    2012-01-01

    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. PMID:22183254

  9. Domain stealing by receptors in a protein transport complex.

    PubMed

    Hulett, Joanne M; Walsh, Peter; Lithgow, Trevor

    2007-09-01

    The mitochondrion is an essential cellular compartment in eukaryotes. The mitochondrial proteins Tom20 and Tom22 are receptors that ensure recognition and binding of proteins imported for mitochondrial biogenesis. Comparison of the sequence for the Tom20 and Tom22 subunits in the yeasts Saccharomyces cerevisiae and Saccharomyces castellii, show a rare case of domain stealing, where in Saccharomyces castellii Tom22 has lost an acidic domain, and Tom20 has gained one. This example of domain stealing is a snapshot of evolution in action and provides excellent evidence that Tom20 and Tom22 are subunits of a single, composite receptor that binds precursor proteins for import into mitochondria. PMID:17586602

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

    PubMed

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

    1999-05-15

    A novel automatic procedure for identifying domains from protein atomic coordinates is presented. The procedure, termed STRUDL (STRUctural Domain Limits), does not take into account information on secondary structures and handles any number of domains made up of contiguous or non-contiguous chain segments. The core algorithm uses the Kernighan-Lin graph heuristic to partition the protein into residue sets which display minimum interactions between them. These interactions are deduced from the weighted Voronoi diagram. The generated partitions are accepted or rejected on the basis of optimized criteria, representing basic expected physical properties of structural domains. The graph heuristic approach is shown to be very effective, it approximates closely the exact solution provided by a branch and bound algorithm for a number of test proteins. In addition, the overall performance of STRUDL is assessed on a set of 787 representative proteins from the Protein Data Bank by comparison to domain definitions in the CATH protein classification. The domains assigned by STRUDL agree with the CATH assignments in at least 81% of the tested proteins. This result is comparable to that obtained previously using PUU (Holm and Sander, Proteins 1994;9:256-268), the only other available algorithm designed to identify domains with any number of non-contiguous chain segments. A detailed discussion of the structures for which our assignments differ from those in CATH brings to light some clear inconsistencies between the concept of structural domains based on minimizing inter-domain interactions and that of delimiting structural motifs that represent acceptable folding topologies or architectures. Considering both concepts as complementary and combining them in a layered approach might be the way forward. PMID:10328269

  11. Sequence and structural analysis of BTB domain proteins

    PubMed Central

    Stogios, Peter J; Downs, Gregory S; Jauhal, Jimmy JS; Nandra, Sukhjeen K; Privé, Gilbert G

    2005-01-01

    Background The BTB domain (also known as the POZ domain) is a versatile protein-protein interaction motif that participates in a wide range of cellular functions, including transcriptional regulation, cytoskeleton dynamics, ion channel assembly and gating, and targeting proteins for ubiquitination. Several BTB domain structures have been experimentally determined, revealing a highly conserved core structure. Results We surveyed the protein architecture, genomic distribution and sequence conservation of BTB domain proteins in 17 fully sequenced eukaryotes. The BTB domain is typically found as a single copy in proteins that contain only one or two other types of domain, and this defines the BTB-zinc finger (BTB-ZF), BTB-BACK-kelch (BBK), voltage-gated potassium channel T1 (T1-Kv), MATH-BTB, BTB-NPH3 and BTB-BACK-PHR (BBP) families of proteins, among others. In contrast, the Skp1 and ElonginC proteins consist almost exclusively of the core BTB fold. There are numerous lineage-specific expansions of BTB proteins, as seen by the relatively large number of BTB-ZF and BBK proteins in vertebrates, MATH-BTB proteins in Caenorhabditis elegans, and BTB-NPH3 proteins in Arabidopsis thaliana. Using the structural homology between Skp1 and the PLZF BTB homodimer, we present a model of a BTB-Cul3 SCF-like E3 ubiquitin ligase complex that shows that the BTB dimer or the T1 tetramer is compatible in this complex. Conclusion Despite widely divergent sequences, the BTB fold is structurally well conserved. The fold has adapted to several different modes of self-association and interactions with non-BTB proteins. PMID:16207353

  12. POU proteins bend DNA via the POU-specific domain.

    PubMed Central

    Verrijzer, C P; van Oosterhout, J A; van Weperen, W W; van der Vliet, P C

    1991-01-01

    POU proteins constitute a family of ubiquitous as well as cell type-specific transcription factors that share the conserved POU DNA binding domain. This domain consists of two distinct subdomains, a POU-specific domain and a POU homeodomain, that are both required for high affinity sequence-specific DNA binding. In a circular permutation assay, several POU proteins, including Oct-1, Oct-2A, Oct-6 and Pit-1, demonstrated a position dependent mobility of the protein-DNA complexes, suggesting induction of DNA bending. This was confirmed by detection of relative bend direction, using pre-bent DNA, and by enhanced ligase mediated cyclization. Bending was caused by interaction with the POU domain. By contrast, binding of the POU homeodomain did not distort the DNA structure, indicating that the POU-specific domain confers DNA bending. Images PMID:1915275

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

    PubMed

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

    2005-01-01

    The Conserved Domain Database (CDD) is the protein classification component of NCBI's Entrez query and retrieval system. CDD is linked to other Entrez databases such as Proteins, Taxonomy and PubMed, and can be accessed at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. CD-Search, which is available at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, is a fast, interactive tool to identify conserved domains in new protein sequences. CD-Search results for protein sequences in Entrez are pre-computed to provide links between proteins and domain models, and computational annotation visible upon request. Protein-protein queries submitted to NCBI's BLAST search service at http://www.ncbi.nlm.nih.gov/BLAST are scanned for the presence of conserved domains by default. While CDD started out as essentially a mirror of publicly available domain alignment collections, such as SMART, Pfam and COG, we have continued an effort to update, and in some cases replace these models with domain hierarchies curated at the NCBI. Here, we report on the progress of the curation effort and associated improvements in the functionality of the CDD information retrieval system. PMID:15608175

  14. The acidic domains of the Toc159 chloroplast preprotein receptor family are intrinsically disordered protein domains

    PubMed Central

    2009-01-01

    Background The Toc159 family of proteins serve as receptors for chloroplast-destined preproteins. They directly bind to transit peptides, and exhibit preprotein substrate selectivity conferred by an unknown mechanism. The Toc159 receptors each include three domains: C-terminal membrane, central GTPase, and N-terminal acidic (A-) domains. Although the function(s) of the A-domain remains largely unknown, the amino acid sequences are most variable within these domains, suggesting they may contribute to the functional specificity of the receptors. Results The physicochemical properties of the A-domains are characteristic of intrinsically disordered proteins (IDPs). Using CD spectroscopy we show that the A-domains of two Arabidopsis Toc159 family members (atToc132 and atToc159) are disordered at physiological pH and temperature and undergo conformational changes at temperature and pH extremes that are characteristic of IDPs. Conclusions Identification of the A-domains as IDPs will be important for determining their precise function(s), and suggests a role in protein-protein interactions, which may explain how these proteins serve as receptors for such a wide variety of preprotein substrates. PMID:20042108

  15. Predicting PDZ domain mediated protein interactions from structure

    PubMed Central

    2013-01-01

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

  16. The ProDom database of protein domain families.

    PubMed Central

    Corpet, F; Gouzy, J; Kahn, D

    1998-01-01

    The ProDom database contains protein domain families generated from the SWISS-PROT database by automated sequence comparisons. It can be searched on the World Wide Web (http://protein.toulouse.inra. fr/prodom.html ) or by E-mail (prodom@toulouse.inra.fr) to study domain arrangements within known families or new proteins. Strong emphasis has been put on the graphical user interface which allows for interactive analysis of protein homology relationships. Recent improvements to the server include: ProDom search by keyword; links to PROSITE and PDB entries; more sensitive ProDom similarity search with BLAST or WU-BLAST; alignments of query sequences with homologous ProDom domain families; and links to the SWISS-MODEL server (http: //www.expasy.ch/swissmod/SWISS-MODEL.html ) for homology based 3-D domain modelling where possible. PMID:9399865

  17. Molekulare Methoden zum Nachweis, zur Quantifizierung und zum Monitoring der Mykotoxinbildung lebensmittelrelevanter Pilze

    NASA Astrophysics Data System (ADS)

    Geisen, Rolf

    Schimmelpilze kommen ubiquitär vor und spielen besonders bei pflanzlichen Lebensmitteln und Rohprodukten eine besondere Rolle als Verderbsorganismen. Es wird geschätzt, dass 20-25 % der jährlichen Produktion an pflanzlichen Produkten durch Schimmelpilze verdorben werden (Smith et al., 1994). Viele der lebensmittelrelevanten Schimmelpilze sind zudem in der Lage, Mykotoxine, toxische Sekundärmetabolite, zu bilden, was das Ausmaß des Problems deutlich macht. Die wichtigsten mykotoxinbildenden Spezies gehören zu den Fusarien (Trichothecene, Fumonisine, Zearalenon), Aspergillen (Aflatoxin, Ochratoxin, Cyclopiazonsäure) und Penicillien (Patulin, Ochratoxin). Für viele Mykotoxine, wie die Aflatoxine, Ochratoxin, Fumonisine und Trichothecene sind Grenzwerte erlassen worden, die die Verkehrsfähigkeit betroffener Produkte regeln. Die Einhaltung der Grenzwerte kann sehr genau durch offizielle chemisch-analytische Methoden, wie HPLC, GC-MS etc. kontrolliert werden. Diese analytischen Methoden sind aber für die Anwendung eines HACCP-Ansatzes zur Kontrolle der Mykotoxinbildung nur bedingt geeignet, da sie Endpunktkontrollen darstellen und nur das über eine längere Zeit gebildete Mykotoxin bestimmen. Sie sagen daher nichts über die biologischen Bedingungen zur Zeit der Bildung durch den Pilz aus.

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

    PubMed Central

    Jernigan, Kristin K.

    2015-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  20. 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. PMID:22046139

  1. Modeling the Evolution of Protein Domain Architectures Using Maximum Parsimony

    PubMed Central

    Fong, Jessica H.; Geer, Lewis Y.; Panchenko, Anna R.; Bryant, Stephen H.

    2007-01-01

    Domains are basic evolutionary units of proteins and most proteins have more than one domain. Advances in domain modeling and collection are making it possible to annotate a large fraction of known protein sequences by a linear ordering of their domains, yielding their architecture. Protein domain architectures link evolutionarily related proteins and underscore their shared functions. Here, we attempt to better understand this association by identifying the evolutionary pathways by which extant architectures may have evolved. We propose a model of evolution in which architectures arise through rearrangements of inferred precursor architectures and acquisition of new domains. These pathways are ranked using a parsimony principle, whereby scenarios requiring the fewest number of independent recombination events, namely fission and fusion operations, are assumed to be more likely. Using a data set of domain architectures present in 159 proteomes that represent all three major branches of the tree of life allows us to estimate the history of over 85% of all architectures in the sequence database. We find that the distribution of rearrangement classes is robust with respect to alternative parsimony rules for inferring the presence of precursor architectures in ancestral species. Analyzing the most parsimonious pathways, we find 87% of architectures to gain complexity over time through simple changes, among which fusion events account for 5.6 times as many architectures as fission. Our results may be used to compute domain architecture similarities, for example, based on the number of historical recombination events separating them. Domain architecture “neighbors” identified in this way may lead to new insights about the evolution of protein function. PMID:17166515

  2. Crystallographic studies on protein misfolding: Domain swapping and amyloid formation in the SH3 domain.

    PubMed

    Cámara-Artigas, Ana

    2016-07-15

    Oligomerization by 3D domain swapping is found in a variety of proteins of diverse size, fold and function. In the early 1960s this phenomenon was postulated for the oligomers of ribonuclease A, but it was not until the 1990s that X-ray diffraction provided the first experimental evidence of this special manner of oligomerization. Nowadays, structural information has allowed the identification of these swapped oligomers in over one hundred proteins. Although the functional relevance of this phenomenon is not clear, this alternative folding of protomers into intertwined oligomers has been related to amyloid formation. Studies on proteins that develop 3D domain swapping might provide some clues on the early stages of amyloid formation. The SH3 domain is a small modular domain that has been used as a model to study the basis of protein folding. Among SH3 domains, the c-Src-SH3 domain emerges as a helpful model to study 3D domain swapping and amyloid formation. PMID:26924596

  3. WAP domain proteins as modulators of mucosal immunity.

    PubMed

    Wilkinson, Thomas S; Roghanian, Ali; Simpson, Alexander John; Sallenave, Jean-Michel

    2011-10-01

    WAP (whey acidic protein) is an important whey protein present in milk of mammals. This protein has characteristic domains, rich in cysteine residues, called 4-DSC (four-disulfide core domain). Other proteins, mainly present at mucosal surfaces, have been shown to also possess these characteristic WAP-4-DSC domains. The present review will focus on two WAP-4-DSC containing proteins, namely SLPI (secretory leucocyte protease inhibitor) and trappin-2/elafin. Although first described as antiproteases able to inhibit in particular host neutrophil proteases [NE (neutrophil elastase), cathepsin-G and proteinase-3] and as such, able to limit maladaptive tissue damage during inflammation, it has become apparent that these molecules have a variety of other functions (direct antimicrobial activity, bacterial opsonization, induction of adaptive immune responses, promotion of tissue repair, etc.). After providing information about the 'classical' antiproteasic role of these molecules, we will discuss the evidence pertaining to their pleiotropic functions in inflammation and immunity. PMID:21936824

  4. Giant protein kinases: domain interactions and structural basis of autoregulation.

    PubMed Central

    Kobe, B; Heierhorst, J; Feil, S C; Parker, M W; Benian, G M; Weiss, K R; Kemp, B E

    1996-01-01

    The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I. Images PMID:9003756

  5. 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

  6. LUD, a new protein domain associated with lactate utilization

    PubMed Central

    2013-01-01

    Background A novel highly conserved protein domain, DUF162 [Pfam: PF02589], can be mapped to two proteins: LutB and LutC. Both proteins are encoded by a highly conserved LutABC operon, which has been implicated in lactate utilization in bacteria. Based on our analysis of its sequence, structure, and recent experimental evidence reported by other groups, we hereby redefine DUF162 as the LUD domain family. Results JCSG solved the first crystal structure [PDB:2G40] from the LUD domain family: LutC protein, encoded by ORF DR_1909, of Deinococcus radiodurans. LutC shares features with domains in the functionally diverse ISOCOT superfamily. We have observed that the LUD domain has an increased abundance in the human gut microbiome. Conclusions We propose a model for the substrate and cofactor binding and regulation in LUD domain. The significance of LUD-containing proteins in the human gut microbiome, and the implication of lactate metabolism in the radiation-resistance of Deinococcus radiodurans are discussed. PMID:24274019

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

    PubMed Central

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

    2011-01-01

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

  8. The BTB domains of the potassium channel tetramerization domain proteins prevalently assume pentameric states.

    PubMed

    Smaldone, Giovanni; Pirone, Luciano; Pedone, Emilia; Marlovits, Thomas; Vitagliano, Luigi; Ciccarelli, Luciano

    2016-06-01

    Potassium channel tetramerization domain-containing (KCTD) proteins are involved in fundamental physio-pathological processes. Here, we report an analysis of the oligomeric state of the Bric-à-brack, Tram-track, Broad complex (BTB) domains of seven distinct KCTDs belonging to five major clades of the family evolution tree. Despite their functional and sequence variability, present electron microscopy data highlight the occurrence of well-defined pentameric states for all domains. Our data also show that these states coexist with alternative forms which include open pentamers. Thermal denaturation analyses conducted using KCTD1 as a model suggest that, in these proteins, different domains cooperate to their overall stability. Finally, negative-stain electron micrographs of KCTD6(BTB) in complex with Cullin3 show the presence of assemblies with a five-pointed pinwheel shape. PMID:27152988

  9. A duplicated motif controls assembly of zona pellucida domain proteins

    NASA Astrophysics Data System (ADS)

    Jovine, Luca; Qi, Huayu; Williams, Zev; Litscher, Eveline S.; Wassarman, Paul M.

    2004-04-01

    Many secreted eukaryotic glycoproteins that play fundamental roles in development, hearing, immunity, and cancer polymerize into filaments and extracellular matrices through zona pellucida (ZP) domains. ZP domain proteins are synthesized as precursors containing C-terminal propeptides that are cleaved at conserved sites. However, the consequences of this processing and the mechanism by which nascent proteins assemble are unclear. By microinjection of mutated DNA constructs into growing oocytes and mammalian cell transfection, we have identified a conserved duplicated motif [EHP (external hydrophobic patch)/IHP (internal hydrophobic patch)] regulating the assembly of mouse ZP proteins. Whereas the transmembrane domain (TMD) of ZP3 can be functionally replaced by an unrelated TMD, mutations in either EHP or IHP do not hinder secretion of full-length ZP3 but completely abolish its assembly. Because mutants truncated before the TMD are not processed, we conclude that the conserved TMD of mammalian ZP proteins does not engage them in specific interactions but is essential for C-terminal processing. Cleavage of ZP precursors results in loss of the EHP, thereby activating secreted polypeptides to assemble by using the IHP within the ZP domain. Taken together, these findings suggest a general mechanism for assembly of ZP domain proteins.

  10. 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. PMID:22383259

  11. 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

  12. Thioaptamers Targeting Dengue Virus Type-2 Envelope Protein Domain III

    PubMed Central

    Gandham, Sai Hari A.; Volk, David E.; Rao, Lokesh G. L.; Neerathilingam, Muniasamy; Gorenstein, David G.

    2014-01-01

    Thioaptamers targeting the dengue-2 virus (DENV-2) envelope protein domain III (EDIII) were developed. EDIII, which contains epitopes for binding neutralizing antibodies, is the putative host-receptor binding domain and is thus an attractive target for development of vaccines, anti-viral therapeutic and diagnostic agents. Thioaptamer DENTA-1 bound to DENV-2 EDIII adjacent to a known neutralizing antibody binding site with a dissociation constant of 154 nM. PMID:25261724

  13. 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.

  14. Pan-Cancer Analysis of Mutation Hotspots in Protein Domains.

    PubMed

    Miller, Martin L; Reznik, Ed; Gauthier, Nicholas P; Aksoy, Bülent Arman; Korkut, Anil; Gao, Jianjiong; Ciriello, Giovanni; Schultz, Nikolaus; Sander, Chris

    2015-09-23

    In cancer genomics, recurrence of mutations in independent tumor samples is a strong indicator of functional impact. However, rare functional mutations can escape detection by recurrence analysis owing to lack of statistical power. We enhance statistical power by extending the notion of recurrence of mutations from single genes to gene families that share homologous protein domains. Domain mutation analysis also sharpens the functional interpretation of the impact of mutations, as domains more succinctly embody function than entire genes. By mapping mutations in 22 different tumor types to equivalent positions in multiple sequence alignments of domains, we confirm well-known functional mutation hotspots, identify uncharacterized rare variants in one gene that are equivalent to well-characterized mutations in another gene, detect previously unknown mutation hotspots, and provide hypotheses about molecular mechanisms and downstream effects of domain mutations. With the rapid expansion of cancer genomics projects, protein domain hotspot analysis will likely provide many more leads linking mutations in proteins to the cancer phenotype. PMID:27135912

  15. 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

  16. The mammalian autophagy initiator complex contains 2 HORMA domain proteins

    PubMed Central

    Michel, Max; Schwarten, Melanie; Decker, Christina; Nagel-Steger, Luitgard; Willbold, Dieter; Weiergräber, Oliver H

    2015-01-01

    ATG101 is an essential component of the ULK complex responsible for initiating cellular autophagy in mammalian cells; its 3-dimensional structure and molecular function, however, are currently unclear. Here we present the X-ray structure of human ATG101. The protein displays an open HORMA domain fold. Both structural properties and biophysical evidence indicate that ATG101 is locked in this conformation, in contrast to the prototypical HORMA domain protein MAD2. Moreover, we discuss a potential mode of dimerization with ATG13 as a fundamental aspect of ATG101 function. PMID:26236954

  17. 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

  18. TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

    PubMed Central

    Reglińska-Matveyev, Natalia; Andersson, Helena M.; Rezende, Suely M.; Dahlbäck, Björn; Crawley, James T. B.; Lane, David A.; Ahnström, Josefin

    2014-01-01

    Protein S is a cofactor for tissue factor pathway inhibitor (TFPI), accelerating the inhibition of activated factor X (FXa). TFPI Kunitz domain 3 residue Glu226 is essential for enhancement of TFPI by protein S. To investigate the complementary functional interaction site on protein S, we screened 44 protein S point, composite or domain swap variants spanning the whole protein S molecule for their TFPI cofactor function using a thrombin generation assay. Of these variants, two protein S/growth arrest–specific 6 chimeras, with either the whole sex hormone–binding globulin (SHBG)-like domain (Val243-Ser635; chimera III) or the SHBG laminin G-type 1 subunit (Ser283-Val459; chimera I), respectively, substituted by the corresponding domain in growth arrest–specific 6, were unable to enhance TFPI. The importance of the protein S SHBG-like domain (and its laminin G-type 1 subunit) for binding and enhancement of TFPI was confirmed in FXa inhibition assays and using surface plasmon resonance. In addition, protein S bound to C4b binding protein showed greatly reduced enhancement of TFPI-mediated inhibition of FXa compared with free protein S. We show that binding of TFPI to the protein S SHBG-like domain enables TFPI to interact optimally with FXa on a phospholipid membrane. PMID:24740810

  19. Fused protein domains inhibit DNA binding by LexA.

    PubMed Central

    Golemis, E A; Brent, R

    1992-01-01

    Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions. Images PMID:1620111

  20. Effective Moment Feature Vectors for Protein Domain Structures

    PubMed Central

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

    2013-01-01

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

  1. Allosteric switching by mutually exclusive folding of protein domains.

    PubMed

    Radley, Tracy L; Markowska, Anna I; Bettinger, Blaine T; Ha, Jeung-Hoi; Loh, Stewart N

    2003-09-19

    Many proteins are built from structurally and functionally distinct domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction. PMID:12963365

  2. BAG4/SODD Protein Contains a Short BAG Domain

    SciTech Connect

    Briknarova, Klara; Takayama, Shinichi; Homma, Sachiko; Baker, Kelly; Cabezas, Edelmira; Hoyt, David W.; Li, Zhen; Satterthwait, Arnold C.; Ely, Kathryn R.

    2002-08-23

    BAG proteins are molecular chaperone regulators that affect diverse cellular pathways. All members share a conserved motif, called the ''BAG domain'' (BD), which binds to Hsp70/Hsc70 family proteins and modulates their activity. We have determined the solution structure of BD from BAG4/SODD (Bcl-2 ? Associated Athanogene / Silencer of Death Domains) by multidimensional nuclear magnetic resonance methods and compared it to the corresponding domain in BAG1 (Briknarova et al., Nature Struct. Biol. 8:349-352). The difference between BDs from these two BAG proteins is striking and the structural comparison defines two subfamilies of mammalian BD-containing proteins. One subfamily includes the closely related BAG3, BAG4 and BAG5 proteins, and the other is represented by BAG1 which contains a structurally and evolutionarily distinct BD. BDs from both BAG1 and BAG4 are three-helix bundles; however, in BAG4, each helix in this bundle is three to four turns shorter than its counterpart in BAG1, which reduces the length of the domain by one-third. BAG4 BD thus represents a prototype of the minimal functional fragment that is capable of binding to Hsc70 and modulating its chaperone activity.

  3. Structural domains and main-chain flexibility in prion proteins.

    PubMed

    Blinov, N; Berjanskii, M; Wishart, D S; Stepanova, M

    2009-02-24

    In this study we describe a novel approach to define structural domains and to characterize the local flexibility in both human and chicken prion proteins. The approach we use is based on a comprehensive theory of collective dynamics in proteins that was recently developed. This method determines the essential collective coordinates, which can be found from molecular dynamics trajectories via principal component analysis. Under this particular framework, we are able to identify the domains where atoms move coherently while at the same time to determine the local main-chain flexibility for each residue. We have verified this approach by comparing our results for the predicted dynamic domain systems with the computed main-chain flexibility profiles and the NMR-derived random coil indexes for human and chicken prion proteins. The three sets of data show excellent agreement. Additionally, we demonstrate that the dynamic domains calculated in this fashion provide a highly sensitive measure of protein collective structure and dynamics. Furthermore, such an analysis is capable of revealing structural and dynamic properties of proteins that are inaccessible to the conventional assessment of secondary structure. Using the collective dynamic simulation approach described here along with a high-temperature simulations of unfolding of human prion protein, we have explored whether locations of relatively low stability could be identified where the unfolding process could potentially be facilitated. According to our analysis, the locations of relatively low stability may be associated with the beta-sheet formed by strands S1 and S2 and the adjacent loops, whereas helix HC appears to be a relatively stable part of the protein. We suggest that this kind of structural analysis may provide a useful background for a more quantitative assessment of potential routes of spontaneous misfolding in prion proteins. PMID:19178154

  4. 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. PMID:26813519

  5. Methods of use of cellulose binding domain proteins

    DOEpatents

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

    1997-09-23

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

  6. Methods of use of cellulose binding domain proteins

    DOEpatents

    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.

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

    PubMed

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

    2002-03-01

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

  8. The nuclear envelope LEM-domain protein emerin

    PubMed Central

    Berk, Jason M; Tifft, Kathryn E; Wilson, Katherine L

    2013-01-01

    Emerin, a conserved LEM-domain protein, is among the few nuclear membrane proteins for which extensive basic knowledge—biochemistry, partners, functions, localizations, posttranslational regulation, roles in development and links to human disease—is available. This review summarizes emerin and its emerging roles in nuclear “lamina” structure, chromatin tethering, gene regulation, mitosis, nuclear assembly, development, signaling and mechano-transduction. We also highlight many open questions, exploration of which will be critical to understand how this intriguing nuclear membrane protein and its “family” influence the genome. PMID:23873439

  9. 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.

  10. Cooperation of phosphoinositides and BAR domain proteins in endosomal tubulation.

    PubMed

    Shinozaki-Narikawa, Naeko; Kodama, Tatsuhiko; Shibasaki, Yoshikazu

    2006-11-01

    Phosphorylated derivatives of phosphatidylinositol (PtdIns) regulate many intracellular events, including vesicular trafficking and actin remodeling, by recruiting proteins to their sites of function. PtdIns(4,5)-bisphosphate [PI(4,5)P2] and related phosphoinositides are mainly synthesized by type I PtdIns-4-phosphate 5-kinases (PIP5Ks). We found that PIP5K induces endosomal tubules in COS-7 cells. ADP-ribosylation factor (ARF) 6 has been shown to act upstream of PIP5K and regulate endocytic transport and tubulation. ARF GAP with coiled-coil, ankyrin repeat, and pleckstrin homology domains 1 (ACAP1) has guanosine triphosphatase-activating protein (GAP) activity for ARF6. While there were few tubules induced by the expression of ACAP1 alone, numerous endosomal tubules were induced by coexpression of PIP5K and ACAP1. ACAP1 has a pleckstrin homology (PH) domain known to bind phosphoinositide and a Bin/amphiphysin/Rvs (BAR) domain that has been reported to detect membrane curvature. Truncated and point mutations in the ACAP1 BAR and PH domains revealed that both BAR and PH domains are required for tubulation. These results suggest that two ARF6 downstream molecules, PIP5K and ACAP1, function together in endosomal tubulation and that phosphoinositide levels may regulate endosomal dynamics. PMID:17010122

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2015-03-27

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

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

    PubMed

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

    2008-05-01

    Formation of rafts and other domains in cell membranes is considered as wetting of proteins by lipids. The membrane is modeled as a continuous elastic medium. Thermodynamic functions of the lipid films that wet proteins are calculated using a mean-field theory of liquid crystals as adapted to biomembranes. This approach yields the conditions necessary for a macroscopic wetting film to form; its thickness could also be determined. It is shown that films of macroscopic thicknesses form around large (tens nanometers in diameter) lipid-protein aggregates; only thin adsorption films form around single proteins or small complexes. The means by which wetting films can facilitate the merger of these aggregates is considered. It is shown that a wetting film prevents a protein from leaving an aggregate. Using experimentally derived values of elastic moduli and spontaneous curvatures as well as height mismatch between aggregates and bulk membrane, we obtained numerical results, which can be compared with the experimental data. PMID:18643096

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

    PubMed Central

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

    2016-01-01

    Summary: The TOPDOM database—originally created as a collection of domains and motifs located consistently on the same side of the membranes in α-helical transmembrane proteins—has been updated and extended by taking into consideration consistently localized domains and motifs in globular proteins, too. By taking advantage of the recently developed CCTOP algorithm to determine the type of a protein and predict topology in case of transmembrane proteins, and by applying a thorough search for domains and motifs as well as utilizing the most up-to-date version of all source databases, we managed to reach a 6-fold increase in the size of the whole database and a 2-fold increase in the number of transmembrane proteins. Availability and implementation: TOPDOM database is available at http://topdom.enzim.hu. The webpage utilizes the common Apache, PHP5 and MySQL software to provide the user interface for accessing and searching the database. The database itself is generated on a high performance computer. Contact: tusnady.gabor@ttk.mta.hu. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153630

  16. 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

  17. A strategy for shuffling numerous Bacillus thuringiensis crystal protein domains.

    PubMed

    Knight, Jacqueline S; Broadwell, Andrew H; Grant, Warwick N; Shoemaker, Charles B

    2004-12-01

    Bacillus thuringiensis that produce Cry1Ba are toxic to Lucilia cuprina Wiedemann blow fly maggots in vivo, and when applied in quantity to sheep fleece, provide up to 6 wk protection against flystrike in the field. These strains also are toxic to Epiphyas postvittana (Walker) light brown apple moth caterpillars. B. thuringiensis expressing Cry1Db are toxic only to E. postvittana. When Cry1Ba and Cry1Db proteins are expressed within Escherichia coli, the recombinant bacteria have the same toxicity profile as the wild-type B. thuringiensis strain. In an effort to develop a Cry protein with improved blow fly toxicity, three different internal regions of Cry1Ba coding DNA, encoding all or part of domains I, II and III respectively were systematically exchanged with the corresponding region from a pool of other Cry protein coding DNAs. The chimeric products were then expressed in recombinant E. coli, and the resulting bacteria assayed for toxicity on L. cuprina and E. postvittana. Clones having insecticide bioactivity were characterized to identify the source of the replacement Cry domain. Despite successfully expressing a large number and variety of chimeric proteins within E. coli, many with measurable insecticidal activity, none of the chimeras had greater potency against L. cuprina than the wild-type Cry1Ba. Chimeric replacements involving domains I and II were rarely active, whereas a much higher proportion of domain III chimeras had some bioactivity. We conclude that shuffling of Cry coding regions through joining at the major conserved sequence motifs is an effective means for the production of a diverse number of chimeric Cry proteins but that such toxins with enhanced bioactive properties will be rare or nonexistent. PMID:15666731

  18. Uncovering Quantitative Protein Interaction Networks for Mouse PDZ Domains using Protein Microarrays

    PubMed Central

    Stiffler, Michael A.; Grantcharova, Viara P.; Sevecka, Mark; MacBeath, Gavin

    2008-01-01

    One of the principle challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach – compatible with genome-wide investigations – that enables us to measure the equilibrium dissociation constant (KD) of recombinant PDZ domains for fluorescently-labeled peptides that represent physiologically-relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the KD for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (KD ≤ 10 μM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network. PMID:16637659

  19. Domain-mediated protein interaction prediction: From genome to network.

    PubMed

    Reimand, Jüri; Hui, Shirley; Jain, Shobhit; Law, Brian; Bader, Gary D

    2012-08-14

    Protein-protein interactions (PPIs), involved in many biological processes such as cellular signaling, are ultimately encoded in the genome. Solving the problem of predicting protein interactions from the genome sequence will lead to increased understanding of complex networks, evolution and human disease. We can learn the relationship between genomes and networks by focusing on an easily approachable subset of high-resolution protein interactions that are mediated by peptide recognition modules (PRMs) such as PDZ, WW and SH3 domains. This review focuses on computational prediction and analysis of PRM-mediated networks and discusses sequence- and structure-based interaction predictors, techniques and datasets for identifying physiologically relevant PPIs, and interpreting high-resolution interaction networks in the context of evolution and human disease. PMID:22561014

  20. Domains of surfactant protein A that affect protein oligomerization, lipid structure and surface tension.

    PubMed

    Palaniyar, N; Ikegami, M; Korfhagen, T; Whitsett, J; McCormack, F X

    2001-05-01

    Surfactant protein A (SP-A) is an abundant protein found in pulmonary surfactant which has been reported to have multiple functions. In this review, we focus on the structural importance of each domain of SP-A in the functions of protein oligomerization, the structural organization of lipids and the surface-active properties of surfactant, with an emphasis on ultrastructural analyses. The N-terminal domain of SP-A is required for disulfide-dependent protein oligomerization, and for binding and aggregation of phospholipids, but there is no evidence that this domain directly interacts with lipid membranes. The collagen-like domain is important for the stability and oligomerization of SP-A. It also contributes shape and dimension to the molecule, and appears to determine membrane spacing in lipid aggregates such as common myelin and tubular myelin. The neck domain of SP-A is primarily involved in protein trimerization, which is critical for many protein functions, but it does not appear to be directly involved in lipid interactions. The globular C-terminal domain of SP-A clearly plays a central role in lipid binding, and in more complex functions such as the formation and/or stabilization of curved membranes. In recent work, we have determined that the maintenance of low surface tension of surfactant in the presence of serum protein inhibitors requires cooperative interactions between the C-terminal and N-terminal domains of the molecule. This effect of SP-A requires a high degree of oligomeric assembly of the protein, and may be mediated by the activity of the protein to alter the form or physical state of surfactant lipid aggregates. PMID:11369537

  1. 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

  2. Targeting a KH-domain protein with RNA decoys.

    PubMed Central

    Makeyev, Aleksandr V; Eastmond, Dawn L; Liebhaber, Stephen A

    2002-01-01

    RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to alphaCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for alphaCP decoy action. Because alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of alphaCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins. PMID:12358435

  3. 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.

  4. A database of domain definitions for proteins with complex interdomain geometry.

    PubMed

    Majumdar, Indraneel; Kinch, Lisa N; Grishin, Nick V

    2009-01-01

    Protein structural domains are necessary for understanding evolution and protein folding, and may vary widely from functional and sequence based domains. Although, various structural domain databases exist, defining domains for some proteins is non-trivial, and definitions of their domain boundaries are not available. Here, we present a novel database of manually defined structural domains for a representative set of proteins from the SCOP "multi-domain proteins" class. (http://prodata.swmed.edu/multidom/). We consider our domains as mobile evolutionary units, which may rearrange during protein evolution. Additionally, they may be visualized as structurally compact and possibly independently folding units. We also found that representing domains as evolutionary and folding units do not always lead to a unique domain definition. However, unlike existing databases, we retain and refine these "alternate" domain definitions after careful inspection of structural similarity, functional sites and automated domain definition methods. We provide domain definitions, including actual residue boundaries, for proteins that well known databases like SCOP and CATH do not attempt to split. Our alternate domain definitions are suitable for sequence and structure searches by automated methods. Additionally, the database can be used for training and testing domain delineation algorithms. Since our domains represent structurally compact evolutionary units, the database may be useful for studying domain properties and evolution. PMID:19352501

  5. 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.

  6. PSI-2: Structural Genomics to Cover Protein Domain Family Space

    PubMed Central

    Dessailly, Benoît H.; Nair, Rajesh; Jaroszewski, Lukasz; Fajardo, J. Eduardo; Kouranov, Andrei; Lee, David; Fiser, Andras; Godzik, Adam; Rost, Burkhard; Orengo, Christine

    2010-01-01

    Summary One major objective of structural genomics efforts, including the NIH-funded Protein Structure Initiative (PSI), has been to increase the structural coverage of protein sequence space. Here, we present the target selection strategy used during the second phase of PSI (PSI-2). This strategy, jointly devised by the bioinformatics groups associated with the PSI-2 large-scale production centres, targets representatives from large, structurally uncharacterised protein domain families, and from structurally uncharacterised subfamilies in very large and diverse families with incomplete structural coverage. These very large families are extremely diverse both structurally and functionally, and are highly over-represented in known proteomes. On the basis of several metrics, we then discuss to what extent PSI-2, during its first three years, has increased the structural coverage of genomes, and contributed structural and functional novelty. Together, the results presented here suggest that PSI-2 is successfully meeting its objectives and provides useful insights into structural and functional space. PMID:19523904

  7. Prediction of human protein-protein interaction by a domain-based approach.

    PubMed

    Zhang, Xiaopan; Jiao, Xiong; Song, Jie; Chang, Shan

    2016-05-01

    Protein-protein interactions (PPIs) are vital to a number of biological processes. With computational methods, plenty of domain information can help us to predict and assess PPIs. In this study, we proposed a domain-based approach for the prediction of human PPIs based on the interactions between the proteins and the domains. In this method, an optimizing model was built with the information from InterDom, 3did, DOMINE and Pfam databases. With this model, for 147 proteins in the integrin adhesome PPI network, 736 probable PPIs have been predicted, and the corresponding confidence probabilities of these PPIs were also calculated. It provides an opportunity to visualize the PPIs by using network graphs, which were constructed with Cytoscape, so that we can indicate underlying pathways possible. PMID:26925814

  8. PROSITE, a protein domain database for functional characterization and annotation.

    PubMed

    Sigrist, Christian J A; Cerutti, Lorenzo; de Castro, Edouard; Langendijk-Genevaux, Petra S; Bulliard, Virginie; Bairoch, Amos; Hulo, Nicolas

    2010-01-01

    PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE is largely used for the annotation of domain features of UniProtKB/Swiss-Prot entries. Among the 983 (DNA-binding) domains, repeats and zinc fingers present in Swiss-Prot (release 57.8 of 22 September 2009), 696 ( approximately 70%) are annotated with PROSITE descriptors using information from ProRule. In order to allow better functional characterization of domains, PROSITE developments focus on subfamily specific profiles and a new profile building method giving more weight to functionally important residues. Here, we describe AMSA, an annotated multiple sequence alignment format used to build a new generation of generalized profiles, the migration of ScanProsite to Vital-IT, a cluster of 633 CPUs, and the adoption of the Distributed Annotation System (DAS) to facilitate PROSITE data integration and interchange with other sources. The latest version of PROSITE (release 20.54, of 22 September 2009) contains 1308 patterns, 863 profiles and 869 ProRules. PROSITE is accessible at: http://www.expasy.org/prosite/. PMID:19858104

  9. PROSITE, a protein domain database for functional characterization and annotation

    PubMed Central

    Sigrist, Christian J. A.; Cerutti, Lorenzo; de Castro, Edouard; Langendijk-Genevaux, Petra S.; Bulliard, Virginie; Bairoch, Amos; Hulo, Nicolas

    2010-01-01

    PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE is largely used for the annotation of domain features of UniProtKB/Swiss-Prot entries. Among the 983 (DNA-binding) domains, repeats and zinc fingers present in Swiss-Prot (release 57.8 of 22 September 2009), 696 (∼70%) are annotated with PROSITE descriptors using information from ProRule. In order to allow better functional characterization of domains, PROSITE developments focus on subfamily specific profiles and a new profile building method giving more weight to functionally important residues. Here, we describe AMSA, an annotated multiple sequence alignment format used to build a new generation of generalized profiles, the migration of ScanProsite to Vital-IT, a cluster of 633 CPUs, and the adoption of the Distributed Annotation System (DAS) to facilitate PROSITE data integration and interchange with other sources. The latest version of PROSITE (release 20.54, of 22 September 2009) contains 1308 patterns, 863 profiles and 869 ProRules. PROSITE is accessible at: http://www.expasy.org/prosite/. PMID:19858104

  10. Analysis of the HD-GYP Domain Cyclic Dimeric GMP Phosphodiesterase Reveals a Role in Motility and the Enzootic Life Cycle of Borrelia burgdorferi ▿ †

    PubMed Central

    Sultan, Syed Z.; Pitzer, Joshua E.; Boquoi, Tristan; Hobbs, Gerry; Miller, Michael R.; Motaleb, M. A.

    2011-01-01

    HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a Km of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi. PMID:21670168

  11. 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. PMID:25820699

  12. Clustering of protein domains for functional and evolutionary studies

    PubMed Central

    Goldstein, Pavle; Zucko, Jurica; Vujaklija, Dušica; Kriško, Anita; Hranueli, Daslav; Long, Paul F; Etchebest, Catherine; Basrak, Bojan; Cullum, John

    2009-01-01

    Background The number of protein family members defined by DNA sequencing is usually much larger than those characterised experimentally. This paper describes a method to divide protein families into subtypes purely on sequence criteria. Comparison with experimental data allows an independent test of the quality of the clustering. Results An evolutionary split statistic is calculated for each column in a protein multiple sequence alignment; the statistic has a larger value when a column is better described by an evolutionary model that assumes clustering around two or more amino acids rather than a single amino acid. The user selects columns (typically the top ranked columns) to construct a motif. The motif is used to divide the family into subtypes using a stochastic optimization procedure related to the deterministic annealing EM algorithm (DAEM), which yields a specificity score showing how well each family member is assigned to a subtype. The clustering obtained is not strongly dependent on the number of amino acids chosen for the motif. The robustness of this method was demonstrated using six well characterized protein families: nucleotidyl cyclase, protein kinase, dehydrogenase, two polyketide synthase domains and small heat shock proteins. Phylogenetic trees did not allow accurate clustering for three of the six families. Conclusion The method clustered the families into functional subtypes with an accuracy of 90 to 100%. False assignments usually had a low specificity score. PMID:19832975

  13. Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.

    PubMed

    Lei, J; Tang, X; Chambers, T C; Pohl, J; Benian, G M

    1994-08-19

    Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated. PMID:8063727

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

    PubMed

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

    1998-09-01

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

  15. A Database of Domain Definitions for Proteins with Complex Interdomain Geometry

    PubMed Central

    Majumdar, Indraneel; Kinch, Lisa N.; Grishin, Nick V.

    2009-01-01

    Protein structural domains are necessary for understanding evolution and protein folding, and may vary widely from functional and sequence based domains. Although, various structural domain databases exist, defining domains for some proteins is non-trivial, and definitions of their domain boundaries are not available. Here, we present a novel database of manually defined structural domains for a representative set of proteins from the SCOP “multi-domain proteins” class. (http://prodata.swmed.edu/multidom/). We consider our domains as mobile evolutionary units, which may rearrange during protein evolution. Additionally, they may be visualized as structurally compact and possibly independently folding units. We also found that representing domains as evolutionary and folding units do not always lead to a unique domain definition. However, unlike existing databases, we retain and refine these “alternate” domain definitions after careful inspection of structural similarity, functional sites and automated domain definition methods. We provide domain definitions, including actual residue boundaries, for proteins that well known databases like SCOP and CATH do not attempt to split. Our alternate domain definitions are suitable for sequence and structure searches by automated methods. Additionally, the database can be used for training and testing domain delineation algorithms. Since our domains represent structurally compact evolutionary units, the database may be useful for studying domain properties and evolution. PMID:19352501

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

    PubMed

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

    1996-03-01

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

  17. West Nile virus methyltransferase domain interacts with protein kinase G

    PubMed Central

    2013-01-01

    Background The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication. PMID:23876037

  18. Targeting the inhibitor of Apoptosis Protein BIR3 binding domains.

    PubMed

    Jaquith, James B

    2014-05-01

    The Inhibitor of Apoptosis Proteins (IAPs) play a critical role in the regulation of cellular apoptosis and cytokine signaling. IAP family members include XIAP, cIAP1, cIAP2, NAIP, survivin, Apollon/Bruce, ML-IAP/livin and TIAP. The IAPs have been targeted using both antisense oligonucleotides and small molecule inhibitors. Several research teams have advanced compounds that bind the highly conserved BIR3 domains of the IAPs into clinical trials, as single agents and in combination with standard of care. This patent review highlights the medicinal chemistry strategies that have been applied to the development of clinical compounds. PMID:24998289

  19. Domain-Swapped Dimer of Pseudomonas aeruginosa Cytochrome c551: Structural Insights into Domain Swapping of Cytochrome c Family Proteins

    PubMed Central

    Nagao, Satoshi; Ueda, Mariko; Osuka, Hisao; Komori, Hirofumi; Kamikubo, Hironari; Kataoka, Mikio; Higuchi, Yoshiki; Hirota, Shun

    2015-01-01

    Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c551 (PA cyt c551), and Hydrogenobacter thermophilus cytochrome c552 (HT cyt c552), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met–heme coordination significantly compared to the monomer. HT cyt c552 forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c551 also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins. PMID:25853415

  20. Enhancing protein stability by adsorption onto raftlike lipid domains.

    PubMed

    Litt, Jeffrey; Padala, Chakradhar; Asuri, Prashanth; Vutukuru, Srinavya; Athmakuri, Krishna; Kumar, Sanat; Dordick, Jonathan; Kane, Ravi S

    2009-05-27

    We demonstrate that the stability of adsorbed proteins can be enhanced by controlling the heterogeneity of the surfaceby creating raftlike domains in a soft liposomal membrane. Recent work has shown that enzymes adsorbed onto highly curved nanoscale supports can be more stable than those adsorbed on flat surfaces with nominally the same chemical structure. This effect has been attributed to a decrease in lateral interenzyme interactions on a curved surface. Exploiting this idea, we asked if adsorbing enzymes onto "patchy" surfaces composed of adsorbing and nonadsorbing regions can be used to reduce lateral interactions even on relatively flat surfaces. We demonstrate that creating domains on which an enzyme can adsorb enhances the stability of that enzyme under denaturing conditions. Furthermore, we demonstrate that the size of these domains has a considerable effect on the degree of stability imparted by adsorption. Such biomimetic raft-inspired systems may find use in applications ranging from biorecognition to the design of novel strategies for the separation of biomolecules and controlling the interaction of multicomponent membrane-bound enzymes. PMID:19385631

  1. Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

    PubMed

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

    2013-12-13

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

  2. J-DOMAIN PROTEINS IN TOMATO AND STRAWBERRY THAT ARE HEAT SHOCK PROTEINS AND ARE EXPRESSED IN REPRODUCTIVE TISSUES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Eucaryotes have a large number of proteins containing a conserved J-domain, corresponding to the N-terminal 75 amino acids of E. coli heat stress protein, DnaJ. DnaJ homologues in eucaryotes are also referred to as Hsp40. In plants, J-domain proteins have been implicated as molecular chaperones in r...

  3. Protein inter-domain linker prediction using Random Forest and amino acid physiochemical properties

    PubMed Central

    2014-01-01

    Background Protein chains are generally long and consist of multiple domains. Domains are distinct structural units of a protein that can evolve and function independently. The accurate prediction of protein domain linkers and boundaries is often regarded as the initial step of protein tertiary structure and function predictions. Such information not only enhances protein-targeted drug development but also reduces the experimental cost of protein analysis by allowing researchers to work on a set of smaller and independent units. In this study, we propose a novel and accurate domain-linker prediction approach based on protein primary structure information only. We utilize a nature-inspired machine-learning model called Random Forest along with a novel domain-linker profile that contains physiochemical and domain-linker information of amino acid sequences. Results The proposed approach was tested on two well-known benchmark protein datasets and achieved 68% sensitivity and 99% precision, which is better than any existing protein domain-linker predictor. Without applying any data balancing technique such as class weighting and data re-sampling, the proposed approach is able to accurately classify inter-domain linkers from highly imbalanced datasets. Conclusion Our experimental results prove that the proposed approach is useful for domain-linker identification in highly imbalanced single- and multi-domain proteins. PMID:25521329

  4. Structure of Yellow Fever Virus Envelope Protein Domain III

    PubMed Central

    Volk, David E.; May, Fiona J.; Gandham, Sai H. A.; Anderson, Anjenique; Von Lindern, Jana J.; Beasley, David W. C.; Barrett, Alan D. T.; Gorenstein, David G.

    2009-01-01

    The structure of recombinant domain III of the envelope protein (rED3) of yellow fever virus (YFV), containing the major neutralization site, was determined using NMR spectroscopy. The amino acid sequence and structure of the YFV-rED3 shows differences from ED3s of other mosquito-borne flaviviruses; in particular, the partially surface-exposed BC loop where methionine-304 and valine-324 were identified as being critical for the structure of the loop. Variations in the structure and surface chemistry of ED3 between flaviviruses affect neutralization sites and may affect host cell receptor interactions and play a role in the observed variations in viral pathogenesis and tissue tropism. PMID:19818466

  5. The BAR Domain Proteins: Molding Membranes in Fission, Fusion, and Phagy

    PubMed Central

    Ren, Gang; Vajjhala, Parimala; Lee, Janet S.; Winsor, Barbara; Munn, Alan L.

    2006-01-01

    The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt α-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes. PMID:16524918

  6. The BAR domain proteins: molding membranes in fission, fusion, and phagy.

    PubMed

    Ren, Gang; Vajjhala, Parimala; Lee, Janet S; Winsor, Barbara; Munn, Alan L

    2006-03-01

    The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt alpha-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes. PMID:16524918

  7. 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.

  8. 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

  9. 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.

  10. ThreaDom: extracting protein domain boundary information from multiple threading alignments

    PubMed Central

    Xue, Zhidong; Xu, Dong; Wang, Yan; Zhang, Yang

    2013-01-01

    Motivation: Protein domains are subunits that can fold and evolve independently. Identification of domain boundary locations is often the first step in protein folding and function annotations. Most of the current methods deduce domain boundaries by sequence-based analysis, which has low accuracy. There is no efficient method for predicting discontinuous domains that consist of segments from separated sequence regions. As template-based methods are most efficient for protein 3D structure modeling, combining multiple threading alignment information should increase the accuracy and reliability of computational domain predictions. Result: We developed a new protein domain predictor, ThreaDom, which deduces domain boundary locations based on multiple threading alignments. The core of the method development is the derivation of a domain conservation score that combines information from template domain structures and terminal and internal alignment gaps. Tested on 630 non-redundant sequences, without using homologous templates, ThreaDom generates correct single- and multi-domain classifications in 81% of cases, where 78% have the domain linker assigned within ±20 residues. In a second test on 486 proteins with discontinuous domains, ThreaDom achieves an average precision 84% and recall 65% in domain boundary prediction. Finally, ThreaDom was examined on 56 targets from CASP8 and had a domain overlap rate 73, 87 and 85% with the target for Free Modeling, Hard multiple-domain and discontinuous domain proteins, respectively, which are significantly higher than most domain predictors in the CASP8. Similar results were achieved on the targets from the most recently CASP9 and CASP10 experiments. Availability: http://zhanglab.ccmb.med.umich.edu/ThreaDom/. Contact: zhng@umich.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23812990

  11. Setting the PAS, the role of circadian PAS domain proteins during environmental adaptation in plants

    PubMed Central

    Vogt, Julia H. M.; Schippers, Jos H. M.

    2015-01-01

    The per-ARNT-sim (PAS) domain represents an ancient protein module that can be found across all kingdoms of life. The domain functions as a sensing unit for a diverse array of signals, including molecular oxygen, small metabolites, and light. In plants, several PAS domain-containing proteins form an integral part of the circadian clock and regulate responses to environmental change. Moreover, these proteins function in pathways that control development and plant stress adaptation responses. Here, we discuss the role of PAS domain-containing proteins in anticipation, and adaptation to environmental changes in plants. PMID:26217364

  12. The CBS domain: a protein module with an emerging prominent role in regulation.

    PubMed

    Baykov, Alexander A; Tuominen, Heidi K; Lahti, Reijo

    2011-11-18

    Regulatory CBS (cystathionine β-synthase) domains exist as two or four tandem copies in thousands of cytosolic and membrane-associated proteins from all kingdoms of life. Mutations in the CBS domains of human enzymes and membrane channels are associated with an array of hereditary diseases. Four CBS domains encoded within a single polypeptide or two identical polypeptides (each having a pair of CBS domains at the subunit interface) form a highly conserved disk-like structure. CBS domains act as autoinhibitory regulatory units in some proteins and activate or further inhibit protein function upon binding to adenosine nucleotides (AMP, ADP, ATP, S-adenosyl methionine, NAD, diadenosine polyphosphates). As a result of the differential effects of the nucleotides, CBS domain-containing proteins can sense cell energy levels. Significant conformational changes are induced in CBS domains by bound ligands, highlighting the structural basis for their effects. PMID:21958115

  13. 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

  14. Different evolutionary patterns of SNPs between domains and unassigned regions in human protein-coding sequences.

    PubMed

    Pang, Erli; Wu, Xiaomei; Lin, Kui

    2016-06-01

    Protein evolution plays an important role in the evolution of each genome. Because of their functional nature, in general, most of their parts or sites are differently constrained selectively, particularly by purifying selection. Most previous studies on protein evolution considered individual proteins in their entirety or compared protein-coding sequences with non-coding sequences. Less attention has been paid to the evolution of different parts within each protein of a given genome. To this end, based on PfamA annotation of all human proteins, each protein sequence can be split into two parts: domains or unassigned regions. Using this rationale, single nucleotide polymorphisms (SNPs) in protein-coding sequences from the 1000 Genomes Project were mapped according to two classifications: SNPs occurring within protein domains and those within unassigned regions. With these classifications, we found: the density of synonymous SNPs within domains is significantly greater than that of synonymous SNPs within unassigned regions; however, the density of non-synonymous SNPs shows the opposite pattern. We also found there are signatures of purifying selection on both the domain and unassigned regions. Furthermore, the selective strength on domains is significantly greater than that on unassigned regions. In addition, among all of the human protein sequences, there are 117 PfamA domains in which no SNPs are found. Our results highlight an important aspect of protein domains and may contribute to our understanding of protein evolution. PMID:26833483

  15. Overexpression and purification of folded domain of prostate cancer related proteins MSMB and PSA.

    PubMed

    Tiwary, Mohini; Agarwal, Nipanshu; Dinda, Amit; Yadav, Subhash C

    2016-05-01

    Overexpression of domains of a human protein using recombinant DNA technology has been challenging because individual domains intend to accumulate as non-soluble aggregate when expressed separately. Studies on identifying right sequences for a domain to be able to fold independently may help understand the folding pattern and underlying protein-engineering events to isolate the functional domains of a protein. In this report, individual domains of prostate cancer related biomarkers; MSMB and PSA were overexpressed in bacterial system and purified in their folded forms using affinity chromatography. The western blotting experiment using domain specific antibodies further confirmed these proteins. The designed nucleotide sequences domains were truncated using fold index software and folding were predicted by phyre2 and I-TASSER software. Other parameters were optimized for their overexpression and purification using Co-NTA affinity chromatography. Purified domains of each protein showed secondary structures such as α + β type for PSA, α/β and β type for the each domains of PSA and MSMB respectively. This is the first report on producing PSA and MSMB individual domains in functional folded forms. This study may help produce the folded domain of many such proteins to be used for better diagnostic purpose. PMID:27038170

  16. A protein domain-based view of the virosphere-host relationship.

    PubMed

    Abroi, Aare

    2015-12-01

    Despite being an important and inseparable part of the biosphere, viruses are too often overlooked in several life sciences, including evolutionary biology, systems biology, and non-marine ecology. In this review, a protein domain-based view of viral proteomes, the proteomes of other organisms and the overlap between them is presented. The data show that in many viral species, viral proteins are not very well annotated with protein domains. Compared with viral proteomes, cellular proteomes are covered quite uniformly with respect to protein domains and show higher coverage. A tremendous number of virally coded domains exist; in fact, the number of protein domains in the characterised virosphere is approaching that found in Archaea, a well-accepted superkingdom. Proteins encoded by viruses contain virosphere-specific domains (i.e., not found in cellular proteomes) and/or many domains shared by viral and cellular proteomes. Virosphere-specific domains are structurally peculiar with respect to different structural measures, making them a clear source of structural and functional novelty. Viral families with RNA genomes tend to harbour more virosphere-specific domains than other viruses. Interestingly, host range preferences of different viral classes are, for the most part, not reflected by domains shared between viruses and different superkingdoms. The role of viruses in the genesis of the cellular domain repertoire is reviewed to bring them more confidently and firmly into the larger biological picture. PMID:26296474

  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 studies of the BTB domain in the Drosophila GAGA and Mod(mdg4) proteins.

    PubMed

    Read, D; Butte, M J; Dernburg, A F; Frasch, M; Kornberg, T B

    2000-10-15

    The BTB/POZ (BTB) domain is an approximately 120 residue sequence that is conserved at the N-terminus of many proteins in both vertebrates and invertebrates. We found that the protein encoded by a lethal allele of the Drosophila modifier of mdg4 [mod(mdg4)] gene has two mutated residues in its BTB domain. The identities of the residues at the positions of these mutations are highly conserved in the BTB domain family of proteins, and when the corresponding mutations were engineered into the BTB domain-containing GAGA protein, the activity of GAGA as a transcription activator in a transient transfection assay was severely reduced. The functional equivalence of the BTB domains was established by showing that the BTB domain of the mod(mdg4) protein can effectively substitute for that of GAGA. PMID:11024164

  19. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain.

    PubMed

    Pearson, M A; Reczek, D; Bretscher, A; Karplus, P A

    2000-04-28

    The ezrin-radixin-moesin (ERM) protein family link actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks their binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain. This extended binding mode suggests a novel mechanism for how different signals could produce varying levels of activation. Sequence conservation suggests a similar regulation of the tumor suppressor merlin. PMID:10847681

  20. A Simple Model of Protein Domain Swapping in Crowded Cellular Environments.

    PubMed

    Woodard, Jaie C; Dunatunga, Sachith; Shakhnovich, Eugene I

    2016-06-01

    Domain swapping in proteins is an important mechanism of functional and structural innovation. However, despite its ubiquity and importance, the physical mechanisms that lead to domain swapping are poorly understood. Here, we present a simple two-dimensional coarse-grained model of protein domain swapping in the cytoplasm. In our model, two-domain proteins partially unfold and diffuse in continuous space. Monte Carlo multiprotein simulations of the model reveal that domain swapping occurs at intermediate temperatures, whereas folded dimers and folded monomers prevail at low temperatures, and partially unfolded monomers predominate at high temperatures. We use a simplified amino acid alphabet consisting of four residue types, and find that the oligomeric state at a given temperature depends on the sequence of the protein. We also show that hinge strain between domains can promote domain swapping, consistent with experimental observations for real proteins. Domain swapping depends nonmonotonically on the protein concentration, with domain-swapped dimers occurring at intermediate concentrations and nonspecific interactions between partially unfolded proteins occurring at high concentrations. For folded proteins, we recover the result obtained in three-dimensional lattice simulations, i.e., that functional dimerization is most prevalent at intermediate temperatures and nonspecific interactions increase at low temperatures. PMID:27276255

  1. 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.

  2. Forced unfolding of protein domains determines cytoskeletal rheology

    NASA Astrophysics Data System (ADS)

    Crocker, John

    2005-03-01

    Cells have recently been shown to have a power-law dynamic shear modulus over wide frequency range; the value of the exponent being non-universal, varying from 0.1-0.25 depending on cell type. This observation has been interpreted as evidence for the Soft Glassy Rheology (SGR) model, a trap-type glass model with an effective granular temperature. We propose a simple, alternative model of cytoskeletal mechanics based on the thermally activated, forced unfolding of domains in proteins cross-linking a stressed semi-flexible polymer gel. It directly relates a cell’s mechanical response to biophysical parameters of the cytoskeleton’s molecular constituents. Simulations indicate that unfolding events in a random network display a collective self-organization, giving rise to an exponential distribution of crosslink stress that can reproduce cell viscoelasticity. The model suggests natural explanations for the observed correlation between cell rheology and intracellular static stress, including those previously explained using the tensegrity concept. Moreover, our model provides insight into potential mechanisms of mechanotransduction as well as cell shape sensing and maintenance.

  3. A multi-objective optimization approach accurately resolves protein domain architectures

    PubMed Central

    Bernardes, J.S.; Vieira, F.R.J.; Zaverucha, G.; Carbone, A.

    2016-01-01

    Motivation: Given a protein sequence and a number of potential domains matching it, what are the domain content and the most likely domain architecture for the sequence? This problem is of fundamental importance in protein annotation, constituting one of the main steps of all predictive annotation strategies. On the other hand, when potential domains are several and in conflict because of overlapping domain boundaries, finding a solution for the problem might become difficult. An accurate prediction of the domain architecture of a multi-domain protein provides important information for function prediction, comparative genomics and molecular evolution. Results: We developed DAMA (Domain Annotation by a Multi-objective Approach), a novel approach that identifies architectures through a multi-objective optimization algorithm combining scores of domain matches, previously observed multi-domain co-occurrence and domain overlapping. DAMA has been validated on a known benchmark dataset based on CATH structural domain assignments and on the set of Plasmodium falciparum proteins. When compared with existing tools on both datasets, it outperforms all of them. Availability and implementation: DAMA software is implemented in C++ and the source code can be found at http://www.lcqb.upmc.fr/DAMA. Contact: juliana.silva_bernardes@upmc.fr or alessandra.carbone@lip6.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26458889

  4. 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

  5. A 127-kDa protein (UV-DDB) binds to the cytoplasmic domain of the Alzheimer's amyloid precursor protein.

    PubMed

    Watanabe, T; Sukegawa, J; Sukegawa, I; Tomita, S; Iijima, K; Oguchi, S; Suzuki, T; Nairn, A C; Greengard, P

    1999-02-01

    Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP695 and identified a protein of approximately 130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S-transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid beta protein secretion, may be involved in the interaction between UV-DDB/p127 and APP. PMID:9930726

  6. 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.

  7. Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.

    PubMed

    Soundararajan, Meera; Willard, Francis S; Kimple, Adam J; Turnbull, Andrew P; Ball, Linda J; Schoch, Guillaume A; Gileadi, Carina; Fedorov, Oleg Y; Dowler, Elizabeth F; Higman, Victoria A; Hutsell, Stephanie Q; Sundström, Michael; Doyle, Declan A; Siderovski, David P

    2008-04-29

    Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules. PMID:18434541

  8. Changes in hydration structure are necessary for collective motions of a multi-domain protein.

    PubMed

    Oroguchi, Tomotaka; Nakasako, Masayoshi

    2016-01-01

    Conformational motions of proteins are necessary for their functions. To date, experimental studies measuring conformational fluctuations of a whole protein structure have revealed that water molecules hydrating proteins are necessary to induce protein functional motions. However, the underlying microscopic mechanism behind such regulation remains unsolved. To clarify the mechanism, multi-domain proteins are good targets because it is obvious that water molecules between domains play an important role in domain motions. Here, we show how changes in hydration structure microscopically correlate with large-amplitude motions of a multi-domain protein, through molecular dynamics simulation supported by structural analyses and biochemical experiments. We first identified collective domain motions of the protein, which open/close an active-site cleft between domains. The analyses on changes in hydration structure revealed that changes in local hydration in the depth of the cleft are necessary for the domain motion and vice versa. In particular, 'wetting'/'drying' at a hydrophobic pocket and 'adsorption'/'dissociation' of a few water molecules at a hydrophilic crevice in the cleft were induced by dynamic rearrangements of hydrogen-bond networks, and worked as a switch for the domain motions. Our results microscopically demonstrated the importance of hydrogen-bond networks of water molecules in understanding energy landscapes of protein motions. PMID:27193111

  9. Changes in hydration structure are necessary for collective motions of a multi-domain protein

    PubMed Central

    Oroguchi, Tomotaka; Nakasako, Masayoshi

    2016-01-01

    Conformational motions of proteins are necessary for their functions. To date, experimental studies measuring conformational fluctuations of a whole protein structure have revealed that water molecules hydrating proteins are necessary to induce protein functional motions. However, the underlying microscopic mechanism behind such regulation remains unsolved. To clarify the mechanism, multi-domain proteins are good targets because it is obvious that water molecules between domains play an important role in domain motions. Here, we show how changes in hydration structure microscopically correlate with large-amplitude motions of a multi-domain protein, through molecular dynamics simulation supported by structural analyses and biochemical experiments. We first identified collective domain motions of the protein, which open/close an active-site cleft between domains. The analyses on changes in hydration structure revealed that changes in local hydration in the depth of the cleft are necessary for the domain motion and vice versa. In particular, ‘wetting’/‘drying’ at a hydrophobic pocket and ‘adsorption’/‘dissociation’ of a few water molecules at a hydrophilic crevice in the cleft were induced by dynamic rearrangements of hydrogen-bond networks, and worked as a switch for the domain motions. Our results microscopically demonstrated the importance of hydrogen-bond networks of water molecules in understanding energy landscapes of protein motions. PMID:27193111

  10. A protein domain-based interactome network for C. elegans early embryogenesis

    PubMed Central

    Boxem, Mike; Maliga, Zoltan; Klitgord, Niels; Li, Na; Lemmens, Irma; Mana, Miyeko; de Lichtervelde, Lorenzo; Mul, Joram D.; van de Peut, Diederik; Devos, Maxime; Simonis, Nicolas; Yildirim, Muhammed A.; Cokol, Murat; Kao, Huey-Ling; de Smet, Anne-Sophie; Wang, Haidong; Schlaitz, Anne-Lore; Hao, Tong; Milstein, Stuart; Fan, Changyu; Tipsword, Mike; Drew, Kevin; Galli, Matilde; Rhrissorrakrai, Kahn; Drechsel, David; Koller, Daphne; Roth, Frederick P.; Iakoucheva, Lilia M.; Dunker, A. Keith; Bonneau, Richard; Gunsalus, Kristin C.; Hill, David E.; Piano, Fabio; Tavernier, Jan; van den Heuvel, Sander; Hyman, Anthony A.; Vidal, Marc

    2008-01-01

    Summary Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or “interactome” networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed new insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms. PMID:18692475

  11. Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions.

    PubMed Central

    Harder, Thomas

    2003-01-01

    Numerous cell membrane associated processes, including signal transduction, membrane sorting, protein processing and virus trafficking take place in membrane subdomains. Protein-protein interactions provide the frameworks necessary to generate biologically functional membrane domains. For example, coat proteins define membrane areas destined for sorting processes, viral proteins self-assemble to generate a budding virus, and adapter molecules organize multimolecular signalling assemblies, which catalyse downstream reactions. The concept of raft lipid-based membrane domains provides a different principle for compartmentalization and segregation of membrane constituents. Accordingly, rafts are defined by the physical properties of the lipid bilayer and function by selective partitioning of membrane lipids and proteins into membrane domains of specific phase behaviour and lipid packing. Here, I will discuss the interplay of these independent principles of protein scaffolds and raft lipid microdomains leading to the generation of biologically functional membrane domains. PMID:12803918

  12. Insulator function and topological domain border strength scale with architectural protein occupancy

    PubMed Central

    2014-01-01

    Background Chromosome conformation capture studies suggest that eukaryotic genomes are organized into structures called topologically associating domains. The borders of these domains are highly enriched for architectural proteins with characterized roles in insulator function. However, a majority of architectural protein binding sites localize within topological domains, suggesting sites associated with domain borders represent a functionally different subclass of these regulatory elements. How topologically associating domains are established and what differentiates border-associated from non-border architectural protein binding sites remain unanswered questions. Results By mapping the genome-wide target sites for several Drosophila architectural proteins, including previously uncharacterized profiles for TFIIIC and SMC-containing condensin complexes, we uncover an extensive pattern of colocalization in which architectural proteins establish dense clusters at the borders of topological domains. Reporter-based enhancer-blocking insulator activity as well as endogenous domain border strength scale with the occupancy level of architectural protein binding sites, suggesting co-binding by architectural proteins underlies the functional potential of these loci. Analyses in mouse and human stem cells suggest that clustering of architectural proteins is a general feature of genome organization, and conserved architectural protein binding sites may underlie the tissue-invariant nature of topologically associating domains observed in mammals. Conclusions We identify a spectrum of architectural protein occupancy that scales with the topological structure of chromosomes and the regulatory potential of these elements. Whereas high occupancy architectural protein binding sites associate with robust partitioning of topologically associating domains and robust insulator function, low occupancy sites appear reserved for gene-specific regulation within topological domains. PMID

  13. IQGAP proteins reveal an atypical phosphoinositide (aPI) binding domain with a pseudo C2 domain fold.

    PubMed

    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

    2012-06-29

    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 (PtdInsP(3)). The binding affinity for PtdInsP(3), 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 PtdInsP(3) 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. PMID:22493426

  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. Correspondence between immunological and functional domains in the transforming protein of Fujinami sarcoma virus.

    PubMed Central

    Stone, J C; Pawson, T

    1985-01-01

    Monoclonal antibodies reactive with either gag or fps portions of the wild-type Fujinami sarcoma virus transforming protein have been used to probe the structure of proteins encoded by mutant genomes constructed in vitro. The pattern of immunoreactivity suggests that the functional domain defined in genetic studies (Stone et al., Cell 37:549-558, 1984) corresponds to a discrete immunological domain in the native, wild-type Fujinami sarcoma virus protein. At least one mutation affecting both the structure and function of the proposed NH2-terminal fps-specific domain encodes a product with high specific activities in kinase assays. Furthermore, a cell line expressing high levels of this mutant protein is only moderately transformed. The striking correspondence between the immunological domain defined here and the functional domain inferred from the results of transfection experiments suggests that this non-kinase-specifying region constitutes a discrete structural as well as functional component of the viral protein. Images PMID:2991592

  16. Site on the human erythrocyte glucose transporter phosphorylated by protein kinase C resides on the protein's hydrophilic domain

    SciTech Connect

    Deziel, M.R.; McReynolds, J.H.; Lippes, H.A.; Jung, C.Y.

    1986-05-01

    A recently published model of the human erythrocyte hexose transporter deduced from the protein's primary structure proposes that the transporter is organized into two membrane domains comprising 77% of the protein's mass and three hydrophilic domains, a short segment that includes the polypeptide's N-terminus and two larger segments, one lying between the membrane domains and the other at the protein's C-terminus. Limited tryptic digestion of the transporter produces two membrane-bound fragments corresponding to the proposed membrane domains and releases a number of soluble peptides. Fast Atom Bombardment Mass Spectroscopic analysis of the released peptides and comparison of the peptide's masses with the transporter's amino acid sequence revealed that tryptic peptides corresponding to at least 63% of the hydrophilic domains' mass were recovered. The site of phosphorylation by protein kinase C, tagged using (/sup 32/P)-ATP, was also released from the transporter under these conditions, (in contrast to sites located within the protein's membrane domains), indicating that this site is located within one of the hydrophilic domains. Tryptic digestion at elevated ionic strength or cleavage with S. Aureus V8 protease results in the recovery of the /sup 32/P label on the carbohydrate-bearing membrane domain that is located near the protein's N-terminus, thus eliminating the C-terminal hydrophilic segment as a possible site of phosphorylation.

  17. Coupled protein domain motion in Taq polymerase revealed by neutron spin-echo spectroscopy

    PubMed Central

    Bu, Zimei; Biehl, Ralf; Monkenbusch, Michael; Richter, Dieter; Callaway, David J. E.

    2005-01-01

    Long-range conformational changes in proteins are ubiquitous in biology for the transmission and amplification of signals; such conformational changes can be triggered by small-amplitude, nanosecond protein domain motion. Understanding how conformational changes are initiated requires the characterization of protein domain motion on these timescales and on length scales comparable to protein dimensions. Using neutron spin-echo spectroscopy (NSE), normal mode analysis, and a statistical-mechanical framework, we reveal overdamped, coupled domain motion within DNA polymerase I from Thermus aquaticus (Taq polymerase). This protein utilizes correlated domain dynamics over 70 Å to coordinate nucleotide synthesis and cleavage during DNA synthesis and repair. We show that NSE spectroscopy can determine the domain mobility tensor, which determines the degree of dynamical coupling between domains. The mobility tensor defines the domain velocity response to a force applied to it or to another domain, just as the sails of a sailboat determine its velocity given the applied wind force. The NSE results provide insights into the nature of protein domain motion that are not appreciated by conventional biophysical techniques. PMID:16306270

  18. The cloning of Grb10 reveals a new family of SH2 domain proteins.

    PubMed

    Ooi, J; Yajnik, V; Immanuel, D; Gordon, M; Moskow, J J; Buchberg, A M; Margolis, B

    1995-04-20

    SH2 domains function to bind proteins containing phosphotyrosine and are components of proteins that are important signal transducers for tyrosine kinases. We have cloned SH2 domain proteins by screening bacterial expression libraries with the tyrosine phosphorylated carboxyterminus of the epidermal growth factor (EGF) receptor. Here we report the identification of a new SH2 domain protein, Grb10. Grb10 is highly related to Grb7, an SH2 domain protein that we have previously identified. In addition to an SH2 domain, Grb7 and Grb10 have a central domain with similarity to a putative C. elegans gene likely to be involved in neuronal migration. At least three forms of Grb10 exist in fibroblasts apparently due to alternate translational start sites. Grb10 undergoes serine but not tyrosine phosphorylation after EGF treatment resulting in a shift mobility in a large fraction of Grb10 molecules. However Grb10 appears to bind poorly to EGF-Receptor and the true binding partner for the Grb10 SH2 domain is unclear. Grb10 maps to mouse chromosome 11 very close to the EGF-Receptor which is remarkably similar to Grb7 that maps near the EGF-Receptor related HER2 receptor. The finding of multiple family members with evolutionarily conserved domains indicates that these SH2 domain proteins are likely to have an important, although as of yet, unidentified function. PMID:7731717

  19. 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

  20. Dynamics and adaptive benefits of protein domain emergence and arrangements during plant genome evolution.

    PubMed

    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

  1. 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

  2. Multiple Protein Interactions Involving Proposed Extracellular Loop Domains of the Tight Junction Protein Occludin

    PubMed Central

    Nusrat, Asma; Brown, G. Thomas; Tom, Jeffrey; Drake, Alex; Bui, Tam T.T.; Quan, Cliff; Mrsny, Randall J.

    2005-01-01

    Occludin is a tetraspan integral membrane protein in epithelial and endothelial tight junction (TJ) structures that is projected to have two extracellular loops. We have used peptides emulating central regions of human occludin's first and second loops, termed O-A:101–121 and O-B:210–228, respectively, to examine potential molecular interactions between these two regions of occludin and other TJ proteins. A superficial biophysical assessment of A:101–121 and O-B:210–228 showed them to have dissimilar solution conformation characteristics. Although O-A:101–121 failed to strongly interact with protein components of the human epithelial intestinal cell line T84, O-B:210–228 selectively associated with occludin, claudin-one and the junctional adhesion molecule (JAM)-A. Further, the presence of O-B:210–228, but not O-A:101–121, impeded the recovery of functional TJ structures. A scrambled peptide sequences of O-B:210–228 failed to influence TJ assembly. These studies demonstrate distinct properties for these two extracellular segments of the occludin protein and provide an improved understanding of how specific domains of occludin may interact with proteins present at TJ structures. PMID:15659655

  3. BEACH-domain proteins act together in a cascade to mediate vacuolar protein trafficking and disease resistance in Arabidopsis.

    PubMed

    Teh, Ooi-kock; Hatsugai, Noriyuki; Tamura, Kentaro; Fuji, Kentaro; Tabata, Ryo; Yamaguchi, Katsushi; Shingenobu, Shuji; Yamada, Masashi; Hasebe, Mitsuyasu; Sawa, Shinichiro; Shimada, Tomoo; Hara-Nishimura, Ikuko

    2015-03-01

    Membrane trafficking to the protein storage vacuole (PSV) is a specialized process in seed plants. However, this trafficking mechanism to PSV is poorly understood. Here, we show that three types of Beige and Chediak-Higashi (BEACH)-domain proteins contribute to both vacuolar protein transport and effector-triggered immunity (ETI). We screened a green fluorescent seed (GFS) library of Arabidopsis mutants with defects in vesicle trafficking and isolated two allelic mutants gfs3 and gfs12 with a defect in seed protein transport to PSV. The gene responsible for the mutant phenotype was found to encode a putative protein belonging to group D of BEACH-domain proteins, which possess kinase domains. Disruption of other BEACH-encoding loci in the gfs12 mutant showed that BEACH homologs acted in a cascading manner for PSV trafficking. The epistatic genetic interactions observed among BEACH homologs were also found in the ETI responses of the gfs12 and gfs12 bchb-1 mutants, which showed elevated avirulent bacterial growth. The GFS12 kinase domain interacted specifically with the pleckstrin homology domain of BchC1. These results suggest that a cascade of multiple BEACH-domain proteins contributes to vacuolar protein transport and plant defense. PMID:25618824

  4. 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.

  5. Protein Domain-Level Landscape of Cancer-Type-Specific Somatic Mutations

    PubMed Central

    Yang, Fan; Petsalaki, Evangelia; Rolland, Thomas; Hill, David E.; Vidal, Marc; Roth, Frederick P.

    2015-01-01

    Identifying driver mutations and their functional consequences is critical to our understanding of cancer. Towards this goal, and because domains are the functional units of a protein, we explored the protein domain-level landscape of cancer-type-specific somatic mutations. Specifically, we systematically examined tumor genomes from 21 cancer types to identify domains with high mutational density in specific tissues, the positions of mutational hotspots within these domains, and the functional and structural context where possible. While hotspots corresponding to specific gain-of-function mutations are expected for oncoproteins, we found that tumor suppressor proteins also exhibit strong biases toward being mutated in particular domains. Within domains, however, we observed the expected patterns of mutation, with recurrently mutated positions for oncogenes and evenly distributed mutations for tumor suppressors. For example, we identified both known and new endometrial cancer hotspots in the tyrosine kinase domain of the FGFR2 protein, one of which is also a hotspot in breast cancer, and found new two hotspots in the Immunoglobulin I-set domain in colon cancer. Thus, to prioritize cancer mutations for further functional studies aimed at more precise cancer treatments, we have systematically correlated mutations and cancer types at the protein domain level. PMID:25794154

  6. 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.

  7. Towards an intelligent system for the automatic assignment of domains in globular proteins.

    PubMed

    Sternberg, M J; Hegyi, H; Islam, S A; Luo, J; Russell, R B

    1995-01-01

    The automatic identification of protein domains from coordinates is the first step in the classification of protein folds and hence is required for databases to guide structure prediction. Most algorithms encode a single concept based and sometimes do not yield assignments that are consistent with the generally accepted perception. Our development of an automatic approach to identify reliably domains from protein coordinates is described. The algorithm is benchmarked against a manual identification of the domains in 284 representative protein chains. The first step is the domain assignment by distance (DAD) algorithm that considers the density of inter-residue contacts represented in a contact matrix. The algorithm yields 85% agreement with the manual assignment. The paper then considers how the reliability of these assignments could be evaluated. Finally the use of structural comparisons using the STAMP algorithm to validate domain assignment is reported on a test case. PMID:7584461

  8. Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates

    PubMed Central

    O’Meally, Robert; Sonnenberg, Jason L.; Cole, Robert N.; Shewmaker, Frank P.

    2015-01-01

    Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (≥100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases. PMID:26317359

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

    PubMed

    Toto, Angelo; Pedersen, Søren W; Karlsson, O Andreas; Moran, Griffin E; Andersson, Eva; Chi, Celestine N; Strømgaard, Kristian; Gianni, Stefano; Jemth, Per

    2016-05-01

    Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 are well studied and include the following three protein ligands: (i)N-methyl-d-aspartate-type ionotropic glutamate receptor subunit GluN2B, (ii) neuronal nitric oxide synthase and (iii) cysteine-rich protein (CRIPT), all of which bind to one or more of the three PDZ domains in PSD-95. While interactions for individual PDZ domains of PSD-95 have been well studied, less is known about the influence of neighbouring domains on the function of the respective individual domain. We therefore performed a systematic study on the ligand-binding kinetics of PSD-95 using constructs of different size for PSD-95 and its ligands. Regarding the canonical peptide-binding pocket and relatively short peptides (up to 15-mer), the PDZ domains in PSD-95 by and large work as individual binding modules. However, in agreement with previous studies, residues outside of the canonical binding pocket modulate the affinity of the ligands. In particular, the dissociation of the 101 amino acid CRIPT from PSD-95 is slowed down at least 10-fold for full-length PSD-95 when compared with the individual PDZ3 domain. PMID:26941280

  10. 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.

  11. 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

  12. Destabilization of Heterologous Proteins Mediated by the GSK3β Phosphorylation Domain of the β-Catenin Protein

    PubMed Central

    Kong, Yuhan; Zhang, Hongyu; Chen, Xian; Zhang, Wenwen; Zhao, Chen; Wang, Ning; Wu, Ningning; He, Yunfeng; Nan, Guoxin; Zhang, Hongmei; Wen, Sheng; Deng, Fang; Liao, Zhan; Wu, Di; Zhang, Junhui; Qin, Xinyue; Haydon, Rex C.; Luu, Hue H.; He, Tong-Chuan; Zhou, Lan

    2014-01-01

    Background and Aims Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3β may play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins. Methods and Results We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. Conclusion Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling. PMID:24335169

  13. DomHR: Accurately Identifying Domain Boundaries in Proteins Using a Hinge Region Strategy

    PubMed Central

    Zhang, Xiao-yan; Lu, Long-jian; Song, Qi; Yang, Qian-qian; Li, Da-peng; Sun, Jiang-ming; Li, Tong-hua; Cong, Pei-sheng

    2013-01-01

    Motivation The precise prediction of protein domains, which are the structural, functional and evolutionary units of proteins, has been a research focus in recent years. Although many methods have been presented for predicting protein domains and boundaries, the accuracy of predictions could be improved. Results In this study we present a novel approach, DomHR, which is an accurate predictor of protein domain boundaries based on a creative hinge region strategy. A hinge region was defined as a segment of amino acids that covers part of a domain region and a boundary region. We developed a strategy to construct profiles of domain-hinge-boundary (DHB) features generated by sequence-domain/hinge/boundary alignment against a database of known domain structures. The DHB features had three elements: normalized domain, hinge, and boundary probabilities. The DHB features were used as input to identify domain boundaries in a sequence. DomHR used a nonredundant dataset as the training set, the DHB and predicted shape string as features, and a conditional random field as the classification algorithm. In predicted hinge regions, a residue was determined to be a domain or a boundary according to a decision threshold. After decision thresholds were optimized, DomHR was evaluated by cross-validation, large-scale prediction, independent test and CASP (Critical Assessment of Techniques for Protein Structure Prediction) tests. All results confirmed that DomHR outperformed other well-established, publicly available domain boundary predictors for prediction accuracy. Availability The DomHR is available at http://cal.tongji.edu.cn/domain/. PMID:23593247

  14. Structure of the GAT domain of the endosomal adapter protein Tom1.

    PubMed

    Xiao, Shuyan; Ellena, Jeffrey F; Armstrong, Geoffrey S; Capelluto, Daniel G S

    2016-06-01

    Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain's association to Tollip's Tom1-binding domain (TBD). In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. PMID:26977434

  15. 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

  16. Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro.

    PubMed

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

    2002-11-15

    Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly. PMID:12171917

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

    PubMed

    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 shaping

  18. Optical Control of Protein–Protein Interactions via Blue Light-Induced Domain Swapping

    PubMed Central

    2015-01-01

    The design of new optogenetic tools for controlling protein function would be facilitated by the development of protein scaffolds that undergo large, well-defined structural changes upon exposure to light. Domain swapping, a process in which a structural element of a monomeric protein is replaced by the same element of another copy of the same protein, leads to a well-defined change in protein structure. We observe domain swapping in a variant of the blue light photoreceptor photoactive yellow protein in which a surface loop is replaced by a well-characterized protein–protein interaction motif, the E-helix. In the domain-swapped dimer, the E-helix sequence specifically binds a partner K-helix sequence, whereas in the monomeric form of the protein, the E-helix sequence is unable to fold into a binding-competent conformation and no interaction with the K-helix is seen. Blue light irradiation decreases the extent of domain swapping (from Kd = 10 μM to Kd = 300 μM) and dramatically enhances the rate, from weeks to <1 min. Blue light-induced domain swapping thus provides a novel mechanism for controlling of protein–protein interactions in which light alters both the stability and the kinetic accessibility of binding-competent states. PMID:25003701

  19. Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides

    SciTech Connect

    Heintz, Udo; Meinhart, Anton; Winkler, Andreas

    2014-03-01

    Crystal structures of two truncated variants of the transcription factor PpsR from R. sphaeroides are presented that enabled the phasing of a triple PAS domain construct. Together, these structures reveal the importance of α-helical PAS extensions for multi-PAS domain-mediated protein oligomerization and function. Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteins are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR from Rhodobacter sphaeroides is such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsR{sub Q-PAS1}) and two (PpsR{sub N-Q-PAS1}) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsR{sub ΔHTH}) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsR{sub ΔHTH} structure reveals how three PAS domains enable the formation of multiple oligomeric states (dimer, tetramer and octamer), highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-terminal extension of the N-domain that

  20. Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells.

    PubMed

    Kwaks, T H J; Sewalt, R G A B; van Blokland, R; Siersma, T J; Kasiem, M; Kelder, A; Otte, A P

    2005-01-12

    Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently. PMID:15607223

  1. Metagenome Analysis of Protein Domain Collocation within Cellulase Genes of Goat Rumen Microbes

    PubMed Central

    Lim, SooYeon; Seo, Jaehyun; Choi, Hyunbong; Yoon, Duhak; Nam, Jungrye; Kim, Heebal; Cho, Seoae; Chang, Jongsoo

    2013-01-01

    In this study, protein domains with cellulase activity in goat rumen microbes were investigated using metagenomic and bioinformatic analyses. After the complete genome of goat rumen microbes was obtained using a shotgun sequencing method, 217,892,109 pair reads were filtered, including only those with 70% identity, 100-bp matches, and thresholds below E−10 using METAIDBA. These filtered contigs were assembled and annotated using blastN against the NCBI nucleotide database. As a result, a microbial community structure with 1431 species was analyzed, among which Prevotella ruminicola 23 bacteria and Butyrivibrio proteoclasticus B316 were the dominant groups. In parallel, 201 sequences related with cellulase activities (EC.3.2.1.4) were obtained through blast searches using the enzyme.dat file provided by the NCBI database. After translating the nucleotide sequence into a protein sequence using Interproscan, 28 protein domains with cellulase activity were identified using the HMMER package with threshold E values below 10−5. Cellulase activity protein domain profiling showed that the major protein domains such as lipase GDSL, cellulase, and Glyco hydro 10 were present in bacterial species with strong cellulase activities. Furthermore, correlation plots clearly displayed the strong positive correlation between some protein domain groups, which was indicative of microbial adaption in the goat rumen based on feeding habits. This is the first metagenomic analysis of cellulase activity protein domains using bioinformatics from the goat rumen. PMID:25049895

  2. Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein.

    PubMed

    Krois, Alexander S; Ferreon, Josephine C; Martinez-Yamout, Maria A; Dyson, H Jane; Wright, Peter E

    2016-03-29

    An important component of the activity of p53 as a tumor suppressor is its interaction with the transcriptional coactivators cyclic-AMP response element-binding protein (CREB)-binding protein (CBP) and p300, which activate transcription of p53-regulated stress response genes and stabilize p53 against ubiquitin-mediated degradation. The highest affinity interactions are between the intrinsically disordered N-terminal transactivation domain (TAD) of p53 and the TAZ1 and TAZ2 domains of CBP/p300. The NMR spectra of simple binary complexes of the TAZ1 and TAZ2 domains with the p53TAD suffer from exchange broadening, but innovations in construct design and isotopic labeling have enabled us to obtain high-resolution structures using fusion proteins, uniformly labeled in the case of the TAZ2-p53TAD fusion and segmentally labeled through transintein splicing for the TAZ1-p53TAD fusion. The p53TAD is bipartite, with two interaction motifs, termed AD1 and AD2, which fold to form short amphipathic helices upon binding to TAZ1 and TAZ2 whereas intervening regions of the p53TAD remain flexible. Both the AD1 and AD2 motifs bind to hydrophobic surfaces of the TAZ domains, with AD2 making more extensive hydrophobic contacts consistent with its greater contribution to the binding affinity. Binding of AD1 and AD2 is synergistic, and structural studies performed with isolated motifs can be misleading. The present structures of the full-length p53TAD complexes demonstrate the versatility of the interactions available to an intrinsically disordered domain containing bipartite interaction motifs and provide valuable insights into the structural basis of the affinity changes that occur upon stress-related posttranslational modification. PMID:26976603

  3. CATHEDRAL: A Fast and Effective Algorithm to Predict Folds and Domain Boundaries from Multidomain Protein Structures

    PubMed Central

    Dallman, Tim; Pearl, Frances M. G; Orengo, Christine A

    2007-01-01

    We present CATHEDRAL, an iterative protocol for determining the location of previously observed protein folds in novel multidomain protein structures. CATHEDRAL builds on the features of a fast secondary-structure–based method (using graph theory) to locate known folds within a multidomain context and a residue-based, double-dynamic programming algorithm, which is used to align members of the target fold groups against the query protein structure to identify the closest relative and assign domain boundaries. To increase the fidelity of the assignments, a support vector machine is used to provide an optimal scoring scheme. Once a domain is verified, it is excised, and the search protocol is repeated in an iterative fashion until all recognisable domains have been identified. We have performed an initial benchmark of CATHEDRAL against other publicly available structure comparison methods using a consensus dataset of domains derived from the CATH and SCOP domain classifications. CATHEDRAL shows superior performance in fold recognition and alignment accuracy when compared with many equivalent methods. If a novel multidomain structure contains a known fold, CATHEDRAL will locate it in 90% of cases, with <1% false positives. For nearly 80% of assigned domains in a manually validated test set, the boundaries were correctly delineated within a tolerance of ten residues. For the remaining cases, previously classified domains were very remotely related to the query chain so that embellishments to the core of the fold caused significant differences in domain sizes and manual refinement of the boundaries was necessary. To put this performance in context, a well-established sequence method based on hidden Markov models was only able to detect 65% of domains, with 33% of the subsequent boundaries assigned within ten residues. Since, on average, 50% of newly determined protein structures contain more than one domain unit, and typically 90% or more of these domains are already

  4. The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain.

    PubMed

    Potisopon, Supanee; Priet, Stéphane; Collet, Axelle; Decroly, Etienne; Canard, Bruno; Selisko, Barbara

    2014-10-01

    Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis. PMID:25209234

  5. The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain

    PubMed Central

    Potisopon, Supanee; Priet, Stéphane; Collet, Axelle; Decroly, Etienne; Canard, Bruno; Selisko, Barbara

    2014-01-01

    Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis. PMID:25209234

  6. Origin and evolution of the cystic fibrosis transmembrane regulator protein R domain

    PubMed Central

    Sebastian, Aswathy; Rishishwar, Lavanya; Wang, Jianrong; Bernard, Karen F.; Conley, Andrew B.; McCarty, Nael A.; Jordan, I. King

    2013-01-01

    The Cystic Fibrosis Transmembrane Conductance Regulator protein (CFTR) is a member of the ABC transporter superfamily. CFTR is distinguished from all other members of this superfamily by its status as an ion channel as well as the presence of its unique regulatory (R) domain. We investigated the origin and subsequent evolution of the R domain along the CFTR evolutionary lineage. The R domain protein coding sequence originated via the loss of a splice donor site at the 3′ end of exon 14, leading to the subsequent read-through and capture of formerly intronic sequence as novel coding sequence. Inclusion of the remaining part of the R domain coding sequence in the CFTR transcript involved a lineage-specific gain of exonic sequence with no homology to protein coding sequences outside of CFTR and loss of two exons conserved among ABC family members. These events occurred at the base of the Gnathostome evolutionary lineage ~550–650 million years ago. The apparent origination of the R domain de novo from previously non-coding sequence is consistent with its lack of sequence similarity to other domains as well as its intrinsically disordered structure, which has important implications for its function. In particular, this lack of structure may provide for a dynamic and inducible regulatory activity based on transient physical interactions with more structured domains of the protein. Since its acquisition along the CFTR evolutionary lineage, the R domain has evolved more rapidly than any other CFTR domain; however, there is no evidence for positive (adaptive) selection in the evolution of the domain. The R domain does show a distinct pattern of relative evolutionary rates compared to other CFTR domains, which sheds additional light on the connection between its function and evolution. The regulatory function of the R domain is dependent upon a fairly small number of sites that are subject to phosphorylation, and these sites were fixed very early in R domain evolution

  7. Origin and evolution of the cystic fibrosis transmembrane regulator protein R domain.

    PubMed

    Sebastian, Aswathy; Rishishwar, Lavanya; Wang, Jianrong; Bernard, Karen F; Conley, Andrew B; McCarty, Nael A; Jordan, I King

    2013-07-10

    The Cystic Fibrosis Transmembrane Conductance Regulator protein (CFTR) is a member of the ABC transporter superfamily. CFTR is distinguished from all other members of this superfamily by its status as an ion channel as well as the presence of its unique regulatory (R) domain. We investigated the origin and subsequent evolution of the R domain along the CFTR evolutionary lineage. The R domain protein coding sequence originated via the loss of a splice donor site at the 3' end of exon 14, leading to the subsequent read-through and capture of formerly intronic sequence as novel coding sequence. Inclusion of the remaining part of the R domain coding sequence in the CFTR transcript involved a lineage-specific gain of exonic sequence with no homology to protein coding sequences outside of CFTR and loss of two exons conserved among ABC family members. These events occurred at the base of the Gnathostome evolutionary lineage ~550-650 million years ago. The apparent origination of the R domain de novo from previously non-coding sequence is consistent with its lack of sequence similarity to other domains as well as its intrinsically disordered structure, which has important implications for its function. In particular, this lack of structure may provide for a dynamic and inducible regulatory activity based on transient physical interactions with more structured domains of the protein. Since its acquisition along the CFTR evolutionary lineage, the R domain has evolved more rapidly than any other CFTR domain; however, there is no evidence for positive (adaptive) selection in the evolution of the domain. The R domain does show a distinct pattern of relative evolutionary rates compared to other CFTR domains, which sheds additional light on the connection between its function and evolution. The regulatory function of the R domain is dependent upon a fairly small number of sites that are subject to phosphorylation, and these sites were fixed very early in R domain evolution and

  8. Assessment of protein domain fusions in human protein interaction networks prediction: application to the human kinetochore model.

    PubMed

    Morilla, Ian; Lees, Jon G; Reid, Adam J; Orengo, Christine; Ranea, Juan A G

    2010-12-31

    In order to understand how biological systems function it is necessary to determine the interactions and associations between proteins. Some proteins, involved in a common biological process and encoded by separate genes in one organism, can be found fused within a single protein chain in other organisms. By detecting these triplets, a functional relationship can be established between the unfused proteins. Here we use a domain fusion prediction method to predict these protein interactions for the human interactome. We observed that gene fusion events are more related to physical interaction between proteins than to other weaker functional relationships such as participation in a common biological pathway. These results suggest that domain fusion is an appropriate method for predicting protein complexes. The most reliable fused domain predictions were used to build protein-protein interaction (PPI) networks. These predicted PPI network models showed the same topological features as real biological networks and different features from random behaviour. We built the PPI domain fusion sub-network model of the human kinetochore and observed that the majority of the predicted interactions have not yet been experimentally characterised in the publicly available PPI repositories. The study of the human kinetochore domain fusion sub-network reveals undiscovered kinetochore proteins with presumably relevant functions, such as hubs with many connections in the kinetochore sub-network. These results suggest that experimentally hidden regions in the predicted PPI networks contain key functional elements, associated with important functional areas, still undiscovered in the human interactome. Until novel experiments shed light on these hidden regions; domain fusion predictions provide a valuable approach for exploring them. PMID:20851221

  9. SET for life: biochemical activities and biological functions of SET domain-containing proteins

    PubMed Central

    Herz, Hans-Martin; Garruss, Alexander; Shilatifard, Ali

    2013-01-01

    SET domain-containing proteins belong to a group of enzymes named after a common domain that utilizes the cofactor S-adenosyl-L-methionine (SAM) to achieve methylation of its substrates. Many SET domain-containing proteins have been shown to display catalytic activity towards particular lysine residues on histones, but emerging evidence also indicates that various non-histone proteins are specifically targeted by this clade of enzymes. Here, we summarize the most recent findings on the biological functions of the major families of SET domain-containing proteins catalyzing the methylation of histones 3 on lysines 4, 9, 27, and 36 (H3K4, H3K9, H3K27, and H3K36) and histone 4 on lysine 20 (H4K20) as well as candidates that have been reported to regulate non-histone substrates. PMID:24148750

  10. 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.

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

    SciTech Connect

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

    2002-11-15

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

  12. Bayesian Modeling of the Yeast SH3 Domain Interactome Predicts Spatiotemporal Dynamics of Endocytosis Proteins

    PubMed Central

    Gfeller, David; Landgraf, Christiane; Panni, Simona; Paoluzi, Serena; Castagnoli, Luisa; Currell, Bridget; Seshagiri, Somasekar; Yu, Haiyuan; Winsor, Barbara; Vidal, Marc; Gerstein, Mark B.; Bader, Gary D.; Volkmer, Rudolf; Cesareni, Gianni; Drubin, David G.; Kim, Philip M.; Sidhu, Sachdev S.; Boone, Charles

    2009-01-01

    SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes. PMID:19841731

  13. Protein reconstitution and three-dimensional domain swapping: Benefits and constraints of covalency

    PubMed Central

    Carey, Jannette; Lindman, Stina; Bauer, Mikael; Linse, Sara

    2007-01-01

    The phenomena of protein reconstitution and three-dimensional domain swapping reveal that highly similar structures can be obtained whether a protein is comprised of one or more polypeptide chains. In this review, we use protein reconstitution as a lens through which to examine the range of protein tolerance to chain interruptions and the roles of the primary structure in related features of protein structure and folding, including circular permutation, natively unfolded proteins, allostery, and amyloid fibril formation. The results imply that noncovalent interactions in a protein are sufficient to specify its structure under the constraints imposed by the covalent backbone. PMID:17962398

  14. The Ubiquitin-associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation*

    PubMed Central

    Budhidarmo, Rhesa; Day, Catherine L.

    2014-01-01

    The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases. PMID:25065467

  15. PDZ motifs in PTP-BL and RIL bind to internal protein segments in the LIM domain protein RIL.

    PubMed

    Cuppen, E; Gerrits, H; Pepers, B; Wieringa, B; Hendriks, W

    1998-03-01

    The specificity of protein-protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells. PMID:9487134

  16. 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.

  17. Retinol Binding Protein-Albumin Domain III Fusion Protein Deactivates Hepatic Stellate Cells

    PubMed Central

    Park, Sangeun; Choi, Soyoung; Lee, Min-Goo; Lim, Chaeseung; Oh, Junseo

    2012-01-01

    Liver fibrosis is characterized by accumulation of extracellular matrix, and activated hepatic stellate cells (HSCs) are the primary source of the fibrotic neomatrix and considered as therapeutic target cells. We previously showed that albumin in pancreatic stellate cells (PSCs), the key cell type for pancreatic fibrogenesis, is directly involved in the formation of vitamin A-containing lipid droplets, inhibiting PSC activation. In this study, we evaluated the anti-fibrotic activity of both albumin and retinol binding protein-albumin domain III fusion protein (R-III), designed for stellate cell-targeted delivery of albumin III, in rat primary HSCs and investigated the underlying mechanism. Forced expression of albumin or R-III in HSCs after passage 2 (activated HSCs) induced lipid droplet formation and deactivated HSCs, whereas point mutations in high-affinity fatty acid binding sites of albumin domain III abolished their activities. Exogenous R-III, but not albumin, was successfully internalized into and deactivated HSC-P2. When HSCs at day 3 after plating (pre-activated HSCs) were cultured in the presence of purified R-III, spontaneous activation of HSCs was inhibited even after passage 2, suggestive of a potential for preventive effect. Furthermore, treatment of HSCs-P2 with R-III led to a significant reduction in both cytoplasmic levels of all-trans retinoic acid and the subsequent retinoic acid signaling. Therefore, our data suggest that albumin deactivates HSCs with reduced retinoic acid levels and that R-III may have therapeutic and preventive potentials on liver fibrosis. PMID:23161170

  18. A folded and functional protein domain in an amyloid-like fibril

    PubMed Central

    Sackewitz, Mirko; von Einem, Sabrina; Hause, Gerd; Wunderlich, Michael; Schmid, Franz-Xaver; Schwarz, Elisabeth

    2008-01-01

    The effect of the polypeptide environment on polyalanine-induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling polyadenylation. Mutation-caused extensions of the natural 10 alanine sequence up to maximally 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy (OPMD). We explored the influence of fibril formation on the structure and function of a one-domain protein linked to the fibril-forming part of PABPN1. The well-characterized, stably folded, one-domain protein, cold-shock protein CspB from Bacillus subtilis, was fused either to the C terminus of the entire N-terminal domain of PABPN1 or directly to peptides consisting of 10 or 17 alanine residues. The fusion protein between the N-terminal domain of PABPN1 and CspB formed fibrils in which the structure and activity of CspB were retained. In the fibrils formed by fusions in which the polyalanine sequence was directly linked to CspB, CspB was unfolded. These results indicate that the folded conformation and the function of a protein domain can be maintained in amyloid-like fibrils, and that the distance between this domain and the fibril plays an important role. PMID:18424511

  19. Putting into Practice Domain-Linear Motif Interaction Predictions for Exploration of Protein Networks

    PubMed Central

    Luck, Katja; Fournane, Sadek; Kieffer, Bruno; Masson, Murielle; Nominé, Yves; Travé, Gilles

    2011-01-01

    PDZ domains recognise short sequence motifs at the extreme C-termini of proteins. A model based on microarray data has been recently published for predicting the binding preferences of PDZ domains to five residue long C-terminal sequences. Here we investigated the potential of this predictor for discovering novel protein interactions that involve PDZ domains. When tested on real negative data assembled from published literature, the predictor displayed a high false positive rate (FPR). We predicted and experimentally validated interactions between four PDZ domains derived from the human proteins MAGI1 and SCRIB and 19 peptides derived from human and viral C-termini of proteins. Measured binding intensities did not correlate with prediction scores, and the high FPR of the predictor was confirmed. Results indicate that limitations of the predictor may arise from an incomplete model definition and improper training of the model. Taking into account these limitations, we identified several novel putative interactions between PDZ domains of MAGI1 and SCRIB and the C-termini of the proteins FZD4, ARHGAP6, NET1, TANC1, GLUT7, MARCH3, MAS, ABC1, DLL1, TMEM215 and CYSLTR2. These proteins are localised to the membrane or suggested to act close to it and are often involved in G protein signalling. Furthermore, we showed that, while extension of minimal interacting domains or peptides toward tandem constructs or longer peptides never suppressed their ability to interact, the measured affinities and inferred specificity patterns often changed significantly. This suggests that if protein fragments interact, the full length proteins are also likely to interact, albeit possibly with altered affinities and specificities. Therefore, predictors dealing with protein fragments are promising tools for discovering protein interaction networks but their application to predict binding preferences within networks may be limited. PMID:22069443

  20. Unusual domain movement in a multidomain protein in the presence of macromolecular crowders.

    PubMed

    Biswas, Saikat; Chowdhury, Pramit K

    2015-08-14

    Domain movements play a fundamental and critical role in the specific biological function that multidomain proteins have evolved to perform. A significant amount of research has been carried out to investigate the effects of macromolecular crowding agents, mostly on single domain proteins, thereby furthering our appreciation for the crowding phenomenon. However similar studies on proteins having multiple domains are relatively scarce. Using the plasma protein human serum albumin (HSA) as the protein of interest, we have probed the influence of dextran based crowding agents (Dextran 6, Dextran 40, and Dextran 70) on the relative movements of domains I and II using FRET, with Trp-214 in domain II acting as the donor and acrylodan (Ac) covalently attached to Cys-34 of domain I as the acceptor. Amongst the higher molecular weight crowders, while both Dextran 70 and Dextran 40 induced a significant decrease in the distance between the aforesaid domains, however for the latter macromolecular crowder (Dextran 40), beyond 50 g L(-1), no change in domain separation was observed even up to concentrations of 175 g L(-1). On the other hand, contrary to our expectations, Dextran 6, having the highest packing density by virtue of it being the smallest crowding agent used, provided an asymmetric excluded volume which resulted in forced elongation of HSA along the Trp-Ac FRET axis. Additionally both chemical and thermal studies performed at varying concentrations of the chemical denaturant, urea, reveal unusual movements of the two domains, an aspect that can have important implications with regard to HSA being an avid transporter of fatty acids, with the binding of latter being known to invoke appreciable domain displacements. We hypothesise that we see a distinct crossover from entropy dominated depletion effects in the case of Dextran 6 to significant enthalpic contribution for Dextran 70 with Dextran 40 lying midway between these two crowders, having characteristics of both

  1. BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

    PubMed

    Gabellini, Chiara; De Luca, Teresa; Trisciuoglio, Daniela; Desideri, Marianna; Di Martile, Marta; Passeri, Daniela; Candiloro, Antonio; Biffoni, Mauro; Rizzo, Maria Giulia; Orlandi, Augusto; Del Bufalo, Donatella

    2013-11-01

    Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis. PMID:23836782

  2. 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.

  3. 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.

  4. Identification of domains in protein structures from the analysis of intramolecular interactions.

    PubMed

    Genoni, Alessandro; Morra, Giulia; Colombo, Giorgio

    2012-03-15

    The subdivision of protein structures into smaller and independent structural domains has a fundamental importance in understanding protein evolution and function and in the development of protein classification methods as well as in the interpretation of experimental data. Due to the rapid growth in the number of solved protein structures, the need for devising new accurate algorithmic methods has become more and more urgent. In this paper, we propose a new computational approach that is based on the concept of domain as a compact and independent folding unit and on the analysis of the residue-residue energy interactions obtainable through classical all-atom force field calculations. In particular, starting from the analysis of the nonbonded interaction energy matrix associated with a protein, our method filters out and selects only those specific subsets of interactions that define possible independent folding nuclei within a complex protein structure. This allows grouping different protein fragments into energy clusters that are found to correspond to structural domains. The strategy has been tested using proper benchmark data sets, and the results have shown that the new approach is fast and reliable in determining the number of domains in a totally ab initio manner and without making use of any training set or knowledge of the systems in exam. Moreover, our method, identifying the most relevant residues for the stabilization of each domain, may complement the results given by other classification techniques and may provide useful information to design and guide new experiments. PMID:22384792

  5. 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

  6. Protein domain of unknown function 3233 is a translocation domain of autotransporter secretory mechanism in gamma proteobacteria.

    PubMed

    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 1/3(rd) 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

  7. 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

  8. 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

  9. PDZ Motifs in PTP-BL and RIL Bind to Internal Protein Segments in the LIM Domain Protein RIL

    PubMed Central

    Cuppen, Edwin; Gerrits, Herlinde; Pepers, Barry; Wieringa, Bé; Hendriks, Wiljan

    1998-01-01

    The specificity of protein–protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells. PMID:9487134

  10. Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities.

    PubMed Central

    Dowler, S; Currie , R A; Campbell , D G; Deak, M; Kular, G; Downes, C P; Alessi, D R

    2000-01-01

    The second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is generated by the action of phosphoinositide 3-kinase (PI 3-kinase), and regulates a plethora of cellular processes. An approach for dissecting the mechanisms by which these processes are regulated is to identify proteins that interact specifically with PtdIns(3,4,5)P(3). The pleckstrin homology (PH) domain has become recognized as the specialized module used by many proteins to interact with PtdIns(3,4,5)P(3). Recent work has led to the identification of a putative phosphatidylinositol 3,4,5-trisphosphate-binding motif (PPBM) at the N-terminal regions of PH domains that interact with this lipid. We have searched expressed sequence tag databases for novel proteins containing PH domains possessing a PPBM. Surprisingly, many of the PH domains that we identified do not bind PtdIns(3,4,5)P(3), but instead possess unexpected and novel phosphoinositide-binding specificities in vitro. These include proteins possessing PH domains that interact specifically with PtdIns(3,4)P(2) [TAPP1 (tandem PH-domain-containing protein-1) and TAPP2], PtdIns4P [FAPP1 (phosphatidylinositol-four-phosphate adaptor protein-1)], PtdIns3P [PEPP1 (phosphatidylinositol-three-phosphate-binding PH-domain protein-1) and AtPH1] and PtdIns(3,5)P(2) (centaurin-beta2). We have also identified two related homologues of PEPP1, termed PEPP2 and PEPP3, that may also interact with PtdIns3P. This study lays the foundation for future work to establish the phospholipid-binding specificities of these proteins in vivo, and their physiological role(s). PMID:11001876

  11. The Pilus Usher Controls Protein Interactions via Domain Masking and is Functional as an Oligomer

    PubMed Central

    Werneburg, Glenn T.; Henderson, Nadine S.; Portnoy, Erica B.; Sarowar, Samema; Hultgren, Scott J.; Li, Huilin; Thanassi, David G.

    2015-01-01

    The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate binding site, but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions, and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria. PMID:26052892

  12. Evolution and Quantitative Comparison of Genome-Wide Protein Domain Distributions

    PubMed Central

    Parikesit, Arli A.; Stadler, Peter F.; Prohaska, Sonja J.

    2011-01-01

    The metabolic and regulatory capabilities of an organism are implicit in its protein content. This is often hard to estimate, however, due to ascertainment biases inherent in the available genome annotations. Its complement of recognizable functional protein domains and their combinations convey essentially the same information and at the same time are much more readily accessible, although protein domain models trained for one phylogenetic group frequently fail on distantly related sequences. Pooling related domain models based on their GO-annotation in combination with de novo gene prediction methods provides estimates that seem to be less affected by phylogenetic biases. We show here for 18 diverse representatives from all eukaryotic kingdoms that a pooled analysis of the tendencies for co-occurrence or avoidance of protein domains is indeed feasible. This type of analysis can reveal general large-scale patterns in the domain co-occurrence and helps to identify lineage-specific variations in the evolution of protein domains. Somewhat surprisingly, we do not find strong ubiquitous patterns governing the evolutionary behavior of specific functional classes. Instead, there are strong variations between the major groups of Eukaryotes, pointing at systematic differences in their evolutionary constraints. PMID:24710298

  13. Effect of Interdomain Linker Length on an Antagonistic Folding-Unfolding Equilibrium between Two Protein Domains

    PubMed Central

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

    2009-01-01

    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 (ΔGX). 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. ΔGX values are determined by measuring the extent to which Co2+ 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 ΔGX 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. PMID:19038264

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

    PubMed

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

    2014-07-01

    With the preponderance of multidomain proteins in eukaryotic genomes, it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces, and the knowledge of the interacting sites is essential to our understanding of the structure-function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multidomain), we predict the interface residues corresponding to domains from the two-domain proteins. We also use information from the three-dimensional structures of individual domains of two-domain proteins to train naïve Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (∼85%) and specific (∼95%) to the domain-domain interfaces. This method is specific to multidomain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain-domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. We believe that these results can be of considerable interest toward rational protein and interaction design, apart from providing us with valuable information on the nature of interactions. PMID:24375512

  15. Activation and assembly of the inflammasomes through conserved protein domain families

    PubMed Central

    2015-01-01

    Inflammasomes are oligomeric protein complexes assembled through interactions among the death domain superfamily members, in particular the CARD and PYD domains. Recent progress has shed lights on how the ASC PYD can polymerize to form filaments using multiple domain:domain interfaces, and how the caspase4 CARD can recognize LPS to activate the non-classical inflammasome pathway. Comprehensive understanding of the molecular mechanisms of inflammasome activation and assembly require more extensive structural and biophysical dissection of the inflammasome components and complexes, in particular additional CARD or PYD filaments. Because of the variations in death domain structures and complexes observed so far, future work will undoubtedly shed lights on the mechanisms of inflammasome assembly as well as more surprises on the versatile structure and function of the death domain superfamily. PMID:25398536

  16. Predicting physiologically relevant SH3 domain mediated protein–protein interactions in yeast

    PubMed Central

    Jain, Shobhit; Bader, Gary D.

    2016-01-01

    Motivation: Many intracellular signaling processes are mediated by interactions involving peptide recognition modules such as SH3 domains. These domains bind to small, linear protein sequence motifs which can be identified using high-throughput experimental screens such as phage display. Binding motif patterns can then be used to computationally predict protein interactions mediated by these domains. While many protein–protein interaction prediction methods exist, most do not work with peptide recognition module mediated interactions or do not consider many of the known constraints governing physiologically relevant interactions between two proteins. Results: A novel method for predicting physiologically relevant SH3 domain-peptide mediated protein–protein interactions in S. cerevisae using phage display data is presented. Like some previous similar methods, this method uses position weight matrix models of protein linear motif preference for individual SH3 domains to scan the proteome for potential hits and then filters these hits using a range of evidence sources related to sequence-based and cellular constraints on protein interactions. The novelty of this approach is the large number of evidence sources used and the method of combination of sequence based and protein pair based evidence sources. By combining different peptide and protein features using multiple Bayesian models we are able to predict high confidence interactions with an overall accuracy of 0.97. Availability and implementation: Domain-Motif Mediated Interaction Prediction (DoMo-Pred) command line tool and all relevant datasets are available under GNU LGPL license for download from http://www.baderlab.org/Software/DoMo-Pred. The DoMo-Pred command line tool is implemented using Python 2.7 and C ++. Contact: gary.bader@utoronto.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26861823

  17. 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 16.8kDa 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. PMID:26946400

  18. Biological implications of SNPs in signal peptide domains of human proteins.

    PubMed

    Jarjanazi, Hamdi; Savas, Sevtap; Pabalan, Noel; Dennis, James W; Ozcelik, Hilmi

    2008-02-01

    Proteins destined for secretion or membrane compartments possess signal peptides for insertion into the membrane. The signal peptide is therefore critical for localization and function of cell surface receptors and ligands that mediate cell-cell communication. About 4% of all human proteins listed in UniProt database have signal peptide domains in their N terminals. A comprehensive literature survey was performed to retrieve functional and disease associated genetic variants in the signal peptide domains of human proteins. In 21 human proteins we have identified 26 disease associated mutations within their signal peptide domains, 14 mutations of which have been experimentally shown to impair the signal peptide function and thus influence protein transportation. We took advantage of SignalP 3.0 predictions to characterize the signal peptide prediction score differences between the mutant and the wild-type alleles of each mutation, as well as 189 previously uncharacterized single nucleotide polymorphisms (SNPs) found to be located in the signal peptide domains of 165 human proteins. Comparisons of signal peptide prediction outcomes of mutations and SNPs, have implicated SNPs potentially impacting the signal peptide function, and thus the cellular localization of the human proteins. The majority of the top candidate proteins represented membrane and secreted proteins that are associated with molecular transport, cell signaling and cell to cell interaction processes of the cell. This is the first study that systematically characterizes genetic variation occurring in the signal peptides of all human proteins. This study represents a useful strategy for prioritization of SNPs occurring within the signal peptide domains of human proteins. Functional evaluation of candidates identified herein may reveal effects on major cellular processes including immune cell function, cell recognition and adhesion, and signal transduction. PMID:17680692

  19. The MDM2 RING domain and central acidic domain play distinct roles in MDM2 protein homodimerization and MDM2-MDMX protein heterodimerization.

    PubMed

    Leslie, Patrick L; Ke, Hengming; Zhang, Yanping

    2015-05-15

    The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization. PMID:25809483

  20. High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors.

    PubMed Central

    Zwilling, S; König, H; Wirth, T

    1995-01-01

    The octamer transcription factors Oct1 and Oct2 are involved in the transcriptional regulation of both lymphoid-specific and ubiquitously expressed genes. Their activity depends critically on their interaction with distinct cellular cofactors. Therefore, we have isolated cDNAs encoding proteins that physically interact with Oct2. Here we describe the analysis of one such clone, representing the murine homologue of high mobility group (HMG) protein 2. We have mapped the interaction domains for both proteins and have shown that HMG2 and Oct2 interact via their HMG domains and POU homeodomains, respectively. This interaction is not restricted to Oct2, as other members of the octamer transcription factor family like Oct1 and Oct6 also interact with HMG2. The interaction with HMG2 results in a marked increase in the sequence-specific DNA binding activity of the Oct proteins. Interestingly, the HMG2 protein is not present in the protein-DNA complex detected by an electrophoretic mobility shift assay. The Oct and HMG2 proteins also interact in vivo. A chimeric protein, in which the strong transactivation domain of VP16 was fused directly to the HMG domains of HMG2, stimulated the activity of an octamer-dependent reporter construct upon cotransfection. Furthermore, the expression of antisense RNA for HMG2 specifically reduces octamer-dependent transcription. These results suggest that one of the functions of HMG2 is to support the octamer transcription factors in their role as transcriptional activators. Images PMID:7720710

  1. 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 ...

  2. 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

  3. A family of RS domain proteins with novel subcellular localization and trafficking

    PubMed Central

    Kavanagh, Steven J.; Schulz, Thomas C.; Davey, Philippa; Claudianos, Charles; Russell, Carrie; Rathjen, Peter D.

    2005-01-01

    We report the sequence, conservation and cell biology of a novel protein, Psc1, which is expressed and regulated within the embryonic pluripotent cell population of the mouse. The Psc1 sequence includes an RS domain and an RNA recognition motif (RRM), and a sequential arrangement of protein motifs that has not been demonstrated for other RS domain proteins. This arrangement was conserved in a second mouse protein (BAC34721). The identification of Psc1 and BAC34721 homologues in vertebrates and related proteins, more widely throughout evolution, defines a new family of RS domain proteins termed acidic rich RS (ARRS) domain proteins. Psc1 incorporated into the nuclear speckles, but demonstrated novel aspects of subcellular distribution including localization to speckles proximal to the nuclear periphery and localization to punctate structures in the cytoplasm termed cytospeckles. Integration of Psc1 into cytospeckles was dependent on the RRM. Cytospeckles were dynamic within the cytoplasm and appeared to traffic into the nucleus. These observations suggest a novel role in RNA metabolism for ARRS proteins. PMID:15741184

  4. N-terminal domains of native multidomain proteins have the potential to assist de novo folding of their downstream domains in vivo by acting as solubility enhancers

    PubMed Central

    Kim, Chul Woo; Han, Kyoung Sim; Ryu, Ki-Sun; Kim, Byung Hee; Kim, Kyun-Hwan; Choi, Seong Il; Seong, Baik L.

    2007-01-01

    The fusion of soluble partner to the N terminus of aggregation-prone polypeptide has been popularly used to overcome the formation of inclusion bodies in the E. coli cytosol. The chaperone-like functions of the upstream fusion partner in the artificial multidomain proteins could occur in de novo folding of native multidomain proteins. Here, we show that the N-terminal domains of three E. coli multidomain proteins such as lysyl-tRNA synthetase, threonyl-tRNA synthetase, and aconitase are potent solubility enhancers for various C-terminal heterologous proteins. The results suggest that the N-terminal domains could act as solubility enhancers for the folding of their authentic C-terminal domains in vivo. Tandem repeat of N-terminal domain or insertion of aspartic residues at the C terminus of the N-terminal domain also increased the solubility of fusion proteins, suggesting that the solubilizing ability correlates with the size and charge of N-terminal domains. The solubilizing ability of N-terminal domains would contribute to the autonomous folding of multidomain proteins in vivo, and based on these results, we propose a model of how N-terminal domains solubilize their downstream domains. PMID:17384228

  5. Use of a Probabilistic Motif Search to Identify Histidine Phosphotransfer Domain-Containing Proteins.

    PubMed

    Surujon, Defne; Ratner, David I

    2016-01-01

    The wealth of newly obtained proteomic information affords researchers the possibility of searching for proteins of a given structure or function. Here we describe a general method for the detection of a protein domain of interest in any species for which a complete proteome exists. In particular, we apply this approach to identify histidine phosphotransfer (HPt) domain-containing proteins across a range of eukaryotic species. From the sequences of known HPt domains, we created an amino acid occurrence matrix which we then used to define a conserved, probabilistic motif. Examination of various organisms either known to contain (plant and fungal species) or believed to lack (mammals) HPt domains established criteria by which new HPt candidates were identified and ranked. Search results using a probabilistic motif matrix compare favorably with data to be found in several commonly used protein structure/function databases: our method identified all known HPt proteins in the Arabidopsis thaliana proteome, confirmed the absence of such motifs in mice and humans, and suggests new candidate HPts in several organisms. Moreover, probabilistic motif searching can be applied more generally, in a manner both readily customized and computationally compact, to other protein domains; this utility is demonstrated by our identification of histones in a range of eukaryotic organisms. PMID:26751210

  6. Use of a Probabilistic Motif Search to Identify Histidine Phosphotransfer Domain-Containing Proteins

    PubMed Central

    Surujon, Defne; Ratner, David I.

    2016-01-01

    The wealth of newly obtained proteomic information affords researchers the possibility of searching for proteins of a given structure or function. Here we describe a general method for the detection of a protein domain of interest in any species for which a complete proteome exists. In particular, we apply this approach to identify histidine phosphotransfer (HPt) domain-containing proteins across a range of eukaryotic species. From the sequences of known HPt domains, we created an amino acid occurrence matrix which we then used to define a conserved, probabilistic motif. Examination of various organisms either known to contain (plant and fungal species) or believed to lack (mammals) HPt domains established criteria by which new HPt candidates were identified and ranked. Search results using a probabilistic motif matrix compare favorably with data to be found in several commonly used protein structure/function databases: our method identified all known HPt proteins in the Arabidopsis thaliana proteome, confirmed the absence of such motifs in mice and humans, and suggests new candidate HPts in several organisms. Moreover, probabilistic motif searching can be applied more generally, in a manner both readily customized and computationally compact, to other protein domains; this utility is demonstrated by our identification of histones in a range of eukaryotic organisms. PMID:26751210

  7. Dual amyloid domains promote differential functioning of the chaplin proteins during Streptomyces aerial morphogenesis

    PubMed Central

    Capstick, David S.; Jomaa, Ahmad; Hanke, Chistopher; Ortega, Joaquin; Elliot, Marie A.

    2011-01-01

    The chaplin proteins are functional amyloids found in the filamentous Streptomyces bacteria. These secreted proteins are required for the aerial development of Streptomyces coelicolor, and contribute to an intricate rodlet ultrastructure that decorates the surfaces of aerial hyphae and spores. S. coelicolor encodes eight chaplin proteins. Previous studies have revealed that only three of these proteins (ChpC, ChpE, and ChpH) are necessary for promoting aerial development, and of these three, ChpH is the primary developmental determinant. Here, we show that the model chaplin, ChpH, contains two amyloidogenic domains: one in the N terminus and one in the C terminus of the mature protein. These domains have different polymerization properties as determined using fluorescence spectroscopy, secondary structure analyses, and electron microscopy. We coupled these in vitro assays with in vivo genetic studies to probe the connection between ChpH amyloidogenesis and its biological function. Using mutational analyses, we demonstrated that both N- and C-terminal amyloid domains of ChpH were required for promoting aerial hypha formation, while the N-terminal domain was dispensable for assembly of the rodlet ultrastructure. These results suggest that there is a functional differentiation of the dual amyloid domains in the chaplin proteins. PMID:21628577

  8. Structure of the GAT domain of the endosomal adapter protein Tom1

    PubMed Central

    Xiao, Shuyan; Ellena, Jeffrey F.; Armstrong, Geoffrey S.; Capelluto, Daniel G.S.

    2016-01-01

    Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. PMID:26977434

  9. 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.

  10. Insights into common functional domains of tospovirus NSm proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Direct demonstration of tospovirus gene function has been impeded by the absence of reliable reverse genetics systems for this virus genus. Use of a Tobacco mosaic virus (TMV)-based expression system has demonstrated that the Tomato spotted wilt virus (TSWV) NSm protein supports cell-to-cell moveme...

  11. Novel Mechanism of Regulation of Tomato Bushy Stunt Virus Replication by Cellular WW-Domain Proteins

    PubMed Central

    Barajas, Daniel; Kovalev, Nikolay; Qin, Jun

    2014-01-01

    ABSTRACT Replication of (+)RNA viruses depends on several co-opted host proteins but is also under the control of cell-intrinsic restriction factors (CIRFs). By using tombusviruses, small model viruses of plants, we dissect the mechanism of inhibition of viral replication by cellular WW-domain-containing proteins, which act as CIRFs. By using fusion proteins between the WW domain and the p33 replication protein, we show that the WW domain inhibits the ability of p33 to bind to the viral RNA and to other p33 and p92 replication proteins leading to inhibition of viral replication in yeast and in a cell extract. Overexpression of WW-domain protein in yeast also leads to reduction of several co-opted host factors in the viral replicase complex (VRC). These host proteins, such as eEF1A, Cdc34 E2 ubiquitin-conjugating enzyme, and ESCRT proteins (Bro1p and Vps4p), are known to be involved in VRC assembly. Simultaneous coexpression of proviral cellular factors with WW-domain protein partly neutralizes the inhibitory effect of the WW-domain protein. We propose that cellular WW-domain proteins act as CIRFs and also as regulators of tombusvirus replication by inhibiting the assembly of new membrane-bound VRCs at the late stage of infection. We suggest that tombusviruses could sense the status of the infected cells via the availability of cellular susceptibility factors versus WW-domain proteins for binding to p33 replication protein that ultimately controls the formation of new VRCs. This regulatory mechanism might explain how tombusviruses could adjust the efficiency of RNA replication to the limiting resources of the host cells during infections. IMPORTANCE Replication of positive-stranded RNA viruses, which are major pathogens of plants, animals, and humans, is inhibited by several cell-intrinsic restriction factors (CIRFs) in infected cells. We define here the inhibitory roles of the cellular Rsp5 ubiquitin ligase and its WW domain in plant-infecting tombusvirus

  12. Identification of a novel human zinc finger protein that specifically interacts with the activation domain of lentiviral Tat proteins.

    PubMed

    Fridell, R A; Harding, L S; Bogerd, H P; Cullen, B R

    1995-06-01

    Transcriptional activation of HIV-1 gene expression by the viral Tat protein requires the interaction of a cellular cofactor with the Tat activation domain. This domain has been shown to consist of the cysteine-rich and core motifs of HIV-1 Tat and is functionally conserved in the distantly related Tat proteins of HIV-2 and EIAV. Using the yeast two-hybrid system, we have identified a novel human gene product, termed HT2A, that specifically and precisely binds to the activation domain of HIV-1 Tat and that can also interact with the HIV-2 and EIAV Tat proteins in vivo. We present data further demonstrating that the interaction between the activation domain of HIV-1 Tat and the HT2A protein can be readily detected in the mammalian cell nucleus. Sequence analysis demonstrates that HT2A is a novel member of the C3HC4 or ring finger family of zinc finger proteins that includes several known oncogenes and transcription factors. Overall, these data suggest that HT2A may play a significant role in mediating the biological activity of the HIV-1 Tat protein in vivo. PMID:7778269

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

    PubMed

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

    2015-01-01

    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

  14. 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

  15. The conserved KNOX domain mediates specificity of tobacco KNOTTED1-type homeodomain proteins.

    PubMed Central

    Sakamoto, T; Nishimura, A; Tamaoki, M; Kuba, M; Tanaka, H; Iwahori, S; Matsuoka, M

    1999-01-01

    Overproduction of the tobacco KNOTTED1-type homeodomain proteins NTH1, NTH15, and NTH23 in transgenic tobacco plants causes mild, severe, and no morphological alterations, respectively. The deduced amino acid sequences of the homeodomains and adjacent ELK domains are highly conserved, and the N-terminal KNOX domains also are moderately conserved. To investigate the contributions of both the conserved and divergent regions to the severity of morphological alterations, we generated chimeric proteins by exchanging different regions of NTH1, NTH15, and NTH23. The severity of the abnormal phenotype was dependent upon the synergistic action of both the N terminus, containing the KNOX domain, and the C terminus, containing the ELK homeodomain. Detailed analysis focusing on the C terminus revealed that the C-terminal half of the ELK domain is more effective in inducing the abnormal phenotypes than are the homeodomains. For the N terminus, severe morphological alterations were induced by exchanging a part of the KNOX domain of NTH1 with the corresponding region of NTH15. This limited region in the KNOX domain of all homeodomain proteins includes a predicted alpha-helical region, but only that in NTH15 is predicted to form a typical amphipathic structure. We discuss the possibility, based on these results, that the secondary structure of the KNOX domain is important for the induction of abnormal morphology in transgenic tobacco plants. PMID:10449577

  16. 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. PMID:12750370

  17. Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain.

    PubMed

    Harper, J F; Huang, J F; Lloyd, S J

    1994-06-14

    CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor. CDPK isoform AK1 from Arabidopsis was expressed in Escherichia coli as a fusion protein sandwiched between glutathione S-transferase and six consecutive histidines at the N- and C-terminal ends, respectively. This fusion, called AK1-6H, was purified and displayed kinase activity which was stimulated up to 127-fold by Ca2+, with a typical specific activity of 2000 nmol min-1 mg-1, using syntide-2 as peptide substrate. A truncation which deletes the calmodulin-like domain, as in mutant delta C-6H, disrupts Ca2+ activation and leaves the enzyme with a basal level of activity. Delta C-6H could be activated 87-fold by preincubation with a purified polyclonal IgG which was raised against a junction domain fusion. A further deletion of the junction domain, as in mutant delta JC, results in a constitutively active enzyme. This indicates that the junction domain in delta C-6H can function as an autoinhibitor. Its function as an autoinhibitor in a full-length enzyme was confirmed by site-specific mutagenesis, as shown by mutant KJM23-6H, which had a six-residue substitution in the junction domain between A422 and A432. Both delta JC and KJM23-6H encoded Ca(2+)-independent enzymes which had specific activities greater than 70% that of a fully active AK1-6H and displayed equivalent Km values for ATP and syntide-2. Inhibition studies on delta JC, using peptides based on the autoinhibitory domains of Ca2+/calmodulin-dependent protein kinases, are consistent with a model where the junction domain contains a similar pseudosubstrate-type autoinhibitor. PMID:8003490

  18. 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

  19. Membrane Anchoring of Aminoacyl-tRNA Synthetases by Convergent Acquisition of a Novel Protein Domain*

    PubMed Central

    Olmedo-Verd, Elvira; Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A. G.; Ribas de Pouplana, Lluis; Luque, Ignacio

    2011-01-01

    Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain. PMID:21965654

  20. 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

  1. 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

  2. Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems

    PubMed Central

    An, Wenlin; Jackson, Rachel E.; Hunter, Paul; Gögel, Stefanie; van Diepen, Michiel; Liu, Karen; Meyer, Martin P.; Eickholt, Britta J.

    2015-01-01

    Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI) with a destabilizing domain (DD) specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models. PMID:26717575

  3. Transmembrane and coiled-coil domain family 1 is a novel protein of the endoplasmic reticulum.

    PubMed

    Zhang, Chao; Kho, Yik-Shing; Wang, Zhe; Chiang, Yan Ting; Ng, Gary K H; Shaw, Pang-Chui; Wang, Yuzhuo; Qi, Robert Z

    2014-01-01

    The endoplasmic reticulum (ER) is a continuous membrane network in eukaryotic cells comprising the nuclear envelope, the rough ER, and the smooth ER. The ER has multiple critical functions and a characteristic structure. In this study, we identified a new protein of the ER, TMCC1 (transmembrane and coiled-coil domain family 1). The TMCC family consists of at least 3 putative proteins (TMCC1-3) that are conserved from nematode to human. We show that TMCC1 is an ER protein that is expressed in diverse human cell lines. TMCC1 contains 2 adjacent transmembrane domains near the C-terminus, in addition to coiled-coil domains. TMCC1 was targeted to the rough ER through the transmembrane domains, whereas the N-terminal region and C-terminal tail of TMCC1 were found to reside in the cytoplasm. Moreover, the cytosolic region of TMCC1 formed homo- or hetero-dimers or oligomers with other TMCC proteins and interacted with ribosomal proteins. Notably, overexpression of TMCC1 or its transmembrane domains caused defects in ER morphology. Our results suggest roles of TMCC1 in ER organization. PMID:24454821

  4. Fusion protein based on Grb2-SH2 domain for cancer therapy

    SciTech Connect

    Saito, Yuriko; Furukawa, Takako; Arano, Yasushi; Fujibayashi, Yasuhisa; Saga, Tsuneo

    2010-08-20

    Research highlights: {yields} Grb2 mediates EGFR signaling through binding to phosphorylate EGFR with SH2 domain. {yields} We generated fusion proteins containing 1 or 2 SH2 domains of Grb2 added with TAT. {yields} The one with 2 SH2 domains (TSSF) interfered ERK phosphorylation. {yields} TSSF significantly delayed the growth of EGFR overexpressing tumor in a mouse model. -- Abstract: Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylated EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR-Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR-Grb2 signaling and could have a potential to treat EGFR-activated cancer.

  5. Structural Basis for Ubiquitin Recognition by the Otu1 Ovarian Tumor Domain Protein

    SciTech Connect

    T Messick; N Russel; A Iwata; K Sarachan; R Shiekhattar; I Shanks; F Reyes-Turcu; K Wilkinson; R Marmorstein

    2011-12-31

    Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of {approx}130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys{sup 48} linkages, having little or no activity on Lys{sup 63}- and Lys{sup 29}-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.

  6. Analysis of the overall structure of the multi-domain amyloid precursor protein (APP).

    PubMed

    Coburger, Ina; Dahms, Sven O; Roeser, Dirk; Gührs, Karl-Heinz; Hortschansky, Peter; Than, Manuel E

    2013-01-01

    The amyloid precursor protein (APP) and its processing by the α-, β- and γ-secretases is widely believed to play a central role during the development of Alzheimer´s disease. The three-dimensional structure of the entire protein, its physiologic function and the regulation of its proteolytic processing remain, however, largely unclear to date. To gain a deeper understanding of the structure of APP that underlies all of its functions, we first cloned and recombinantly expressed different constructs in E. coli. Using limited proteolysis followed by mass spectrometry and Edman degradation as well as analytical gel permeation chromatography coupled static light scattering, we experimentally analyzed the structural domain boundaries and determined that the large ectodomain of APP consists of exactly two rigidly folded domains - the E1-domain (Leu18-Ala190) and the E2-domain (Ser295-Asp500). Both, the acidic domain (AcD) connecting E1 and E2 as well as the juxtamembrane region (JMR) connecting E2 to the single transmembrane helix are highly flexible and extended. We identified in-between the E1-domain and the AcD an additional domain of conservation and partial flexibility that we termed extension domain (ED, Glu191-Glu227). Using Bio-layer interferometry, pull-down assays and analytical gel filtration experiments we demonstrated that the E1-domain does not tightly interact with the E2-domain, both in the presence and in the absence of heparin. APP hence forms an extended molecule that is flexibly tethered to the membrane. Its multi-domain architecture enables together with the many known functionalities the concomitant performance of several, independent functions, which might be regulated by cellular, compartment specific pH-changes. PMID:24324731

  7. 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

  8. 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

  9. 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

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

    PubMed Central

    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

    2015-01-01

    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. PMID:25864124

  11. Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein

    PubMed Central

    Mi, Michael Y; Zhang, Jiying; He, Yukai

    2005-01-01

    Background A critical step in the production of new HIV virions involves the TAT protein binding to the TAR element. The TAT protein contains in close proximity its TAR RNA binding domain and protein transduction domain (PTD). The PTD domain of TAT has been identified as being instrumental in the protein's ability to cross mammalian cell and nuclear membranes. All together, this information led us to form the hypothesis that a protein containing the TAR RNA binding domain could compete with the native full length TAT protein and effectively block the TAR RNA binding site in transduced HIV infected cells. Results We synthesized a short peptide named Tat-P, which contained the TAR RNA binding and PTD domains to examine whether the peptide has the potential of inhibiting TAT dependent HIV replication. We investigated the inhibiting effects of Tat-P in vitro using a HIV derived lentiviral vector model. We found that the TAT PTD domain not only efficiently transduced test cells, but also effectively inhibited the production of lentiviral particles in a TAT dependent manner. These results were also supported by data derived from the TAT activated LTR-luciferase expression model and RNA binding assays. Conclusion Tat-P may become part of a category of anti-HIV drugs that competes with full length TAT proteins to inhibit HIV replication. In addition, this study indicates that the HIV derived lentiviral vector system is a safe and reliable screening method for anti-HIV drugs, especially for those targeting the interaction of TAT and TAR RNAs. PMID:16293193

  12. The Canine Papillomavirus and Gamma HPV E7 Proteins Use an Alternative Domain to Bind and Destabilize the Retinoblastoma Protein

    PubMed Central

    Wang, Jingang; Zhou, Dan; Prabhu, Anjali; Schlegel, Richard; Yuan, Hang

    2010-01-01

    The high-risk HPV E6 and E7 proteins cooperate to immortalize primary human cervical cells and the E7 protein can independently transform fibroblasts in vitro, primarily due to its ability to associate with and degrade the retinoblastoma tumor suppressor protein, pRb. The binding of E7 to pRb is mediated by a conserved Leu-X-Cys-X-Glu (LXCXE) motif in the conserved region 2 (CR2) of E7 and this domain is both necessary and sufficient for E7/pRb association. In the current study, we report that the E7 protein of the malignancy-associated canine papillomavirus type 2 encodes an E7 protein that has serine substituted for cysteine in the LXCXE motif. In HPV, this substitution in E7 abrogates pRb binding and degradation. However, despite variation at this critical site, the canine papillomavirus E7 protein still bound and degraded pRb. Even complete deletion of the LXSXE domain of canine E7 failed to interfere with binding to pRb in vitro and in vivo. Rather, the dominant binding site for pRb mapped to the C-terminal domain of canine E7. Finally, while the CR1 and CR2 domains of HPV E7 are sufficient for degradation of pRb, the C-terminal region of canine E7 was also required for pRb degradation. Screening of HPV genome sequences revealed that the LXSXE motif of the canine E7 protein was also present in the gamma HPVs and we demonstrate that the gamma HPV-4 E7 protein also binds pRb in a similar way. It appears, therefore, that the type 2 canine PV and gamma-type HPVs not only share similar properties with respect to tissue specificity and association with immunosuppression, but also the mechanism by which their E7 proteins interact with pRb. PMID:20824099

  13. Molecular interfaces of the galactose-binding protein Tectonin domains in host-pathogen interaction.

    PubMed

    Low, Diana Hooi Ping; Frecer, Vladimir; Le Saux, Agnès; Srinivasan, Ganesh Anand; Ho, Bow; Chen, Jianzhu; Ding, Jeak Ling

    2010-03-26

    Beta-propeller proteins function in catalysis, protein-protein interaction, cell cycle regulation, and innate immunity. The galactose-binding protein (GBP) from the plasma of the horseshoe crab, Carcinoscorpius rotundicauda, is a beta-propeller protein that functions in antimicrobial defense. Studies have shown that upon binding to Gram-negative bacterial lipopolysaccharide (LPS), GBP interacts with C-reactive protein (CRP) to form a pathogen-recognition complex, which helps to eliminate invading microbes. However, the molecular basis of interactions between GBP and LPS and how it interplays with CRP remain largely unknown. By homology modeling, we showed that GBP contains six beta-propeller/Tectonin domains. Ligand docking indicated that Tectonin domains 6 to 1 likely contain the LPS binding sites. Protein-protein interaction studies demonstrated that Tectonin domain 4 interacts most strongly with CRP. Hydrogen-deuterium exchange mass spectrometry mapped distinct sites of GBP that interact with LPS and with CRP, consistent with in silico predictions. Furthermore, infection condition (lowered Ca(2+) level) increases GBP-CRP affinity by 1000-fold. Resupplementing the system with a physiological level of Ca(2+) did not reverse the protein-protein affinity to the basal state, suggesting that the infection-induced complex had undergone irreversible conformational change. We propose that GBP serves as a bridging molecule, participating in molecular interactions, GBP-LPS and GBP-CRP, to form a stable pathogen-recognition complex. The interaction interfaces in these two partners suggest that Tectonin domains can differentiate self/nonself, crucial to frontline defense against infection. In addition, GBP shares architectural and functional homologies to a human protein, hTectonin, suggesting its evolutionarily conservation for approximately 500 million years, from horseshoe crab to human. PMID:20118243

  14. Requirement of the FATC domain of protein kinase Tel1 for localization to DNA ends and target protein recognition

    PubMed Central

    Ogi, Hiroo; Goto, Greicy H.; Ghosh, Avik; Zencir, Sevil; Henry, Everett; Sugimoto, Katsunori

    2015-01-01

    Two large phosphatidylinositol 3-kinase–related protein kinases (PIKKs), ATM and ATR, play a central role in the DNA damage response pathway. PIKKs contain a highly conserved extreme C-terminus called the FRAP-ATM-TRRAP-C-terminal (FATC) domain. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. In this study, we characterized functions of the FATC domain of Tel1 by introducing substitution or truncation mutations. One substitution mutation, termed tel1-21, and a truncation mutation, called tel1-ΔC, did not significantly affect the expression level. The tel1-21 mutation impaired the cellular response to DNA damage and conferred moderate telomere maintenance defect. In contrast, the tel1-ΔC mutation behaved like a null mutation, conferring defects in both DNA damage response and telomere maintenance. Tel1-21 protein localized to DNA ends as effectively as wild-type Tel1 protein, whereas Tel1-ΔC protein failed. Introduction of a hyperactive TEL1-hy mutation suppressed the tel1-21 mutation but not the tel1-ΔC mutation. In vitro analyses revealed that both Tel1-21 and Tel1-ΔC proteins undergo efficient autophosphorylation but exhibit decreased kinase activities toward the exogenous substrate protein, Rad53. Our results show that the FATC domain of Tel1 mediates localization to DNA ends and contributes to phosphorylation of target proteins. PMID:26246601

  15. Assessing the Metabolic Diversity of Streptococcus from a Protein Domain Point of View

    PubMed Central

    Koehorst, Jasper J.; Martins dos Santos, Vitor A. P.; Schaap, Peter J.

    2015-01-01

    Understanding the diversity and robustness of the metabolism of bacteria is fundamental for understanding how bacteria evolve and adapt to different environments. In this study, we characterised 121 Streptococcus strains and studied metabolic diversity from a protein domain perspective. Metabolic pathways were described in terms of the promiscuity of domains participating in metabolic pathways that were inferred to be functional. Promiscuity was defined by adapting existing measures based on domain abundance and versatility. The approach proved to be successful in capturing bacterial metabolic flexibility and species diversity, indicating that it can be described in terms of reuse and sharing functional domains in different proteins involved in metabolic activity. Additionally, we showed striking differences among metabolic organisation of the pathogenic serotype 2 Streptococcus suis and other strains. PMID:26366735

  16. 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.

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

    PubMed

    Sadiq, Irfan; Keren, Ido; Citovsky, Vitaly

    2016-02-19

    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

  18. Remarkable alkaline stability of an engineered protein A as immunoglobulin affinity ligand: C domain having only one amino acid substitution

    PubMed Central

    Minakuchi, Kazunobu; Murata, Dai; Okubo, Yuji; Nakano, Yoshiyuki; Yoshida, Shinichi

    2013-01-01

    Protein A affinity chromatography is the standard purification process for the capture of therapeutic antibodies. The individual IgG-binding domains of protein A (E, D, A, B, C) have highly homologous amino acid sequences. From a previous report, it has been assumed that the C domain has superior resistance to alkaline conditions compared to the other domains. We investigated several properties of the C domain as an IgG-Fc capture ligand. Based on cleavage site analysis of a recombinant protein A using a protein sequencer, the C domain was found to be the only domain to have neither of the potential alkaline cleavage sites. Circular dichroism (CD) analysis also indicated that the C domain has good physicochemical stability. Additionally, we evaluated the amino acid substitutions at the Gly-29 position of the C domain, as the Z domain (an artificial B domain) acquired alkaline resistance through a G29A mutation. The G29A mutation proved to increase the alkaline resistance of the C domain, based on BIACORE analysis, although the improvement was significantly smaller than that observed for the B domain. Interestingly, a number of other amino acid mutations at the same position increased alkaline resistance more than did the G29A mutation. This result supports the notion that even a single mutation on the originally alkali-stable C domain would improve its alkaline stability. An engineered protein A based on this C domain is expected to show remarkable performance as an affinity ligand for immunoglobulin. PMID:23868198

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

    PubMed Central

    Vinogradova, Maia V.; Malanina, Galina G.; Waitzman, Joshua S.; Rice, Sarah E.; Fletterick, Robert J.

    2013-01-01

    Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca2+-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca2+ signaling since Ca2+- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface. PMID:23805258

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

    PubMed

    Vinogradova, Maia V; Malanina, Galina G; Waitzman, Joshua S; Rice, Sarah E; Fletterick, Robert J

    2013-01-01

    Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca(2+)-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca(2+) signaling since Ca(2+)- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface. PMID:23805258

  1. Activation of Nanoscale Allosteric Protein Domain Motion Revealed by Neutron Spin Echo Spectroscopy

    PubMed Central

    Farago, Bela; Li, Jianquan; Cornilescu, Gabriel; Callaway, David J.E.; Bu, Zimei

    2010-01-01

    NHERF1 is a multidomain scaffolding protein that assembles 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 the membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 Ångstroms 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 submicrosecond timescales upon forming a complex with ezrin. We show that a much-simplified coarse-grained model suffices to describe interdomain motion of a multidomain 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. Our results demonstrate that the dynamic propagation of allosteric signals to distal sites involves changes in long-range coupled domain motions on submicrosecond timescales, and that these coupled motions can be distinguished and characterized by NSE. PMID:21081097

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

    PubMed Central

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

    2014-01-01

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

  3. Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex

    PubMed Central

    Lobingier, Braden T.; Merz, Alexey J.

    2012-01-01

    Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal Habc domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function. PMID:23051737

  4. Protein domain mapping by internal labeling and single particle electron microscopy.

    PubMed

    Ciferri, Claudio; Lander, Gabriel C; Nogales, Eva

    2015-11-01

    In recent years, electron microscopy (EM) and single particle analysis have emerged as essential tools for investigating the architecture of large biological complexes. When high resolution is achievable, crystal structure docking and de-novo modeling allows for precise assignment of individual protein domain sequences. However, the achievable resolution may limit the ability to do so, especially when small or flexible complexes are under study. In such cases, protein labeling has emerged as an important complementary tool to characterize domain architecture and elucidate functional mechanistic details. All labeling strategies proposed to date are either focused on the identification of the position of protein termini or require multi-step labeling strategies, potentially interfering with the final labeling efficiency. Here we describe a strategy for determining the position of internal protein domains within EM maps using a recombinant one-step labeling approach named Efficient Mapping by Internal Labeling (EMIL). EMIL takes advantage of the close spatial proximity of the GFP's N- and C-termini to generate protein chimeras containing an internal GFP at desired locations along the main protein chain. We apply this method to characterize the subunit domain localization of the human Polycomb Repressive Complex 2. PMID:26431894

  5. Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase.

    PubMed Central

    Leberer, E; Wu, C; Leeuw, T; Fourest-Lieuvin, A; Segall, J E; Thomas, D Y

    1997-01-01

    Ste20p from Saccharomyces cerevisiae belongs to the Ste20p/p65PAK family of protein kinases which are highly conserved from yeast to man and regulate conserved mitogen-activated protein kinase pathways. Ste20p fulfills multiple roles in pheromone signaling, morphological switching and vegetative growth and binds Cdc42p, a Rho-like small GTP binding protein required for polarized morphogenesis. We have analyzed the functional consequences of mutations that prevent binding of Cdc42p to Ste20p. The complete amino-terminal, non-catalytic half of Ste20p, including the conserved Cdc42p binding domain, was dispensable for heterotrimeric G-protein-mediated pheromone signaling. However, the Cdc42p binding domain was necessary for filamentous growth in response to nitrogen starvation and for an essential function that Ste20p shares with its isoform Cla4p during vegetative growth. Moreover, the Cdc42p binding domain was required for cell-cell adhesion during conjugation. Subcellular localization of wild-type and mutant Ste20p fused to green fluorescent protein showed that the Cdc42p binding domain is needed to direct localization of Ste20p to regions of polarized growth. These results suggest that Ste20p is regulated in different developmental pathways by different mechanisms which involve heterotrimeric and small GTP binding proteins. PMID:9009270

  6. Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting HER2 protein extracellular domain

    PubMed Central

    Banappagari, Sashikanth; McCall, Alecia; Fontenot, Krystal; Vicente, M. Graca H.; Gujar, Amit; Satyanarayanajois, Seetharama

    2013-01-01

    Among the EGFRs, HER2 is a major heterodimer partner and also has important implications in the formation of particular tumors. Interaction of HER2 protein with other EGFR proteins can be modulated by small molecule ligands and, hence, these protein-protein interactions play a key role in biochemical reactions related to control of cell growth. A peptidomimetic (compound 5-1) that binds to HER2 protein extracellular domain and inhibits protein-protein interactions of EGFRs was conjugated with BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene). Conjugation of BODIPY to the peptidomimetic was investigated by different approaches. The conjugate was characterized for its ability to bind to HER2 overexpressing SKBR-3 and BT-474 cells. Furthermore, cellular uptake of conjugate of BODIPY was studied in the presence of membrane tracker and Lyso tracker using confocal microscopy. Our results suggested that fluorescently labeled compound 5-7 binds to the extracellular domain and stays in the membrane for nearly 24 h. After 24 h there is an indication of internalization of the conjugate. Inhibition of protein-protein interaction and downstream signaling effect of compound 5-1 was also studied by proximity ligation assay and western blot analysis. Results suggested that compound 5-1 inhibits protein-protein interactions of HER2-HER3 and phosphorylation of HER2 in a time-dependent manner. PMID:23688700

  7. Identification of multiple proteins expressed in murine embryos as binding partners for the WW domains of the ubiquitin-protein ligase Nedd4.

    PubMed Central

    Jolliffe, C N; Harvey, K F; Haines, B P; Parasivam, G; Kumar, S

    2000-01-01

    Nedd4 is a member of a growing family of ubiquitin-protein ligases which consist of a lipid-binding domain, two to four WW domains and a C-terminal ubiquitin-protein ligase domain. The Nedd4 mRNA levels are developmentally regulated and Nedd4 protein is highly expressed in many mouse embryonic tissues. In this study we have used a far-Western screen to identify embryonic proteins that interact with the WW domains in mouse Nedd4. We report here identification of eight Nedd4 WW-domain-interacting proteins from mouse embryonic cDNA expression libraries. Two of the proteins are novel, while two have been identified previously as ligands for a WW domain. All of these proteins contain one or more PY motifs. In seven of the eight proteins, these PY motifs are necessary for their interaction with the WW domains of Nedd4. Using site-directed mutagenesis, and by using individual WW domains of Nedd4 as probes for far-Western analysis, we show that the three WW domains in Nedd4 interact with varying affinities with the PY motifs present in various Nedd4-binding proteins. These results provide evidence that Nedd4 can potentially interact with multiple proteins, possibly simultaneously, through its WW domains. PMID:11042109

  8. Calmodulin-binding domains in Alzheimer's disease proteins: extending the calcium hypothesis.

    PubMed

    O'Day, Danton H; Myre, Michael A

    2004-08-01

    The calcium hypothesis of Alzheimer's disease (AD) invokes the disruption of calcium signaling as the underlying cause of neuronal dysfunction and ultimately apoptosis. As a primary calcium signal transducer, calmodulin (CaM) responds to cytosolic calcium fluxes by binding to and regulating the activity of target CaM-binding proteins (CaMBPs). Ca(2+)-dependent CaMBPs primarily contain domains (CaMBDs) that can be classified into motifs based upon variations on the basic amphiphilic alpha-helix domain involving conserved hydrophobic residues at positions 1-10, 1-14 or 1-16. In contrast, an IQ or IQ-like domain often mediates Ca(2+)-independent CaM-binding. Based on these attributes, a search for CaMBDs reveals that many of the proteins intimately linked to AD may be calmodulin-binding proteins, opening new avenues for research on this devastating disease. PMID:15249195

  9. 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

  10. 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

  11. Expression of the mouse PR domain protein Prdm8 in the developing central nervous system.

    PubMed

    Komai, Tae; Iwanari, Hiroko; Mochizuki, Yasuhiro; Hamakubo, Takao; Shinkai, Yoichi

    2009-10-01

    It was first shown in the PR (PRDI-BF1 and RIZ homology) domain family proteins that the PR domain has homology to the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain, a catalytic domain of the histone lysine methyltransferases. Recently, there are many reports that the PR domain proteins have important roles in development and/or cell differentiation. In this report, we show the expression patterns of one of the mouse PR domain proteins, Prdm8, in the developing central nervous system. In the developing retina, Prdm8 expression was detected in postmitotic neurons in the inner nuclear layer and the ganglion cell layer, and its expression became restricted predominantly to the rod bipolar cells when retinogenesis was completed. In the developing spinal cord, Prdm8 was expressed first in the progenitor populations of ventral interneurons and motor neurons, and later in a subpopulation of interneurons. In the developing brain, Prdm8 expression was observed in postmitotic neurons in the intermediate zone and the cortical plate. In the postnatal brain, Prdm8 was expressed mainly in layer 4 neurons of the cerebral cortex. These results show that Prdm8 expression is tightly regulated in a spatio-temporal manner during neural development and mainly restricted to postmitotic neurons, except in the spinal cord. PMID:19616129

  12. Structure of the catalytic domain of Plasmodium falciparum ARF GTPase-activating protein (ARFGAP)

    SciTech Connect

    Cook, William J.; Senkovich, Olga; Chattopadhyay, Debasish

    2012-03-26

    The crystal structure of the catalytic domain of the ADP ribosylation factor GTPase-activating protein (ARFGAP) from Plasmodium falciparum has been determined and refined to 2.4 {angstrom} resolution. Multiwavelength anomalous diffraction (MAD) data were collected utilizing the Zn{sup 2+} ion bound at the zinc-finger domain and were used to solve the structure. The overall structure of the domain is similar to those of mammalian ARFGAPs. However, several amino-acid residues in the area where GAP interacts with ARF1 differ in P. falciparum ARFGAP. Moreover, a number of residues that form the dimer interface in the crystal structure are unique in P. falciparum ARFGAP.

  13. Sip1, a novel RS domain-containing protein essential for pre-mRNA splicing.

    PubMed

    Zhang, W J; Wu, J Y

    1998-02-01

    Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5' and 3' splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors. PMID:9447963

  14. 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. PMID:25664627

  15. Physical Instability of a Therapeutic Fc Fusion Protein: Domain Contributions to Conformational and Colloidal Stability†

    PubMed Central

    Fast, Jonas L; Cordes, Amanda A; Carpenter, John F; Randolph, Theodore W

    2009-01-01

    Protein therapeutics made up of artificially combined proteins or protein domains, so called fusion proteins, are a novel and growing class of biopharmaceuticals. We have studied abatacept (Orencia®), a fusion protein that is constructed of a modified IgG Fc domain and the soluble part of the T-cell receptor CTLA-4. In accelerated degradation studies conducted at at 40 °C, a pH shift from 7.5 to 6.0 yields significantly faster aggregation kinetics, as measured by size-exclusion chromatography. To understand how the fusion domains and their interactions contribute to this result, we considered aggregation in light of the modified Lumry-Eyring reaction pathway. Protein conformational stabilities against chaotropes and temperature were measured. The structural consequences of these perturbations were observed by a variety of experimental techniques, including differential scanning calorimetry, circular dichroism, and intrinsic fluorescence. Abatacept’s colloidal stability was studied by measuring zeta potentials and osmotic second virial coefficients, as well as by modeling electrostatic potentials on the protein’s surface. The domains of abatacept exhibit different conformational stabilities that are highly pH dependent, whereas abatacept was weakly colloidally unstable at pH 6 or pH 7.5. These results are ascribed to conformational instability of the CTLA-4 and CH2 domains, which unfold to form a molten globule-like structure that is aggregation-prone. We suggest the instability against aggregation is determined by the least stable domains. PMID:19899812

  16. MutationAligner: a resource of recurrent mutation hotspots in protein domains in cancer.

    PubMed

    Gauthier, Nicholas Paul; Reznik, Ed; Gao, Jianjiong; Sumer, Selcuk Onur; Schultz, Nikolaus; Sander, Chris; Miller, Martin L

    2016-01-01

    The MutationAligner web resource, available at http://www.mutationaligner.org, enables discovery and exploration of somatic mutation hotspots identified in protein domains in currently (mid-2015) more than 5000 cancer patient samples across 22 different tumor types. Using multiple sequence alignments of protein domains in the human genome, we extend the principle of recurrence analysis by aggregating mutations in homologous positions across sets of paralogous genes. Protein domain analysis enhances the statistical power to detect cancer-relevant mutations and links mutations to the specific biological functions encoded in domains. We illustrate how the MutationAligner database and interactive web tool can be used to explore, visualize and analyze mutation hotspots in protein domains across genes and tumor types. We believe that MutationAligner will be an important resource for the cancer research community by providing detailed clues for the functional importance of particular mutations, as well as for the design of functional genomics experiments and for decision support in precision medicine. MutationAligner is slated to be periodically updated to incorporate additional analyses and new data from cancer genomics projects. PMID:26590264

  17. 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.

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26027519

  19. MutationAligner: a resource of recurrent mutation hotspots in protein domains in cancer

    PubMed Central

    Gauthier, Nicholas Paul; Reznik, Ed; Gao, Jianjiong; Sumer, Selcuk Onur; Schultz, Nikolaus; Sander, Chris; Miller, Martin L.

    2016-01-01

    The MutationAligner web resource, available at http://www.mutationaligner.org, enables discovery and exploration of somatic mutation hotspots identified in protein domains in currently (mid-2015) more than 5000 cancer patient samples across 22 different tumor types. Using multiple sequence alignments of protein domains in the human genome, we extend the principle of recurrence analysis by aggregating mutations in homologous positions across sets of paralogous genes. Protein domain analysis enhances the statistical power to detect cancer-relevant mutations and links mutations to the specific biological functions encoded in domains. We illustrate how the MutationAligner database and interactive web tool can be used to explore, visualize and analyze mutation hotspots in protein domains across genes and tumor types. We believe that MutationAligner will be an important resource for the cancer research community by providing detailed clues for the functional importance of particular mutations, as well as for the design of functional genomics experiments and for decision support in precision medicine. MutationAligner is slated to be periodically updated to incorporate additional analyses and new data from cancer genomics projects. PMID:26590264

  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. ADAR Proteins: Double-stranded RNA and Z-DNA Binding Domains

    PubMed Central

    Barraud, Pierre; Allain, Frédéric H.-T

    2012-01-01

    Adenosine deaminases acting on RNA (ADARs) 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 are reviewing 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 double-stranded RNA and Z-DNA binding domains. PMID:21728134

  2. 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. PMID:22434723

  3. 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

  4. Mapping flexible protein domains at subnanometer resolution with the atomic force microscope.

    PubMed

    Müller, D J; Fotiadis, D; Engel, A

    1998-06-23

    The mapping of flexible protein domains with the atomic force microscope is reviewed. Examples discussed are the bacteriorhodopsin from Halobacterium salinarum, the head-tail-connector from phage phi29, and the hexagonally packed intermediate layer from Deinococcus radiodurans which all were recorded in physiological buffer solution. All three proteins undergo reversible structural changes that are reflected in standard deviation maps calculated from aligned topographs of individual protein complexes. Depending on the lateral resolution (up to 0.8 nm) flexible surface regions can ultimately be correlated with individual polypeptide loops. In addition, multivariate statistical classification revealed the major conformations of the protein surface. PMID:9678604

  5. 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. PMID:25772550

  6. PDP-CON: prediction of domain/linker residues in protein sequences using a consensus approach.

    PubMed

    Chatterjee, Piyali; Basu, Subhadip; Zubek, Julian; Kundu, Mahantapas; Nasipuri, Mita; Plewczynski, Dariusz

    2016-04-01

    The prediction of domain/linker residues in protein sequences is a crucial task in the functional classification of proteins, homology-based protein structure prediction, and high-throughput structural genomics. In this work, a novel consensus-based machine-learning technique was applied for residue-level prediction of the domain/linker annotations in protein sequences using ordered/disordered regions along protein chains and a set of physicochemical properties. Six different classifiers-decision tree, Gaussian naïve Bayes, linear discriminant analysis, support vector machine, random forest, and multilayer perceptron-were exhaustively explored for the residue-level prediction of domain/linker regions. The protein sequences from the curated CATH database were used for training and cross-validation experiments. Test results obtained by applying the developed PDP-CON tool to the mutually exclusive, independent proteins of the CASP-8, CASP-9, and CASP-10 databases are reported. An n-star quality consensus approach was used to combine the results yielded by different classifiers. The average PDP-CON accuracy and F-measure values for the CASP targets were found to be 0.86 and 0.91, respectively. The dataset, source code, and all supplementary materials for this work are available at https://cmaterju.org/cmaterbioinfo/ for noncommercial use. PMID:26969678

  7. Functional classification of CATH superfamilies: a domain-based approach for protein function annotation

    PubMed Central

    Das, Sayoni; Lee, David; Sillitoe, Ian; Dawson, Natalie L.; Lees, Jonathan G.; Orengo, Christine A.

    2015-01-01

    Motivation: Computational approaches that can predict protein functions are essential to bridge the widening function annotation gap especially since <1.0% of all proteins in UniProtKB have been experimentally characterized. We present a domain-based method for protein function classification and prediction of functional sites that exploits functional sub-classification of CATH superfamilies. The superfamilies are sub-classified into functional families (FunFams) using a hierarchical clustering algorithm supervised by a new classification method, FunFHMMer. Results: FunFHMMer generates more functionally coherent groupings of protein sequences than other domain-based protein classifications. This has been validated using known functional information. The conserved positions predicted by the FunFams are also found to be enriched in known functional residues. Moreover, the functional annotations provided by the FunFams are found to be more precise than other domain-based resources. FunFHMMer currently identifies 110 439 FunFams in 2735 superfamilies which can be used to functionally annotate > 16 million domain sequences. Availability and implementation: All FunFam annotation data are made available through the CATH webpages (http://www.cathdb.info). The FunFHMMer webserver (http://www.cathdb.info/search/by_funfhmmer) allows users to submit query sequences for assignment to a CATH FunFam. Contact: sayoni.das.12@ucl.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26139634

  8. Expression and purification of HER2 extracellular domain proteins in Schneider2 insect cells.

    PubMed

    Kanthala, Shanthi; Mill, Christopher P; Riese, David J; Jaiswal, Mihir; Jois, Seetharama

    2016-09-01

    Overexpression of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu) results in ligand independent activation of kinase signaling and is found in about 30% of human breast cancers, and is correlated with a more aggressive tumor phenotype. The HER2 extracellular domain (ECD) consists of four domains - I, II, III and IV. Although the role of each domain in the dimerization and activation of the receptor has been extensively studied, the role of domain IV (DIV) is not clearly understood yet. In our previous studies, we reported peptidomimetic molecules inhibit HER2:HER3 heterodimerization. In order to study the binding interactions of peptidomimetics with HER2 DIV in detail, properly folded recombinant HER2 protein in pure form is important. We have expressed and purified HER2 ECD and DIV proteins in the Drosophila melanogaster Schneider2 (S2) cell line. Using the commercial Drosophila expression system (DES), we transfected S2 cells with plasmids designed to direct the expression of secreted recombinant HER2 ECD and DIV proteins. The secreted proteins were purified from the conditioned medium by filtration, ultrafiltration, dialysis and nickel affinity chromatography techniques. The purified HER2 proteins were then analyzed using Western blot, mass spectrometry and circular dichroism (CD) spectroscopy. PMID:26363121

  9. [Cloning and expression analysis of a LIM-domain protein gene from cotton (Gossypium hirsuturm L.)].

    PubMed

    Luo, Ming; Xiao, Yue-Hua; Hou, Lei; Luo, Xiao-Ying; Li, De-Mou; Pei, Yan

    2003-02-01

    LIM-domain protein plays an important role in various cellular processes, including construction of cytoskeleton, transcription control and signal transduction. Based on cotton fiber EST database and contig analysis, the coding region of a cotton LIM-domain protein gene (GhLIM1) was obtained by RT-PCR from 4DPA (day post anthesis) ovule with fiber. The cloned fragment of 848 bp contains an open reading frame of 570 bp, coding for a polypeptide of 189 amino acids. It was demonstrated that the deduced GhLIM1 protein was highly homologous to the LIM-domain protein of sunflower (Helianthus annuus), tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Two intact LIM-domains, with the conserved sequence of a double zinc-finger structure (C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-24-C-X2-H), were found in the GhLIM1 protein. RT-PCR and Northern blot analysis showed that GhLIM1 gene expressed in root, shoot tip, hypocotyls, bud, leaf, anther, ovule and fiber (4DPA, 12DPA, 18DPA). However it was preferentially expressed in the shoot tip, fiber and ovule. It was proposed that the express of GhLIM1 gene is related to cotton fiber development. PMID:12776607

  10. 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

  11. 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

  12. Pellino Proteins Contain a Cryptic FHA Domain that Mediates Interaction with Phosphorylated IRAK1

    SciTech Connect

    Lin, Chun-Chi; Huoh, Yu-San; Schmitz, Karl R.; Jensen, Liselotte E.; Ferguson, Kathryn M.

    2009-03-23

    Pellino proteins are RING E3 ubiquitin ligases involved in signaling events downstream of the Toll and interleukin-1 (IL-1) receptors, key initiators of innate immune and inflammatory responses. Pellino proteins associate with and ubiquitinate proteins in these pathways, including the interleukin-1 receptor associated kinase-1 (IRAK1). We determined the X-ray crystal structure of a Pellino2 fragment lacking only the RING domain. This structure reveals that the IRAK1-binding region of Pellino proteins consists largely of a previously unidentified forkhead-associated (FHA) domain. FHA domains are well-characterized phosphothreonine-binding modules, and this cryptic example in Pellino2 can drive interaction of this protein with phosphorylated IRAK1. The Pellino FHA domain is decorated with an unusual appendage or wing composed of two long inserts that lie within the FHA homology region. Delineating how this E3 ligase associates with substrates, and how these interactions are regulated by phosphorylation, is crucial for a complete understanding of Toll/IL-1 receptor signaling.

  13. 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. PMID:25356683

  14. Beyond the E-Value: Stratified Statistics for Protein Domain Prediction.

    PubMed

    Ochoa, Alejandro; Storey, John D; Llinás, Manuel; Singh, Mona

    2015-11-01

    E-values have been the dominant statistic for protein sequence analysis for the past two decades: from identifying statistically significant local sequence alignments to evaluating matches to hidden Markov models describing protein domain families. Here we formally show that for "stratified" multiple hypothesis testing problems-that is, those in which statistical tests can be partitioned naturally-controlling the local False Discovery Rate (lFDR) per stratum, or partition, yields the most predictions across the data at any given threshold on the FDR or E-value over all strata combined. For the important problem of protein domain prediction, a key step in characterizing protein structure, function and evolution, we show that stratifying statistical tests by domain family yields excellent results. We develop the first FDR-estimating algorithms for domain prediction, and evaluate how well thresholds based on q-values, E-values and lFDRs perform in domain prediction using five complementary approaches for estimating empirical FDRs in this context. We show that stratified q-value thresholds substantially outperform E-values. Contradicting our theoretical results, q-values also outperform lFDRs; however, our tests reveal a small but coherent subset of domain families, biased towards models for specific repetitive patterns, for which weaknesses in random sequence models yield notably inaccurate statistical significance measures. Usage of lFDR thresholds outperform q-values for the remaining families, which have as-expected noise, suggesting that further improvements in domain predictions can be achieved with improved modeling of random sequences. Overall, our theoretical and empirical findings suggest that the use of stratified q-values and lFDRs could result in improvements in a host of structured multiple hypothesis testing problems arising in bioinformatics, including genome-wide association studies, orthology prediction, and motif scanning. PMID:26575353

  15. Domain function dissection and catalytic properties of Listeria monocytogenes p60 protein with bacteriolytic activity.

    PubMed

    Yu, Minfeng; Zuo, Jinrong; Gu, Hao; Guo, Minliang; Yin, Yuelan

    2015-12-01

    The major extracellular protein p60 of Listeria monocytogenes (Lm-p60) is an autolysin that can hydrolyze the peptidoglycan of bacterial cell wall and has been shown to be required for L. monocytogenes virulence. The predicted three-dimensional structure of Lm-p60 showed that Lm-p60 could be split into two independent structural domains at the amino acid residue 270. Conserved motif analysis showed that V30, D207, S395, and H444 are the key amino acid residues of the corresponding motifs. However, not only the actual functions of these two domains but also the catalytic properties of Lm-p60 are unclear. We try to express recombinant Lm-p60 and identify the functions of two domains by residue substitution (V30A, D207A, S395A, and H444A) and peptide truncation. The C-terminal domain was identified as catalytic element and N-terminal domain as substrate recognition and binding element. Either N-terminal domain truncation or C-terminal domain truncation presents corresponding biological activity. The catalytic activity of Lm-p60 with a malfunctioned substrate-binding domain was decreased, while the substrate binding was not affected by a mulfunctioned catalytic domain. With turbidimetric method, we determined the optimal conditions for the bacteriolytic activity of Lm-p60 against Micrococcus lysodeikficus. The assay for the effect of Lm-p60 on the bacteriolytic activity of lysozyme revealed that the combined use of Lm-p60 protein with lysozyme showed a strong synergistic effect on the bacteriolytic activity. PMID:26363556

  16. The Fusion Protein Specificity of the Parainfluenza Virus Hemagglutinin-Neuraminidase Protein Is Not Solely Defined by the Primary Structure of Its Stalk Domain

    PubMed Central

    Ito, Morihiro; Ohtsuka, Junpei; Hara, Kenichiro; Komada, Hiroshi; Nishio, Machiko; Nosaka, Tetsuya

    2015-01-01

    ABSTRACT Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain. IMPORTANCE It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular

  17. Telomere Capping Proteins are Structurally Related to RPA with an additional Telomere-Specific Domain

    SciTech Connect

    Gelinas, A.; Paschini, M; Reyes, F; Heroux, A; Batey, R; Lundblad, V; Wuttke, D

    2009-01-01

    Telomeres must be capped to preserve chromosomal stability. The conserved Stn1 and Ten1 proteins are required for proper capping of the telomere, although the mechanistic details of how they contribute to telomere maintenance are unclear. Here, we report the crystal structures of the C-terminal domain of the Saccharomyces cerevisiae Stn1 and the Schizosaccharomyces pombe Ten1 proteins. These structures reveal striking similarities to corresponding subunits in the replication protein A complex, further supporting an evolutionary link between telomere maintenance proteins and DNA repair complexes. Our structural and in vivo data of Stn1 identify a new domain that has evolved to support a telomere-specific role in chromosome maintenance. These findings endorse a model of an evolutionarily conserved mechanism of DNA maintenance that has developed as a result of increased chromosomal structural complexity.

  18. SMP-domain proteins at membrane contact sites: Structure and function.

    PubMed

    Reinisch, Karin M; De Camilli, Pietro

    2016-08-01

    SMP-domains are found in proteins that localize to membrane contact sites. Elucidation of the properties of these proteins gives clues as to the molecular bases underlying processes that occur at such sites. Described here are recent discoveries concerning the structure, function, and regulation of the Extended-Synaptotagmin proteins and ERMES complex subunits, SMP-domain proteins at endoplasmic reticulum (ER)-plasma membrane and ER-mitochondrial contacts, respectively. They act as tethers contributing to the architecture of these sites and as lipid transporters that convey glycerolipids between apposed membranes. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. PMID:26686281

  19. The interactome of a PTB domain-containing adapter protein, Odin, revealed by SILAC

    PubMed Central

    Zhong, Jun; Chaerkady, Raghothama; Kandasamy, Kumaran; Gucek, Marjan; Cole, Robert N.; Pandey, Akhilesh

    2011-01-01

    Signal transduction pathways are tightly controlled by positive and negative regulators. We have previously identified Odin (also known as ankyrin repeat and sterile alpha motif domain containing 1A; gene symbol AKNS1A) as a negative regulator of growth factor signaling; however, the mechanisms through which Odin regulates these pathways remain to be elucidated. To determine how Odin negatively regulates growth factor signaling, we undertook a proteomic approach to systematically identify proteins that interact with Odin using the SILAC strategy. In this study, we identified 18 molecules that were specifically associated in a protein complex with Odin. Our study established that the complete family of 14-3-3 proteins occur in a protein complex with Odin, which is also supported by earlier reports that identified a few members of the 14-3-3 family as Odin interactors. Among the novel protein interactors of Odin were CD2-associated protein, SH3 domain kinase binding protein 1 and DAB2 interacting protein. We confirmed 8 of the eighteen interactions identified in the Odin protein complex by co-immunoprecipitation experiments. Finally, a literature-based network analysis revealed that Odin interacting partners are involved in various cellular processes, some of which are key molecules in regulating receptor endocytosis. PMID:21081186

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

    PubMed Central

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

    2012-01-01

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

  1. A Novel Catalytic Function of Synthetic IgG-Binding Domain (Z Domain) from Staphylococcal Protein A: Light Emission with Coelenterazine.

    PubMed

    Inouye, Satoshi; Sahara-Miura, Yuiko

    2014-01-01

    The synthetic IgG-binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine-utilizing luciferase. The Z domain derivatives (ZZ-gCys, Z-gCys and Z-domain) were purified and the luminescence properties were characterized by comparing with coelenterazine-utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and the order of the initial maximum intensity of luminescence was ZZ-gCys (100%) > Z-gCys (36.8%) > Z-domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ-gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for coelenterazine analogues. In contrast, the luminescence properties of Z-gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG-binding region of the Z domain. PMID:24138575

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

    PubMed

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

    2012-12-01

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

  3. Addition of missing loops and domains to protein models by x-ray solution scattering.

    PubMed Central

    Petoukhov, Maxim V; Eady, Nigel A J; Brown, Katherine A; Svergun, Dmitri I

    2002-01-01

    Inherent flexibility and conformational heterogeneity in proteins can often result in the absence of loops and even entire domains in structures determined by x-ray crystallographic or NMR methods. X-ray solution scattering offers the possibility of obtaining complementary information regarding the structures of these disordered protein regions. Methods are presented for adding missing loops or domains by fixing a known structure and building the unknown regions to fit the experimental scattering data obtained from the entire particle. Simulated annealing was used to minimize a scoring function containing the discrepancy between the experimental and calculated patterns and the relevant penalty terms. In low-resolution models where interface location between known and unknown parts is not available, a gas of dummy residues represents the missing domain. In high-resolution models where the interface is known, loops or domains are represented as interconnected chains (or ensembles of residues with spring forces between the C(alpha) atoms), attached to known position(s) in the available structure. Native-like folds of missing fragments can be obtained by imposing residue-specific constraints. After validation in simulated examples, the methods have been applied to add missing loops or domains to several proteins where partial structures were available. PMID:12496082

  4. Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation

    PubMed Central

    Mitterer, Valentin; Murat, Guillaume; Réty, Stéphane; Blaud, Magali; Delbos, Lila; Stanborough, Tamsyn; Bergler, Helmut; Leulliot, Nicolas; Kressler, Dieter; Pertschy, Brigitte

    2016-01-01

    Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins. PMID:26831757

  5. Opening of holes in liposomal membranes is induced by proteins possessing the FERM domain.

    PubMed

    Takeda, Shuichi; Saitoh, Akihiko; Furuta, Mayumi; Satomi, Nao; Ishino, Atsushi; Nishida, Gakushi; Sudo, Hiroaki; Hotani, Hirokazu; Takiguchi, Kingo

    2006-09-22

    The destabilization of vesicles caused by interactions between lipid bilayers and proteins was studied by direct, real-time observation using high-intensity dark-field microscopy. We previously reported that talin, a cytoskeletal submembranous protein, can reversibly open stable large holes in giant liposomes made of neutral and acidic phospholipids. Talin and other proteins belonging to the band 4.1 superfamily have the FERM domain at their N-terminal and interact with lipid membranes via that domain. Here, we observed that band 4.1, ezrin and moesin, members of the band 4.1 superfamily, are also able to open stable holes in liposomes. However, truncation of their C-terminal domains, which can interact with the N-terminal FERM domain, impaired their hole opening activities. Oligomeric states of ezrin affected the capability of the membrane hole formation. Phosphatidylinositol bisphosphate (PIP2), which binds to the FERM domain and disrupts the interaction between the N and C termini of the band 4.1 superfamily, down-regulates their membrane opening activity. These results suggest that the intermolecular interaction plays a key role in the observed membrane hole formation. PMID:16934293

  6. Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication.

    PubMed

    Gu, Haidong

    2016-02-12

    Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that establishes latent infection in ganglia neurons. Its unique life cycle requires a balanced "conquer and compromise" strategy to deal with the host anti-viral defenses. One of HSV-1 α (immediate early) gene products, infected cell protein 0 (ICP0), is a multifunctional protein that interacts with and modulates a wide range of cellular defensive pathways. These pathways may locate in different cell compartments, which then migrate or exchange factors upon stimulation, for the purpose of a concerted and effective defense. ICP0 is able to simultaneously attack multiple host pathways by either degrading key restrictive factors or modifying repressive complexes. This is a viral protein that contains an E3 ubiquitin ligase, translocates among different cell compartments and interacts with major defensive complexes. The multiple functional domains of ICP0 can work independently and at the same time coordinate with each other. Dissecting the functional domains of ICP0 and delineating the coordination of these domains will help us understand HSV-1 pathogenicity as well as host defense mechanisms. This article focuses on describing individual ICP0 domains, their biochemical properties and their implication in HSV-1 infection. By putting individual domain functions back into the picture of host anti-viral defense network, this review seeks to elaborate the complex interactions between HSV-1 and its host. PMID:26870669

  7. Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication

    PubMed Central

    Gu, Haidong

    2016-01-01

    Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that establishes latent infection in ganglia neurons. Its unique life cycle requires a balanced “conquer and compromise” strategy to deal with the host anti-viral defenses. One of HSV-1 α (immediate early) gene products, infected cell protein 0 (ICP0), is a multifunctional protein that interacts with and modulates a wide range of cellular defensive pathways. These pathways may locate in different cell compartments, which then migrate or exchange factors upon stimulation, for the purpose of a concerted and effective defense. ICP0 is able to simultaneously attack multiple host pathways by either degrading key restrictive factors or modifying repressive complexes. This is a viral protein that contains an E3 ubiquitin ligase, translocates among different cell compartments and interacts with major defensive complexes. The multiple functional domains of ICP0 can work independently and at the same time coordinate with each other. Dissecting the functional domains of ICP0 and delineating the coordination of these domains will help us understand HSV-1 pathogenicity as well as host defense mechanisms. This article focuses on describing individual ICP0 domains, their biochemical properties and their implication in HSV-1 infection. By putting individual domain functions back into the picture of host anti-viral defense network, this review seeks to elaborate the complex interactions between HSV-1 and its host. PMID:26870669

  8. The Zn-finger domain of RIZ protein promotes MCF-7 cell proliferation.

    PubMed

    Rossi, Mariangela; Abbondanza, Ciro; D'Arcangelo, Andrea; Gazzerro, Patrizia; Medici, Nicola; Moncharmont, Bruno; Puca, Giovanni Alfredo

    2004-11-25

    In order to understand the oncogenic properties of retinoblastoma-interacting zinc-finger (RIZ) gene products, we produced an MCF-7-derived cell line expressing a fusion protein containing the zinc-finger (aa 359-497) domain of RIZ protein (MCF-7/znf). The Zn-finger domain contains three of the eight putative Zn-finger motifs and is located in proximity of the E1A-like domain containing the Rb protein-binding motif. The MCF-7/znf cells showed a higher growth rate than the parental or the control cell lines, both in hormone-deprived conditions or upon estrogen stimulation. Furthermore, they were less sensitive to the growth inhibitory effect of anti-estrogens and showed a higher level of expression of cyclin D1 and A. The expressed Zn-finger domain recombinant product was localized in the nucleus and in the nucleoli and its expression modified the pattern of actin staining in the cytoplasm. In conclusion the presented results indicated that the Zn-finger domain could be endowed with the putative oncogenic activity of RIZ2 gene product. PMID:15488642

  9. Knowledge-guided inference of domain–domain interactions from incomplete protein–protein interaction networks

    PubMed Central

    Liu, Mei; Chen, Xue-wen; Jothi, Raja

    2009-01-01

    Motivation: Protein-protein interactions (PPIs), though extremely valuable towards a better understanding of protein functions and cellular processes, do not provide any direct information about the regions/domains within the proteins that mediate the interaction. Most often, it is only a fraction of a protein that directly interacts with its biological partners. Thus, understanding interaction at the domain level is a critical step towards (i) thorough understanding of PPI networks; (ii) precise identification of binding sites; (iii) acquisition of insights into the causes of deleterious mutations at interaction sites; and (iv) most importantly, development of drugs to inhibit pathological protein interactions. In addition, knowledge derived from known domain–domain interactions (DDIs) can be used to understand binding interfaces, which in turn can help discover unknown PPIs. Results: Here, we describe a novel method called K-GIDDI (knowledge-guided inference of DDIs) to narrow down the PPI sites to smaller regions/domains. K-GIDDI constructs an initial DDI network from cross-species PPI networks, and then expands the DDI network by inferring additional DDIs using a divide-and-conquer biclustering algorithm guided by Gene Ontology (GO) information, which identifies partial-complete bipartite sub-networks in the DDI network and makes them complete bipartite sub-networks by adding edges. Our results indicate that K-GIDDI can reliably predict DDIs. Most importantly, K-GIDDI's novel network expansion procedure allows prediction of DDIs that are otherwise not identifiable by methods that rely only on PPI data. Contact: xwchen@ku.edu Availability: http://www.ittc.ku.edu/∼xwchen/domainNetwork/ddinet.html Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19667081

  10. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins

    PubMed Central

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners. PMID:22536173

  11. Domain cooperativity in multidomain proteins: what can we learn from molecular alignment in anisotropic media?

    PubMed

    Yuwen, Tairan; Post, Carol Beth; Skrynnikov, Nikolai R

    2011-09-01

    Many proteins have modular design with multiple globular domains connected via flexible linkers. As a simple model of such system, we study a tandem construct consisting of two identical SH3 domains and a variable-length Gly/Ser linker. When the linker is short, this construct represents a dumbbell-shaped molecule with limited amount of domain-domain mobility. Due to its elongated shape, this molecule efficiently aligns in steric alignment media. As the length of the linker increases, the two domains become effectively uncoupled and begin to behave as independent entities. Consequently, their degree of alignment drops, approaching that found in the (near-spherical) isolated SH3 domains. To model the dependence of alignment parameters on the length of the interdomain linker, we have generated in silico a series of conformational ensembles representing SH3 tandems with different linker length. These ensembles were subsequently used as input for alignment prediction software PALES. The predicted alignment tensors were compared with the results of experimental measurements using a series of tandem-SH3 samples in PEG/hexanol alignment media. This comparison broadly confirmed the expected trends. At the same time, it has been found that the isolated SH3 domain aligns much stronger than expected. This finding can be attributed to complex morphology of the PEG/hexanol media and/or to weak site-specific interactions between the protein and the media. In the latter case, there are strong indications that electrostatic interactions may play a role. The fact that PEG/hexanol does not behave as a simple steric media should serve as a caution for studies that use PALES as a quantitative prediction tool (especially for disordered proteins). Further progress in this area depends on our ability to accurately model the anisotropic media and its site-specific interactions with protein molecules. Once this ability is improved, it should be possible to use the alignment parameters as a

  12. The RNA triphosphatase domain of L protein of Rinderpest virus exhibits pyrophosphatase and tripolyphosphatase activities.

    PubMed

    Singh, Piyush Kumar; Subbarao, Shaila Melkote

    2016-10-01

    L protein of the Rinderpest virus, an archetypal paramyxovirus possesses RNA-dependent RNA polymerase activity which transcribes the genome into mRNAs as well as replicates the RNA genome. The protein also possesses RNA triphosphatase (RTPase), guanylyltransferase (GTase) and methyltransferase enzyme activities responsible for capping the mRNAs in a conventional pathway similar to that of the host pathway. Subsequent to the earlier characterization of the GTase activity of L protein and identification of the RTPase domain of the L protein, we report here, additional enzymatic activities associated with the RTPase domain. We have characterized the pyrophosphatase and tripolyphosphatase activities of the L-RTPase domain which are metal-dependent and proceed much faster than the RTPase activity. Interestingly, the mutant proteins E1645A and E1647A abrogated the pyrophosphatase and tripolyphosphatase significantly, indicating a strong overlap of the active sites of these activities with that of RTPase. We discuss the likely role of GTase-associated L protein pyrophosphatase in the polymerase function. We also discuss a possible biological role for the tripolyphosphatase activity hitherto considered insignificant for the viruses possessing such activity. PMID:27170418

  13. Strength limit of entropic elasticity in beta-sheet protein domains

    NASA Astrophysics Data System (ADS)

    Keten, Sinan; Buehler, Markus J.

    2008-12-01

    Elasticity and strength of individual beta-sheet protein domains govern key biological functions and the mechanical properties of biopolymers including spider silk, amyloids, and muscle fibers. The worm-like-chain (WLC) model is commonly used to describe the entropic elasticity of polypeptides and other biomolecules. However, force spectroscopy experiments have shown pronounced deviations from the ideal WLC behavior, leading to controversial views about the appropriate elastic description of proteins at nanoscale. Here we report a simple model that explains the physical mechanism that leads to the breakdown of the WLC idealization in experiments by using only two generic parameters of the protein domain, the H-bond energy and the protein backbone’s persistence length. We show that a rupture initiation condition characterized by the free energy release rate of H-bonds characterizes the limit of WLC entropic elasticity of beta-sheet protein domains and the onset of rupture. Our findings reveal that strength and elasticity are coupled and cannot be treated separately. The predictions of the model are compared with atomic force microscopy experiments of protein rupture.

  14. A novel Aurelia aurita protein mesoglein contains DSL and ZP domains.

    PubMed

    Matveev, I V; Shaposhnikova, T G; Podgornaya, O I

    2007-09-01

    Body of the scyphoid jellyfish Aurelia aurita consists of 2 epithelia -- epidermis and gastroderm. The layers are separated by a thick layer of extracellular matrix -- mesoglea. A. aurita has a lot of cells in the mesoglea unlike many other Cnidarians. The major protein of the mesoglea with apparent molecular mass of 47 kDa was detected by SDS-PAGE. A partial mRNA of the protein 1421 bp long was cloned and sequenced. The search for homologous nucleotide and protein sequences shows that the mRNA sequence is novel. Deduced amino acid sequence of 416 aa contains zona pellucida (ZP) domain and Delta/Serrate/Lag-2 (DSL) domain. The protein was named mesoglein. According to reverse transcription PCR analysis it is expressed in the mature medusa exclusively in the mesogleal cells. Mesoglein belongs to the lowest phyla among ZP domain-containing proteins. The protein is supposed to be a structural element of the mesoglea extracellular matrix. PMID:17583446

  15. Protein 4.1R core domain structure and insights into regulation of cytoskeletal organization.

    PubMed

    Han, B G; Nunomura, W; Takakuwa, Y; Mohandas, N; Jap, B K

    2000-10-01

    The crystal structure of the core domain (N-terminal 30 kDa domain) of cytoskeletal protein 4.1R has been determined and shows a cloverleaf-like architecture. Each lobe of the cloverleaf contains a specific binding site for either band 3, glycophorin C/D or p55. At a central region of the molecule near where the three lobes are joined are two separate calmodulin (CaM) binding regions. One of these is composed primarily of an alpha-helix and is Ca 2+ insensitive; the other takes the form of an extended structure and its binding with CaM is dramatically enhanced by the presence of Ca 2+, resulting in the weakening of protein 4.1R binding to its target proteins. This novel architecture, in which the three lobes bind with three membrane associated proteins, and the location of calmodulin binding sites provide insight into how the protein 4.1R core domain interacts with membrane proteins and dynamically regulates cell shape in response to changes in intracellular Ca2+ levels. PMID:11017195

  16. 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

  17. NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs.

    PubMed

    Mineev, Konstantin S; Goncharuk, Sergey A; Kuzmichev, Pavel K; Vilar, Marçal; Arseniev, Alexander S

    2015-08-18

    P75NTR is a type I integral membrane protein that plays a key role in neurotrophin signaling. However, structural data for the receptor in various functional states are sparse and controversial. In this work, we studied the spatial structure and mobility of the transmembrane and intracellular parts of p75NTR, incorporated into lipid-protein nanodiscs of various sizes and compositions, by solution NMR spectroscopy. Our data reveal a high level of flexibility and disorder in the juxtamembrane chopper domain of p75NTR, which results in the motions of the receptor death domain being uncoupled from the motions of the transmembrane helix. Moreover, none of the intracellular domains of p75NTR demonstrated a propensity to interact with the membrane or to self-associate under the experimental conditions. The obtained data are discussed in the context of the receptor activation mechanism. PMID:26287629

  18. Expression, purification, and characterization of human osteoclastic protein-tyrosine phosphatase catalytic domain in Escherichia coli.

    PubMed

    Jiang, Huan; Sui, Yuan; Cui, Yue; Lin, Peng; Li, Wannan; Xing, Shu; Wang, Deli; Hu, Min; Fu, Xueqi

    2015-03-01

    Osteoclastic protein tyrosine phosphatase (PTP-oc) is a structurally unique transmembrane protein tyrosine phosphatase (PTP) that contains only a relatively small intracellular PTP catalytic domain, does not have an extracellular domain, and lacks a signal peptide proximal to the NH2 terminus. The present study reports the expression, purification, and characterization of the intracellular catalytic domain of PTP-oc (ΔPTP-oc). ΔPTP-oc was expressed in Escherichia coli cells as a fusion with a six-histidine tag and was purified via nickel affinity chromatography. When with para-nitrophenylphosphate (p-NPP) as a substrate, ΔPTP-oc exhibited classical Michaelis-Menten kinetics. Its responses to temperature and ionic strength were similar to those of other PTPs. The optimal pH value of ΔPTP-oc is approximately 7.0, unlike other PTPs, whose optimal pH values are approximately 5.0. PMID:25462809

  19. PR Domain-containing Protein 7 (PRDM7) Is a Histone 3 Lysine 4 Trimethyltransferase*

    PubMed Central

    Blazer, Levi L.; Lima-Fernandes, Evelyne; Gibson, Elisa; Eram, Mohammad S.; Loppnau, Peter; Arrowsmith, Cheryl H.; Schapira, Matthieu; Vedadi, Masoud

    2016-01-01

    PR domain-containing protein 7 (PRDM7) is a primate-specific histone methyltransferase that is the result of a recent gene duplication of PRDM9. The two proteins are highly homologous, especially in the catalytic PR/SET domain, where they differ by only three amino acid residues. Here we report that PRDM7 is an efficient methyltransferase that selectively catalyzes the trimethylation of H3 lysine 4 (H3K4) both in vitro and in cells. Through selective mutagenesis we have dissected the functional roles of each of the three divergent residues between the PR domains of PRDM7 and PRDM9. These studies indicate that after a single serine to tyrosine mutation at residue 357 (S357Y), PRDM7 regains the substrate specificities and catalytic activities similar to its evolutionary predecessor, including the ability to efficiently methylate H3K36. PMID:27129774

  20. Formation of irreversibly bound annexin A1 protein domains on POPC/POPS solid supported membranes.

    PubMed

    Faiss, Simon; Kastl, Katja; Janshoff, Andreas; Steinem, Claudia

    2008-01-01

    The specific interaction of annexin A1 with phospholipid bilayers is scrutinized by means of scanning force and fluorescence microscopy, quartz crystal microbalance, ellipsometry, and modeled by dynamic Monte Carlo simulations. It was found that POPC/POPS bilayers exhibit phase separation in POPC- and POPS-enriched domains as a function of Ca2+ concentration. Annexin A1 interacts with POPC/POPS bilayers by forming irreversibly bound protein domains with monolayer thickness on POPS-enriched nanodomains, while the attachment of proteins to the POPC-enriched regions is fully reversible. A thorough kinetic analysis of the process reveals that both, the binding constant of annexin A1 at the POPC-rich areas as well as the irreversible adsorption rate to the POPS-rich domains increases with calcium ion concentration. Based on the thermodynamic and kinetic data, a possible mechanism of the annexin A1 membrane interaction can be proposed. PMID:18237543

  1. Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number.

    PubMed

    Myre, Michael A; O'Day, Danton H

    2004-09-17

    Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number. PMID:15325281

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

    PubMed Central

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

    2015-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.11897.001 PMID:26714107

  3. 3D topology and arrangement of proteins inside ceramide-rich domains

    NASA Astrophysics Data System (ADS)

    Imhäuser, Christian; Gulbins, Heike; Gulbins, Erich; Lipinski, Hans-Gerd

    2010-04-01

    Clustering of receptor and signalling molecules (such as CD95 or CD40) within ceramide-enriched membrane domains results in a very high density of these proteins facilitating activation of associated enzymes, the exclusion of inhibitory molecules and/or the recruitment of further signalling molecules to transmit the signal into the cells. However, at present the mechanisms of receptor clustering and the exact distribution of proteins within the ceramide-enriched domains are unknown. Therefore, we generated digital images from anti-CD95 stimulated JYcells that were stained with FITC-coupled anti-ceramide and Cy3-labelled anti-CD95 antibodies. We developed image processing methods to determine the spatial distribution of proteins in ceramide-enriched membrane domains and visualized them by volume rendering and surface models. After image preprocessing with appropriate filters for contrast enhancement, noise reduction and logarithmic scaling, 3D models were generated using adapted volume and surface reconstruction. To detect the colocalization of CD95 and ceramide molecules we developed several different methods rasterizing 3D data of each channel into cells and counting intensity values above a specified colour threshold value. The colocalization voxel was set either by normalized product of totals (product intensity) or depending on binarization. In addition, a cross-covariance function to quantify the colocalization was determined and embedded as a 3D object. These computerized techniques allowed for a quantitative analysis of the spatial arrangement of proteins in ceramide-rich domains of living cells.

  4. Crystal structure of a beta-finger domain of Prp8 reveals analogy to ribosomal proteins

    SciTech Connect

    Yang, K.; Heroux, A.; Zhang, L.; Zhao, R.

    2008-09-16

    Prp8 stands out among hundreds of splicing factors as a key regulator of spliceosome activation and a potential cofactor of the splicing reaction. We present here the crystal structure of a 274-residue domain (residues 1,822-2,095) near the C terminus of Saccharomyces cerevisiae Prp8. The most striking feature of this domain is a {beta}-hairpin finger protruding out of the protein (hence, this domain will be referred to as the {beta}-finger domain), resembling many globular ribosomal proteins with protruding extensions. Mutations throughout the {beta}-finger change the conformational equilibrium between the first and the second catalytic step. Mutations at the base of the {beta}-finger affect U4/U6 unwinding-mediated spliceosome activation. Prp8 may insert its {beta}-finger into the first-step complex (U2/U5/U6/pre-mRNA) or U4/U6.U5 tri-snRNP and stabilize these complexes. Mutations on the {beta}-finger likely alter these interactions, leading to the observed mutant phenotypes. Our results suggest a possible mechanism of how Prp8 regulates spliceosome activation. These results also demonstrate an analogy between a spliceosomal protein and ribosomal proteins that insert extensions into folded rRNAs and stabilize the ribosome.

  5. Higher-order assemblies of BAR domain proteins for shaping membranes.

    PubMed

    Suetsugu, Shiro

    2016-06-01

    Most cellular organelles contain lipid bilayer membranes. The earliest characterization of cellular organelles was performed by electron microscopy observation of such membranes. However, the precise mechanisms for shaping the membrane in particular subcellular organelles is poorly understood. Classically, the overall cellular shape, i.e. the shape of the plasma membrane, was thought to be governed by the reorganization of cytoskeletal components such as actin and microtubules. The plasma membrane contains various submicron structures such as clathrin-coated pits, caveolae, filopodia and lamellipodia. These subcellular structures are either invaginations or protrusions and are associated with the cytoskeleton. Therefore, it could be hypothesized that there are membrane-binding proteins that cooperates with cytoskeleton in shaping of plasma membrane organelles. Proteins with the Bin-Amphiphysin-Rvs (BAR) domain connect a variety of membrane shapes to actin filaments. The BAR domains themselves bend the membranes by their rigidity and then mold the membranes into tubules through their assembly as spiral polymers, which are thought to be involved in the various submicron structures. Membrane tubulation by polymeric assembly of the BAR domains is supposed to be regulated by binding proteins, binding lipids and the mechanical properties of the membrane. This review gives an overview of BAR protein assembly, describes the significance of the assembly and discusses how to study the assembly in the context of membrane and cellular morphology. The technical problems encountered in microscopic observation of BAR domain assembly are also discussed. PMID:26884618

  6. Kits and methods of detection using cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.

    1998-04-14

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

  7. Kits and methods of detection using cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, Oded

    1998-01-01

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

  8. A misassembled transmembrane domain of a polytopic protein associates with signal peptide peptidase

    PubMed Central

    2004-01-01

    The endoplasmic reticulum (ER) exerts a quality control over newly synthesized proteins and a variety of components have been implicated in the specific recognition of aberrant or misfolded polypeptides. We have exploited a site-specific cross-linking approach to search for novel ER components that may specifically recognize the misassembled transmembrane domains present in truncated polytopic proteins. We find that a single probe located in the transmembrane domain of a truncated opsin fragment is cross-linked to several ER proteins. These components are distinct from subunits of the Sec61 complex and represent a ‘post-translocon’ environment. In this study, we identify one of these post-translocon cross-linking partners as the signal peptide peptidase (SPP). We find that the interaction of truncated opsin chains with SPP is mediated by its second transmembrane domain, and propose that this interaction may contribute to the recognition of misassembled transmembrane domains during membrane protein quality control at the ER. PMID:15373738

  9. Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase (HN) protein

    SciTech Connect

    Yuan Ping; Leser, George P.; Demeler, Borries; Lamb, Robert A.; Jardetzky, Theodore S.

    2008-09-01

    The mechanism by which the paramyxovirus hemagglutinin-neuraminidase (HN) protein couples receptor binding to activation of virus entry remains to be fully understood, but the HN stalk is thought to play an important role in the process. We have characterized ectodomain constructs of the parainfluenza virus 5 HN to understand better the underlying architecture and oligomerization properties that may influence HN functions. The PIV 5 neuraminidase (NA) domain is monomeric whereas the ectodomain forms a well-defined tetramer. The HN stalk also forms tetramers and higher order oligomers with high {alpha}-helical content. Together, the data indicate that the globular NA domains form weak intersubunit interactions at the end of the HN stalk tetramer, while stabilizing the stalk and overall oligomeric state of the ectodomain. Electron microscopy of the HN ectodomain reveals flexible arrangements of the NA and stalk domains, which may be important for understanding how these two HN domains impact virus entry.

  10. The Src Homology 3 Domain Is Required for Junctional Adhesion Molecule Binding to the Third PDZ Domain of the Scaffolding Protein ZO-1

    SciTech Connect

    Nomme, Julian; Fanning, Alan S.; Caffrey, Michael; Lye, Ming F.; Anderson, James M.; Lavie, Arnon

    2012-01-20

    Tight junctions are cell-cell contacts that regulate the paracellular flux of solutes and prevent pathogen entry across cell layers. The assembly and permeability of this barrier are dependent on the zonula occludens (ZO) membrane-associated guanylate kinase (MAGUK) proteins ZO-1, -2, and -3. MAGUK proteins are characterized by a core motif of protein-binding domains that include a PDZ domain, a Src homology 3 (SH3) domain, and a region of homology to guanylate kinase (GUK); the structure of this core motif has never been determined for any MAGUK. To better understand how ZO proteins organize the assembly of protein complexes we have crystallized the entire PDZ3-SH3-GUK core motif of ZO-1. We have also crystallized this core motif in complex with the cytoplasmic tail of the ZO-1 PDZ3 ligand, junctional adhesion molecule A (JAM-A) to determine how the activity of different domains is coordinated. Our study shows a new feature for PDZ class II ligand binding that implicates the two highly conserved Phe{sup -2} and Ser{sup -3} residues of JAM. Our x-ray structures and NMR experiments also show for the first time a role for adjacent domains in the binding of ligands to PDZ domains in the MAGUK proteins family.

  11. The structure of the Ca{sup 2+}-binding , glycosylated F-spondin domain of F-spondin- A C2-domain variant in an extracellular matrix protein.

    SciTech Connect

    Tan, K.; Lawler, J.

    2011-05-10

    F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin{_}N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin. We present the crystal structure of human F-spondin FS domain at 1.95{angstrom} resolution. The structure reveals a Ca{sup 2+}-binding C2 domain variant with an 8-stranded antiparallel {beta}-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain. The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca{sup 2+}- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.

  12. Crystallization of an engineered RUN domain of Rab6-interacting protein 1/DENND5

    SciTech Connect

    Fernandes, Humberto; Franklin, Edward; Khan, Amir R.

    2011-08-29

    Effectors of the Rab small GTPases are large multi-domain proteins which have proved difficult to express in soluble form in Escherichia coli. Generally, effectors are recruited to a distinct subcellular compartment by active (GTP-bound) Rabs, which are linked to membranes by one or two prenylated Cys residues at their C-termini. Following recruitment via their Rab-binding domain (RBD), effectors carry out various aspects of vesicle formation, transport, tethering and fusion through their other domains. Previously, successful purification of the RUN-PLAT tandem domains (residues 683-1061) of the 1263-residue Rab6-interacting protein 1 (R6IP1) required co-expression with Rab6, as attempts to solubly express the effector alone were unsuccessful. R6IP1 is also known as DENN domain-containing protein 5 (DENND5) and is expressed as two isoforms, R6IP1A/B (DENND5A/B), which differ by 24 amino acids at the N-terminus. Here, a deletion in R6IP1 was engineered to enable soluble expression and to improve the quality of the crystals grown in complex with Rab6. A large 23-residue loop linking two -helices in the RUN1 domain was removed and replaced with a short linker. This loop resides on the opposite face to the Rab6-binding site and is not conserved in the RUN-domain family. In contrast to wild-type R6IP1-Rab6 crystals, which took several weeks to grow to full size, the engineered R6IP1 (RPdel)-Rab6 crystals could be grown in a matter of days.

  13. Metagenome-based screening reveals worldwide distribution of LOV-domain proteins.

    PubMed

    Pathak, Gopal P; Losi, Aba; Gärtner, Wolfgang

    2012-01-01

    Metagenomes from various environments were screened for sequences homologous to light, oxygen, voltage (LOV)-domain proteins. LOV domains are flavin binding, blue-light (BL)-sensitive photoreceptors present in 10-15% of deposited prokaryotic genomes. The LOV domain has been selected, since BL is an ever present and sometimes harmful environmental factor for microbial communities. The majority of the metagenome material originated from the Sargasso Sea Project and from open-ocean sampling. In total, more than 40 million open reading frames were investigated for LOV-domain sequences. Most sequences were identified from aquatic material, but they were also found in metagenomes from soil and extreme environments, e.g. hypersaline ponds, acidic mine drainage or wastewater treatment facilities. A total of 578 LOV domains was assigned by three criteria: (1) the highly conserved core region, (2) the presence of minimally 14 essential amino acids and (3) a minimal length of 80 amino acids. More than three quarters of these identified genes showed a sequence divergence of more than 20% from database-deposited LOV domains from known organisms, indicating the large variation of this photoreceptor motif. The broad occurrence of LOV domains in metagenomes emphasizes their important physiological role for light-induced signal transduction, stress adaptation and survival mechanisms. PMID:22044076

  14. Functional analysis of the transmembrane domain in paramyxovirus F protein-mediated membrane fusion

    PubMed Central

    Bissonnette, Mei Lin Z.; Donald, Jason E.; DeGrado, William F.; Jardetzky, Theodore S.; Lamb, Robert A.

    2009-01-01

    To enter cells enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 (PIV5) mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the PIV5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion. PMID:19121325

  15. Purification of proteins containing zinc finger domains using Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Voráčková, Irena; Suchanová, Šárka; Ulbrich, Pavel; Diehl, William E.; Ruml, Tomáš

    2011-01-01

    Heterologous proteins are frequently purified by Immobilized Metal Ion Affinity Chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e. CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state. PMID:21600288

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

    PubMed

    Khan, Waqasuddin; Duffy, Fergal; Pollastri, Gianluca; Shields, Denis C; Mooney, Catherine

    2013-01-01

    Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif) containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58).Next, we trained a bidirectional recurrent neural network (BRNN) using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72) showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods) clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors. PMID:24019881

  17. Targeting of a distinctive protein-serine phosphatase to the protein kinase-like domain of the atrial natriuretic peptide receptor.

    PubMed Central

    Chinkers, M

    1994-01-01

    Protein kinase-related domains of unknown function are present in the JAK family of protein tyrosine kinases and in receptor/guanylyl cyclases. I used the yeast two-hybrid system to screen for proteins interacting with the kinase-like domain of the atrial natriuretic peptide (ANP) receptor/guanylyl cyclase. A yeast strain was constructed expressing a fusion of this kinase-like domain to the lexA DNA-binding domain and containing a HIS3 gene under the control of lexA upstream activating sequences. These yeast cells were transformed with a plasmid library of mouse embryo cDNA fragments fused to the VP16 transcriptional activation domain. Cells containing VP16-fusion proteins interacting with the lexA-kinase-like domain fusion protein were selected by growth in the absence of histidine. A partial-length cDNA clone isolated by using this approach encoded a protein that interacted specifically with the ANP-receptor protein kinase-like domain both in yeast cells and in vitro. Tissue-specific expression of a 2.2-kb mRNA hybridizing to this cDNA paralleled the known pattern of ANP-receptor mRNA expression. A full-length cDNA clone isolated from a rat lung library was predicted to encode a 55-kDa protein containing at its amino terminus a targeting domain that binds to the ANP-receptor kinase-like domain and containing at its carboxyl terminus a putative protein-serine phosphatase domain. This protein is a possible candidate for the phosphatase involved in desensitizing the ANP receptor. Targeting of regulatory proteins may be an important function of protein kinase-like domains. Images PMID:7972012

  18. Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063.

    PubMed

    Gourlay, Louise J; Peano, Clelia; Deantonio, Cecilia; Perletti, Lucia; Pietrelli, Alessandro; Villa, Riccardo; Matterazzo, Elena; Lassaux, Patricia; Santoro, Claudio; Puccio, Simone; Sblattero, Daniele; Bolognesi, Martino

    2015-11-01

    The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography. PMID:26527140

  19. Crystal Structure of Trimeric Carbohydrate Recognition and Neck Domains of Surfactant Protein A

    SciTech Connect

    Head,J.; Mealy, T.; McCormack, F.; Seaton, B.

    2003-01-01

    Surfactant protein A (SP-A), one of four proteins associated with pulmonary surfactant, binds with high affinity to alveolar phospholipid membranes, positioning the protein at the first line of defense against inhaled pathogens. SP-A exhibits both calcium-dependent carbohydrate binding, a characteristic of the collectin family, and specific interactions with lipid membrane components. The crystal structure of the trimeric carbohydrate recognition domain and neck domain of SP-A was solved to 2.1-{angstrom} resolution with multiwavelength anomalous dispersion phasing from samarium. Two metalbinding sites were identified, one in the highly conserved lectin site and the other 8.5 {angstrom} away. The interdomain carbohydrate recognition domain-neck angle is significantly less in SP-A than in the homologous collectins, surfactant protein D, and mannose-binding protein. This conformational difference may endow the SP-A trimer with a more extensive hydrophobic surface capable of binding lipophilic membrane components. The appearance of this surface suggests a putative binding region for membrane-derived SP-A ligands such as phosphatidylcholine and lipid A, the endotoxic lipid component of bacterial lipopolysaccharide that mediates the potentially lethal effects of Gram-negative bacterial infection.

  20. Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain

    PubMed Central

    Gruber, Tobias; Balbach, Jochen

    2015-01-01

    The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state. PMID:26368922

  1. Expansion and Function of Repeat Domain Proteins During Stress and Development in Plants.

    PubMed

    Sharma, Manisha; Pandey, Girdhar K

    2015-01-01

    The recurrent repeats having conserved stretches of amino acids exists across all domains of life. Subsequent repetition of single sequence motif and the number and length of the minimal repeating motifs are essential characteristics innate to these proteins. The proteins with tandem peptide repeats are essential for providing surface to mediate protein-protein interactions for fundamental biological functions. Plants are enriched in tandem repeat containing proteins typically distributed into various families. This has been assumed that the occurrence of multigene repeats families in plants enable them to cope up with adverse environmental conditions and allow them to rapidly acclimatize to these conditions. The evolution, structure, and function of repeat proteins have been studied in all kingdoms of life. The presence of repeat proteins is particularly profuse in multicellular organisms in comparison to prokaryotes. The precipitous expansion of repeat proteins in plants is presumed to be through internal tandem duplications. Several repeat protein gene families have been identified in plants. Such as Armadillo (ARM), Ankyrin (ANK), HEAT, Kelch-like repeats, Tetratricopeptide (TPR), Leucine rich repeats (LRR), WD40, and Pentatricopeptide repeats (PPR). The structure and functions of these repeat proteins have been extensively studied in plants suggesting a critical role of these repeating peptides in plant cell physiology, stress and development. In this review, we illustrate the structural, functional, and evolutionary prospects of prolific repeat proteins in plants. PMID:26793205

  2. Caspase recruitment domain-containing protein 9 signaling in innate immunity and inflammation.

    PubMed

    Roth, Susanne; Ruland, Jürgen

    2013-06-01

    Caspase recruitment domain-containing protein (Card)9 is a nonredundant adapter protein that functions in the innate immune system in the assembly of multifunctional signaling complexes. Together with B cell lymphoma (Bcl)10 and the paracaspase, mucosa-associated lymphoid tissue lymphoma translocation protein (Malt)1, Card9 links spleen-tyrosine kinase (Syk)-coupled C-type lectin receptors to inflammatory responses. Card9 signaling also responds to intracellular danger sensors, such as retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) and nucleotide-oligomerization domain (Nod)2. Card9 complexes are engaged upon fungal, bacterial, or viral recognition, and they are essential for host protection. Moreover, Card9 polymorphisms are commonly associated with human inflammatory diseases. Here, we discuss the molecular regulation and the physiological functions of Card9 in host defense and immune homeostasis, and provide a framework for the therapeutic targeting of Card9 signaling in immune-mediated diseases. PMID:23523010

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

    PubMed

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

    2016-09-01

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

  4. Affinity purification of antibodies using immobilized FB domain of protein A.

    PubMed

    Solomon, B; Raviv, O; Leibman, E; Fleminger, G

    1992-04-24

    A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A. PMID:1517325

  5. The Amyloid Precursor Protein Forms Plasmalemmal Clusters via Its Pathogenic Amyloid-β Domain

    PubMed Central

    Schreiber, Arne; Fischer, Sebastian; Lang, Thorsten

    2012-01-01

    The amyloid precursor protein (APP) is a large, ubiquitous integral membrane protein with a small amyloid-β (Aβ) domain. In the human brain, endosomal processing of APP produces neurotoxic Aβ-peptides, which are involved in Alzheimer's disease. Here, we show that the Aβ sequence exerts a physiological function when still present in the unprocessed APP molecule. From the extracellular site, Aβ concentrates APP molecules into plasmalemmal membrane protein clusters. Moreover, Aβ stabilization of clusters is a prerequisite for their targeting to endocytic clathrin structures. Therefore, we conclude that the Aβ domain directly mediates a central step in APP trafficking, driving its own conversion into neurotoxic peptides. PMID:22455924

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

    SciTech Connect

    Singh, Pratibha; Savithri, H.S.

    2015-08-15

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

  7. Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition.

    PubMed

    Jun, S; Wallen, R V; Goriely, A; Kalionis, B; Desplan, C

    1998-11-10

    Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes. PMID:9811867

  8. A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain

    NASA Technical Reports Server (NTRS)

    Wang, W.; Takezawa, D.; Narasimhulu, S. B.; Reddy, A. S. N.; Poovaiah, B. W.

    1996-01-01

    Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

  9. Properties of the C-terminal domain of 4.1 proteins.

    PubMed

    Scott, C; Phillips, G W; Baines, A J

    2001-07-01

    At the C-terminus of all known 4.1 proteins is a sequence domain unique to these proteins, known as the C-terminal domain (CTD). Mammalian CTDs are associated with a growing number of protein-protein interactions, although such activities have yet to be associated with invertebrate CTDs. Mammalian CTDs are generally defined by sequence alignment as encoded by exons 18-21. Comparison of known vertebrate 4.1 proteins with invertebrate (Caenorhabditis elegans and Drosophila melanogaster) 4.1 proteins indicates that mammalian 4.1 exon 19 represents a vertebrate adaptation that extends the sequence of the CTD with a Ser/Thr-rich sequence. The CTD was first described as a 22/24-kDa domain by chymotryptic digestion of erythrocyte 4.1 (4.1R) [Leto, T.L. & Marchesi, V.T. (1984) J. Biol. Chem. 259, 4603-4608]. Here we show that in 4.1R the 22/24-kDa fragment is not stable but rapidly processed to a 15-kDa fragment by chymotrypsin. The 15-kDa fragment is extremely stable, being resistant to overnight digestion in chymotrypsin on ice. Analysis of this fragment indicates that it is derived from residues 709-858 (SwissProt accession no. P48193), and represents the CTD of 4.1R. The fragment behaves as a globular monomer in solution. Secondary-structure predictions indicate that this domain is composed of five or six beta strands with an alpha helix before the most C-terminal of these. Together these data indicate that the CTD probably represents an independent folding structure which has gained function since the divergence of vertebrates from invertebrates. PMID:11432737

  10. Functional Domain Analysis of the Remorin Protein LjSYMREM1 in Lotus japonicus

    PubMed Central

    Madsen, Esben B.; Ye, Juanying; Popp, Claudia; Antolín-Llovera, Meritxell; Grossmann, Christina; Jensen, Ole N.; Schüßler, Arthur; Parniske, Martin; Ott, Thomas

    2012-01-01

    In legumes rhizobial infection during root nodule symbiosis (RNS) is controlled by a conserved set of receptor proteins and downstream components. MtSYMREM1, a protein of the Remorin family in Medicago truncatula, was shown to interact with at least three receptor-like kinases (RLKs) that are essential for RNS. Remorins are comprised of a conserved C-terminal domain and a variable N-terminal region that defines the six different Remorin groups. While both N- and C-terminal regions of Remorins belonging to the same phylogenetic group are similar to each other throughout the plant kingdom, the N-terminal domains of legume-specific group 2 Remorins show exceptional high degrees of sequence divergence suggesting evolutionary specialization of this protein within this clade. We therefore identified and characterized the MtSYMREM1 ortholog from Lotus japonicus (LjSYMREM1), a model legume that forms determinate root nodules. Here, we resolved its spatio-temporal regulation and showed that over-expression of LjSYMREM1 increases nodulation on transgenic roots. Using a structure-function approach we show that protein interactions including Remorin oligomerization are mainly mediated and stabilized by the Remorin C-terminal region with its coiled-coil domain while the RLK kinase domains transiently interact in vivo and phosphorylate a residue in the N-terminal region of the LjSYMREM1 protein in vitro. These data provide novel insights into the mechanism of this putative molecular scaffold protein and underline its importance during rhizobial infection. PMID:22292047

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

    SciTech Connect

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

    2009-01-01

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

  12. Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6*

    PubMed Central

    Zhang, Zhixian; He, Feng; Constantine, Ryan; Baker, Matthew L.; Baehr, Wolfgang; Schmid, Michael F.; Wensel, Theodore G.; Agosto, Melina A.

    2015-01-01

    The cGMP phosphodiesterase of rod photoreceptor cells, PDE6, is the key effector enzyme in phototransduction. Two large catalytic subunits, PDE6α and -β, each contain one catalytic domain and two non-catalytic GAF domains, whereas two small inhibitory PDE6γ subunits allow tight regulation by the G protein transducin. The structure of holo-PDE6 in complex with the ROS-1 antibody Fab fragment was determined by cryo-electron microscopy. The ∼11 Å map revealed previously unseen features of PDE6, and each domain was readily fit with high resolution structures. A structure of PDE6 in complex with prenyl-binding protein (PrBP/δ) indicated the location of the PDE6 C-terminal prenylations. Reconstructions of complexes with Fab fragments bound to N or C termini of PDE6γ revealed that PDE6γ stretches from the catalytic domain at one end of the holoenzyme to the GAF-A domain at the other. Removal of PDE6γ caused dramatic structural rearrangements, which were reversed upon its restoration. PMID:25809480

  13. Human heterochromatin proteins form large domains containing KRAB-ZNF genes

    PubMed Central

    Vogel, Maartje J.; Guelen, Lars; de Wit, Elzo; Hupkes, Daniel Peric; Lodén, Martin; Talhout, Wendy; Feenstra, Marike; Abbas, Ben; Classen, Anne-Kathrin; van Steensel, Bas

    2006-01-01

    Heterochromatin is important for gene regulation and chromosome structure, but the genes that are occupied by heterochromatin proteins in the mammalian genome are largely unknown. We have adapted the DamID method to systematically identify target genes of the heterochromatin proteins HP1 and SUV39H1 in human and mouse cells. Unexpectedly, we found that CBX1 (formerly HP1β) and SUV39H1 bind to genes encoding KRAB domain containing zinc finger (KRAB-ZNF) transcriptional repressors. These genes constitute one of the largest gene families and are organized in clusters in the human genome. Preference of CBX1 for this gene family was observed in both human and mouse cells. High-resolution mapping on human chromosome 19 revealed that CBX1 coats large domains 0.1–4 Mb in size, which coincide with the position of KRAB-ZNF gene clusters. These domains show an intricate CBX1 binding pattern: While CBX1 is globally elevated throughout the domains, it is absent from the promoters and binds more strongly to the 3′ ends of KRAB-ZNF genes. KRAB-ZNF domains contain large numbers of LINE elements, which may contribute to CBX1 recruitment. These results uncover a surprising link between heterochromatin and a large family of regulatory genes in mammals. We suggest a role for heterochromatin in the evolution of the KRAB-ZNF gene family. PMID:17038565

  14. Human heterochromatin proteins form large domains containing KRAB-ZNF genes.

    PubMed

    Vogel, Maartje J; Guelen, Lars; de Wit, Elzo; Peric-Hupkes, Daniel; Lodén, Martin; Talhout, Wendy; Feenstra, Marike; Abbas, Ben; Classen, Anne-Kathrin; van Steensel, Bas

    2006-12-01

    Heterochromatin is important for gene regulation and chromosome structure, but the genes that are occupied by heterochromatin proteins in the mammalian genome are largely unknown. We have adapted the DamID method to systematically identify target genes of the heterochromatin proteins HP1 and SUV39H1 in human and mouse cells. Unexpectedly, we found that CBX1 (formerly HP1beta) and SUV39H1 bind to genes encoding KRAB domain containing zinc finger (KRAB-ZNF) transcriptional repressors. These genes constitute one of the largest gene families and are organized in clusters in the human genome. Preference of CBX1 for this gene family was observed in both human and mouse cells. High-resolution mapping on human chromosome 19 revealed that CBX1 coats large domains 0.1-4 Mb in size, which coincide with the position of KRAB-ZNF gene clusters. These domains show an intricate CBX1 binding pattern: While CBX1 is globally elevated throughout the domains, it is absent from the promoters and binds more strongly to the 3' ends of KRAB-ZNF genes. KRAB-ZNF domains contain large numbers of LINE elements, which may contribute to CBX1 recruitment. These results uncover a surprising link between heterochromatin and a large family of regulatory genes in mammals. We suggest a role for heterochromatin in the evolution of the KRAB-ZNF gene family. PMID:17038565

  15. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    SciTech Connect

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  16. Polarized Defense Against Fungal Pathogens Is Mediated by the Jacalin-Related Lectin Domain of Modular Poaceae-Specific Proteins.

    PubMed

    Weidenbach, Denise; Esch, Lara; Möller, Claudia; Hensel, Goetz; Kumlehn, Jochen; Höfle, Caroline; Hückelhoven, Ralph; Schaffrath, Ulrich

    2016-04-01

    Modular proteins are an evolutionary answer to optimize performance of proteins that physically interact with each other for functionality. Using a combination of genetic and biochemical experiments, we characterized the rice protein OsJAC1, which consists of a jacalin-related lectin (JRL) domain predicted to bind mannose-containing oligosaccharides, and a dirigent domain which might function in stereoselective coupling of monolignols. Transgenic overexpression of OsJAC1 in rice resulted in quantitative broad-spectrum resistance against different pathogens including bacteria, oomycetes, and fungi. Overexpression of this gene or its wheat ortholog TAJA1 in barley enhanced resistance against the powdery mildew fungus. Both protein domains of OsJAC1 are required to establish resistance as indicated by single or combined transient expression of individual domains. Expression of artificially separated and fluorescence-tagged protein domains showed that the JRL domain is sufficient for targeting the powdery mildew penetration site. Nevertheless, co-localization of the lectin and the dirigent domain occurred. Phylogenetic analyses revealed orthologs of OsJAC1 exclusively within the Poaceae plant family. Dicots, by contrast, only contain proteins with either JRL or dirigent domain(s). Altogether, our results identify OsJAC1 as a representative of a novel type of resistance protein derived from a plant lineage-specific gene fusion event for better function in local pathogen defense. PMID:26708413

  17. LRR Conservation Mapping to Predict Functional Sites within Protein Leucine-Rich Repeat Domains

    PubMed Central

    Helft, Laura; Reddy, Vignyan; Chen, Xiyang; Koller, Teresa; Federici, Luca; Fernández-Recio, Juan; Gupta, Rishabh; Bent, Andrew

    2011-01-01

    Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains. PMID:21789174

  18. Shrimp single WAP domain (SWD)-containing protein exhibits proteinase inhibitory and antimicrobial activities.

    PubMed

    Amparyup, Piti; Donpudsa, Suchao; Tassanakajon, Anchalee

    2008-01-01

    Single WAP domain (SWD)-containing proteins are small proteins with a C-terminal region containing a single whey acidic protein (WAP) domain. In the present study, the cDNAs representing three isoforms of SWD proteins (SWDPm1, SWDPm2 and SWDPm3) were identified from hemocytes of the black tiger shrimp, Penaeus monodon. The deduced peptides revealed that they contain a putative signal peptide of 24 amino acids and encode for a mature peptide of 69, 68 and 56 amino acids, respectively, which contain typical characters similar to those of the shrimp SWD proteins (type III crustin) with a Pro-Arg region and a WAP domain towards the C-terminus. Tissue distribution analysis by RT-PCR showed that all three SWDPm transcripts were primarily found in hemocytes. Transcript expression of SWDPm1 was down-regulated upon injection with Staphylococcus aureus whilst there was no change of SWDPm2 and SWDPm3 expression. In contrast, white spot syndrome virus (WSSV) injection resulted in a biphasic response with up-regulation of SWDPm1 and SWDPm2 transcripts at 6h followed by significant down-regulation by 24h after infection. Genomic organization of the SWDPm2 gene revealed the presence of three exons interrupted by two introns. To characterize the biological functions of the SWD protein, the mature SWDPm2 protein encoding cDNA was cloned and expressed in Escherichia coli. Purified recombinant (r)SWDPm2 exhibits antibacterial activity against several Gram-positive, but not Gram-negative, bacteria and is a competitive inhibitor of subtilisin A with an inhibition constant (Ki) of 1.98nM. Thus, rSWDPm2 may contribute to the inhibitory regulation of subtilisin A from bacterial infection and P. monodon SWD protein likely function as immune effectors in defense against invasion of shrimp pathogens. PMID:18602420

  19. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

    PubMed Central

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-01-01

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

  20. The CW domain, a new histone recognition module in chromatin proteins.

    PubMed

    Hoppmann, Verena; Thorstensen, Tage; Kristiansen, Per Eugen; Veiseth, Silje Veie; Rahman, Mohummad Aminur; Finne, Kenneth; Aalen, Reidunn B; Aasland, Rein

    2011-05-18

    Post-translational modifications of the N-terminal histone tails, including lysine methylation, have key roles in regulation of chromatin and gene expression. A number of protein modules have been identified that recognize differentially modified histone tails and provide their proteins with the capacity to sense such modifications. Here, we identify the CW domain of plant and animal chromatin-related proteins as a novel module that recognizes different methylated states of lysine 4 on histone H3 (H3K4me). The solution structure of the CW domain of the Arabidopsis ASH1 HOMOLOG2 (ASHH2) histone methyltransferase provides insight into how different CW domains can distinguish different methylated histone tails. We provide evidence that ASHH2 is acting on H3K4me-marked genes, allowing for ASHH2-dependent H3K36 tri-methylation, which contributes to sustained expression of tissue-specific and developmentally regulated genes. This suggests that ASHH2 is a combined 'reader' and 'writer' of the histone code. We propose that different CW domains, dependent on their specificity for different H3K4 methylations, are important for epigenetic memory or participate in switching between permissive and repressive chromatin states. PMID:21522130

  1. The pilus usher controls protein interactions via domain masking and is functional as an oligomer

    SciTech Connect

    Werneburg, Glenn T.; Li, Huilin; Henderson, Nadine S.; Portnoy, Erica B.; Sarowar, Samema; Hultgren, Scott J.; Thanassi, David G.

    2015-06-08

    The chaperone/usher (CU) pathway is responsible for biogenesis of organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Assembly and secretion of pili by the CU pathway requires a dedicated periplasmic chaperone and a multidomain outer membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate binding site, but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which served as a central switch controlling usher activation. In addition, we demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions, and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.

  2. The pilus usher controls protein interactions via domain masking and is functional as an oligomer

    DOE PAGESBeta

    Werneburg, Glenn T.; Li, Huilin; Henderson, Nadine S.; Portnoy, Erica B.; Sarowar, Samema; Hultgren, Scott J.; Thanassi, David G.

    2015-06-08

    The chaperone/usher (CU) pathway is responsible for biogenesis of organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Assembly and secretion of pili by the CU pathway requires a dedicated periplasmic chaperone and a multidomain outer membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate binding site, but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which served as a central switch controlling usher activation. In addition, we demonstratemore » that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions, and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.« less

  3. Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy

    PubMed Central

    Carlsson, Emil; Thwaite, Joanne E.; Jenner, Dominic C.; Spear, Abigail M.; Flick-Smith, Helen; Atkins, Helen S.; Ding, Jeak Ling

    2016-01-01

    Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence. PMID:27391310

  4. fastSCOP: a fast web server for recognizing protein structural domains and SCOP superfamilies.

    PubMed

    Tung, Chi-Hua; Yang, Jinn-Moon

    2007-07-01

    The fastSCOP is a web server that rapidly identifies the structural domains and determines the evolutionary superfamilies of a query protein structure. This server uses 3D-BLAST to scan quickly a large structural classification database (SCOP1.71 with <95% identity with each other) and the top 10 hit domains, which have different superfamily classifications, are obtained from the hit lists. MAMMOTH, a detailed structural alignment tool, is adopted to align these top 10 structures to refine domain boundaries and to identify evolutionary superfamilies. Our previous works demonstrated that 3D-BLAST is as fast as BLAST, and has the characteristics of BLAST (e.g. a robust statistical basis, effective search and reliable database search capabilities) in large structural database searches based on a structural alphabet database and a structural alphabet substitution matrix. The classification accuracy of this server is approximately 98% for 586 query structures and the average execution time is approximately 5. This server was also evaluated on 8700 structures, which have no annotations in the SCOP; the server can automatically assign 7311 (84%) proteins (9420 domains) to the SCOP superfamilies in 9.6 h. These results suggest that the fastSCOP is robust and can be a useful server for recognizing the evolutionary classifications and the protein functions of novel structures. The server is accessible at http://fastSCOP.life.nctu.edu.tw. PMID:17485476

  5. Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy.

    PubMed

    Carlsson, Emil; Thwaite, Joanne E; Jenner, Dominic C; Spear, Abigail M; Flick-Smith, Helen; Atkins, Helen S; Byrne, Bernadette; Ding, Jeak Ling

    2016-01-01

    Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence. PMID:27391310

  6. Solution structure of villin 14T, a domain conserved among actin-severing proteins.

    PubMed Central

    Markus, M. A.; Nakayama, T.; Matsudaira, P.; Wagner, G.

    1994-01-01

    The solution structure of the N-terminal domain of the actin-severing protein villin has been determined by multidimensional heteronuclear resonance spectroscopy. Villin is a member of a family of actin-severing proteins that regulate the organization of actin in the eukaryotic cytoskeleton. Members of this family are built from 3 or 6 homologous repeats of a structural domain of approximately 130 amino acids that is unrelated to any previously known structure. The N-terminal domain of villin (14T) contains a central beta-sheet with 4 antiparallel strands and a fifth parallel strand at one edge. This sheet is sandwiched between 2 helices on one side and a 2-stranded parallel beta-sheet with another helix on the other side. The strongly conserved sequence characteristic of the protein family corresponds to internal hydrophobic residues. Calcium titration experiments suggest that there are 2 binding sites for Ca2+, a stronger site near the N-terminal end of the longest helix, with a Kd of 1.8 +/- 0.4 mM, and a weaker site near the C-terminal end of the same helix, with a Kd of 11 +/- 2 mM. Mutational and biochemical studies of this domain in several members of the family suggest that the actin monomer binding site is near the parallel strand at the edge of the central beta-sheet. PMID:8142900

  7. A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

    PubMed Central

    Glanzer, Jason G.; Carnes, Katie A.; Soto, Patricia; Liu, Shengqin; Parkhurst, Lawrence J.; Oakley, Gregory G.

    2013-01-01

    Replication protein A (RPA), essential for DNA replication, repair and DNA damage signalling, possesses six ssDNA-binding domains (DBDs), including DBD-F on the N-terminus of the largest subunit, RPA70. This domain functions as a binding site for p53 and other DNA damage and repair proteins that contain amphipathic alpha helical domains. Here, we demonstrate direct binding of both ssDNA and the transactivation domain 2 of p53 (p53TAD2) to DBD-F, as well as DBD-F-directed dsDNA strand separation by RPA, all of which are inhibited by fumaropimaric acid (FPA). FPA binds directly to RPA, resulting in a conformational shift as determined through quenching of intrinsic tryptophan fluorescence in full length RPA. Structural analogues of FPA provide insight on chemical properties that are required for inhibition. Finally, we confirm the inability of RPA possessing R41E and R43E mutations to bind to p53, destabilize dsDNA and quench tryptophan fluorescence by FPA, suggesting that protein binding, DNA modulation and inhibitor binding all occur within the same site on DBD-F. The disruption of p53–RPA interactions by FPA may disturb the regulatory functions of p53 and RPA, thereby inhibiting cellular pathways that control the cell cycle and maintain the integrity of the human genome. PMID:23267009

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

    PubMed

    Meagher, Martin; Enemark, Eric J

    2016-07-01

    The crystal structure of the N-terminal domain of the Pyrococcus furiosus minichromosome maintenance (MCM) protein as a double hexamer is described. The MCM complex is a ring-shaped helicase that unwinds DNA at the replication fork of eukaryotes and archaea. Prior to replication initiation, the MCM complex assembles as an inactive double hexamer at specific sites of DNA. The presented structure is highly consistent with previous MCM double-hexamer structures and shows two MCM hexamers with a head-to-head interaction mediated by the N-terminal domain. Minor differences include a diminished head-to-head interaction and a slightly reduced inter-hexamer rotation. PMID:27380371

  9. The use of orthogonal projection to visualize mosaic domains from topographic data collected on protein crystals

    SciTech Connect

    Lovelace, J.J.; Borgstahl, G.E.O.

    2010-07-23

    Owing to the relatively low intensity of diffraction and resulting low contrast topographic images, and also the richly detailed domain structure, it can be difficult to extract individual mosaic domains (size and shape) for protein crystals. Here, orthogonal projection was tested and found to be a superior approach over previous methods to extract mosaic domain information from a fine-sliced topographic sequence of images. The topographic sequence was collected at room temperature from a lysozyme crystal with a low mosaicity. Orthogonal projection can be applied to image sequences that are spatially invariant and composed of linearly additive image formation processes. The domains were determined by using a particle swarm optimizer to fit Gaussians to the integrated intensity profile of the reflection as a function of angle, although any basis function could have been used. This optimization method was more amenable to automation and converged to the global minimum more efficiently. The number of domains can normally be determined by visually inspecting the integrated intensity profile; this only needs to be done once for each crystal, because the number of domains remains constant during collection, and not for each reflection. The results can be used to provide a picture of the internal structure of the crystal and may be useful to aid in the improvement of crystal growth techniques.

  10. Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains

    PubMed Central

    Herrera, Raul; Anderson, Charles; Kumar, Krishan; Molina-Cruz, Alvaro; Nguyen, Vu; Burkhardt, Martin; Reiter, Karine; Shimp, Richard; Howard, Randall F.; Srinivasan, Prakash; Nold, Michael J.; Ragheb, Daniel; Shi, Lirong; DeCotiis, Mark; Aebig, Joan; Lambert, Lynn; Rausch, Kelly M.; Muratova, Olga; Jin, Albert; Reed, Steven G.; Sinnis, Photini; Barillas-Mury, Carolina; Duffy, Patrick E.; MacDonald, Nicholas J.

    2015-01-01

    The extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N- and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine. PMID:26169272

  11. Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains.

    PubMed

    Herrera, Raul; Anderson, Charles; Kumar, Krishan; Molina-Cruz, Alvaro; Nguyen, Vu; Burkhardt, Martin; Reiter, Karine; Shimp, Richard; Howard, Randall F; Srinivasan, Prakash; Nold, Michael J; Ragheb, Daniel; Shi, Lirong; DeCotiis, Mark; Aebig, Joan; Lambert, Lynn; Rausch, Kelly M; Muratova, Olga; Jin, Albert; Reed, Steven G; Sinnis, Photini; Barillas-Mury, Carolina; Duffy, Patrick E; MacDonald, Nicholas J; Narum, David L

    2015-10-01

    The extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N- and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine. PMID:26169272

  12. Structure and Function of the SWIRM Domain, a Conserved Protein Module Found in Chromatin Regulatory Complexes

    SciTech Connect

    Da,G.; Lenkart, J.; Zhao, K.; Shiekhattar, R.; Cairns, B.; Marmorstein, R.

    2006-01-01

    The SWIRM domain is a module found in the Swi3 and Rsc8 subunits of SWI/SNF-family chromatin remodeling complexes, and the Ada2 and BHC110/LSD1 subunits of chromatin modification complexes. Here we report the high-resolution crystal structure of the SWIRM domain from Swi3 and characterize the in vitro and in vivo function of the SWIRM domains from Saccharomyces cerevisiae Swi3 and Rsc8. The Swi3 SWIRM forms a four-helix bundle containing a pseudo 2-fold axis and a helix-turn-helix motif commonly found in DNA-binding proteins. We show that the Swi3 SWIRM binds free DNA and mononucleosomes with high and comparable affinity and that a subset of Swi3 substitution mutants that display growth defects in vivo also show impaired DNA-binding activity in vitro, consistent with a nucleosome targeting function of this domain. Genetic and biochemical studies also reveal that the Rsc8 and Swi3 SWIRM domains are essential for the proper assembly and in vivo functions of their respective complexes. Together, these studies identify the SWIRM domain as an essential multifunctional module for the regulation of gene expression.

  13. Resin embedded multicycle imaging (REMI): a tool to evaluate protein domains.

    PubMed

    Busse, B L; Bezrukov, L; Blank, P S; Zimmerberg, J

    2016-01-01

    Protein complexes associated with cellular processes comprise a significant fraction of all biology, but our understanding of their heterogeneous organization remains inadequate, particularly for physiological densities of multiple protein species. Towards resolving this limitation, we here present a new technique based on resin-embedded multicycle imaging (REMI) of proteins in-situ. By stabilizing protein structure and antigenicity in acrylic resins, affinity labels were repeatedly applied, imaged, removed, and replaced. In principle, an arbitrarily large number of proteins of interest may be imaged on the same specimen with subsequent digital overlay. A series of novel preparative methods were developed to address the problem of imaging multiple protein species in areas of the plasma membrane or volumes of cytoplasm of individual cells. For multiplexed examination of antibody staining we used straightforward computational techniques to align sequential images, and super-resolution microscopy was used to further define membrane protein colocalization. We give one example of a fibroblast membrane with eight multiplexed proteins. A simple statistical analysis of this limited membrane proteomic dataset is sufficient to demonstrate the analytical power contributed by additional imaged proteins when studying membrane protein domains. PMID:27499335

  14. Resin embedded multicycle imaging (REMI): a tool to evaluate protein domains

    PubMed Central

    Busse, B. L.; Bezrukov, L.; Blank, P. S.; Zimmerberg, J.

    2016-01-01

    Protein complexes associated with cellular processes comprise a significant fraction of all biology, but our understanding of their heterogeneous organization remains inadequate, particularly for physiological densities of multiple protein species. Towards resolving this limitation, we here present a new technique based on resin-embedded multicycle imaging (REMI) of proteins in-situ. By stabilizing protein structure and antigenicity in acrylic resins, affinity labels were repeatedly applied, imaged, removed, and replaced. In principle, an arbitrarily large number of proteins of interest may be imaged on the same specimen with subsequent digital overlay. A series of novel preparative methods were developed to address the problem of imaging multiple protein species in areas of the plasma membrane or volumes of cytoplasm of individual cells. For multiplexed examination of antibody staining we used straightforward computational techniques to align sequential images, and super-resolution microscopy was used to further define membrane protein colocalization. We give one example of a fibroblast membrane with eight multiplexed proteins. A simple statistical analysis of this limited membrane proteomic dataset is sufficient to demonstrate the analytical power contributed by additional imaged proteins when studying membrane protein domains. PMID:27499335

  15. Expansion and Function of Repeat Domain Proteins During Stress and Development in Plants

    PubMed Central

    Sharma, Manisha; Pandey, Girdhar K.

    2016-01-01

    The recurrent repeats having conserved stretches of amino acids exists across all domains of life. Subsequent repetition of single sequence motif and the number and length of the minimal repeating motifs are essential characteristics innate to these proteins. The proteins with tandem peptide repeats are essential for providing surface to mediate protein–protein interactions for fundamental biological functions. Plants are enriched in tandem repeat containing proteins typically distributed into various families. This has been assumed that the occurrence of multigene repeats families in plants enable them to cope up with adverse environmental conditions and allow them to rapidly acclimatize to these conditions. The evolution, structure, and function of repeat proteins have been studied in all kingdoms of life. The presence of repeat proteins is particularly profuse in multicellular organisms in comparison to prokaryotes. The precipitous expansion of repeat proteins in plants is presumed to be through internal tandem duplications. Several repeat protein gene families have been identified in plants. Such as Armadillo (ARM), Ankyrin (ANK), HEAT, Kelch-like repeats, Tetratricopeptide (TPR), Leucine rich repeats (LRR), WD40, and Pentatricopeptide repeats (PPR). The structure and functions of these repeat proteins have been extensively studied in plants suggesting a critical role of these repeating peptides in plant cell physiology, stress and development. In this review, we illustrate the structural, functional, and evolutionary prospects of prolific repeat proteins in plants. PMID:26793205

  16. Transmembrane Protein (Perfringolysin O) Association with Ordered Membrane Domains (Rafts) Depends Upon the Raft-Associating Properties of Protein-Bound Sterol

    PubMed Central

    Lin, Qingqing; London, Erwin

    2013-01-01

    Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds both to sterols that tend to localize in ordered domains (e.g., cholesterol), and to those that do not (e.g., coprostanol), but it does not bind to epicholesterol, a raft-promoting 3α-OH sterol. Using a fluorescence resonance energy transfer assay in model membrane vesicles containing coexisting ordered and disordered lipid domains, both TM and non-TM forms of PFO were found to concentrate in ordered domains in vesicles containing high and low-Tm lipids plus cholesterol or 1:1 (mol/mol) cholesterol/epicholesterol, whereas they concentrate in disordered domains in vesicles containing high-Tm and low-Tm lipids plus 1:1 (mol/mol) coprostanol/epicholesterol. Combined with previous studies this behavior indicates that TM protein association with ordered domains is dependent upon both the association of the protein-bound sterol with ordered domains and hydrophobic match between TM segments and rafts. PMID:24359745

  17. MLL repression domain interacts with histone deacetylases, the polycomb group proteins HPC2 and BMI-1, and the corepressor C-terminal-binding protein

    PubMed Central

    Xia, Zhen-Biao; Anderson, Melanie; Diaz, Manuel O.; Zeleznik-Le, Nancy J.

    2003-01-01

    The MLL (mixed-lineage leukemia) gene is involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia. We previously identified a transcriptional repression domain in MLL, which contains a region with homology to DNA methyltransferase. In chromosomal translocations, the MLL repression domain is retained in the leukemogenic fusion protein and is required for transforming activity of MLL fusion proteins. We explored the mechanism of action of the MLL repression domain. Histone deacetylase 1 interacts with the MLL repression domain, partially mediating its activity; binding of Cyp33 to the adjacent MLL-PHD domain potentiates this binding. Because the MLL repression domain activity was only partially relieved with the histone deacetylase inhibitor trichostatin A, we explored other protein interactions with this domain. Polycomb group proteins HPC2 and BMI-1 and the corepressor C-terminal-binding protein also bind the MLL repression domain. Expression of exogenous BMI-1 potentiates MLL repression domain activity. Functional antagonism between Mll and Bmi-1 has been shown genetically in murine knockout models for Mll and Bmi-1. Our new data suggest a model whereby recruitment of BMI-1 to the MLL protein may be able to modulate its function. Furthermore, repression mediated by histone deacetylases and that mediated by polycomb group proteins may act either independently or together for MLL function in vivo. PMID:12829790

  18. Analysis of the Protein Domain and Domain Architecture Content in Fungi and Its Application in the Search of New Antifungal Targets

    PubMed Central

    Barrera, Alejandro; Alastruey-Izquierdo, Ana; Martín, María J.; Cuesta, Isabel; Vizcaíno, Juan Antonio

    2014-01-01

    Over the past several years fungal infections have shown an increasing incidence in the susceptible population, and caused high mortality rates. In parallel, multi-resistant fungi are emerging in human infections. Therefore, the identification of new potential antifungal targets is a priority. The first task of this study was to analyse the protein domain and domain architecture content of the 137 fungal proteomes (corresponding to 111 species) available in UniProtKB (UniProt KnowledgeBase) by January 2013. The resulting list of core and exclusive domain and domain architectures is provided in this paper. It delineates the different levels of fungal taxonomic classification: phylum, subphylum, order, genus and species. The analysis highlighted Aspergillus as the most diverse genus in terms of exclusive domain content. In addition, we also investigated which domains could be considered promiscuous in the different organisms. As an application of this analysis, we explored three different ways to detect potential targets for antifungal drugs. First, we compared the domain and domain architecture content of the human and fungal proteomes, and identified those domains and domain architectures only present in fungi. Secondly, we looked for information regarding fungal pathways in public repositories, where proteins containing promiscuous domains could be involved. Three pathways were identified as a result: lovastatin biosynthesis, xylan degradation and biosynthesis of siroheme. Finally, we classified a subset of the studied fungi in five groups depending on their occurrence in clinical samples. We then looked for exclusive domains in the groups that were more relevant clinically and determined which of them had the potential to bind small molecules. Overall, this study provides a comprehensive analysis of the available fungal proteomes and shows three approaches that can be used as a first step in the detection of new antifungal targets. PMID:25033262

  19. Annotation of Protein Domains Reveals Remarkable Conservation in the Functional Make up of Proteomes Across Superkingdoms

    PubMed Central

    Nasir, Arshan; Naeem, Aisha; Khan, Muhammad Jawad; Lopez-Nicora, Horacio D.; Caetano-Anollés, Gustavo

    2011-01-01

    The functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. The molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden Markov models of structural recognition. Here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. We find there is a remarkable conservation in the distribution of domains with respect to the molecular functions they perform in the three superkingdoms of life. In general, most of the protein repertoire is spent in functions related to metabolic processes but there are significant differences in the usage of domains for regulatory and extra-cellular processes both within and between superkingdoms. Our results support the hypotheses that the proteomes of superkingdom Eukarya evolved via genome expansion mechanisms that were directed towards innovating new domain architectures for regulatory and extra/intracellular process functions needed for example to maintain the integrity of multicellular structure or to interact with environmental biotic and abiotic factors (e.g., cell signaling and adhesion, immune responses, and toxin production). Proteomes of microbial superkingdoms Archaea and Bacteria retained fewer numbers of domains and maintained simple and smaller protein repertoires. Viruses appear to play an important role in the evolution of superkingdoms. We finally identify few genomic outliers that deviate significantly from the conserved functional design. These include Nanoarchaeum equitans, proteobacterial symbionts of insects with extremely reduced genomes, Tenericutes and Guillardia theta. These organisms spend most of their domains on information functions, including translation and transcription, rather than on metabolism and harbor a domain repertoire characteristic of

  20. 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.

  1. Reassessing Domain Architecture Evolution of Metazoan Proteins: Major Impact of Gene Prediction Errors

    PubMed Central

    Nagy, Alinda; Szláma, György; Szarka, Eszter; Trexler, Mária; Bányai, László; Patthy, László

    2011-01-01

    In view of the fact that appearance of novel protein domain architectures (DA) is closely associated with biological innovations, there is a growing interest in the genome-scale reconstruction of the evolutionary history of the domain architectures of multidomain proteins. In such analyses, however, it is usually ignored that a significant proportion of Metazoan sequences analyzed is mispredicted and that this may seriously affect the validity of the conclusions. To estimate the contribution of errors in gene prediction to differences in DA of predicted proteins, we have used the high quality manually curated UniProtKB/Swiss-Prot database as a reference. For genome-scale analysis of domain architectures of predicted proteins we focused on RefSeq, EnsEMBL and NCBI's GNOMON predicted sequences of Metazoan species with completely sequenced genomes. Comparison of the DA of UniProtKB/Swiss-Prot sequences of worm, fly, zebrafish, frog, chick, mouse, rat and orangutan with those of human Swiss-Prot entries have identified relatively few cases where orthologs had different DA, although the percentage with different DA increased with evolutionary distance. In contrast with this, comparison of the DA of human, orangutan, rat, mouse, chicken, frog, zebrafish, worm and fly RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences with those of the corresponding/orthologous human Swiss-Prot entries identified a significantly higher proportion of domain architecture differences than in the case of the comparison of Swiss-Prot entries. Analysis of RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences with DAs different from those of their Swiss-Prot orthologs confirmed that the higher rate of domain architecture differences is due to errors in gene prediction, the majority of which could be corrected with our FixPred protocol. We have also demonstrated that contamination of databases with incomplete, abnormal or mispredicted sequences introduces a bias in DA

  2. Reassessing domain architecture evolution of metazoan proteins: major impact of gene prediction errors.

    PubMed

    Nagy, Alinda; Szláma, György; Szarka, Eszter; Trexler, Mária; Bányai, László; Patthy, László

    2011-01-01

    In view of the fact that appearance of novel protein domain architectures (DA) is closely associated with biological innovations, there is a growing interest in the genome-scale reconstruction of the evolutionary history of the domain architectures of multidomain proteins. In such analyses, however, it is usually ignored that a significant proportion of Metazoan sequences analyzed is mispredicted and that this may seriously affect the validity of the conclusions. To estimate the contribution of errors in gene prediction to differences in DA of predicted proteins, we have used the high quality manually curated UniProtKB/Swiss-Prot database as a reference. For genome-scale analysis of domain architectures of predicted proteins we focused on RefSeq, EnsEMBL and NCBI's GNOMON predicted sequences of Metazoan species with completely sequenced genomes. Comparison of the DA of UniProtKB/Swiss-Prot sequences of worm, fly, zebrafish, frog, chick, mouse, rat and orangutan with those of human Swiss-Prot entries have identified relatively few cases where orthologs had different DA, although the percentage with different DA increased with evolutionary distance. In contrast with this, comparison of the DA of human, orangutan, rat, mouse, chicken, frog, zebrafish, worm and fly RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences with those of the corresponding/orthologous human Swiss-Prot entries identified a significantly higher proportion of domain architecture differences than in the case of the comparison of Swiss-Prot entries. Analysis of RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences with DAs different from those of their Swiss-Prot orthologs confirmed that the higher rate of domain architecture differences is due to errors in gene prediction, the majority of which could be corrected with our FixPred protocol. We have also demonstrated that contamination of databases with incomplete, abnormal or mispredicted sequences introduces a bias in DA

  3. Adsorption thermodynamics of two-domain antifreeze proteins: theory and Monte Carlo simulations.

    PubMed

    Narambuena, Claudio F; Sanchez Varretti, Fabricio O; Ramirez-Pastor, Antonio J

    2016-09-21

    In this paper we develop the statistical thermodynamics of two-domain antifreeze proteins adsorbed on ice. We use a coarse-grained model and a lattice network in order to represent the protein and ice, respectively. The theory is obtained by combining the exact analytical expression for the partition function of non-interacting linear k-mers adsorbed in one dimension, and its extension to higher dimensions. The total and partial adsorption isotherms, and the coverage and temperature dependence of the Helmholtz free energy and configurational entropy are given. The formalism reproduces the classical Langmuir equation, leads to the exact statistical thermodynamics of molecules adsorbed in one dimension, and provides a close approximation for two-dimensional systems. Comparisons with analytical data obtained using the modified Langmuir model (MLM) and Monte Carlo simulations in the grand canonical ensemble were performed in order to test the validity of the theoretical predictions. In the MC calculations, the different mechanisms proposed in the literature to describe the adsorption of two-domain antifreeze proteins on ice were analyzed. Indistinguishable results were obtained in all cases, which verifies the thermodynamic equivalence of these mechanisms and allows the choice of the most suitable mechanism for theoretical studies of equilibrium properties. Even though a good qualitative agreement is obtained between MLM and MC data, it is found that the new theoretical framework offers a more accurate description of the phenomenon of adsorption of two-domain antifreeze proteins. PMID:27539563

  4. KCNQ1 channel modulation by KCNE proteins via the voltage-sensing domain

    PubMed Central

    Nakajo, Koichi; Kubo, Yoshihiro

    2015-01-01

    The gating of the KCNQ1 potassium channel is drastically regulated by auxiliary subunit KCNE proteins. KCNE1, for example, slows the activation kinetics of KCNQ1 by two orders of magnitude. Like other voltage-gated ion channels, the opening of KCNQ1 is regulated by the voltage-sensing domain (VSD; S1–S4 segments). Although it has been known that KCNE proteins interact with KCNQ1 via the pore domain, some recent reports suggest that the VSD movement may be altered by KCNE. The altered VSD movement of KCNQ1 by KCNE proteins has been examined by site-directed mutagenesis, the scanning cysteine accessibility method (SCAM), voltage clamp fluorometry (VCF) and gating charge measurements. These accumulated data support the idea that KCNE proteins interact with the VSDs of KCNQ1 and modulate the gating of the KCNQ1 channel. In this review, we will summarize recent findings and current views of the KCNQ1 modulation by KCNE via the VSD. In this context, we discuss our recent findings that KCNE1 may alter physical interactions between the S4 segment (VSD) and the S5 segment (pore domain) of KCNQ1. Based on these findings from ourselves and others, we propose a hypothetical mechanism for how KCNE1 binding alters the VSD movement and the gating of the channel. PMID:25603957

  5. KCNQ1 channel modulation by KCNE proteins via the voltage-sensing domain.

    PubMed

    Nakajo, Koichi; Kubo, Yoshihiro

    2015-06-15

    The gating of the KCNQ1 potassium channel is drastically regulated by auxiliary subunit KCNE proteins. KCNE1, for example, slows the activation kinetics of KCNQ1 by two orders of magnitude. Like other voltage-gated ion channels, the opening of KCNQ1 is regulated by the voltage-sensing domain (VSD; S1-S4 segments). Although it has been known that KCNE proteins interact with KCNQ1 via the pore domain, some recent reports suggest that the VSD movement may be altered by KCNE. The altered VSD movement of KCNQ1 by KCNE proteins has been examined by site-directed mutagenesis, the scanning cysteine accessibility method (SCAM), voltage clamp fluorometry (VCF) and gating charge measurements. These accumulated data support the idea that KCNE proteins interact with the VSDs of KCNQ1 and modulate the gating of the KCNQ1 channel. In this review, we will summarize recent findings and current views of the KCNQ1 modulation by KCNE via the VSD. In this context, we discuss our recent findings that KCNE1 may alter physical interactions between the S4 segment (VSD) and the S5 segment (pore domain) of KCNQ1. Based on these findings from ourselves and others, we propose a hypothetical mechanism for how KCNE1 binding alters the VSD movement and the gating of the channel. PMID:25603957

  6. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.

    PubMed Central

    Henry, L J; Xia, D; Wilke, M E; Deisenhofer, J; Gerard, R D

    1994-01-01

    The adenovirus fiber protein is used for attachment of the virus to a specific receptor on the cell surface. Structurally, the protein consists of a long, thin shaft that protrudes from the vertex of the virus capsid and terminates in a globular domain termed the knob. To verify that the knob is the domain which interacts with the cellular receptor, we have cloned and expressed the knob from adenovirus type 5 together with a single repeat of the shaft in Escherichia coli. The protein was purified by conventional chromatography and functionally characterized for its interaction with the adenovirus receptor. The recombinant knob domain bound about 4,700 sites per HeLa cell with an affinity of 3 x 10(9) M-1 and blocked adenovirus infection of human cells. Antibodies raised against the knob also blocked virus infection. By gel filtration and X-ray diffraction analysis of protein crystals, the knob was shown to consist of a homotrimer of 21-kDa subunits. The results confirm that the trimeric knob is the ligand for attachment to the adenovirus receptor. Images PMID:8035520

  7. Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family1[C][W

    PubMed Central

    Roppolo, Daniele; Boeckmann, Brigitte; Pfister, Alexandre; Boutet, Emmanuel; Rubio, Maria C.; Dénervaud-Tendon, Valérie; Vermeer, Joop E.M.; Gheyselinck, Jacqueline; Xenarios, Ioannis; Geldner, Niko

    2014-01-01

    CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells. PMID:24920445

  8. NMR structure of the forkhead-associated domain from the Arabidopsis receptor kinase-associated protein phosphatase

    PubMed Central

    Lee, Gui-in; Ding, Zhaofeng; Walker, John C.; Van Doren, Steven R.

    2003-01-01

    Forkhead-associated (FHA) domains are phosphoprotein-binding modules found in diverse signaling proteins that bind partners phosphorylated on threonine or serine. Kinase-associated protein phosphatase from Arabidopsis employs its FHA domain for negative regulation of receptor-like kinase signaling pathways, which are important in plant development. The solution structure of the free state of kinase-interacting FHA domain (KI-FHA) of kinase-associated protein phosphatase has been determined with high precision and accuracy using residual dipolar couplings. KI-FHA is a sandwich of a five-stranded mixed β-sheet with a six-stranded antiparallel β-sheet. Despite homology only in the recognition loops, this fold is shared with FHA domains from checkpoint proteins from yeast and humans, as well as with nonhomologous MH2 domains of Smad tumor suppressors. A shared pattern of hydrophobicity throughout FHA domains and Smad MH2 domains may stabilize the core of the β-sandwich. Evolutionary trace analysis of FHA domains suggests class-specific residues in the recognition loops that could tune their phosphoprotein-binding specificity. This surface agrees with that of KI-FHA in contact with a phosphothreonine peptide ligand. Evolutionary trace analysis also predicts an unexpected swath of class-specific residues on another face of FHA domains. Protein interactions with these faces may affect assembly of transmembrane signaling complexes in plants, and in other FHA domain-containing assemblies. PMID:14500786

  9. Removal of the BH4 domain from Bcl-2 protein triggers an autophagic process that impairs tumor growth.

    PubMed

    Trisciuoglio, Daniela; De Luca, Teresa; Desideri, Marianna; Passeri, Daniela; Gabellini, Chiara; Scarpino, Stefania; Liang, Chengyu; Orlandi, Augusto; Del Bufalo, Donatella

    2013-03-01

    Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2) protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4) domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. In particular, upon different autophagic stimuli in vitro, overexpression of bcl-2 protein deleted of BH4 domain induces autophagosome accumulation, conversion of microtubule-associated protein 1 light chain 3B-II, reduced expression of p62/SQSTM1 protein, and thereby enhanced autophagic flux. The relevance of Beclin-1 is evidenced by the fact that 1) the autophagy-promoting and growth-inhibiting properties are partially rescued by Beclin-1 knockdown in cells expressing bcl-2 protein lacking the BH4 domain, 2) Beclin-1 only interacts with wild-type but not with deleted bcl-2, and 3) BH4 domain removal from bcl-2 protein does not influence in vitro and in vivo growth of tumor cells expressing low levels of endogenous Beclin-1. These results provide new insight into molecular mechanism of bcl-2 functions and represent a rationale for the development of agents interfering with the BH4 domain of bcl-2 protein. PMID:23479509

  10. Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth12

    PubMed Central

    Trisciuoglio, Daniela; De Luca, Teresa; Desideri, Marianna; Passeri, Daniela; Gabellini, Chiara; Scarpino, Stefania; Liang, Chengyu; Orlandi, Augusto; Del Bufalo, Donatella

    2013-01-01

    Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2) protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4) domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. In particular, upon different autophagic stimuli in vitro, overexpression of bcl-2 protein deleted of BH4 domain induces autophagosome accumulation, conversion of microtubule-associated protein 1 light chain 3B-II, reduced expression of p62/SQSTM1 protein, and thereby enhanced autophagic flux. The relevance of Beclin-1 is evidenced by the fact that 1) the autophagy-promoting and growth-inhibiting properties are partially rescued by Beclin-1 knockdown in cells expressing bcl-2 protein lacking the BH4 domain, 2) Beclin-1 only interacts with wild-type but not with deleted bcl-2, and 3) BH4 domain removal from bcl-2 protein does not influence in vitro and in vivo growth of tumor cells expressing low levels of endogenous Beclin-1. These results provide new insight into molecular mechanism of bcl-2 functions and represent a rationale for the development of agents interfering with the BH4 domain of bcl-2 protein. PMID:23479509

  11. Novel insights on ENTH domain-containing proteins in apicomplexan parasites.

    PubMed

    Kibria, K M Kaderi; Hossain, Mohammad Uzzal; Oany, Arafat Rahman; Ahmad, Shah Adil Ishtiyaq

    2016-06-01

    The phylum Apicomplexa includes a large group of early branching eukaryotes having significant medical and economical importance. The molecular machinery responsible for protein trafficking is poorly understood in these apicomplexans. One of the most important proteins involved in clathrin-mediated protein trafficking is Epsin, which contains ENTH domain, a conserved domain crucial for membrane bending leading to vesicle formation. We undertook homology searching and phylogenetic analyses to produce a rigorously annotated set of Epsin homologs retrieved from diverse apicomplexan genomes. Genomic and phylogenetic comparisons revealed that apicomplexans contain unusual Epsin homologs that are distinct from those observed in mammals and yeast. Although there are four Epsin genes in mammalian system and five in the yeast genome, apicomplexan parasites consist only a single Epsin gene. The apicomplexan Epsin contains the conserved ENTH domain consisting of phosphoinositide (PtdIns)-binding sites which indicate about their functional significance in the formation of vesicles; however, the absence of ubiquitin-interacting motif (UIM) suggests a possible different mechanism for protein trafficking. The existence of dileucine motif in Plasmodium, Cryptosporidum parvum and Eimeria tenella Epsins might solve their functionality while lacking a lot of conserved motifs as this motif is known to interact with different adaptor protein complexes (AP1, AP2 and AP3). Other Epsin homologs are also shown to have different peptide motifs reported for possible interaction with α-ear appendage, γ-ear appendage and EH domain present in different adaptors. Bioinformatic and phylogenetic analyses suggest that the apicomplexan Epsins have unusual functionality from that of the mammalian Epsins. This detailed study may greatly facilitate future molecular cell biological investigation for the role of Epsins in these parasites. PMID:26922178

  12. Evidence-Based Structural Model of the Staphylococcal Repressor Protein: Separation of Functions into Different Domains

    PubMed Central

    Nyíri, Kinga; Kőhegyi, Bianka; Micsonai, András; Kardos, József; Vertessy, Beata G.

    2015-01-01

    Horizontal transfer of mobile genetic elements within Staphylococci is of high biomedical significance as such elements are frequently responsible for virulence and toxic effects. Staphylococcus-encoded repressor proteins regulate the replication of these mobile genetic elements that are located within the so-called pathogenicity islands. Here, we report structural and functional characterization of one such repressor protein, namely the Stl protein encoded by the pathogenicity island SaPIbov1. We create a 3D structural model and based on this prediction, we investigate the different functionalities of truncated and point mutant constructs. Results suggest that a helix-turn-helix motif governs the interaction of the Stl protein with its cognate DNA site: point mutations within this motif drastically decrease DNA-binding ability, whereas the interaction with the Stl-binding partner protein dUTPase is unperturbed by these point mutations. The 3D model also suggested the potential independent folding of a carboxy-terminal domain. This suggestion was fully verified by independent experiments revealing that the carboxy-terminal domain does not bind to DNA but is still capable of binding to and inhibiting dUTPase. A general model is proposed, which suggests that among the several structurally different repressor superfamilies Stl-like Staphylococcal repressor proteins belong to the helix-turn-helix transcription factor group and the HTH motif is suggested to reside within N-terminal segment. PMID:26414067

  13. Capsid protease domain as a tool for assessing protein-domain folding during organelle import of nascent polypeptides in living cells.

    PubMed

    Kang, Kongsoo; Takahara, Michiyo; Sakaue, Haruka; Sakaguchi, Masao

    2016-05-01

    Various proteins synthesized by ribosomes are imported into specific organelles. To elucidate the behavior of protein domains during import, we developed a folding probe, in which the capsid protease (CP) domain of the Semliki Forest virus was connected to enhanced green fluorescent protein (EGFP). The probe was fused to appropriate N-terminal organelle-targeting signal sequences and expressed in cultured cells. When the entire CP-domain was present in the cytosol, it became folded and cleaved off the following EGFP-domain. Once cleaved, EGFP stability was not affected by upstream sequences. Based on EGFP localization, we estimated the extent of CP-domain folding in the cytosolic space. When fused to mitochondrial hydrophobic multispanning membrane protein ABCB10, more than half of the EGFP remained in the cytoplasm, whereas most of the CP-portion was in the mitochondrial fraction. When fused to the endoplasmic reticulum (ER) signal, the cleaved EGFP was observed only in the ER fraction, confirming that the CP-domain cannot fold on the cytoplasmic side during cotranslational ER translocation. Thus, import of the ABCB10 molecule was not as tightly coupled with chain elongation as ER translocation. Use of this probe to quantitatively examine stop-translocation at the ER translocon in living cells revealed that positively charged residues on the translocating nascent chain stall at the ER translocon. PMID:26711239

  14. Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

    PubMed Central

    Weng, Z; Thomas, S M; Rickles, R J; Taylor, J A; Brauer, A W; Seidel-Dugan, C; Michael, W M; Dreyfuss, G; Brugge, J S

    1994-01-01

    Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through

  15. ACBD3 Interaction with TBC1 Domain 22 Protein Is Differentially Affected by Enteroviral and Kobuviral 3A Protein Binding

    PubMed Central

    Greninger, Alexander L.; Knudsen, Giselle M.; Betegon, Miguel; Burlingame, Alma L.; DeRisi, Joseph L.

    2013-01-01

    ABSTRACT Despite wide sequence divergence, multiple picornaviruses use the Golgi adaptor acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GCP60) to recruit phosphatidylinositol 4-kinase class III beta (PI4KIIIβ/PI4KB), a factor required for viral replication. The molecular basis of this convergent interaction and the cellular function of ACBD3 are not fully understood. Using affinity purification-mass spectrometry, we identified the putative Rab33 GTPase-activating proteins TBC1D22A and TBC1D22B as ACBD3-interacting factors. Fine-scale mapping of binding determinants within ACBD3 revealed that the interaction domains for TBC1D22A/B and PI4KB are identical. Affinity purification confirmed that PI4KB and TBC1D22A/B interactions with ACBD3 are mutually exclusive, suggesting a possible regulatory mechanism for recruitment of PI4KB. The C-terminal Golgi dynamics (GOLD) domain of ACBD3 has been previously shown to bind the 3A replication protein from Aichi virus. We find that the 3A proteins from several additional picornaviruses, including hepatitis A virus, human parechovirus 1, and human klassevirus, demonstrate an interaction with ACBD3 by mammalian two-hybrid assay; however, we also find that the enterovirus and kobuvirus 3A interactions with ACBD3 are functionally distinct with respect to TBC1D22A/B and PI4KB recruitment. These data reinforce the notion that ACBD3 organizes numerous cellular functionalities and that RNA virus replication proteins likely modulate these interactions by more than one mechanism. PMID:23572552

  16. The role of palmitoylation and transmembrane domain in sorting of transmembrane adaptor proteins.

    PubMed

    Chum, Tomáš; Glatzová, Daniela; Kvíčalová, Zuzana; Malínský, Jan; Brdička, Tomáš; Cebecauer, Marek

    2016-01-01

    Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins. PMID:26585312

  17. TM9 family proteins control surface targeting of glycine-rich transmembrane domains.

    PubMed

    Perrin, Jackie; Le Coadic, Marion; Vernay, Alexandre; Dias, Marco; Gopaldass, Navin; Ouertatani-Sakouhi, Hajer; Cosson, Pierre

    2015-07-01

    TM9 family proteins (also named Phg1 proteins) have been previously shown to control cell adhesion by determining the cell surface localization of adhesion proteins such as the Dictyostelium SibA protein. Here, we show that the glycine-rich transmembrane domain (TMD) of SibA is sufficient to confer Phg1A-dependent surface targeting to a reporter protein. Accordingly, in Dictyostelium phg1A-knockout (KO) cells, proteins with glycine-rich TMDs were less efficiently transported out of the endoplasmic reticulum (ER) and to the cell surface. Phg1A, as well as its human ortholog TM9SF4 specifically associated with glycine-rich TMDs. In human cells, genetic inactivation of TM9SF4 resulted in an increased retention of glycine-rich TMDs in the endoplasmic reticulum, whereas TM9SF4 overexpression enhanced their surface localization. The bulk of the TM9SF4 protein was localized in the Golgi complex and a proximity-ligation assay suggested that it might interact with glycine-rich TMDs. Taken together, these results suggest that one of the main roles of TM9 proteins is to serve as intramembrane cargo receptors controlling exocytosis and surface localization of a subset of membrane proteins. PMID:25999474

  18. TM9 family proteins control surface targeting of glycine-rich transmembrane domains

    PubMed Central

    Perrin, Jackie; Le Coadic, Marion; Vernay, Alexandre; Dias, Marco; Gopaldass, Navin; Ouertatani-Sakouhi, Hajer; Cosson, Pierre

    2015-01-01

    ABSTRACT TM9 family proteins (also named Phg1 proteins) have been previously shown to control cell adhesion by determining the cell surface localization of adhesion proteins such as the Dictyostelium SibA protein. Here, we show that the glycine-rich transmembrane domain (TMD) of SibA is sufficient to confer Phg1A-dependent surface targeting to a reporter protein. Accordingly, in Dictyostelium phg1A-knockout (KO) cells, proteins with glycine-rich TMDs were less efficiently transported out of the endoplasmic reticulum (ER) and to the cell surface. Phg1A, as well as its human ortholog TM9SF4 specifically associated with glycine-rich TMDs. In human cells, genetic inactivation of TM9SF4 resulted in an increased retention of glycine-rich TMDs in the endoplasmic reticulum, whereas TM9SF4 overexpression enhanced their surface localization. The bulk of the TM9SF4 protein was localized in the Golgi complex and a proximity-ligation assay suggested that it might interact with glycine-rich TMDs. Taken together, these results suggest that one of the main roles of TM9 proteins is to serve as intramembrane cargo receptors controlling exocytosis and surface localization of a subset of membrane proteins. PMID:25999474

  19. The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles

    PubMed Central

    Ziegler, Christopher M.; Eisenhauer, Philip; Bruce, Emily A.; Weir, Marion E.; King, Benjamin R.; Klaus, Joseph P.; Krementsov, Dimitry N.; Shirley, David J.; Ballif, Bryan A.; Botten, Jason

    2016-01-01

    Arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. Persistently-infected rodents harbor high levels of defective interfering (DI) particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. Little is known about what drives the production of DI particles. We show that neither the PPXY late domain encoded within the lymphocytic choriomeningitis virus (LCMV) matrix protein nor a functional endosomal sorting complex transport (ESCRT) pathway is absolutely required for the generation of standard infectious virus particles. In contrast, DI particle release critically requires the PPXY late domain and is ESCRT-dependent. Additionally, the terminal tyrosine in the PPXY motif is reversibly phosphorylated and our findings indicate that this posttranslational modification may regulate DI particle formation. Thus we have uncovered a new role for the PPXY late domain and a possible mechanism for its regulation. PMID:27010636

  20. A Novel Protein Kinase-Like Domain in a Selenoprotein, Widespread in the Tree of Life

    PubMed Central

    Dudkiewicz, Małgorzata; Szczepińska, Teresa; Grynberg, Marcin; Pawłowski, Krzysztof

    2012-01-01

    Selenoproteins serve important functions in many organisms, usually providing essential oxidoreductase enzymatic activity, often for defense against toxic xenobiotic substances. Most eukaryotic genomes possess a small number of these proteins, usually not more than 20. Selenoproteins belong to various structural classes, often related to oxidoreductase function, yet a few of them are completely uncharacterised. Here, the structural and functional prediction for the uncharacterised selenoprotein O (SELO) is presented. Using bioinformatics tools, we predict that SELO protein adopts a three-dimensional fold similar to protein kinases. Furthermore, we argue that despite the lack of conservation of the “classic” catalytic aspartate residue of the archetypical His-Arg-Asp motif, SELO kinases might have retained catalytic phosphotransferase activity, albeit with an atypical active site. Lastly, the role of the selenocysteine residue is considered and the possibility of an oxidoreductase-regulated kinase function for SELO is discussed. The novel kinase prediction is discussed in the context of functional data on SELO orthologues in model organisms, FMP40 a.k.a.YPL222W (yeast), and ydiU (bacteria). Expression data from bacteria and yeast suggest a role in oxidative stress response. Analysis of genomic neighbourhoods of SELO homologues in the three domains of life points toward a role in regulation of ABC transport, in oxidative stress response, or in basic metabolism regulation. Among bacteria possessing SELO homologues, there is a significant over-representation of aquatic organisms, also of aerobic ones. The selenocysteine residue in SELO proteins occurs only in few members of this protein family, including proteins from Metazoa, and few small eukaryotes (Ostreococcus, stramenopiles). It is also demonstrated that enterobacterial mchC proteins involved in maturation of bactericidal antibiotics, microcins, form a distant subfamily of the SELO proteins. The new protein

  1. Analysis of periplasmic sensor domains from Anaeromyxobacter dehalogenans 2CP-C: structure of one sensor domain from a histidine kinase and another from a chemotaxis protein.

    PubMed

    Pokkuluri, P Raj; Dwulit-Smith, Jeff; Duke, Norma E; Wilton, Rosemarie; Mack, Jamey C; Bearden, Jessica; Rakowski, Ella; Babnigg, Gyorgy; Szurmant, Hendrik; Joachimiak, Andrzej; Schiffer, Marianne

    2013-10-01

    Anaeromyxobacter dehalogenans is a δ-proteobacterium found in diverse soils and sediments. It is of interest in bioremediation efforts due to its dechlorination and metal-reducing capabilities. To gain an understanding on A. dehalogenans' abilities to adapt to diverse environments we analyzed its signal transduction proteins. The A. dehalogenans genome codes for a large number of sensor histidine kinases (HK) and methyl-accepting chemotaxis proteins (MCP); among these 23 HK and 11 MCP proteins have a sensor domain in the periplasm. These proteins most likely contribute to adaptation to the organism's surroundings. We predicted their three-dimensional folds and determined the structures of two of the periplasmic sensor domains by X-ray diffraction. Most of the domains are predicted to have either PAS-like or helical bundle structures, with two predicted to have solute-binding protein fold, and another predicted to have a 6-phosphogluconolactonase like fold. Atomic structures of two sensor domains confirmed the respective fold predictions. The Adeh_2942 sensor (HK) was found to have a helical bundle structure, and the Adeh_3718 sensor (MCP) has a PAS-like structure. Interestingly, the Adeh_3718 sensor has an acetate moiety bound in a binding site typical for PAS-like domains. Future work is needed to determine whether Adeh_3718 is involved in acetate sensing by A. dehalogenans. PMID:23897711

  2. In vitro dimerization of the bovine papillomavirus E5 protein transmembrane domain

    PubMed Central

    Oates, Joanne; Hicks, Matthew; Dafforn, Timothy R.; DiMaio, Daniel; Dixon, Ann M.

    2013-01-01

    The E5 protein from bovine papillomavirus is a type II membrane protein and the product of the smallest known oncogene. E5 causes tumor formation by binding and activating the platelet derived growth factor beta receptor (PDGFβR). In order to productively interact with the receptor it is thought that E5 binds as a dimer. However, wild-type E5 and various mutants have also been shown to form trimers, tetramers and even higher order oligomers. The residues in E5 that drive and stabilize a dimeric state are also still in question. At present, two different models for the E5 dimer exist in the literature, one symmetric and one asymmetric. There is universal agreement, however, that the transmembrane (TM) domain plays a vital role in stabilizing the functional oligomer, indeed mutation of various TM domain residues can abolish E5 function. In order to better resolve the role of the E5 TM domain in function, we have undertaken the first quantitative in vitro characterization of the E5 TM domain in detergent micelles and liposomes. Circular and linear dichroism analyses verify that the TM domain adopts a stable α-helical structure and is able to partition efficiently across lipid bilayers. SDS-PAGE and analytical ultracentrifugation confirm for the first time that the TM domain of E5 forms a strong dimer with a standard state free energy of dissociation of 5.0 kcal mol−1. We have used our new results to interpret existing models of E5 dimer formation and provide a direct link between TM helix interactions and E5 function. PMID:18672907

  3. Osteogenesis induced by frizzled-related protein (FRZB) is linked to the netrin-like domain.

    PubMed

    Thysen, Sarah; Cailotto, Frederic; Lories, Rik

    2016-05-01

    Abnormal Wnt signaling is associated with bone mass disorders. Frizzled-related protein (FRZB, also known as secreted frizzled-related protein-3 (SFRP3)) is a Wnt modulator that contains an amino-terminal cysteine-rich domain (CRD) and a carboxy-terminal Netrin-like (NTN) motif. Frzb(-/-) mice show increased cortical thickness. However, the direct effect of FRZB on osteogenic differentiation and the involvement of the structural domains herein are not fully understood. In this study, we observed that stable overexpression of Frzb in MC3T3-E1 cells increased calcium deposition and osteoblast markers compared with control. Western blot analysis showed that the increased osteogenesis was associated with reduced canonical, but increased non-canonical Wnt signaling. On the contrary, loss of Frzb induced the opposite effects on osteogenesis and Wnt signaling. To translationally validate the positive effects of FRZB on primary human cells, we treated human periosteal and human bone marrow stromal cells with conditioned medium from MC3T3-E1 cells overexpressing Frzb and observed an increase in Alizarin red staining. We further studied the effect of the domains. FrzbNTN overexpression induced similar effects on osteogenesis as full-length Frzb, whereas FrzbCRD overexpressing cells mimicked loss of Frzb experiments. The CRD is considered as the Wnt binding domain, but the NTN domain also has important effects on bone biology. FRZB and other SFRPs or their specific domains may hold surprising potential as therapeutics for bone and joint disorders considering that excess of SFRPs has effects that are not expected under physiological, endogenous expression conditions. PMID:26927515

  4. Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain.

    PubMed

    Harprecht, Christina; Okifo, Oghenefejiro; Robbins, Kevin J; Motwani, Tina; Alexandrescu, Andrei T; Teschke, Carolyn M

    2016-05-20

    The I-domain is a genetic insertion in the phage P22 coat protein that chaperones its folding and stability. Of 11 acidic residues in the I-domain, seven participate in stabilizing electrostatic interactions with basic residues across elements of secondary structure, fastening the β-barrel fold. A hydrogen-bonded salt bridge between Asp-302 and His-305 is particularly interesting as Asp-302 is the site of a temperature-sensitive-folding mutation. The pKa of His-305 is raised to 9.0, indicating the salt bridge stabilizes the I-domain by ∼4 kcal/mol. Consistently, urea denaturation experiments indicate the stability of the WT I-domain decreases by 4 kcal/mol between neutral and basic pH. The mutants D302A and H305A remove the pH dependence of stability. The D302A substitution destabilizes the I-domain by 4 kcal/mol, whereas H305A had smaller effects, on the order of 1-2 kcal/mol. The destabilizing effects of D302A are perpetuated in the full-length coat protein as shown by a higher sensitivity to protease digestion, decreased procapsid assembly rates, and impaired phage production in vivo By contrast, the mutants have only minor effects on capsid expansion or stability in vitro The effects of the Asp-302-His-305 salt bridge are thus complex and context-dependent. Substitutions that abolish the salt bridge destabilize coat protein monomers and impair capsid self-assembly, but once capsids are formed the effects of the substitutions are overcome by new quaternary interactions between subunits. PMID:27006399

  5. FMN binding and photochemical properties of plant putative photoreceptors containing two LOV domains, LOV/LOV proteins.

    PubMed

    Kasahara, Masahiro; Torii, Mayumi; Fujita, Akimitsu; Tainaka, Kengo

    2010-11-01

    LOV domains function as blue light-sensing modules in various photoreceptors in plants, fungi, algae, and bacteria. A LOV/LOV protein (LLP) has been found from Arabidopsis thaliana (AtLLP) as a two LOV domain-containing protein. However, its function remains unknown. We isolated cDNA clones coding for an LLP homolog from tomato (Solanum lycopersicum) and two homologs from the moss Physcomitrella patens. The tomato LLP (SlLLP) contains two LOV domains (LOV1 and LOV2 domains), as in AtLLP. Most of the amino acids required for association with chromophore are conserved in both LOV domains, except that the amino acid at the position equivalent to the cysteine essential for cysteinyl adduct formation is glycine in the LOV1 domain as in AtLLP. When expressed in Escherichia coli, SlLLP binds FMN and undergoes a self-contained photocycle upon irradiation of blue light. Analyses using mutant SlLLPs revealed that SlLLP binds FMN in both LOV domains, although the LOV1 domain does not show spectral changes on irradiation. However, when Gly(66) in the LOV1 domain, which is located at the position equivalent to the essential cysteine of LOV domains, is replaced by cysteine, the mutated LOV1 domain shows light-induced spectral changes. In addition, all four LOV domains of P. patens LLPs (PpLLP1 and PpLLP2) show the typical features of LOV domains, including the reactive cysteine in each. This study shows that plants have a new LOV domain-containing protein family with the typical biochemical and photochemical properties of other LOV domain-containing proteins such as the phototropins. PMID:20826774

  6. Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity

    PubMed Central

    Weger-Lucarelli, James; Aliota, Matthew T.; Wlodarchak, Nathan; Kamlangdee, Attapon; Swanson, Ryan

    2015-01-01

    ABSTRACT Alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicity in vitro and in vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination in Aedes aegypti mosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis. IMPORTANCE Chikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat

  7. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95.

    PubMed

    Kornau, H C; Schenker, L T; Kennedy, M B; Seeburg, P H

    1995-09-22

    The N-methyl-D-aspartate (NMDA) receptor subserves synaptic glutamate-induced transmission and plasticity in central neurons. The yeast two-hybrid system was used to show that the cytoplasmic tails of NMDA receptor subunits interact with a prominent postsynaptic density protein PSD-95. The second PDZ domain in PSD-95 binds to the seven-amino acid, COOH-terminal domain containing the terminal tSXV motif (where S is serine, X is any amino acid, and V is valine) common to NR2 subunits and certain NR1 splice forms. Transcripts encoding PSD-95 are expressed in a pattern similar to that of NMDA receptors, and the NR2B subunit co-localizes with PSD-95 in cultured rat hippocampal neurons. The interaction of these proteins may affect the plasticity of excitatory synapses. PMID:7569905

  8. LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins

    SciTech Connect

    Sosa, Brian A.; Rothballer, Andrea; Kutay, Ulrike; Schwartz, Thomas U.

    2012-08-31

    Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansive grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.

  9. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    SciTech Connect

    Lee, A L

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the {delta}-Al-{var_epsilon} activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a {beta}{alpha}{beta}-{beta}{alpha}{beta} pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel {beta}-sheet. In addition {sup 15}N T{sub 1}, T{sub 2}, and {sup 15}N/{sup 1}H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone {sup 1}H, {sup 13}C, and {sup 15}N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and {sup 15}N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

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

    PubMed

    Shrive, Annette K; Moeller, Jesper B; Burns, Ian; Paterson, Jenny M; Shaw, Amy J; Schlosser, Anders; Sorensen, Grith L; Greenhough, Trevor J; Holmskov, Uffe

    2014-01-31

    The high resolution crystal structures of a recombinant fragment of the C-terminal fibrinogen-like recognition domain of FIBCD1, a vertebrate receptor that binds chitin, have been determined. The overall tetrameric structure shows similarity in structure and aggregation to the horseshoe crab innate immune protein tachylectin 5A. The high affinity ligand N-acetylmannosamine (ManNAc) binds in the S1 site, predominantly via the acetyl group with the oxygen and acetamide nitrogen hydrogen-bonded to the protein and the methyl group inserted into a hydrophobic pocket. The binding of the ManNAc pyranose ring differs markedly between the two independent subunits, but in all structures the binding of the N-acetyl group is conserved. In the native structure, a crystal contact results in one of the independent protomers binding the first GlcNAc of the Asn(340) N-linked glycan on the other independent protomer. In the ligand-bound structure this GlcNAc is replaced by the higher affinity ligand ManNAc. In addition, a sulfate ion has been modeled into the electron density at a location similar to the S3 binding site in L-ficolin, whereas in the native structure an acetate ion has been placed in the S1 N-acetyl binding site, and a sulfate ion has been placed adjacent to this site. These ion binding sites are ideally placed to receive the N-acetyl and sulfate groups of sulfated GalNAc residues of glycosaminoglycans such as chondroitin and dermatan sulfate. Together, these structures give insight into important determinants of ligand selectivity, demonstrating versatility in recognition and binding while maintaining conservation in N-acetyl and calcium binding. PMID:24293368

  11. Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

    SciTech Connect

    Wilbur, Jeremy D.; Hwang, Peter K.; Brodsky, Frances M.; Fletterick, Robert J.

    2010-03-01

    Variable packing interaction related to the conformational flexibility within the huntingtin-interacting protein 1 coiled coil domain. Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington’s disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coil domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.

  12. Function of the MAPK scaffold protein, Ste5, requires a cryptic PH domain

    PubMed Central

    Garrenton, Lindsay S.; Young, Susan L.; Thorner, Jeremy

    2006-01-01

    Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered Gβγ freed upon pheromone receptor occupancy, thereby initiating downstream signaling. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic α-helix (PM motif) are not sufficient for Ste5 action. Rather, Ste5 contains a pleckstrin-homology (PH) domain (residues 388–518) that is essential for its membrane recruitment and function. Altering residues (R407S K411S) equivalent to those that mediate phosphoinositide binding in other PH domains abolishes Ste5 function. The isolated PH domain, but not a R407S K411S derivative, binds phosphoinositides in vitro. Ste5(R407S K411S) is expressed normally, retains Gβγ and Ste11 binding, and oligomerizes, yet is not recruited to the membrane in response to pheromone. Artificial membrane tethering of Ste5(R407S K411S) restores signaling. R407S K411S loss-of-function mutations abrogate the constitutive activity of gain-of-function Ste5 alleles, including one (P44L) that increases membrane affinity of the PM motif. Thus, the PH domain is essential for stable membrane recruitment of Ste5, and this association is critical for initiation of downstream signaling because it allows Ste5-bound Ste11 (MAPKKK) to be activated by membrane-bound Ste20 (MAPKKKK). PMID:16847350

  13. Native mass spectrometry and ion mobility characterize the orange carotenoid protein functional domains.

    PubMed

    Zhang, Hao; Liu, Haijun; Lu, Yue; Wolf, Nathan R; Gross, Michael L; Blankenship, Robert E

    2016-06-01

    Orange Carotenoid Protein (OCP) plays a unique role in protecting many cyanobacteria from light-induced damage. The active form of OCP is directly involved in energy dissipation by binding to the phycobilisome (PBS), the major light-harvesting complex in cyanobacteria. There are two structural modules in OCP, an N-terminal domain (NTD), and a C-terminal domain (CTD), which play different functional roles during the OCP-PBS quenching cycle. Because of the quasi-stable nature of active OCP, structural analysis of active OCP has been lacking compared to its inactive form. In this report, partial proteolysis was used to generate two structural domains, NTD and CTD, from active OCP. We used multiple native mass spectrometry (MS) based approaches to interrogate the structural features of the NTD and the CTD. Collisional activation and ion mobility analysis indicated that the NTD releases its bound carotenoid without forming any intermediates and the CTD is resistant to unfolding upon collisional energy ramping. The unfolding intermediates observed in inactive intact OCP suggest that it is the N-terminal extension and the NTD-CTD loop that lead to the observed unfolding intermediates. These combined approaches extend the knowledge of OCP photo-activation and structural features of OCP functional domains. Combining native MS, ion mobility, and collisional activation promises to be a sensitive new approach for studies of photosynthetic protein-pigment complexes. PMID:26921809

  14. Ring-like oligomers of Synaptotagmins and related C2 domain proteins

    PubMed Central

    Zanetti, Maria N; Bello, Oscar D; Wang, Jing; Coleman, Jeff; Cai, Yiying; Sindelar, Charles V; Rothman, James E; Krishnakumar, Shyam S

    2016-01-01

    We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which could potentially regulate neurotransmitter release. Here we report that analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, using electron microscopy combined with targeted mutations, we show that under physiologically relevant conditions, both the Syt1 ring assembly and its rapid disruption by Ca2+ involve the well-established functional surfaces on the C2B domain that are important for synaptic transmission. Our data suggests that ring formation may be triggered at an early step in synaptic vesicle docking and positions Syt1 to synchronize neurotransmitter release to Ca2+ influx. DOI: http://dx.doi.org/10.7554/eLife.17262.001 PMID:27434670

  15. The cytoplasmic domain of simian immunodeficiency virus transmembrane protein modulates infectivity.

    PubMed Central

    Chakrabarti, L; Emerman, M; Tiollais, P; Sonigo, P

    1989-01-01

    A striking characteristic of the simian immunodeficiency virus (SIV) and of the human immunodeficiency virus type 2 (HIV-2) is the presence of a nonsense mutation in the env gene resulting in the synthesis of a truncated transmembrane protein lacking the cytoplasmic domain. By mutagenesis of an infectious molecular clone of SIVmac142, we investigated the function of the cytoplasmic domain and the significance of the env nonsense mutation. When the nonsense codon (TAG) was replaced by a glutamine codon (CAG), the virus infected HUT78 cells with markedly delayed kinetics. This negative effect was counterselected in vitro as reversion of the slow phenotype frequently occurred. The sequencing of one revertant revealed the presence of a new stop codon three nucleotides 5' to the original mutation. Deletions or an additional nonsense mutation introduced 3' to the original stop codon did not modify SIV infectivity. In contrast, the same deletions or nonsense mutation introduced in the clone in which the stop codon was replaced by CAG abolished infectivity. These results indicated that the envelope domain located 3' to the stop codon is not necessary for in vitro replication. However, the presence of this domain in SIV transmembrane protein leads to a reduced infectivity. This negative effect might correspond to a function controlling the rate of spread of the virus during in vivo infection. Images PMID:2778881

  16. Electrostatic interactions of Hsp-organizing protein tetratricopeptide domains with Hsp70 and Hsp90: computational analysis and protein engineering.

    PubMed

    Kajander, Tommi; Sachs, Jonathan N; Goldman, Adrian; Regan, Lynne

    2009-09-11

    The Hsp-organizing protein (HOP) binds to the C termini of the chaperones Hsp70 and Hsp90, thus bringing them together so that substrate proteins can be passed from Hsp70 to Hsp90. Because Hsp90 is essential for the correct folding and maturation of many oncogenic proteins, it has become a significant target for anti-cancer drug design. HOP binds to Hsp70 and Hsp90 via two independent tetratricopeptide (TPR) domains, TPR1 and TPR2A, respectively. We have analyzed ligand binding using Poisson-Boltzmann continuum electrostatic calculations, free energy perturbation, molecular dynamics simulations, and site-directed mutagenesis to delineate the contribution of different interactions to the affinity and specificity of the TPR-peptide interactions. We found that continuum electrostatic calculations could be used to guide protein design by removing unfavorable interactions to increase binding affinity, with an 80-fold increase in affinity for TPR2A. Contributions at buried charged residues, however, were better predicted by free energy perturbation calculations. We suggest using a combination of the two approaches for increasing the accuracy of results, with free energy perturbation calculations used only at selected buried residues of the ligand binding pocket. Finally we present the crystal structure of TPR2A in complex with its non-cognate Hsp70 ligand, which provides insight on the origins of specificity in TPR domain-peptide recognition. PMID:19586912

  17. Spectral-domain optical coherence phase microscopy for label-free multiplexed protein microarray assay

    PubMed Central

    Joo, Chulmin; Özkumur, Emre; Ünlü, M. Selim; de Boer, Johannes F.

    2009-01-01

    Quantitative measurement of affinities and kinetics of various biomolecular interactions such as protein-protein, protein-DNA and receptor-ligand is central to our understanding of basic molecular and cellular functions and is useful for therapeutic evaluation. Here, we describe a laser-scanning quantitative imaging method, referred to as spectral-domain optical coherence phase microscopy, as an optical platform for label-free detection of biomolecular interactions. The instrument is based on a confocal interferometric microscope that enables depth-resolved quantitative phase measurements on sensor surface with high spatial resolution and phase stability. We demonstrate picogram per square millimeter surface mass sensitivity, and show its sensing capability by presenting static and dynamic detection of multiplexed protein microarray as immobilized antigens capture their corresponding antibodies. PMID:19674885

  18. Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis

    PubMed Central

    Li, Fangfang; Meng, Fanping; Jin, Quanxin; Sun, Changyuan; Li, Yingxin; Li, Honghua; Jin, Songzhu

    2014-01-01

    Single-chain variable domain fragment (scFv) 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion protein was expressed in Pichia pastoris. The affinity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunofluorescence staining. The ability of the fusion protein to block myasthenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for specific immunosuppressive therapy of myasthenia gravis. PMID:25206900

  19. NMR Solution Structure and DNA Binding Model of the DNA Binding Domain of Competence Protein A

    PubMed Central

    Hobbs, Carey A.; Bobay, Benjamin G.; Thompson, Richele J.; Perego, Marta; Cavanagh, John

    2010-01-01

    Competence protein A (ComA) is a response regulator protein involved in the development of genetic competence in the Gram-positive spore forming bacterium Bacillus subtilis, as well as the regulation of the production of degradative enzymes and antibiotic synthesis. ComA belongs to the NarL family of proteins which are characterized by a C-terminal transcriptional activator domain that consists of a bundle of four helices, where the second and third helices (α8 and α9) form a helix-turn-helix DNA binding domain. Using NMR spectroscopy, the high resolution three-dimensional solution structure of the C-terminal DNA-binding domain of ComA (ComAC) has been determined. In addition, surface plasmon resonance and NMR protein-DNA titration experiments allowed for the analysis of the interaction of ComAC with its target DNA sequences. Combining the solution structure and biochemical data, a model of ComAC bound to the ComA recognition sequences on the srfA promoter has been developed. The model shows that for DNA binding, ComA uses the conserved helix-turn-helix motif present in other NarL family members. However, the model also reveals that ComA may use a slightly different part of the helix-turn-helix motif and there appears to be some associated domain re-orientation. These observations suggest a basis for DNA binding specificity within the NarL family. PMID:20302877

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

    SciTech Connect

    Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

    2007-08-07

    DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

  1. RNA-binding proteins with prion-like domains in ALS and FTLD-U.

    PubMed

    Gitler, Aaron D; Shorter, James

    2011-01-01

    Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) is a debilitating, and universally fatal, neurodegenerative disease that devastates upper and lower motor neurons. The causes of ALS are poorly understood. A central role for RNA-binding proteins and RNA metabolism in ALS has recently emerged. The RNA-binding proteins, TDP-43 and FUS, are principal components of cytoplasmic inclusions found in motor neurons of ALS patients and mutations in TDP-43 and FUS are linked to familial and sporadic ALS. Pathology and genetics also connect TDP-43 and FUS with frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). It was unknown whether mechanisms of FUS aggregation and toxicity were similar or different to those of TDP-43. To address this issue, we have employed yeast models and pure protein biochemistry to define mechanisms underlying TDP-43 and FUS aggregation and toxicity, and to identify genetic modifiers relevant for human disease. We have identified prion-like domains in FUS and TDP-43 and provide evidence that these domains are required for aggregation. Our studies have defined key similarities as well as important differences between the two proteins. Collectively, however, our findings lead us to suggest that FUS and TDP-43, though similar RNA-binding proteins, likely aggregate and confer disease phenotypes via distinct mechanisms. PMID:21847013

  2. Characterization and Evolution of the Cell Cycle-Associated Mob Domain-Containing Proteins in Eukaryotes

    PubMed Central

    Vitulo, Nicola; Vezzi, Alessandro; Galla, Giulio; Citterio, Sandra; Marino, Giada; Ruperti, Benedetto; Zermiani, Monica; Albertini, Emidio; Valle, Giorgio; Barcaccia, Gianni

    2007-01-01

    The MOB family includes a group of cell cycle-associated proteins highly conserved throughout eukaryotes, whose founding members are implicated in mitotic exit and co-ordination of cell cycle progression with cell polarity and morphogenesis. Here we report the characterization and evolution of the MOB domain-containing proteins as inferred from the 43 eukaryotic genomes so far sequenced. We show that genes for Mob-like proteins are present in at least 41 of these genomes, confirming the universal distribution of this protein family and suggesting its prominent biological function. The phylogenetic analysis reveals five distinct MOB domain classes, showing a progressive expansion of this family from unicellular to multicellular organisms, reaching the highest number in mammals. Plant Mob genes appear to have evolved from a single ancestor, most likely after the loss of one or more genes during the early stage of Viridiplantae evolutionary history. Three of the Mob classes are widespread among most of the analyzed organisms. The possible biological and molecular function of Mob proteins and their role in conserved signaling pathways related to cell proliferation, cell death and cell polarity are also presented and critically discussed. PMID:19468312

  3. InterPro: an integrated documentation resource for protein families, domains and functional sites.

    PubMed

    Mulder, Nicola J; Apweiler, Rolf; Attwood, Terri K; Bairoch, Amos; Bateman, Alex; Binns, David; Biswas, Margaret; Bradley, Paul; Bork, Peer; Bucher, Phillip; Copley, Richard; Courcelle, Emmanuel; Durbin, Richard; Falquet, Laurent; Fleischmann, Wolfgang; Gouzy, Jerome; Griffith-Jones, Sam; Haft, Daniel; Hermjakob, Henning; Hulo, Nicolas; Kahn, Daniel; Kanapin, Alexander; Krestyaninova, Maria; Lopez, Rodrigo; Letunic, Ivica; Orchard, Sandra; Pagni, Marco; Peyruc, David; Ponting, Chris P; Servant, Florence; Sigrist, Christian J A

    2002-09-01

    The exponential increase in the submission of nucleotide sequences to the nucleotide sequence database by genome sequencing centres has resulted in a need for rapid, automatic methods for classification of the resulting protein sequences. There are several signature and sequence cluster-based methods for protein classification, each resource having distinct areas of optimum application owing to the differences in the underlying analysis methods. In recognition of this, InterPro was developed as an integrated documentation resource for protein families, domains and functional sites, to rationalise the complementary efforts of the individual protein signature database projects. The member databases - PRINTS, PROSITE, Pfam, ProDom, SMART and TIGRFAMs - form the InterPro core. Related signatures from each member database are unified into single InterPro entries. Each InterPro entry includes a unique accession number, functional descriptions and literature references, and links are made back to the relevant member database(s). Release 4.0 of InterPro (November 2001) contains 4,691 entries, representing 3,532 families, 1,068 domains, 74 repeats and 15 sites of post-translational modification (PTMs) encoded by different regular expressions, profiles, fingerprints and hidden Markov models (HMMs). Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (2,141,621 InterPro hits from 586,124 SWISS-PROT and TrEMBL protein sequences). The database is freely accessible for text- and sequence-based searches. PMID:12230031

  4. The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    PubMed Central

    Brunati, Martina; Perucca, Simone; Han, Ling; Cattaneo, Angela; Consolato, Francesco; Andolfo, Annapaola; Schaeffer, Céline; Olinger, Eric; Peng, Jianhao; Santambrogio, Sara; Perrier, Romain; Li, Shuo; Bokhove, Marcel; Bachi, Angela; Hummler, Edith; Devuyst, Olivier; Wu, Qingyu; Jovine, Luca; Rampoldi, Luca

    2015-01-01

    Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins. DOI: http://dx.doi.org/10.7554/eLife.08887.001 PMID:26673890

  5. Different Transmembrane Domains Associate with Distinct Endoplasmic Reticulum Components during Membrane Integration of a Polytopic Protein

    PubMed Central

    Meacock, Suzanna L.; Lecomte, Fabienne J.L.; Crawshaw, Samuel G.; High, Stephen

    2002-01-01

    We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the α and β subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61α and Sec61β during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the “stage” of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide. PMID:12475939

  6. Export of autotransported proteins proceeds through an oligomeric ring shaped by C-terminal domains.

    PubMed

    Veiga, Esteban; Sugawara, Etsuko; Nikaido, Hiroshi; de Lorenzo, Víctor; Fernández, Luis Angel

    2002-05-01

    An investigation was made into the oligomerization, the ability to form pores and the secretion-related properties of the 45 kDa C-terminal domain of the IgA protease (C-IgAP) from Neisseria gonorrhoeae. This protease is the best studied example of the autotransporters (ATs), a large family of exoproteins from Gram-negative bacteria that includes numerous virulence factors from human pathogens. These proteins contain an N-terminal passenger domain that em bodies the secreted polypeptide, while the C-domain inserts into the outer membrane (OM) and trans locates the linked N-module into the extracellular medium. Here we report that purified C-IgAP forms an oligomeric complex of approximately 500 kDa with a ring-like structure containing a central cavity of approximately 2 nm diameter that is the conduit for the export of the N-domains. These data overcome the previous model for ATs, which postulated the passage of the N-module through the hydrophilic channel of the beta-barrel of each monomeric C-domain. Our results advocate a secretion mechanism not unlike other bacterial export systems, such as the secretins or fimbrial ushers, which rely on multimeric complexes assembled in the OM. PMID:11980709

  7. Structure-Function Analysis of the Mcl-1 Protein Identifies a Novel Senescence-regulating Domain.

    PubMed

    Demelash, Abeba; Pfannenstiel, Lukas W; Tannenbaum, Charles S; Li, Xiaoxia; Kalady, Matthew F; DeVecchio, Jennifer; Gastman, Brian R

    2015-09-01

    Unlike other antiapoptotic Bcl-2 family members, Mcl-1 also mediates resistance to cancer therapy by uniquely inhibiting chemotherapy-induced senescence (CIS). In general, Bcl-2 family members regulate apoptosis at the level of the mitochondria through a common prosurvival binding groove. Through mutagenesis, we determined that Mcl-1 can inhibit CIS even in the absence of its apoptotically important mitochondrion-localizing domains. This finding prompted us to generate a series of Mcl-1 deletion mutants from both the N and C termini of the protein, including one that contained a deletion of all of the Bcl-2 homology domains, none of which impacted anti-CIS capabilities. Through subsequent structure-function analyses of Mcl-1, we identified a previously uncharacterized loop domain responsible for the anti-CIS activity of Mcl-1. The importance of the loop domain was confirmed in multiple tumor types, two in vivo models of senescence, and by demonstrating that a peptide mimetic of the loop domain can effectively inhibit the anti-CIS function of Mcl-1. The results from our studies appear to be highly translatable because we discerned an inverse relationship between the expression of Mcl-1 and of various senescence markers in cancerous human tissues. In summary, our findings regarding the unique structural properties of Mcl-1 provide new approaches for targeted cancer therapy. PMID:26205817

  8. Viroporin potential of the lentivirus lytic peptide (LLP) domains of the HIV-1 gp41 protein

    PubMed Central

    Costin, Joshua M; Rausch, Joshua M; Garry, Robert F; Wimley, William C

    2007-01-01

    Background Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development. Virally induced changes in membrane ionic permeability induced by lytic viruses of many families contribute to cytopathogenesis. HIV-1 induces disturbances in plasma membrane ion transport. The carboxyl terminus of TM (gp41) contains potential amphipathic α-helical motifs identified through their structural similarities to naturally occurring cytolytic peptides. These sequences have been dubbed lentiviral lytic peptides (LLP) -1, -2, and -3. Results Peptides corresponding to the LLP domains (from a clade B virus) partition into lipid membranes, fold into α-helices and disrupt model membrane permeability. A peptide corresponding to the LLP-1 domain of a clade D HIV-1 virus, LLP-1D displayed similar activity to the LLP-1 domain of the clade B virus in all assays, despite a lack of amino acid sequence identity. Conclusion These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin. Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs. PMID:18028545

  9. New class of blue animal pigments based on Frizzled and Kringle protein domains.

    PubMed

    Bulina, Maria E; Lukyanov, Konstantin A; Yampolsky, Ilia V; Chudakov, Dmitry M; Staroverov, Dmitry B; Shcheglov, Alexander S; Gurskaya, Nadya G; Lukyanov, Sergey

    2004-10-15

    The nature of coloration in many marine animals remains poorly investigated. Here we studied the blue pigment of a scyfoid jellyfish Rhizostoma pulmo and determined it to be a soluble extracellular 30-kDa chromoprotein with a complex absorption spectrum peaking at 420, 588, and 624 nm. Furthermore, we cloned the corresponding cDNA and confirmed its identity by immunoblotting and mass spectrometry experiments. The chromoprotein, named rpulFKz1, consists of two domains, a Frizzled cysteine-rich domain and a Kringle domain, inserted into one another. Generally, Frizzleds are members of a basic Wnt signal transduction pathway investigated intensely with regard to development and cancerogenesis. Kringles are autonomous structural domains found throughout the blood clotting and fibrinolytic proteins. Neither Frizzled and Kringle domains association with any type of coloration nor Kringle intrusion into Frizzled sequence was ever observed. Thus, rpulFKz1 represents a new class of animal pigments, whose chromogenic group remains undetermined. The striking homology between a chromoprotein and members of the signal transduction pathway provides a novel node in the evolution track of growth factor-mediated morphogenesis compounds. PMID:15297465

  10. ClpB N-terminal domain plays a regulatory role in protein disaggregation

    PubMed Central

    Rosenzweig, Rina; Farber, Patrick; Velyvis, Algirdas; Rennella, Enrico; Latham, Michael P.; Kay, Lewis E.

    2015-01-01

    ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB–substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD–substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD–substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD–substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation. PMID:26621746

  11. A novel route for immobilization of proteins to silica particles incorporating silaffin domains.

    PubMed

    Nam, Dong Hyun; Won, Keehoon; Kim, Yong Hwan; Sang, Byung In

    2009-01-01

    In the diatom Cylindrotheca fusiformis, modified peptides called silaffin polypeptides are responsible for silica deposition in vivo at ambient conditions. Recently, it was discovered that the synthetic R5 peptide, the repeat unit of silaffin polypeptide without post-translational modification, was capable of precipitating silica in vitro and at ambient conditions. Herein, chimeric proteins were generated by incorporating synthetic silaffin R5 peptides and related unmodified silaffin domains (R1-R7) from Cylindrotheca fusiformis onto green fluorescent protein (GFP) by recombinant DNA technology and their ability to cause silicification was also examined. GFP chimeric proteins showed silicification at very low concentrations (600-700 microg/mL) when compared with adding excess amounts of R5 peptides (10 mg/mL) as previously reported. Sensitive to pH conditions, only the GFP-R1 chimera showed silicification activity at pH 8.0. The protein immobilization efficiencies of these chimeras were unexpectedly high ranging from 75 to 85%, with the R1 silaffin-protein construct showing excellent immobilization efficiency and a constant molar ratio of silica to protein ranging from 250 to 350 over a wide pH range. The average silica particle sizes had a tendency to decrease as pH increased to basic conditions. This study demonstrated the production of nanoscale immobilized protein, fabricated via silaffin-fused proteins. PMID:19774662

  12. Ultrafast differential flexibility of Cro-protein binding domains of two operator DNAs with different sequences.

    PubMed

    Choudhury, Susobhan; Ghosh, Basusree; Singh, Priya; Ghosh, Raka; Roy, Siddhartha; Pal, Samir Kumar

    2016-07-21

    The nature of the interface of specific protein-DNA complexes has attracted immense interest in contemporary molecular biology. Although extensive studies on the role of flexibility of DNA in the specific interaction in the genetic regulatory activity of lambda Cro (Cro-protein) have been performed, the exploration of quantitative features remains deficient. In this study, we have mutated (site directed mutagenesis: SDM) Cro-protein at the 37th position with a cysteine residue (G37C) retaining the functional integrity of the protein and labelled the cysteine residue, which is close to the interface, with a fluorescent probe (AEDANS), for the investigation of its interface with operator DNAs (OR3 and OR2). We have employed picosecond resolved polarization gated fluorescence spectroscopy and the well known strategy of solvation dynamics for the exploration of physical motions of the fluorescent probes and associated environments, respectively. Even though this particular probe on the protein (AEDANS) shows marginal changes in its structural flexibility upon interaction with the DNAs, a non-covalent DNA bound probe (DAPI), which binds to the minor groove, shows a major differential alteration in the dynamical flexibility in the OR3-Cro complex when compared to that of the OR2 complex with the Cro-protein. We attempt to correlate the observed significant structural fluctuation of the Cro-protein binding domain of OR3 for the specificity of the protein to the operator DNA. PMID:27326896

  13. Dictyostelium nucleomorphin is a member of the BRCT-domain family of cell cycle checkpoint proteins.

    PubMed

    Myre, Michael A; O'Day, Danton H

    2004-11-18

    A search of the Dictyostelium genome project database (http://dictybase.org/db/cgi-bin/blast.pl) with nucleomorphin, a protein that regulates the nuclear number, predicted it to be encoded by a larger gene containing a putative breast cancer carboxy-terminus domain (BRCT). Using RT-PCR, Northern and Western blotting we have identified a differentially expressed, 2318 bp cDNA encoding a protein isoform of Dictyostelium NumA with an apparent molecular weight of 70 kDa that we have called NumB. It contains a single amino-terminal BRCT-domain spanning residues 125-201. Starvation of shaking cultures reduces NumA expression by approximately 88+/-5.6%, whereas NumB expression increases approximately 35+/-3.5% from vegetative levels. NumC, a third isoform that is also expressed during development but not growth, remains to be characterized. These findings suggest NumB may be a member of the BRCT-domain containing cell cycle checkpoint proteins. PMID:15535983

  14. Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis

    PubMed Central

    Molino, Diana; Nola, Sébastien; Lam, Sin Man; Verraes, Agathe; Proux-Gillardeaux, Véronique; Boncompain, Gaëlle; Perez, Franck; Wenk, Markus; Shui, Guanghou; Danglot, Lydia; Galli, Thierry

    2015-01-01

    Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI–anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis. PMID:26196023

  15. Linking the functions of unrelated proteins using a novel directed evolution domain insertion method

    PubMed Central

    Edwards, Wayne R.; Busse, Kathy; Allemann, Rudolf K.; Jones, D. Dafydd

    2008-01-01

    We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the separate protein activities, thus providing a general strategy for constructing molecular switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 β-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b562 with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were observed in loops, several also occurred close to the termini of α-helices and β-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains. PMID:18559359

  16. A Variable Light Domain Fluorogen Activating Protein Homodimerizes to Activate Dimethylindole Red†

    PubMed Central

    Senutovitch, Nina; Stanfield, Robyn L.; Bhattacharyya, Shantanu; Rule, Gordon S.; Wilson, Ian A.; Armitage, Bruce A.; Waggoner, Alan S.; Berget, Peter B.

    2012-01-01

    Novel fluorescent tools such as green fluorescent protein analogs and Fluorogen Activating Proteins (FAPs) are useful in biological imaging to track protein dynamics in real-time with low fluorescence background. FAPs are single-chain variable fragments (scFvs) selected from a yeast surface display library that produce fluorescence upon binding a specific dye or fluorogen that is normally not fluorescent when present in solution. FAPs generally consist of human immunoglobulin variable heavy (VH) and variable light (VL) domains covalently attached via a glycine and serine rich linker. Previously, we determined that the yeast surface clone, VH-VL M8, could bind and activate the fluorogen dimethylindole red (DIR), but that the fluorogen activation properties were localized to the M8VL domain. We report here that both NMR and X-ray diffraction methods indicate the M8VL forms non-covalent, anti-parallel homodimers that are the fluorogen activating species. The M8VL homodimers activate DIR by restriction of internal rotation of the bound dye. These structural results, together with directed evolution experiments of both VH-VL M8 and M8VL, led us to rationally design tandem, covalent homodimers of M8VL domains joined by a flexible linker that have a high affinity for DIR and good quantum yield. PMID:22390683

  17. A Variable Light Domain Fluorogen Activating Protein Homodimerizes To Activate Dimethylindole Red

    SciTech Connect

    Senutovitch, Nina; Stanfield, Robyn L.; Bhattacharyya, Shantanu; Rule, Gordon S.; Wilson, Ian A.; Armitage, Bruce A.; Waggoner, Alan S.; Berget, Peter B.

    2012-07-11

    Novel fluorescent tools such as green fluorescent protein analogues and fluorogen activating proteins (FAPs) are useful in biological imaging for tracking protein dynamics in real time with a low fluorescence background. FAPs are single-chain variable fragments (scFvs) selected from a yeast surface display library that produce fluorescence upon binding a specific dye or fluorogen that is normally not fluorescent when present in solution. FAPs generally consist of human immunoglobulin variable heavy (V{sub H}) and variable light (V{sub L}) domains covalently attached via a glycine- and serine-rich linker. Previously, we determined that the yeast surface clone, V{sub H}-V{sub L} M8, could bind and activate the fluorogen dimethylindole red (DIR) but that the fluorogen activation properties were localized to the M8V{sub L} domain. We report here that both nuclear magnetic resonance and X-ray diffraction methods indicate the M8V{sub L} forms noncovalent, antiparallel homodimers that are the fluorogen activating species. The M8V{sub L} homodimers activate DIR by restriction of internal rotation of the bound dye. These structural results, together with directed evolution experiments with both V{sub H}-V{sub L} M8 and M8V{sub L}, led us to rationally design tandem, covalent homodimers of M8V{sub L} domains joined by a flexible linker that have a high affinity for DIR and good quantum yields.

  18. A novel mammalian HORMA domain-containing protein, HORMAD1, preferentially associates with unsynapsed meiotic chromosomes.

    PubMed

    Fukuda, Tomoyuki; Daniel, Katrin; Wojtasz, Lukasz; Toth, Attila; Höög, Christer

    2010-01-15

    HORMA domain-containing proteins regulate interactions between homologous chromosomes (homologs) during meiosis in a wide range of eukaryotes. We have identified a mouse HORMA domain-containing protein, HORMAD1, and biochemically and cytologically shown it to be associated with the meiotic chromosome axis. HORMAD1 first accumulates on the chromosomes during the leptotene to zygotene stages of meiotic prophase I. As germ cells progress into the pachytene stage, HORMAD1 disappears from the synapsed chromosomal regions. However, once the chromosomes desynapse during the diplotene stage, HORMAD1 again accumulates on the chromosome axis of the desynapsed homologs. HORMAD1 thus preferentially localizes to unsynapsed or desynapsed chromosomal regions during the prophase I stage of meiosis. Analysis of mutant strains lacking different components of the synaptonemal complex (SC) revealed that establishment of the SC is required for the displacement of HORMAD1 from the chromosome axis. Our results therefore strongly suggest that also mammalian cells use a HORMA domain-containing protein as part of a surveillance system that monitors synapsis or other interactions between homologs. PMID:19686734

  19. Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

    PubMed Central

    Guryanov, Sergey G.; Selivanova, Olga M.; Nikulin, Alexey D.; Enin, Gennady A.; Melnik, Bogdan S.; Kretov, Dmitry A.; Serdyuk, Igor N.; Ovchinnikov, Lev P.

    2012-01-01

    YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions. PMID:22590640

  20. Functional characterization of spectrin-actin-binding domains in 4.1 family of proteins.

    PubMed

    Gimm, J Aura; An, Xiuli; Nunomura, Wataru; Mohandas, Narla

    2002-06-11

    Protein 4.1R is the prototypical member of a protein family that includes 4.1G, 4.1B, and 4.1N. 4.1R plays a crucial role in maintaining membrane mechanical integrity by binding cooperatively to spectrin and actin through its spectrin-actin-binding (SAB) domain. While the binary interaction between 4.1R and spectrin has been well characterized, the actin binding site in 4.1R remains unidentified. Moreover, little is known about the interaction of 4.1R homologues with spectrin and actin. In the present study, we showed that the 8 aa motif (LKKNFMES) within the 10 kDa spectrin-actin-binding domain of 4.1R plays a critical role in binding of 4.1R to actin. Recombinant 4.1R SAB domain peptides with mutations in this motif showed a marked decrease in their ability to form ternary complexes with spectrin and actin. Binary protein-protein interaction studies revealed that this decrease resulted from the inability of mutant SAB peptides to bind to actin filaments while affinity for spectrin was unchanged. We also documented that the 14 C-terminal residues of the 21 amino acid cassette encoded by exon 16 in conjunction with residues 27-43 encoded by exon 17 constituted a fully functional minimal spectrin-binding motif. Finally, we showed that 4.1N SAB domain was unable to form a ternary complex with spectrin and actin, while 4.1G and 4.1B SAB domains were able to form such a complex but less efficiently than 4.1R SAB. This was due to a decrease in the ability of 4.1G and 4.1B SAB domain to interact with actin but not with spectrin. These data enabled us to propose a model for the 4.1R-spectrin-actin ternary complex which may serve as a general paradigm for regulation of spectrin-based cytoskeleton interaction in various cell types. PMID:12044158

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

    PubMed Central

    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.). PMID:25972845

  2. 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.). PMID:25972845

  3. The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

    PubMed Central

    Tienari, P J; De Strooper, B; Ikonen, E; Simons, M; Weidemann, A; Czech, C; Hartmann, T; Ida, N; Multhaup, G; Masters, C L; Van Leuven, F; Beyreuther, K; Dotti, C G

    1996-01-01

    We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP. Images PMID:8895567

  4. Valosin-containing protein (VCP/p97) is capable of unfolding polyubiquitinated proteins through its ATPase domains.

    PubMed

    Song, Changcheng; Wang, Qing; Song, Changzheng; Rogers, Thomas J

    2015-07-31

    Valosin-containing protein (VCP or p97) is required for the proteasomal degradation of polyubiquitinated proteins. However, the molecular mechanism for VCP to process the polyubiquitinated proteins remains unclear. Here, we show that VCP can unfold polyubiquitinated proteins. It preferably unfolds the pentaubiquitin-over monoubiquin-conjugated dihydrofolate reductase (Ub5-DHFR or Ub-DHFR) in a dose dependent manner. In addition, the unfolding activity of VCP does not depend on its ATPase activity, on the contrary, ATP and its non-hydrolysable analogs suppress the unfolding of Ub5-DHFR. The structural and functional analysis showed that either D1 or D2 domain of VCP is sufficient to carry out this unfolding activity. The structure of the substrates also affects its unfolding by VCP. VCP is unable to unfold Ub5-DHFR in a tight structure when it binds with methotrexate, a folate analog with high affinity to DHFR. Thus, these results support that VCP is capable of unfolding polyubiquitinated proteins and suggest that VCP may facilitate the proteasomal degradation of polyubiquitinated proteins through its unfolding activity. PMID:26043696

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

    SciTech Connect

    Zhang, Yanfeng; Gao, Xiaoli; Michael Garavito, R.

    2011-04-22

    Highlights: {yields} Crystal structure of the intracellular domain of (pro)renin receptor (PRR-IC) as MBP fusion protein at 2.0 A (maltose-free) and 2.15 A (maltose-bound). {yields} MBP fusion protein is a dimer in crystals in the presence and absence of maltose. {yields} PRR-IC domain is responsible for the dimerization of the fusion protein. {yields} Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermolecular interactions