WorldWideScience

Sample records for px domain proteins

  1. Deletion of a novel protein kinase with PX and FYVE-related domains increases the rate of differentiation of Trypanosoma brucei.

    Science.gov (United States)

    Vassella, E; Krämer, R; Turner, C M; Wankell, M; Modes, C; van den Bogaard, M; Boshart, M

    2001-07-01

    Growth control of African trypanosomes in the mammalian host is coupled to differentiation of a non-dividing life cycle stage, the stumpy bloodstream form. We show that a protein kinase with novel domain architecture is important for growth regulation. Zinc finger kinase (ZFK) has a kinase domain related to RAC and S6 kinases flanked by a FYVE-related zinc finger and a phox (PX) homology domain. To investigate the function of the kinase during cyclical development, a stable transformation procedure for bloodstream forms of differentiation-competent (pleomorphic) Trypanosoma brucei strains was established. Deletion of both allelic copies of ZFK by homologous recombination resulted in reduced growth of bloodstream-form parasites in culture, which was correlated with an increased rate of differentiation to the non-dividing stumpy form. Growth and differentiation rates were returned to wild-type level by ectopic ZFK expression. The phenotype is stage-specific, as growth of procyclic (insect form) trypanosomes was unaffected, and Deltazfk/Deltazfk clones were able to undergo full cyclical development in the tsetse fly vector. Deletion of ZFK in a differentiation-defective (monomorphic) strain of T. brucei did not change its growth rate in the bloodstream stage. This suggests a function of ZFK associated with the trypanosomes' decision between either cell cycle progression, as slender bloodstream form, or differentiation to the non-dividing stumpy form.

  2. EFFECTS OF PROTEIN-XANTHOPHYLL (PX CONCENTRATE OF ALFALFA ADDITIVE TO CRUDE PROTEIN-REDUCED DIETS ON NITROGEN EXCRETION, GROWTH PERFORMANCE AND MEAT QUALITY OF PIGS

    Directory of Open Access Journals (Sweden)

    Eugeniusz GRELA

    2009-06-01

    Full Text Available The infl uence of protein-xanthophyll (PX concentrate of alfalfa supplement to crude protein-reduced diets was examined in relation to nitrogen excretion, performance parameters and pig meat quality. The investigations included 60 growers (PL x PLW x Duroc crossbreeds assigned to 3 groups. The conclusion is that there is a large potential to decrease nitrogen emission to the environment by 10% lowering of dietary crude protein intake along with reduced animal growth rate and elevated mixture utilization. Inclusion of a protein-xanthophyll concentrate (PX of alfalfa to the diet is likely to diminish disadvantageous productive parameters arising from limiting of total crude protein level in relation to the requirements of pigs feeding norms [1993]. At the same time, it improves feed nitrogen utilization and reduces noxious odour emissions from a piggery. The components of a protein-xanthophyll concentrate (PX contribute to increased liver and kidney weight.

  3. Protein domain prediction

    NARCIS (Netherlands)

    Ingolfsson, Helgi; Yona, Golan

    2008-01-01

    Domains are considered to be the building blocks of protein structures. A protein can contain a single domain or multiple domains, each one typically associated with a specific function. The combination of domains determines the function of the protein, its subcellular localization and the interacti

  4. Cellulose binding domain proteins

    Science.gov (United States)

    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.

  5. Expansion of protein domain repeats.

    Directory of Open Access Journals (Sweden)

    Asa K Björklund

    2006-08-01

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

  6. Protein domain organisation: adding order

    Directory of Open Access Journals (Sweden)

    Kummerfeld Sarah K

    2009-01-01

    Full Text Available Abstract Background Domains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit. Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved. Results We identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance. To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation. Conclusion Taking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected

  7. A peptidoglycan recognition protein (PxPGRP-SA) regulating the expression of antimicrobial peptides in Plutella xylostella (Lepidoptera: Plutellidae)%一个调控抗菌肽表达的小菜蛾肽聚糖识别蛋白(PxPGRP-SA)

    Institute of Scientific and Technical Information of China (English)

    郑志华; 许小霞; 余静; 高延富; 张玉清; 欧阳莉娜; 金丰良

    2016-01-01

    [目的]肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)是昆虫免疫系统中一类重要的模式识别蛋白.本研究旨在阐明经苏云金芽孢杆菌Bacillus thuringiensis侵染后,小菜蛾Plutella xylostella PGRP-SA基因(命名为PxPGRP-SA)在体内的表达模式和对抗菌肽基因的表达调控.[方法]本研究利用实时荧光定量PCR (qRT-PCR)技术分析B.thuringiensis侵染小菜蛾幼虫后PxPGRP-SA的转录模式,通过RNAi技术结合抗血清封闭实验检测PxPGRP-SA对小菜蛾抗茵肽基因的表达调控作用.[结果]qRT-PCR检测表明,小菜蛾4龄幼虫在注射具有活性的B.thuringiensis 6 h后,PxPGRP-SA在脂肪体和血细胞中表达量迅速上升,其中脂肪体中的表达量在注射24 h后达到高峰,而在血细胞中的表达量在18 h后达到高峰.RNAi沉默小菜蛾4龄幼虫PxPGRP-SA的转录后,可显著降低小菜蛾脂肪体中cecropin,moricin-2,lysozyme和defensin4个抗茵肽基因及Dorsal和Sp(a)tzle基因的mRNA转录水平;注射anti-PxPGRP-SA封闭小菜蛾体内PxPGRP-SA的活性后,也可降低小菜蛾脂肪体中4个抗菌肽基因的mRNA转录水平;PxPGRP-SA转录沉默后,同时导致添食B.thuringiensis的小菜蛾幼虫的存活率明显降低.[结论]PxPGRP-SA参与了小菜蛾体内抗茵肽cecropin,moricin-2,lysozyme和defensin基因的表达调控,并在免疫防御B.thuringiensis的侵染过程中起了重要的作用.%[Aim] Peptidoglycan recognition proteins (PGRPs) are one kind of important pattern recognition proteins in insect immune system.In this study,we aim to investigate the expression patterns of PGRP-SA from Plutella xylostella (designated as PxPGRP-SA) and its involvement in the regulation of the expression of antimicrobial peptide genes in P.xylostella after infection by Bacillus thuringiensis.[Methods] The transcription pattern of PxPGRP-SA in P.xylostella larvae infected by B.thuringiensis was analyzed by quantitative real-time reverse transcription

  8. Ubiquitin domain proteins in disease

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  9. Evolution studied through protein structural domains

    OpenAIRE

    YANG, SONG

    2007-01-01

    A protein structural domain is defined as a compact, spatially distinct part of a protein that can fold independently of neighboring sequences. Since the number of protein domains is limited, and protein domains are evolutionarily more conserved than protein sequences, protein domains play an important role in our understanding of the structure, function and evolution of proteins. As fundamental evolutionary units, protein domains are associated with a variety of evolutionary processes such a...

  10. Cellulose binding domain fusion proteins

    Science.gov (United States)

    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.

  11. The evolutionary history of protein domains viewed by species phylogeny

    National Research Council Canada - National Science Library

    Yang, Song; Bourne, Philip E

    2009-01-01

    .... The evolutionary history of protein domains, including the origin of protein domains, the identification of domain loss, transfer, duplication and combination with other domains to form new proteins...

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

    Directory of Open Access Journals (Sweden)

    Susan Khor

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

  13. Emergence of novel domains in proteins

    OpenAIRE

    Toll-Riera, Macarena; Albà Soler, Mar

    2013-01-01

    Background Proteins are composed of a combination of discrete, well-defined, sequence domains, associated with specific functions that have arisen at different times during evolutionary history. The emergence of novel domains is related to protein functional diversification and adaptation. But currently little is known about how novel domains arise and how they subsequently evolve. Results To gain insights into the impact of recently emerged domains in protein evolution we have identified all...

  14. Membrane binding domains

    OpenAIRE

    Hurley, James H.

    2006-01-01

    Eukaryotic signaling and trafficking proteins are rich in modular domains that bind cell membranes. These binding events are tightly regulated in space and time. The structural, biochemical, and biophysical mechanisms for targeting have been worked out for many families of membrane binding domains. This review takes a comparative view of seven major classes of membrane binding domains, the C1, C2, PH, FYVE, PX, ENTH, and BAR domains. These domains use a combination of specific headgroup inter...

  15. Multiple graph regularized protein domain ranking

    KAUST Repository

    Wang, Jim Jing-Yan

    2012-11-19

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

  16. J domain independent functions of J proteins.

    Science.gov (United States)

    Ajit Tamadaddi, Chetana; Sahi, Chandan

    2016-07-01

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

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

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2004-01-01

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

  18. Structural principles governing domain motions in proteins

    NARCIS (Netherlands)

    Hayward, S

    1999-01-01

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

  19. Structural principles governing domain motions in proteins

    NARCIS (Netherlands)

    Hayward, S

    1999-01-01

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

  20. UBA domain containing proteins in fission yeast

    DEFF Research Database (Denmark)

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

    2003-01-01

    The ubiquitin-proteasome pathway for intracellular proteolysis is involved in a series of cellular and molecular functions, including the degradation of bulk proteins, cell cycle control, DNA repair, antigen presentation, vesicle transport and the regulation of signal transudation pathways and tr....... The proteins display remarkable differences in their domain organisation, indicating that these potential ubiquitin binding proteins are involved in various cell activities....

  1. Using context to improve protein domain identification

    Directory of Open Access Journals (Sweden)

    Llinás Manuel

    2011-03-01

    Full Text Available Abstract Background Identifying domains in protein sequences is an important step in protein structural and functional annotation. Existing domain recognition methods typically evaluate each domain prediction independently of the rest. However, the majority of proteins are multidomain, and pairwise domain co-occurrences are highly specific and non-transitive. Results Here, we demonstrate how to exploit domain co-occurrence to boost weak domain predictions that appear in previously observed combinations, while penalizing higher confidence domains if such combinations have never been observed. Our framework, Domain Prediction Using Context (dPUC, incorporates pairwise "context" scores between domains, along with traditional domain scores and thresholds, and improves domain prediction across a variety of organisms from bacteria to protozoa and metazoa. Among the genomes we tested, dPUC is most successful at improving predictions for the poorly-annotated malaria parasite Plasmodium falciparum, for which over 38% of the genome is currently unannotated. Our approach enables high-confidence annotations in this organism and the identification of orthologs to many core machinery proteins conserved in all eukaryotes, including those involved in ribosomal assembly and other RNA processing events, which surprisingly had not been previously known. Conclusions Overall, our results demonstrate that this new context-based approach will provide significant improvements in domain and function prediction, especially for poorly understood genomes for which the need for additional annotations is greatest. Source code for the algorithm is available under a GPL open source license at http://compbio.cs.princeton.edu/dpuc/. Pre-computed results for our test organisms and a web server are also available at that location.

  2. Domain structure of Lassa virus L protein.

    Science.gov (United States)

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

    2011-01-01

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

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

    OpenAIRE

    Song Yang; Philip E. Bourne

    2009-01-01

    BACKGROUND: Protein structural domains are evolutionary units whose relationships can be detected over long evolutionary distances. The evolutionary history of protein domains, including the origin of protein domains, the identification of domain loss, transfer, duplication and combination with other domains to form new proteins, and the formation of the entire protein domain repertoire, are of great interest. METHODOLOGY/PRINCIPAL FINDINGS: A methodology is presented for providing a parsimon...

  4. Neural network models of protein domain evolution

    OpenAIRE

    Sylvia Nagl

    2000-01-01

    Protein domains are complex adaptive systems, and here a novel procedure is presented that models the evolution of new functional sites within stable domain folds using neural networks. Neural networks, which were originally developed in cognitive science for the modeling of brain functions, can provide a fruitful methodology for the study of complex systems in general. Ethical implications of developing complex systems models of biomolecules are discussed, with particular reference to molecu...

  5. Inferring Evolutionary Scenarios for Protein Domain Compositions

    Science.gov (United States)

    Wiedenhoeft, John; Krause, Roland; Eulenstein, Oliver

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

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

    Science.gov (United States)

    2013-05-07

    that approximately 80% of eukaryotic proteins and 67% of prokaryotic proteins have multiple domains [13,14]. Most annotation databases characterize...domain annotations, Domain-domain interactions, Protein-protein interaction networks Background The living cell is a dynamic, interconnected system...detailed in Methods. Here, we illustrate its application on a well- annotated single- cell organism. We created a merged set of protein-domain annotations

  7. Evaluating, Comparing, and Interpreting Protein Domain Hierarchies

    Science.gov (United States)

    2014-01-01

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

  8. I-mfa domain proteins specifically interact with SERTA domain proteins and repress their transactivating functions.

    Science.gov (United States)

    Kusano, Shuichi; Shiimura, Yuki; Eizuru, Yoshito

    2011-09-01

    The I-mfa domain proteins I-mfa and HIC are considered to be candidate tumor suppressor genes and have been shown to be involved in transcriptional regulation. We show here that I-mfa and HIC specifically interact with SEI-1 through their C-terminal I-mfa domains in vivo. This interaction affects the intracellular localization of I-mfa and requires the region of SEI-1 between 30 and 90 amino acids, which includes its SERTA domain, and results in repression of its intrinsic transcriptional activity. I-mfa also decreases the levels of the SEI-1·DP-1 complex and endogenous Fbxw7 mRNA, the expression of which is coregulated by E2F·DP-1 and SEI-1 in an interaction-dependent manner in vitro. In addition, I-mfa also specifically interacts with other SERTA domain-containing proteins, including SEI-2, SEI-3, SERTAD3 and SERTAD4, through its I-mfa domain in vivo. This interaction also affects the intracellular localization of I-mfa and represses the intrinsic transcriptional activities of SEI-2 and SERTAD3, which are also involved in the E2F-dependent transcription. These data reveal for the first time that I-mfa domain proteins interact with SERTA domain proteins and negatively regulate their transcriptional activity. Because SEI-1, SEI-2 and SERTAD3, whose intrinsic transcriptional activities are repressed by I-mfa, are suggested to be oncogenes, I-mfa domain proteins may be involved in their oncogenic functions by negatively regulating their transcriptional activities.

  9. Recovering protein-protein and domain-domain interactions from aggregation of IP-MS proteomics of coregulator complexes.

    Directory of Open Access Journals (Sweden)

    Amin R Mazloom

    2011-12-01

    Full Text Available Coregulator proteins (CoRegs are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP followed by mass spectrometry (MS applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/.

  10. Deciphering protein-protein interactions. Part II. Computational methods to predict protein and domain interaction partners

    National Research Council Canada - National Science Library

    Shoemaker, Benjamin A; Panchenko, Anna R

    2007-01-01

    .... In this review we describe different approaches to predict protein interaction partners as well as highlight recent achievements in the prediction of specific domains mediating protein-protein interactions...

  11. A segmental labeling strategy for unambiguous determination of domain-domain interactions of large multi-domain proteins

    Energy Technology Data Exchange (ETDEWEB)

    Chen Jianglei; Wang Jianjun, E-mail: jjwang@med.wayne.edu [Wayne State University, Department of Biochemistry and Molecular Biology, School of Medicine (United States)

    2011-08-15

    NMR structural determination of large multi-domain proteins is a challenging task due to significant spectral overlap with a particular difficulty in unambiguous identification of domain-domain interactions. Segmental labeling is a NMR strategy that allows for isotopically labeling one domain and leaves the other domain unlabeled. This significantly simplifies spectral overlaps and allows for quick identification of domain-domain interaction. Here, a novel segmental labeling strategy is presented for detection of inter-domain NOEs. To identify domain-domain interactions in human apolipoprotein E (apoE), a multi-domain, 299-residues {alpha}-helical protein, on-column expressed protein ligation was utilized to generate a segmental-labeled apoE samples in which the N-terminal (NT-) domain was {sup 2}H(99%)/{sup 15}N-labeled whereas the C-terminal (CT-) domain was either {sup 15}N- or {sup 15}N/{sup 13}C-labeled. 3-D {sup 15}N-edited NOESY spectra of these segmental-labeled apoE samples allow for direct observation of the inter-domain NOEs between the backbone amide protons of the NT-domain and the aliphatic protons of the CT-domain. This straightforward approach permits unambiguous identification of 78 inter-domain NOEs, enabling accurate definition of the relative positions of both the NT- and the CT-domains and determination of the NMR structure of apoE.

  12. Support vector machine for predicting protein interactions using domain scores

    Institute of Scientific and Technical Information of China (English)

    PENG Xin-jun; WANG Yi-fei

    2009-01-01

    Protein-protein interactions play a crucial role in the cellular process such as metabolic pathways and immunological recognition. This paper presents a new domain score-based support vector machine (SVM) to infer protein interactions, which can be used not only to explore all possible domain interactions by the kernel method, but also to reflect the evolutionary conservation of domains in proteins by using the domain scores of proteins. The experimental result on the Saccharomyces cerevisiae dataset demonstrates that this approach can predict protein-protein interactions with higher performances compared to the existing approaches.

  13. Multi-domain proteins in the three kingdoms of life: orphan domains and other unassigned regions.

    Science.gov (United States)

    Ekman, Diana; Björklund, Asa K; Frey-Skött, Johannes; Elofsson, Arne

    2005-04-22

    Comparative studies of the proteomes from different organisms have provided valuable information about protein domain distribution in the kingdoms of life. Earlier studies have been limited by the fact that only about 50% of the proteomes could be matched to a domain. Here, we have extended these studies by including less well-defined domain definitions, Pfam-B and clustered domains, MAS, in addition to Pfam-A and SCOP domains. It was found that a significant fraction of these domain families are homologous to Pfam-A or SCOP domains. Further, we show that all regions that do not match a Pfam-A or SCOP domain contain a significantly higher fraction of disordered structure. These unstructured regions may be contained within orphan domains or function as linkers between structured domains. Using several different definitions we have re-estimated the number of multi-domain proteins in different organisms and found that several methods all predict that eukaryotes have approximately 65% multi-domain proteins, while the prokaryotes consist of approximately 40% multi-domain proteins. However, these numbers are strongly dependent on the exact choice of cut-off for domains in unassigned regions. In conclusion, all eukaryotes have similar fractions of multi-domain proteins and disorder, whereas a high fraction of repeating domain is distinguished only in multicellular eukaryotes. This implies a role for repeats in cell-cell contacts while the other two features are important for intracellular functions.

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

    CERN Document Server

    Kahraman, Osman; Haselwandter, Christoph A

    2016-01-01

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

  15. Eukaryotic protein domains as functional units of cellular evolution

    DEFF Research Database (Denmark)

    Jin, Jing; Xie, Xueying; Chen, Chen

    2009-01-01

    Modular protein domains are functional units that can be modified through the acquisition of new intrinsic activities or by the formation of novel domain combinations, thereby contributing to the evolution of proteins with new biological properties. Here, we assign proteins to groups with related...... biological processes. Evolutionary jumps are associated with a domain that coordinately acquires a new intrinsic function and enters new domain clubs, thereby providing the modified domain with access to a new cellular microenvironment. We also coordinately analyzed the covalent and noncovalent interactions...... that domains, and the proteins in which they reside, are selected during evolution through reciprocal interactions with protein domains in their local microenvironment. Based on this scheme, we propose a mechanism by which Tudor domains may have evolved to support different modes of epigenetic regulation...

  16. Roc, a Ras/GTPase domain in complex proteins

    NARCIS (Netherlands)

    Bosgraaf, Leonard; Haastert, Peter J.M. van

    2003-01-01

    We identified a novel group of the Ras/GTPase superfamily, termed Roc, that is present as domain in complex proteins together with other domains, including leucine-rich repeats (LRRs), ankyrin repeats, WD40 repeats, kinase domains, RasGEF and RhoGAP domains. Roc is always succeeded by a novel 300–40

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2013-08-23

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

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

    OpenAIRE

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

    2012-01-01

    Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of rep...

  20. Protein domain boundary prediction by combining support vector machine and domain guess by size algorithm

    Institute of Scientific and Technical Information of China (English)

    Dong Qiwen; Wang Xiaolong; Lin Lei

    2007-01-01

    Successful prediction of protein domain boundaries provides valuable information not only for the computational structure prediction of multi-domain proteins but also for the experimental structure determination. A novel method for domain boundary prediction has been presented, which combines the support vector machine with domain guess by size algorithm. Since the evolutional information of multiple domains can be detected by position specific score matrix, the support vector machine method is trained and tested using the values of position specific score matrix generated by PSI-BLAST. The candidate domain boundaries are selected from the output of support vector machine, and are then inputted to domain guess by size algorithm to give the final results of domain boundary prediction. The experimental results show that the combined method outperforms the individual method of both support vector machine and domain guess by size.

  1. Research of BH3 domain protein inducing cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    FENG Wan-yu; LIU Yang; ZHANG Zhi-cheng

    2008-01-01

    Objective BH3 domain protein plays an important role in control mechanism of cell apoptosis. The article mainly discusses its mechanism of promoting cell apoptosis and control. Methods The article analyzed and evaluated the mechanism of BH3 domain protein promoting cell apoptosis by internal and overseas literature. Results Activation of BH3 domain protein could promote the increase of mitochondrial membrane permeability, then it would start mitoehondrial apoptosis pathway, and at the last the cell apoptosis. Conclusions BH3 domain protein is the necessary condition of starting cell apoptosis. Its activation can cause cell apoptosis.

  2. Coverage of protein domain families with structural protein-protein interactions: current progress and future trends.

    Science.gov (United States)

    Goncearenco, Alexander; Shoemaker, Benjamin A; Zhang, Dachuan; Sarychev, Alexey; Panchenko, Anna R

    2014-01-01

    Protein interactions have evolved into highly precise and regulated networks adding an immense layer of complexity to cellular systems. The most accurate atomistic description of protein binding sites can be obtained directly from structures of protein complexes. The availability of structurally characterized protein interfaces significantly improves our understanding of interactomes, and the progress in structural characterization of protein-protein interactions (PPIs) can be measured by calculating the structural coverage of protein domain families. We analyze the coverage of protein domain families (defined according to CDD and Pfam databases) by structures, structural protein-protein complexes and unique protein binding sites. Structural PPI coverage of currently available protein families is about 30% without any signs of saturation in coverage growth dynamics. Given the current growth rates of domain databases and structural PPI deposition, complete domain coverage with PPIs is not expected in the near future. As a result of this study we identify families without any protein-protein interaction evidence (listed on a supporting website http://www.ncbi.nlm.nih.gov/Structure/ibis/coverage/) and propose them as potential targets for structural studies with a focus on protein interactions. Published by Elsevier Ltd.

  3. The Epc-N domain: a predicted protein-protein interaction domain found in select chromatin associated proteins

    Directory of Open Access Journals (Sweden)

    Perry Jason

    2006-01-01

    Full Text Available Abstract Background An underlying tenet of the epigenetic code hypothesis is the existence of protein domains that can recognize various chromatin structures. To date, two major candidates have emerged: (i the bromodomain, which can recognize certain acetylation marks and (ii the chromodomain, which can recognize certain methylation marks. Results The Epc-N (Enhancer of Polycomb-N-terminus domain is formally defined herein. This domain is conserved across eukaryotes and is predicted to form a right-handed orthogonal four-helix bundle with extended strands at both termini. The types of amino acid residues that define the Epc-N domain suggest a role in mediating protein-protein interactions, possibly specifically in the context of chromatin binding, and the types of proteins in which it is found (known components of histone acetyltransferase complexes strongly suggest a role in epigenetic structure formation and/or recognition. There appear to be two major Epc-N protein families that can be divided into four unique protein subfamilies. Two of these subfamilies (I and II may be related to one another in that subfamily I can be viewed as a plant-specific expansion of subfamily II. The other two subfamilies (III and IV appear to be related to one another by duplication events in a primordial fungal-metazoan-mycetozoan ancestor. Subfamilies III and IV are further defined by the presence of an evolutionarily conserved five-center-zinc-binding motif in the loop connecting the second and third helices of the four-helix bundle. This motif appears to consist of a PHD followed by a mononuclear Zn knuckle, followed by a PHD-like derivative, and will thus be referred to as the PZPM. All non-Epc-N proteins studied thus far that contain the PZPM have been implicated in histone methylation and/or gene silencing. In addition, an unusual phyletic distribution of Epc-N-containing proteins is observed. Conclusion The data suggest that the Epc-N domain is a protein-protein

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

    Science.gov (United States)

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

    1999-01-01

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

  5. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2010-08-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  8. Unusual cold denaturation of a small protein domain.

    Science.gov (United States)

    Buchner, Ginka S; Shih, Natalie; Reece, Amy E; Niebling, Stephan; Kubelka, Jan

    2012-08-21

    A thermal unfolding study of the 45-residue α-helical domain UBA(2) using circular dichroism is presented. The protein is highly thermostable and exhibits a clear cold unfolding transition with the onset near 290 K without denaturant. Cold denaturation in proteins is rarely observed in general and is quite unique among small helical protein domains. The cold unfolding was further investigated in urea solutions, and a simple thermodynamic model was used to fit all thermal and urea unfolding data. The resulting thermodynamic parameters are compared to those of other small protein domains. Possible origins of the unusual cold unfolding of UBA(2) are discussed.

  9. 14-3-3 Proteins, FHA Domains and BRCT Domains in the DNA Damage Response

    OpenAIRE

    Mohammad, Duaa H.; Yaffe, Michael B.

    2009-01-01

    The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as p...

  10. Synthesis of an allergy inducing tetrasaccharide "4P-X".

    Science.gov (United States)

    Moriya, Takashi; Nagahata, Naoki; Odaka, Rei; Nakamura, Hirohide; Yoshikawa, Jun; Kurashima, Katsumi; Saito, Tadao

    2017-02-01

    4P-X (β-D-galactopyranosyl-(1 → 4)-β-D-galactopyranosyl-(1 → 6)-[β-D-galactopyranosyl-(1 → 4)]-β-D-glucopyranose) is included in galacto-oligosaccharides (GOSs) produced by β-galactosidase derived from Bacillus circulans. 4P-X has been known to induce particularly strong allergies. High purity 4P-X is essential for use as a standard to quantify the amount of 4P-X in GOSs; however, the isolation of high purity 4P-X has never been reported. In this study, we achieved the synthesis of 4P-X by a combination of organic and enzymatic chemical syntheses in a short time. This is the first report of isolated, high purity 4P-X.

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

    OpenAIRE

    Sillitoe, I; Dawson, N.; Thornton, J; Orengo, C.

    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 compreh...

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

    DEFF Research Database (Denmark)

    Lenaerts, Tom; Ferkinghoff-Borg, Jesper; Stricher, Francois

    2008-01-01

    that directly relates protein structural dynamics to information exchange between functional sites is still lacking. Results: Here we introduce a method to analyze protein dynamics within the framework of information theory and show that signal transduction within proteins can be considered as a particular...... distal communication is achieved. We illustrate the approach by analyzing information transfer by the SH2 domain of Fyn tyrosine kinase, obtained from Monte Carlo dynamics simulations. Our analysis reveals that the Fyn SH2 domain forms a noisy communication channel that couples residues located...... by crossing the core of the SH2 domain. Conclusion: As a result, our method provides a means to directly map the exchange of biological information on the structure of protein domains, making it clear how binding triggers conformational changes in the protein structure. As such it provides a structural road...

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

    Science.gov (United States)

    Arthur, Christopher P; Stowell, Michael H B

    2007-06-01

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

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

    Science.gov (United States)

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

    2015-10-23

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

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

    Directory of Open Access Journals (Sweden)

    Marc Lenoir

    2015-10-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2012-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Daniel Taylor

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

  19. Narrowing the conformational space sampled by two-domain proteins with paramagnetic probes in both domains

    Energy Technology Data Exchange (ETDEWEB)

    Dasgupta, Soumyasri; Hu Xiaoyu [University of Florence, Magnetic Resonance Center (CERM) (Italy); Keizers, Peter H. J.; Liu Weimin [Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories (Netherlands); Luchinat, Claudio, E-mail: luchinat@cerm.unifi.it; Nagulapalli, Malini [University of Florence, Magnetic Resonance Center (CERM) (Italy); Overhand, Mark [Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories (Netherlands); Parigi, Giacomo [University of Florence, Magnetic Resonance Center (CERM) (Italy); Sgheri, Luca [Istituto per le Applicazioni del Calcolo, Sezione di Firenze, CNR (Italy); Ubbink, Marcellus [Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories (Netherlands)

    2011-11-15

    Calmodulin is a two-domain protein which in solution can adopt a variety of conformations upon reorientation of its domains. The maximum occurrence (MO) of a set of calmodulin conformations that are representative of the overall conformational space possibly sampled by the protein, has been calculated from the paramagnetism-based restraints. These restraints were measured after inclusion of a lanthanide binding tag in the C-terminal domain to supplement the data obtained by substitution of three paramagnetic lanthanide ions to the calcium ion in the second calcium binding loop of the N-terminal domain. The analysis shows that the availability of paramagnetic restraints arising from metal ions placed on both domains, reduces the MO of the conformations to different extents, thereby helping to identify those conformations that can be mostly sampled by the protein.

  20. An ambiguity principle for assigning protein structural domains

    Science.gov (United States)

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

    2017-01-01

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

  1. An ambiguity principle for assigning protein structural domains.

    Science.gov (United States)

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

    2017-01-01

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

  2. Protein-protein interaction domains of Bacillus subtilis DivIVA

    NARCIS (Netherlands)

    van Baarle, S.; Celik, I.N.; Kaval, K.G.; Bramkamp, M.; Hamoen, L.W.; Halbedel, S.

    2013-01-01

    DivIVA proteins are curvature sensitive membrane binding proteins that recruit other proteins to the poles and the division septum. They consist of a conserved N-terminal lipid binding domain fused to a less conserved C-terminal domain. DivIVA homologues interact with different proteins involved in

  3. Protein-protein interaction domains of Bacillus subtilis DivIVA

    NARCIS (Netherlands)

    S. van Baarle; I.N. Celik; K.G. Kaval; M. Bramkamp; L.W. Hamoen; S. Halbedel

    2012-01-01

    DivIVA proteins are curvature sensitive membrane binding proteins that recruit other proteins to the poles and the division septum. They consist of a conserved N-terminal lipid binding domain fused to a less conserved C-terminal domain. DivIVA homologues interact with different proteins involved in

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

    Directory of Open Access Journals (Sweden)

    Isambert Hervé

    2007-11-01

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

  5. PX Andromedae superhumps and variable eclipse depth

    CERN Document Server

    Stanishev, V; Boffin, H M J; Genkov, V

    2002-01-01

    Results of a photometric study of the SW Sex novalike PX And are presented. The periodogram analysis of the observations obtained in October 2000 reveals the presence of three signals with periods of 0.142, 4.8 and 0.207 days. The first two periods are recognized as "negative superhumps" and the corresponding retrograde precession period of the accretion disk. The origin of the third periodic signal remains unknown. The observations in September-October 2001 point only to the presence of "negative superhumps" and possibly to the precession period. The origin of the "negative superhumps" is discussed and two possible mechanisms are suggested. All light curves show strong flickering activity and power spectra with a typical red noise shape. PX And shows eclipses with highly variable shape and depth. The analysis suggests that the eclipse depth is modulated with the precession period and two possible explanations of this phenomenon are discussed. An improved orbital ephemeris is also determined: T_min[HJD]=49238...

  6. Plasma glutathione peroxidase (GSH-Px) concentration is elevated in rheumatoid arthritis: a case-control study.

    Science.gov (United States)

    Jacobson, Glenn A; Ives, Stephen J; Narkowicz, Christian; Jones, Graeme

    2012-11-01

    Plasma glutathione peroxidase (GSH-Px) by enzyme-linked immunosorbent assay (ELISA) offers a complimentary measurement approach to traditional GSH-Px activity methods. The aim was to investigate whether GSH-Px measured by ELISA in rheumatoid arthritis patients was elevated compared to controls. This was a case-control study with rheumatoid arthritis patients recruited from private practice and gender and age-matched controls randomly selected from the electoral role. GSH-Px concentration was measured by ELISA. Plasma malondialdehyde was used as a measure of oxidative stress, and antioxidant capacity was measured based on reduction of Cu(++) to Cu(+) by antioxidants in the sample. Disease severity was measured using the Health Assessment Questionnaire-Disability Index (HAQ-DI) and C-reactive protein was measured using an immunoturbidometric method. A total of 74 patients were recruited, consisting of 35 rheumatoid arthritis cases and 39 healthy controls. There were no differences between rheumatoid arthritis cases and controls for oxidative stress and antioxidant capacity; however, GSH-Px concentration was markedly elevated in the rheumatoid arthritis sufferers (85.9 ± 147.7 versus 17.3 ± 13.0 mg/L, respectively; mean ± SD; p arthritis demonstrated increased GSH-Px consistent with an adaptive upregulation of GSH-Px to protect against oxidative stress.

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

    Directory of Open Access Journals (Sweden)

    Samir Merabet

    2011-10-01

    Full Text Available 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.

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

    OpenAIRE

    Indraneel Majumdar; 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....

  9. Phylogenetic Analysis of RhoGAP Domain-Containing Proteins

    Institute of Scientific and Technical Information of China (English)

    Marcelo M.Brand(a)o; Karina L.Silva-Brand(a)o; Fernando F.Costa; Sara T.O.Saad

    2006-01-01

    Proteins containing an Rho GTPase-activating protein (RhoGAP) domain work as molecular switches involved in the regulation of diverse cellular functions. The ability of these GTPases to regulate a wide number of cellular processes comes from their interactions with multiple effectors and inhibitors, including the RhoGAP family, which stimulates their intrinsic GTPase activity. Here, a phylogenetic approach was applied to study the evolutionary relationship among 59 RhoGAP domain-containing proteins. The sequences were aligned by their RhoGAP domains and the phylogenetic hypotheses were generated using Maximum Parsimony and Bayesian analyses. The character tracing of two traits, GTPase activity and presence of other domains, indicated a significant phylogenetic signal for both of them.

  10. A protein domain interaction interface database: InterPare

    Directory of Open Access Journals (Sweden)

    Lee Jungsul

    2005-08-01

    Full Text Available Abstract Background Most proteins function by interacting with other molecules. Their interaction interfaces are highly conserved throughout evolution to avoid undesirable interactions that lead to fatal disorders in cells. Rational drug discovery includes computational methods to identify the interaction sites of lead compounds to the target molecules. Identifying and classifying protein interaction interfaces on a large scale can help researchers discover drug targets more efficiently. Description We introduce a large-scale protein domain interaction interface database called InterPare http://interpare.net. It contains both inter-chain (between chains interfaces and intra-chain (within chain interfaces. InterPare uses three methods to detect interfaces: 1 the geometric distance method for checking the distance between atoms that belong to different domains, 2 Accessible Surface Area (ASA, a method for detecting the buried region of a protein that is detached from a solvent when forming multimers or complexes, and 3 the Voronoi diagram, a computational geometry method that uses a mathematical definition of interface regions. InterPare includes visualization tools to display protein interior, surface, and interaction interfaces. It also provides statistics such as the amino acid propensities of queried protein according to its interior, surface, and interface region. The atom coordinates that belong to interface, surface, and interior regions can be downloaded from the website. Conclusion InterPare is an open and public database server for protein interaction interface information. It contains the large-scale interface data for proteins whose 3D-structures are known. As of November 2004, there were 10,583 (Geometric distance, 10,431 (ASA, and 11,010 (Voronoi diagram entries in the Protein Data Bank (PDB containing interfaces, according to the above three methods. In the case of the geometric distance method, there are 31,620 inter-chain domain-domain

  11. DOG 1.0: illustrator of protein domain structures

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  12. Protein domains and architectural innovation in plant-associated Proteobacteria

    Directory of Open Access Journals (Sweden)

    Downie J Allan

    2005-02-01

    Full Text Available Abstract Background Evolution of new complex biological behaviour tends to arise by novel combinations of existing building blocks. The functional and evolutionary building blocks of the proteome are protein domains, the function of a protein being dependent on its constituent domains. We clustered completely-sequenced proteomes of prokaryotes on the basis of their protein domain content, as defined by Pfam (release 16.0. This revealed that, although there was a correlation between phylogeny and domain content, other factors also have an influence. This observation motivated an investigation of the relationship between an organism's lifestyle and the complement of domains and domain architectures found within its proteome. Results We took a census of all protein domains and domain combinations (architectures encoded in the completely-sequenced proteobacterial genomes. Nine protein domain families were identified that are found in phylogenetically disparate plant-associated bacteria but are absent from non-plant-associated bacteria. Most of these are known to play a role in the plant-associated lifestyle, but they also included domain of unknown function DUF1427, which is found in plant symbionts and pathogens of the alpha-, beta- and gamma-Proteobacteria, but not known in any other organism. Further, several domains were identified as being restricted to phytobacteria and Eukaryotes. One example is the RolB/RolC glucosidase family, which is found only in Agrobacterium species and in plants. We identified the 0.5% of Pfam protein domain families that were most significantly over-represented in the plant-associated Proteobacteria with respect to the background frequencies in the whole set of available proteobacterial proteomes. These included guanylate cyclase, domains implicated in aromatic catabolism, cellulase and several domains of unknown function. We identified 459 unique domain architectures found in phylogenetically diverse plant pathogens

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

    Science.gov (United States)

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

    2015-12-01

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

  14. The trypanosome Pumilio domain protein PUF5.

    Directory of Open Access Journals (Sweden)

    Bhaskar Anand Jha

    Full Text Available PUF proteins are a conserved family of RNA binding proteins found in all eukaryotes examined so far. This study focussed on PUF5, one of 11 PUF family members encoded in the Trypanosoma brucei genome. Native PUF5 is present at less than 50000 molecules per cell in both bloodstream and procyclic form trypanosomes. C-terminally myc-tagged PUF5 was mainly found in the cytoplasm and could be cross-linked to RNA. PUF5 knockdown by RNA interference had no effect on the growth of bloodstream forms. Procyclic forms lacking PUF5 grew normally, but expression of PUF5 bearing a 21 kDa tandem affinity purification tag inhibited growth. Knockdown of PUF5 did not have any effect on the ability of trypanosomes to differentiate from the mammalian to the insect form of the parasite.

  15. InterProScan: protein domains identifier.

    Science.gov (United States)

    Quevillon, E; Silventoinen, V; Pillai, S; Harte, N; Mulder, N; Apweiler, R; Lopez, R

    2005-07-01

    InterProScan [E. M. Zdobnov and R. Apweiler (2001) Bioinformatics, 17, 847-848] is a tool that combines different protein signature recognition methods from the InterPro [N. J. Mulder, R. Apweiler, T. K. Attwood, A. Bairoch, A. Bateman, D. Binns, P. Bradley, P. Bork, P. Bucher, L. Cerutti et al. (2005) Nucleic Acids Res., 33, D201-D205] consortium member databases into one resource. At the time of writing there are 10 distinct publicly available databases in the application. Protein as well as DNA sequences can be analysed. A web-based version is accessible for academic and commercial organizations from the EBI (http://www.ebi.ac.uk/InterProScan/). In addition, a standalone Perl version and a SOAP Web Service [J. Snell, D. Tidwell and P. Kulchenko (2001) Programming Web Services with SOAP, 1st edn. O'Reilly Publishers, Sebastopol, CA, http://www.w3.org/TR/soap/] are also available to the users. Various output formats are supported and include text tables, XML documents, as well as various graphs to help interpret the results.

  16. Pleiotropic roles of cold shock domain proteins in plants

    Directory of Open Access Journals (Sweden)

    Kentaro eSasaki

    2012-01-01

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

  17. Pumilio Puf domain RNA-binding proteins in Arabidopsis.

    Science.gov (United States)

    Abbasi, Nazia; Park, Youn-Il; Choi, Sang-Bong

    2011-03-01

    Pumilio proteins are a class of RNA-binding proteins harboring Puf domains (or PUM-HD; Pumilio-Homology Domain), named after the founding members, Pumilio (from Drosophila melanogaster) and FBF (Fem-3 mRNA-Binding Factor from Caenorhabditis elegans). The domains contain multiple tandem repeats each of which recognizes one RNA base and is comprised of 35-39 amino acids. Puf domain proteins have been reported in organisms ranging from single-celled yeast to higher multicellular eukaryotes, such as humans and plants. In yeast and animals, they are involved in a variety of posttranscriptional RNA metabolism including RNA decay, RNA transport, rRNA processing and translational repression. However, their roles in plants are largely unknown. Recently, we have characterized the first member of the Puf family of RNA-binding proteins, APUM23, in Arabidopsis. Here, we discuss and summarize the diverse roles and targets of Puf proteins previously reported in other organisms and then highlight the potential regulatory roles of Puf proteins in Arabidopsis, using our recent study as an example.

  18. Elucidating the Interacting Domains of Chandipura Virus Nucleocapsid Protein

    Directory of Open Access Journals (Sweden)

    Kapila Kumar

    2013-01-01

    Full Text Available The nucleocapsid (N protein of Chandipura virus (CHPV plays a crucial role in viral life cycle, besides being an important structural component of the virion through proper organization of its interactions with other viral proteins. In a recent study, the authors had mapped the associations among CHPV proteins and shown that N protein interacts with four of the viral proteins: N, phosphoprotein (P, matrix protein (M, and glycoprotein (G. The present study aimed to distinguish the regions of CHPV N protein responsible for its interactions with other viral proteins. In this direction, we have generated the structure of CHPV N protein by homology modeling using SWISS-MODEL workspace and Accelrys Discovery Studio client 2.55 and mapped the domains of N protein using PiSQRD. The interactions of N protein fragments with other proteins were determined by ZDOCK rigid-body docking method and validated by yeast two-hybrid and ELISA. The study revealed a unique binding site, comprising of amino acids 1–30 at the N terminus of the nucleocapsid protein (N1 that is instrumental in its interactions with N, P, M, and G proteins. It was also observed that N2 associates with N and G proteins while N3 interacts with N, P, and M proteins.

  19. Dynamics of domain coverage of the protein sequence universe

    Science.gov (United States)

    2012-01-01

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

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

    KAUST Repository

    Abdel Messih, Mario A.

    2012-09-07

    Motivation: Burgeoning sequencing technologies have generated massive amounts of genomic and proteomic data. Annotating the functions of proteins identified in this data has become a big and crucial problem. Various computational methods have been developed to infer the protein functions based on either the sequences or domains of proteins. The existing methods, however, ignore the recurrence and the order of the protein domains in this function inference. Results: We developed two new methods to infer protein functions based on protein domain recurrence and domain order. Our first method, DRDO, calculates the posterior probability of the Gene Ontology terms based on domain recurrence and domain order information, whereas our second method, DRDO-NB, relies on the nave Bayes methodology using the same domain architecture information. Our large-scale benchmark comparisons show strong improvements in the accuracy of the protein function inference achieved by our new methods, demonstrating that domain recurrence and order can provide important information for inference of protein functions. The Author(s) 2012. Published by Oxford University Press.

  1. BAG4/SODD Protein Contains a Short BAG Domain

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. Methods of use of cellulose binding domain proteins

    Science.gov (United States)

    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.

  3. Development of energy scanning software to facilitate MAD experiment at PX-BL21, Indus-2 synchrotron

    Science.gov (United States)

    Dwivedi, Abhilash; Kumar, Ashwani; Ghosh, Biplab; Poswal, H. K.; Makde, Ravindra; Sharma, Surinder M.

    2017-05-01

    The protein crystallography beamline (PX-BL 21) at Indus-2 synchrotron (RRCAT, Indore) is dedicated for structure determination of macromolecules. For solving the phase problem in structure determination of macromolecules, MAD (Multi-Wavelength Anomalous Diffraction) scan is one of the well established methods. This scan requires a precise measurement of absorption data at or near the absorption edge of doped heavy atom for different wavelengths of X-rays. Software for absorption edge scanning using fluorescence detector and double crystal monochromator has been developed and deployed at PX-BL21. MAD experiment of a protein sample has been recorded and analyzed. With this important development, PX-BL21 is now fully equipped to perform anomalous diffraction experiments with single crystal protein samples.

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

    Science.gov (United States)

    Stachowiak, Jeanne C; Hayden, Carl C; Sanchez, Mari Angelica A; Wang, Julia; Bunker, Bruce C; Voigt, James A; Sasaki, Darryl Y

    2011-02-15

    We demonstrate the construction of novel protein-lipid assemblies through the design of a lipid-like molecule, DPIDA, endowed with tail-driven affinity for specific lipid membrane phases and head-driven affinity for specific proteins. In studies performed on giant unilamellar vesicles (GUVs) with varying mole fractions of dipalymitoylphosphatidylcholine (DPPC), cholesterol, and diphytanoylphosphatidyl choline (DPhPC), DPIDA selectively partitioned into the more ordered phases, either solid or liquid-ordered (L(o)) depending on membrane composition. Fluorescence imaging established the phase behavior of the resulting quaternary lipid system. Fluorescence correlation spectroscopy confirmed the fluidity of the L(o) phase containing DPIDA. In the presence of CuCl(2), the iminodiacetic acid (IDA) headgroup of DPIDA forms the Cu(II)-IDA complex that exhibits a high affinity for histidine residues. His-tagged proteins were bound specifically to domains enriched in DPIDA, demonstrating the capacity to target protein binding selectively to both solid and L(o) phases. Steric pressure from the crowding of surface-bound proteins transformed the domains into tubules with persistence lengths that depended on the phase state of the lipid domains.

  5. Purification and characterization of coacervate-forming cuticular proteins from Papilio xuthus pupae.

    Science.gov (United States)

    Yamanaka, Masahiro; Ishizaki, Yumi; Nakagawa, Taro; Taoka, Azuma; Fukumori, Yoshihiro

    2013-07-01

    The Papilio xuthus (Lepidoptera: Papilionidae) pupa expresses novel soluble proteins that undergo reversible temperature-dependent coacervate-formation. We purified two coacervate-forming proteins, PX-1 and PX-4, from the wings of pharate adults. PX-1 and PX-4 form coacervates upon warming. Transmission electron microscopy analysis revealed that these proteins assemble ordered bead-like ultrastructures. We cloned and sequenced PX-1 and PX-4 cDNAs. The PX-1 and PX-4 amino acid sequences contain many hydrophobic residues and show homologies to insect cuticular proteins. Moreover, when recombinant PX-1 and PX-4 were overexpressed in Escherichia coli, both recombinant proteins exhibited temperature-dependent coacervation. Furthermore, analyses of truncated mutants of PX-1 suggest that both the Val/Pro-rich region and Gly/lle-rich regions of PX-1 are involved in such coacervation.

  6. Structure and Membrane Binding Properties of the Endosomal Tetratricopeptide Repeat (TPR) Domain-containing Sorting Nexins SNX20 and SNX21.

    Science.gov (United States)

    Clairfeuille, Thomas; Norwood, Suzanne J; Qi, Xiaying; Teasdale, Rohan D; Collins, Brett M

    2015-06-01

    Sorting nexins (SNX) orchestrate membrane trafficking and signaling events required for the proper distribution of proteins within the endosomal network. Their phox homology (PX) domain acts as a phosphoinositide (PI) recognition module that targets them to specific endocytic membrane domains. The modularity of SNX proteins confers a wide variety of functions from signaling to membrane deformation and cargo binding, and many SNXs are crucial modulators of endosome dynamics and are involved in a myriad of physiological and pathological processes such as neurodegenerative diseases, cancer, and inflammation. Here, we have studied the poorly characterized SNX20 and its paralogue SNX21, which contain an N-terminal PX domain and a C-terminal PX-associated B (PXB) domain of unknown function. The two proteins share similar PI-binding properties and are recruited to early endosomal compartments by their PX domain. The crystal structure of the SNX21 PXB domain reveals a tetratricopeptide repeat (TPR)-fold, a module that typically binds short peptide motifs, with three TPR α-helical repeats. However, the C-terminal capping helix adopts a highly unusual and potentially self-inhibitory topology. SAXS solution structures of SNX20 and SNX21 show that these proteins adopt a compact globular architecture, and membrane interaction analyses indicate the presence of overlapping PI-binding sites that may regulate their intracellular localization. This study provides the first structural analysis of this poorly characterized subfamily of SNX proteins, highlighting a likely role as endosome-associated scaffolds.

  7. A Bayesian Sampler for Optimization of Protein Domain Hierarchies

    Science.gov (United States)

    2014-01-01

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

  8. Membrane shape instabilities induced by BAR domain proteins

    Science.gov (United States)

    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.

  9. SLIDE, the Protein Interacting Domain of Imitation Switch Remodelers, Binds DDT-Domain Proteins of Different Subfamilies in Chromatin Remodeling Complexes

    Institute of Scientific and Technical Information of China (English)

    Jiaqiang Dong; Zheng Gao; Shujing Liu; Guang Li; Zhongnan Yang; Hai Huang; Lin Xu

    2013-01-01

    The Imitation Switch (ISWI) type adenosine triphosphate (ATP)-dependent chromatin remodeling factors are conserved proteins in eukaryotes, and some of them are known to form stable remodeling complexes with members from a family of proteins, termed DDT-domain proteins. Although it is well documented that ISWIs play important roles in different biological processes in many eukaryotic species, the molecular basis for protein interactions in ISWI complexes has not been fully addressed. Here, we report the identification of interaction domains for both ISWI and DDT-domain proteins. By analyzing CHROMATIN REMODELING11 (CHR11) and RINGLET1 (RLT1), an Arabidopsis thaliana ISWI (AtISWI) and AtDDT-domain protein, respectively, we show that the SLIDE domain of CHR11 and the DDT domain together with an adjacent sequence of RLT1 are responsible for their binding. The Arabidopsis genome contains at least 12 genes that encode DDT-domain proteins, which could be grouped into five subfamilies based on the sequence similarity. The SLIDE domain of AtISWI is able to bind members from different AtDDT subfamilies. Moreover, a human ISWI protein SNF2H is capable of binding AtDDT-domain proteins through its SLIDE domain, suggesting that binding to DDT-domain proteins is a conserved biochemical function for the SLIDE domain of ISWIs in eukaryotes.

  10. PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95

    DEFF Research Database (Denmark)

    Møller, Thor C; Wirth, Volker F; Roberts, Nina Ingerslev;

    2013-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present...

  11. [Fluorescent fusion proteins with 10th human fibronectin domain].

    Science.gov (United States)

    Petrovskaia, L E; Gapizov, S Sh; Shingarova, L N; Kriukova, E A; Boldyreva, E F; Iakimov, S A; Svirshchevskaia, E V; Lukashev, E P; Dolgikh, D A; Kirpichnikov, M P

    2014-01-01

    In the current paper we describe a new type of hybrid molecules including red fluorescent protein mCherry and 10th type III human fibronectin domain (10Fn3) - one of the alternative scaffold proteins which can be used for the construction of antibody mimics with various binding specificity. We have constructed different gene variants encoding for the hybrid fluorescent protein and studied their expression in Escherichia coli cells. It was shown that N-terminal position of mCherry and modification of its N-terminal amino acid sequence promotes efficientbacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construction we have obtained the hybrid fluorescent protein ChIBF, containing alphaVbeta3-integrin binding vari- ant of 10Fn3, and demonstrated the possibility of its utilization for the visualization of alphaVbeta3-integrin at the surface of MDCK epithelial cells by confocal microscopy.

  12. Generation of mice lacking DUF1220 protein domains

    DEFF Research Database (Denmark)

    Keeney, J G; O'Bleness, M S; Anderson, N

    2015-01-01

    Sequences encoding DUF1220 protein domains show the most extreme human lineage-specific copy number increase of any coding region in the genome and have been linked to human brain evolution. In addition, DUF1220 copy number (dosage) has been implicated in influencing brain size within the human...... species, both in normal populations and in individuals associated with brain size pathologies (1q21-associated microcephaly and macrocephaly). More recently, increasing dosage of a subtype of DUF1220 has been linked with increasing severity of the primary symptoms of autism. Despite these intriguing...... associations, a function for these domains has not been described. As a first step in addressing this question, we have developed the first transgenic model of DUF1220 function by removing the single DUF1220 domain (the ancestral form) encoded in the mouse genome. In a hypothesis generating exercise...

  13. Binding of Y-box proteins to RNA: involvement of different protein domains.

    Science.gov (United States)

    Ladomery, M; Sommerville, J

    1994-01-01

    Eukaryotic Y-box proteins are reported to interact with a wide variety of nucleic acid structures to act as transcription factors and mRNA masking proteins. The modular structure of Y-box proteins includes a highly conserved N-terminal cold-shock domain (CSD, equivalent to the bacterial cold-shock proteins) plus four basic C-terminal domains containing arginine clusters and aromatic residues. In addition, the basic domains are separated by acidic regions which contain several potential sites for serine/threonine phosphorylation. The interaction of Y-box proteins, isolated from Xenopus oocytes (FRGY2 type), with RNA molecules has been studied by UV crosslinking and protein fragmentation. We have identified two distinct binding activities. The CSD interacts preferentially with the polypurines poly(A,G) and poly(G) but not poly(A), this activity being sensitive to 5 mM MgCl2 but not to 5 mM spermidine. In the presence of 1 mM MgCl2 or 1 mM spermidine, the basic domains interact preferentially with poly(C,U), this activity being sensitive to 0.5 M NaCl. Binding of the basic domains is also sensitive to low concentrations of heparin. The basic domains can be crosslinked individually to labelled RNA. These results are discussed with reference to the various specificities noted in the binding of Y-box proteins to RNA and DNA. Images PMID:7530842

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

    Science.gov (United States)

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

    2012-09-01

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

  15. Energetic pathway sampling in a protein interaction domain

    DEFF Research Database (Denmark)

    Hultqvist, Greta; Haq, S. Raza; Punekar, Avinash S.;

    2013-01-01

    The affinity and specificity of protein-ligand interactions are influenced by energetic crosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interaction pathways...... changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one...

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

    Science.gov (United States)

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

    2017-02-17

    Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements which control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith EC, et al. Trimeric transmembrane domain interactions in paramyxovirus fusion proteins. 2013. J Biol Chem. 288, 35726). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability.

  17. Deployment of membrane fusion protein domains during fusion.

    Science.gov (United States)

    Bentz, J; Mittal, A

    2000-01-01

    It is clear that both viral and intracellular membrane fusion proteins contain a minimal set of domains which must be deployed at the appropriate time during the fusion process. An account of these domains and their functions is given here for the four best-described fusion systems: influenza HA, sendai virus F1, HIV gp120/41 and the neuronal SNARE core composed of synaptobrevin (syn), syntaxin (stx) and the N- and C-termini of SNAP25 (sn25), together with the Ca(2+)binding protein synaptotagmin (syt). Membrane fusion begins with the binding of the virion or vesicle to the target membrane via receptors. The committed step in influenza HA- mediated fusion begins with an aggregate of HAs (at least eight) with some of their HA2 N-termini, a.k.a. fusion peptides, embedded into the viral bilayer (Bentz, 2000 a). The hypothesis presented in Bentz (2000 b) is that the conformational change of HA to the extended coiled coil extracts the fusion peptides from the viral bilayer. When this extraction occurs from the center of the site of restricted lipid flow, it exposes acyl chains and parts of the HA transmembrane domains to the aqueous media, i.e. a hydrophobic defect is formed. This is the 'transition state' of the committed step of fusion. It is stabilized by a 'dam' of HAs, which are inhibited from diffusing away by the rest of the HAs in the aggregate and because that would initially expose more acyl chains to water. Recruitment of lipids from the apposed target membrane can heal this hydrophobic defect, initiating lipid mixing and fusion. The HA transmembrane domains are required to be part of the hydrophobic defect, because the HA aggregate must be closely packed enough to restrict lipid flow. This hypothesis provides a simple and direct coupling between the energy released by the formation of the coiled coil to the energy needed to create and stabilize the high energy intermediates of fusion. Several of these essential domains have been described for the viral fusion

  18. The Rapamycin-Binding Domain of the Protein Kinase mTOR is a Destabilizing Domain*

    Science.gov (United States)

    Edwards, Sarah R.; Wandless, Thomas J.

    2013-01-01

    Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding domain (FRB) of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to ten-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retain the ability to inhibit mTOR, albeit with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wildtype FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems. PMID:17350953

  19. Clustering of protein domains for functional and evolutionary studies

    Directory of Open Access Journals (Sweden)

    Long Paul F

    2009-10-01

    Full Text Available Abstract 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.

  20. Continuous monitoring of membrane protein micro-domain association during cell signaling

    CERN Document Server

    Huang, Heng

    2011-01-01

    Central to understanding membrane bound cell signaling is to quantify how the membrane ultra-structure consisting of transient spatial domains modulates signaling and how the signaling influences this ultra-structure. Yet, measuring the association of membrane proteins with domains in living, intact cells poses considerable challenges. Here, we describe a non-destructive method to quantify protein-lipid domain and protein cytoskeleton interactions in single, intact cells enabling continuous monitoring of the protein domains interaction over time during signaling.

  1. LOV Domain-Containing F-Box Proteins:Light-Dependent Protein Degradation Modules in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Shogo Ito; Young Hun Song; Takato Imaizumi

    2012-01-01

    Plants constantly survey the surrounding environment using several sets of photoreceptors.They can sense changes in the quantity (=intensity) and quality (=wavelength) of light and use this information to adjust their physiological responses,growth,and developmental patterns.In addition to the classical photoreceptors,such as phytochromes,cryptochromes,and phototropins,ZEITLUPE (ZTL),FLAVIN-BINDING,KELCH REPEAT,F-BOX 1 (FKF1),and LOV KELCH PROTEIN 2 (LKP2) proteins have been recently identified as blue-light photoreceptors that are important for regulation of the circadian clock and photoperiodic flowering.The ZTL/FKF1/LKP2 protein family possesses a unique combination of domains:a blue-light-absorbing LOV (Light,Oxygen,or Voltage) domain along with domains involved in protein degradation.Here,we summarize recent advances in our understanding of the function of the Arabidopsis ZTL/FKF1/LKP2 proteins.We summarize the distinct photochemical properties of their LOV domains and discuss the molecular mechanisms by which the ZTL/FKF1/LKP2 proteins regulate the circadian clock and photoperiodic flowering by controlling blue-light-dependent protein degradation.

  2. Internal Indpendent Assessment Report - CASTLE-PX SQA

    Energy Technology Data Exchange (ETDEWEB)

    Whitney, D. M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Dancy, L. L. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Pope, V. L. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-04-01

    This IIA assessed the flow down of institutional 830 Software Quality Assurance requirements through three required document templates to the CASTLE-PX software effort and the implementation of those SQA requirements. The templates flow down the DOE O 414.1D consensus standard requirements for Safety Software. This assessment did not include the flow down of NAP-24, Weapon Quality Policy, requirements. The assessment focused on the CASTLE-PX project’s software development and release processes. It did not assess Pantex’s acceptance or usage of the software. The assessment resulted in 3 Deficiencies, 5 Observations, 1 Recommendation, and 3 Strengths. Overall the CASTLE-PX team demonstrated it values quality and has worked to integrate quality practices into its software development processes. Improvement in documentation will enhance their SQA implementation.

  3. Protein-protein interaction domains of Bacillus subtilis DivIVA.

    Science.gov (United States)

    van Baarle, Suey; Celik, Ilkay Nazli; Kaval, Karan Gautam; Bramkamp, Marc; Hamoen, Leendert W; Halbedel, Sven

    2013-03-01

    DivIVA proteins are curvature-sensitive membrane binding proteins that recruit other proteins to the poles and the division septum. They consist of a conserved N-terminal lipid binding domain fused to a less conserved C-terminal domain. DivIVA homologues interact with different proteins involved in cell division, chromosome segregation, genetic competence, or cell wall synthesis. It is unknown how DivIVA interacts with these proteins, and we used the interaction of Bacillus subtilis DivIVA with MinJ and RacA to investigate this. MinJ is a transmembrane protein controlling division site selection, and the DNA-binding protein RacA is crucial for chromosome segregation during sporulation. Initial bacterial two-hybrid experiments revealed that the C terminus of DivIVA appears to be important for recruiting both proteins. However, the interpretation of these results is limited since it appeared that C-terminal truncations also interfere with DivIVA oligomerization. Therefore, a chimera approach was followed, making use of the fact that Listeria monocytogenes DivIVA shows normal polar localization but is not biologically active when expressed in B. subtilis. Complementation experiments with different chimeras of B. subtilis and L. monocytogenes DivIVA suggest that MinJ and RacA bind to separate DivIVA domains. Fluorescence microscopy of green fluorescent protein-tagged RacA and MinJ corroborated this conclusion and suggests that MinJ recruitment operates via the N-terminal lipid binding domain, whereas RacA interacts with the C-terminal domain. We speculate that this difference is related to the cellular compartments in which MinJ and RacA are active: the cell membrane and the cytoplasm, respectively.

  4. ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

    Science.gov (United States)

    Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

    2010-10-01

    The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion.

  5. Sox2 uses multiple domains to associate with proteins present in Sox2-protein complexes.

    Directory of Open Access Journals (Sweden)

    Jesse L Cox

    Full Text Available Master regulators, such as Sox2, Oct4 and Nanog, control complex gene networks necessary for the self-renewal and pluripotency of embryonic stem cells (ESC. These master regulators associate with co-activators and co-repressors to precisely control their gene targets. Recent studies using proteomic analysis have identified a large, diverse group of co-activators and co-repressors that associate with master regulators, including Sox2. In this report, we examined the size distribution of nuclear protein complexes containing Sox2 and its associated proteins HDAC1, Sall4 and Lin28. Interestingly, we determined that Sox2 and HDAC1 associate with protein complexes that vary greatly in size; whereas, Lin28 primarily associates with smaller complexes, and Sall4 primarily associates with larger complexes. Additionally, we examined the domains of Sox2 necessary to mediate its association with its partner proteins Sall4, HDAC1 and HDAC2. We determined that Sox2 uses multiple and distinct domains to associate with its partner proteins. We also examined the domains of Sox2 necessary to mediate its self-association, and we determined that Sox2 self-association is mediated through multiple domains. Collectively, these studies provide novel insights into how Sox2 is able to associate with a wide array of nuclear proteins that control gene transcription.

  6. The transmembrane domain of TACE regulates protein ectodomain shedding

    Institute of Scientific and Technical Information of China (English)

    Xiaojin Li; Liliana Pérez; Zui Pan; Huizhou Fan

    2007-01-01

    Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme (TACE), which causes the release of their ectodomains. An ADAM (a disintegrin and metalloprotease domain) family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain (TM) of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylino-sitol (GPI)-binding polypeptide failed to restore shedding of transforming growth factor-α (TGF-α), tumor necrosis factor-α (TNF-α) and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor (PDGFR) also resulted in severe loss of TGF-α shedding, but had no effects on the cleavage of TNF-α and L-selectin. Replacement of the TM in TGF-a with that of L-selectin enabled TGF-a shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.

  7. C2 Domains as Protein-Protein Interaction Modules in the Ciliary Transition Zone

    Directory of Open Access Journals (Sweden)

    Kim Remans

    2014-07-01

    Full Text Available RPGR-interacting protein 1 (RPGRIP1 is mutated in the eye disease Leber congenital amaurosis (LCA and its structural homolog, RPGRIP1-like (RPGRIP1L, is mutated in many different ciliopathies. Both are multidomain proteins that are predicted to interact with retinitis pigmentosa G-protein regulator (RPGR. RPGR is mutated in X-linked retinitis pigmentosa and is located in photoreceptors and primary cilia. We solved the crystal structure of the complex between the RPGR-interacting domain (RID of RPGRIP1 and RPGR and demonstrate that RPGRIP1L binds to RPGR similarly. RPGRIP1 binding to RPGR affects the interaction with PDEδ, the cargo shuttling factor for prenylated ciliary proteins. RPGRIP1-RID is a C2 domain with a canonical β sandwich structure that does not bind Ca2+ and/or phospholipids and thus constitutes a unique type of protein-protein interaction module. Judging from the large number of C2 domains in most of the ciliary transition zone proteins identified thus far, the structure presented here seems to constitute a cilia-specific module that is present in multiprotein transition zone complexes.

  8. PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95

    DEFF Research Database (Denmark)

    Møller, Thor C; Wirth, Volker F; Roberts, Nina Ingerslev;

    2013-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present...... with colocalization of the full-length proteins in cells and with previous studies, we suggest that the range of relevant interactions might extend to interactions with K i = 450 µM in the in vitro assays. Within this range, we identify novel PSD-95 interactions with the chemokine receptor CXCR2, the neuropeptide Y...

  9. PDZ Domain Proteins: ‘Dark Matter' of the Plant Proteome?

    Institute of Scientific and Technical Information of China (English)

    John Gardiner; Robyn Overall; Jan Marc

    2011-01-01

    PDZ domain proteins in metazoans function in diverse roles,and in conjunction with PDZ domain-binding proteins form macromolecular complexes for signaling at synapses and cell junctions.Bioinformatics approaches using the SMART tool indicate there are only a modest number of Arabidopsis PDZ proteins.However,there are hundreds of proteins predicted to possess PDZ domain-binding motifs,suggesting that there are many PDZ domain proteins not detectable by conventional bioinformatic approaches.Our Scansite analysis of PDZ domain-binding proteins indicates that PDZ domain proteins may play key roles in cytoskeletal organization including actin microfilaments,microtubules,and nuclear cytoskeletal proteins,and in the organization of macromolecular complexes involved in cell-to-cell signaling,transport,and cell wall formation.

  10. Critical interaction domains between bloom syndrome protein and RAD51.

    Science.gov (United States)

    Bergeron, Krystal L; Murphy, Eileen L; Brown, Lily W; Almeida, Karen H

    2011-01-01

    The American Cancer Society's 2009 statistics estimate that 1 out of every 4 deaths is cancer related. Genomic instability is a common feature of cancerous states, and an increase in genomic instability is the diagnostic feature of Bloom Syndrome. Bloom Syndrome, a rare disorder characterized by a predisposition to cancer, is caused by mutations of the BLM gene. This study focuses on the partnerships of BLM protein to RAD51, a Homologous Recombination repair protein essential for survival. A systematic set of BLM deletion fragments were generated to refine the protein binding domains of BLM to RAD51 and determine interacting regions of BLM and ssDNA. Results show that RAD51 and ssDNA interact in overlapping regions; BLM₁₀₀₋₂₁₄ and BLM₁₃₁₇₋₁₃₆₇. The overlapping nature of these regions suggests a preferential binding for one partner that could function to regulate homologous recombination and therefore helps to clarify the role of BLM in maintaining genomic stability.

  11. Proteins with GGDEF and EAL domains regulate Pseudomonas putida biofilm formation and dispersal

    DEFF Research Database (Denmark)

    Gjermansen, Morten; Ragas, Paula Cornelia; Tolker-Nielsen, Tim

    2006-01-01

    H protein, which contains an EAL domain, strongly inhibited biofilm formation. Induction of YhjH expression in P. putida cells situated in established biofilms led to rapid dispersion of the biofilms. These results support the emerging theme that GGDEF-domain and EAL-domain proteins are involved...

  12. Structure-function analysis of the NB-ARC domain of plant disease resistance proteins

    NARCIS (Netherlands)

    van Ooijen, G.; Mayr, G.; Kasiem, M.M.A.; Albrecht, M.; Cornelissen, B.J.C.; Takken, F.L.W.

    2008-01-01

    Resistance (R) proteins in plants are involved in pathogen recognition and subsequent activation of innate immune responses. Most resistance proteins contain a central nucleotide-binding domain. This so-called NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. The NB-ARC domain is a fun

  13. Preparation and functional analysis of recombinant protein transduction domain-metallothionein fusion proteins.

    Science.gov (United States)

    Lim, Kwang Suk; Won, Young-Wook; Park, Yong Soo; Kim, Yong-Hee

    2010-08-01

    In order for proteins to be used as pharmaceuticals, delivery technologies need to be developed to overcome biochemical and anatomical barriers to protein drug transport, to protect proteins from systemic degradation, and to target the drug action to specific sites. Protein transduction domains (PTDs) are used for the non-specific transduction of bio-active cargo, such as proteins, genes, and particles, through cellular membranes to overcome biological barriers. Metallothionein (MT) is a low molecular weight intra-cellular protein that consists of 61 amino acids, including 20 cysteine residues, and is over-expressed under stressful conditions. Although MT has the potential to improve the viability of islet cells and cardiomyocytes by inhibiting diabetic-induced apoptosis and by removing reactive oxygen species (ROS), and thereby prevent or reduce diabetes and diabetic complications, all MT applications have been made for gene therapy or under induced over-expression of endogenous MT. To overcome the drawbacks of ineffective intra-cellular MT protein uptake, a human MT gene was cloned and fused with protein transduction domains (PTDs), such as HIV-1 Tat and undeca-arginine, in a bacterial expression vector to produce PTD-MT fusion proteins. The expression and purification of three types of proteins were optimized by adding Zn ions to maintain their stability and functionality mimicking intra-cellular stable conformation of MT as a Zn-MT cluster. The Zn-MT cluster showed better stability than MT in vitro. PTD-MT fusion proteins strongly protected Ins-1 beta cells against oxidative stress and apoptosis induced by glucolipotoxicity with or without hypoxia, and also protected H9c2 cardiomyocytes against hyperglycemia-induced apoptosis with or without hypoxia. PTD-MT recombinant fusion proteins may be useful protein therapeutics for the treatment or prevention of diabetes and diabetes-related complications.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  15. Improving protein-protein interaction article classification using biological domain knowledge.

    Science.gov (United States)

    Chen, Yifei; Guo, Hongjian; Liu, Feng; Manderick, Bernard

    2015-01-01

    Interaction Article Classification (IAC) is a specific text classification application in biological domain that tries to find out which articles describe Protein-Protein Interactions (PPIs) to help extract PPIs from biological literature more efficiently. However, the existing text representation and feature weighting schemes commonly used for text classification are not well suited for IAC. We capture and utilise biological domain knowledge, i.e. gene mentions also known as protein or gene names in the articles, to address the problem. We put forward a new gene mention order-based approach that highlights the important role of gene mentions to represent the texts. Furthermore, we also incorporate the information concerning gene mentions into a novel feature weighting scheme called Gene Mention-based Term Frequency (GMTF). By conducting experiments, we show that using the proposed representation and weighting schemes, our Interaction Article Classifier (IACer) performs better than other leading systems for the moment.

  16. Protein-protein Interaction Between Domains of PDZ and BAR from PICK1

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Two DNA fragments encoding PDZ domain(21-110 residues) and BAR domain( 150-360 residues) from PICK1 (1-416 residues) were amplified by PCR and then introduced into vectors, pET-32M and pMAL-c2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins: His-tagged PDZ (PDZ domain) and maltose binding protein-BAR( MBP-BAR domain) respectively, as confirmed by both SDS-PAGE and Western blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches ( 16 ±0. 5 )%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR(bait) and PDZ(prey) in vivo was also examined with a yeast two-hybrid system.

  17. Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

    Science.gov (United States)

    Ogawara, Hiroshi

    2016-09-01

    PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

  18. Structural organization and interactions of transmembrane domains in tetraspanin proteins

    Directory of Open Access Journals (Sweden)

    DeGrado William F

    2005-06-01

    Full Text Available Abstract 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 (abcdefgn. 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

  19. Phosphorylation and Functional Properties of the IIA Domain of the Lactose Transport Protein of Streptococcus thermophilus

    NARCIS (Netherlands)

    Gunnewijk, M.G W; Postma, P.W.; Poolman, B.

    1999-01-01

    The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIALacS) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) in various organisms, of which IIAGlc of Esc

  20. The structure in solution of the b domain of protein disulfide isomerase

    NARCIS (Netherlands)

    Kemmink, J; Dijkstra, K; Mariani, M; Scheek, RM; Penka, E; Nilges, M; Darby, NJ

    1999-01-01

    Protein disulfide isomerase (PDI) is a multifunctional protein of the endoplasmic reticulum, which catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. It consists of four domains designated a, b, b' and a'. Both a and a' domains contain an active site with

  1. The WWE domain: a common interaction module in protein ubiquitination and ADP ribosylation.

    Science.gov (United States)

    Aravind, L

    2001-05-01

    Sequence profile analysis was used to detect a conserved globular domain in several proteins including deltex, Trip12 and poly-ADP-ribose polymerase homologs. It was named the WWE domain after its most conserved residues and is predicted to mediate specific protein-protein interactions in ubiquitin and ADP-ribose conjugation systems.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-01-01

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

  3. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures

    Science.gov (United States)

    Marchler-Bauer, Aron; Bo, Yu; Han, Lianyi; He, Jane; Lanczycki, Christopher J.; Lu, Shennan; Chitsaz, Farideh; Derbyshire, Myra K.; Geer, Renata C.; Gonzales, Noreen R.; Gwadz, Marc; Hurwitz, David I.; Lu, Fu; Marchler, Gabriele H.; Song, James S.; Thanki, Narmada; Wang, Zhouxi; Yamashita, Roxanne A.; Zhang, Dachuan; Zheng, Chanjuan; Geer, Lewis Y.; Bryant, Stephen H.

    2017-01-01

    NCBI's Conserved Domain Database (CDD) aims at annotating biomolecular sequences with the location of evolutionarily conserved protein domain footprints, and functional sites inferred from such footprints. An archive of pre-computed domain annotation is maintained for proteins tracked by NCBI's Entrez database, and live search services are offered as well. CDD curation staff supplements a comprehensive collection of protein domain and protein family models, which have been imported from external providers, with representations of selected domain families that are curated in-house and organized into hierarchical classifications of functionally distinct families and sub-families. CDD also supports comparative analyses of protein families via conserved domain architectures, and a recent curation effort focuses on providing functional characterizations of distinct subfamily architectures using SPARCLE: Subfamily Protein Architecture Labeling Engine. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml. PMID:27899674

  4. A Positive Energy Theorem for $P(X, \\phi)$ Theories

    CERN Document Server

    Elder, Benjamin; Khoury, Justin; Tolley, Andrew J

    2014-01-01

    We descibe a positive energy theorem for Einstein gravity coupled to scalar fields with first-derivative interactions, so-called $P(X, \\phi)$ theories. We offer two independent derivations of this result. The first method introduces an auxiliary field to map the theory to a lagrangian describing two canonical scalar fields, where one can apply a positive energy result of Boucher and Townsend. The second method works directly at the $P(X, \\phi)$ level and uses spinorial arguments introduced by Witten. The latter approach follows that of arXiv:1310.1663, but the end result is less restrictive. We point to the technical step where our derivation deviates from theirs. One of the more interesting implications of our analysis is to show it is possible to have positive energy in cases where dispersion relations following from locality and S-Matrix analyticity are violated.

  5. Length variations amongst protein domain superfamilies and consequences on structure and function.

    Directory of Open Access Journals (Sweden)

    Sankaran Sandhya

    Full Text Available BACKGROUND: Related protein domains of a superfamily can be specified by proteins of diverse lengths. The structural and functional implications of indels in a domain scaffold have been examined. METHODOLOGY: In this study, domain superfamilies with large length variations (more than 30% difference from average domain size, referred as 'length-deviant' superfamilies and 'length-rigid' domain superfamilies (<10% length difference from average domain size were analyzed for the functional impact of such structural differences. Our delineated dataset, derived from an objective algorithm, enables us to address indel roles in the presence of peculiar structural repeats, functional variation, protein-protein interactions and to examine 'domain contexts' of proteins tolerant to large length variations. Amongst the top-10 length-deviant superfamilies analyzed, we found that 80% of length-deviant superfamilies possess distant internal structural repeats and nearly half of them acquired diverse biological functions. In general, length-deviant superfamilies have higher chance, than length-rigid superfamilies, to be engaged in internal structural repeats. We also found that approximately 40% of length-deviant domains exist as multi-domain proteins involving interactions with domains from the same or other superfamilies. Indels, in diverse domain superfamilies, were found to participate in the accretion of structural and functional features amongst related domains. With specific examples, we discuss how indels are involved directly or indirectly in the generation of oligomerization interfaces, introduction of substrate specificity, regulation of protein function and stability. CONCLUSIONS: Our data suggests a multitude of roles for indels that are specialized for domain members of different domain superfamilies. These specialist roles that we observe and trends in the extent of length variation could influence decision making in modeling of new superfamily

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

    Science.gov (United States)

    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.

  7. Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

    Science.gov (United States)

    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.

  8. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    Energy Technology Data Exchange (ETDEWEB)

    Simarro, Maria [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Gimenez-Cassina, Alfredo [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Kedersha, Nancy [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A. [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Rhee, Kirsten [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Tisdale, Sarah; Danial, Nika [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Benarafa, Charaf [Theodor Kocher Institute, University of Bern, 3012 Bern (Switzerland); Orduna, Anonio [Unidad de Investigacion, Hospital Clinico Universitario de Valladolid, 47005 Valladolid (Spain); Anderson, Paul, E-mail: panderson@rics.bwh.harvard.edu [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States)

    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.

  9. A Method to Identify p62's UBA Domain Interacting Proteins

    Directory of Open Access Journals (Sweden)

    Pridgeon Julia W.

    2003-01-01

    Full Text Available The UBA domain is a conserved sequence motif among polyubiquitin binding proteins. For the first time, we demonstrate a systematic, high throughput approach to identification of UBA domain-interacting proteins from a proteome-wide perspective. Using the rabbit reticulocyte lysate in vitro expression cloning system, we have successfully identified eleven proteins that interact with p62’s UBA domain, and the majority of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer’s disease. Therefore, p62 may play a novel regulatory role through its UBA domain. Our approach provides an easy route to the characterization of UBA domain interacting proteins and its application will unfold the important roles that the UBA domain plays.

  10. Small Molecule-Induced Domain Swapping as a Mechanism for Controlling Protein Function and Assembly

    Science.gov (United States)

    Karchin, Joshua M.; Ha, Jeung-Hoi; Namitz, Kevin E.; Cosgrove, Michael S.; Loh, Stewart N.

    2017-01-01

    Domain swapping is the process by which identical proteins exchange reciprocal segments to generate dimers. Here we introduce induced domain swapping (INDOS) as a mechanism for regulating protein function. INDOS employs a modular design consisting of the fusion of two proteins: a recognition protein that binds a triggering molecule, and a target protein that undergoes a domain swap in response to binding of the triggering ligand. The recognition protein (FK506 binding protein) is inserted into functionally-inactivated point mutants of two target proteins (staphylococcal nuclease and ribose binding protein). Binding of FK506 to the FKBP domain causes the target domain to first unfold, then refold via domain swap. The inactivating mutations become ‘swapped out’ in the dimer, increasing nuclease and ribose binding activities by 100-fold and 15-fold, respectively, restoring them to near wild-type values. INDOS is intended to convert an arbitrary protein into a functional switch, and is the first example of rational design in which a small molecule is used to trigger protein domain swapping and subsequent activation of biological function. PMID:28287617

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

    Science.gov (United States)

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

    2007-01-01

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

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spec...

  13. Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

    Energy Technology Data Exchange (ETDEWEB)

    Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N. (UW)

    2012-03-15

    The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

  14. Genetic, Structural, and Molecular Insights into the Function of Ras of Complex Proteins Domains

    OpenAIRE

    Civiero, Laura; Dihanich, Sybille; Lewis, Patrick A.; Greggio, Elisa

    2014-01-01

    Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson’s disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role ...

  15. Identification of novel human WW domain-containing proteins by cloning of ligand targets.

    Science.gov (United States)

    Pirozzi, G; McConnell, S J; Uveges, A J; Carter, J M; Sparks, A B; Kay, B K; Fowlkes, D M

    1997-06-06

    A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-alpha, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-10-16

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

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

    Science.gov (United States)

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

    2011-01-01

    Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell–cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated. PMID:22117215

  18. Altered Specificity of DNA-Binding Proteins with Transition Metal Dimerization Domains

    Science.gov (United States)

    Cuenoud, Bernard; Schepartz, Alanna

    1993-01-01

    The bZIP motif is characterized by a leucine zipper domain that mediates dimerization and a basic domain that contacts DNA. A series of transition metal dimerization domains were used to alter systematically the relative orientation of basic domain peptides. Both the affinity and the specificity of the peptide-DNA interaction depend on domain orientation. These results indicate that the precise configuration linking the domains is important; dimerization is not always sufficient for DNA binding. This approach to studying the effect of orientation on protein function complements mutagenesis and could be used in many systems.

  19. The impact of a carbon nanotube on the cholesterol domain localized on a protein surface

    CERN Document Server

    Gburski, Zygmunt; Raczynski, Przemyslaw; 10.1016/j.ssc.2009.12.005

    2011-01-01

    The influence of a single walled carbon nanotube on the structure of a cholesterol cluster (domain) developed over the surface of the endothelial protein 1LQV has been investigated using the classical molecular dynamics (MD) simulation technique. We have observed a substantial impact of carbon nanotube on the arrangement of the cholesterol domain. The carbon nanotube can drag out cholesterol molecules, remarkable reducing the volume of the domain settled down on the protein.

  20. Identification of divergent protein domains by combining HMM-HMM comparisons and co-occurrence detection.

    Directory of Open Access Journals (Sweden)

    Amel Ghouila

    Full Text Available Identification of protein domains is a key step for understanding protein function. Hidden Markov Models (HMMs have proved to be a powerful tool for this task. The Pfam database notably provides a large collection of HMMs which are widely used for the annotation of proteins in sequenced organisms. This is done via sequence/HMM comparisons. However, this approach may lack sensitivity when searching for domains in divergent species. Recently, methods for HMM/HMM comparisons have been proposed and proved to be more sensitive than sequence/HMM approaches in certain cases. However, these approaches are usually not used for protein domain discovery at a genome scale, and the benefit that could be expected from their utilization for this problem has not been investigated. Using proteins of P. falciparum and L. major as examples, we investigate the extent to which HMM/HMM comparisons can identify new domain occurrences not already identified by sequence/HMM approaches. We show that although HMM/HMM comparisons are much more sensitive than sequence/HMM comparisons, they are not sufficiently accurate to be used as a standalone complement of sequence/HMM approaches at the genome scale. Hence, we propose to use domain co-occurrence--the general domain tendency to preferentially appear along with some favorite domains in the proteins--to improve the accuracy of the approach. We show that the combination of HMM/HMM comparisons and co-occurrence domain detection boosts protein annotations. At an estimated False Discovery Rate of 5%, it revealed 901 and 1098 new domains in Plasmodium and Leishmania proteins, respectively. Manual inspection of part of these predictions shows that it contains several domain families that were missing in the two organisms. All new domain occurrences have been integrated in the EuPathDomains database, along with the GO annotations that can be deduced.

  1. Identification of divergent protein domains by combining HMM-HMM comparisons and co-occurrence detection.

    Science.gov (United States)

    Ghouila, Amel; Florent, Isabelle; Guerfali, Fatma Zahra; Terrapon, Nicolas; Laouini, Dhafer; Yahia, Sadok Ben; Gascuel, Olivier; Bréhélin, Laurent

    2014-01-01

    Identification of protein domains is a key step for understanding protein function. Hidden Markov Models (HMMs) have proved to be a powerful tool for this task. The Pfam database notably provides a large collection of HMMs which are widely used for the annotation of proteins in sequenced organisms. This is done via sequence/HMM comparisons. However, this approach may lack sensitivity when searching for domains in divergent species. Recently, methods for HMM/HMM comparisons have been proposed and proved to be more sensitive than sequence/HMM approaches in certain cases. However, these approaches are usually not used for protein domain discovery at a genome scale, and the benefit that could be expected from their utilization for this problem has not been investigated. Using proteins of P. falciparum and L. major as examples, we investigate the extent to which HMM/HMM comparisons can identify new domain occurrences not already identified by sequence/HMM approaches. We show that although HMM/HMM comparisons are much more sensitive than sequence/HMM comparisons, they are not sufficiently accurate to be used as a standalone complement of sequence/HMM approaches at the genome scale. Hence, we propose to use domain co-occurrence--the general domain tendency to preferentially appear along with some favorite domains in the proteins--to improve the accuracy of the approach. We show that the combination of HMM/HMM comparisons and co-occurrence domain detection boosts protein annotations. At an estimated False Discovery Rate of 5%, it revealed 901 and 1098 new domains in Plasmodium and Leishmania proteins, respectively. Manual inspection of part of these predictions shows that it contains several domain families that were missing in the two organisms. All new domain occurrences have been integrated in the EuPathDomains database, along with the GO annotations that can be deduced.

  2. Modulation of catalytic activity in multi-domain protein tyrosine phosphatases.

    Directory of Open Access Journals (Sweden)

    Lalima L Madan

    Full Text Available Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1 domains, while the membrane-distal (D2 domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A. While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.

  3. A novel domain-based method for predicting the functional classes of proteins

    Institute of Scientific and Technical Information of China (English)

    YU Xiaojing; LIN Jiancheng; SHI Tieliu; LI Yixue

    2004-01-01

    Prediction of protein functions from known genomic sequences is an important mission of bioinformatics. One approach is to classify proteins into functional categories. We have therefore developed a method based on protein domain composition and the maximum likelihood estimation (MLE) algorithm to classify proteins according to functions. Using the Saccharomyces cerevisiae genome, we compared the effectiveness of the MLE approach with that of an intuitive and simple method. The MLE method outperformed the simple method, achieving an estimated specificity of 75.45% and an estimated sensitivity of 40.26%. These results indicate that domain is an important feature of proteins and is closely related to protein function.

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

    Science.gov (United States)

    Allan, Rudi Kenneth; Ratajczak, Thomas

    2011-07-01

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

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

    Science.gov (United States)

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

    2017-01-10

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

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

    Science.gov (United States)

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

    2012-02-01

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

  7. The Evolutionary Relationship of the Domain Architectures in the RhoGEF-containing Proteins

    Institute of Scientific and Technical Information of China (English)

    Qing-Lan Sun; Hong-Jun Zhou; Kui Lin

    2005-01-01

    Domain insertions and deletions lead to variations in the domain architectures of the proteins from their common ancestor. In this work, we investigated four groups of the RhoGEF-containing proteins from different organisms with domain architectures RhoGEF-PH-SH3, SH3-RhoGEF-PH, RhoGEF-PH, and SH3-RhoGEF defined in the Pfam database. The phylogenetic trees were constructed using each individual domain and/or the combinations of all the domains. The phylogenetic analysis suggests that RhoGEF-PH-SH3 and SH3-RhoGEF-PH might have evolved from RhoGEF-PH through the insertion of SH3 independently, while SH3-RhoGEF of proteins in fruit fly might have evolved from SH3-RhoGEF-PH by the degeneration of PH domain.

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

    Energy Technology Data Exchange (ETDEWEB)

    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 (Helsinki); (Penn)

    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.

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

    Science.gov (United States)

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

    2014-03-01

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

  10. Molecular basis for TPR domain-mediated regulation of protein phosphatase 5.

    Science.gov (United States)

    Yang, Jing; Roe, S Mark; Cliff, Matthew J; Williams, Mark A; Ladbury, John E; Cohen, Patricia T W; Barford, David

    2005-01-12

    Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C-terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR-mediated phosphatase inhibition and Hsp90- and arachidonic acid-induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C-terminal alphaJ helix that contacts a region of the Hsp90-binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR-phosphatase domain interface.

  11. TIR-domain-containing protein repertoire of nine anthozoan species reveals coral-specific expansions and uncharacterized proteins.

    Science.gov (United States)

    Poole, Angela Z; Weis, Virginia M

    2014-10-01

    The intracellular toll/interleukin-1 receptor (TIR) domain plays an important role in vertebrate immunity, but the evolution and function of invertebrate TIR-domain-containing proteins is not fully understood. This study characterized and compared the TIR-domain-containing protein repertoire of nine cnidarians in class Anthozoa. A diverse set of proteins, including MyD88 (myeloid differentiation primary response protein 88), toll-like receptor (TLR)-like, interleukin-1 receptor (IL-1R)-like, and TIR-only proteins are present in the species surveyed. Increased numbers of TIR-only proteins were observed in corals compared to anemones, especially in the Acroporid and Pocilloporid coral families. This expansion could be linked to diversity of the microbial community on or in hosts and managing both positive and negative associations. Phylogenetic analysis indicates there are two groups of proteins with IL-1R-like domain architecture in anthozoans that potentially evolved independently of the vertebrate family. Bacterial-like TIR_2 domain proteins are also present, including one sequence with novel domain architecture. Overall this work promotes a better understanding of the anthozoan immune repertoire, which is important in the context learning about ancestral immune pathways and host-microbe interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    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.

  14. Chaperone ligand-discrimination by the TPR-domain protein Tah1.

    Science.gov (United States)

    Millson, Stefan H; Vaughan, Cara K; Zhai, Chao; Ali, Maruf M U; Panaretou, Barry; Piper, Peter W; Pearl, Laurence H; Prodromou, Chrisostomos

    2008-07-15

    Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

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

    Directory of Open Access Journals (Sweden)

    Maggie P Wear

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Tong, Junsen; Yang, Huiseon [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Eom, Soo Hyun [School of Life Sciences, Steitz Center for Structural Biology, and Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of); Chun, ChangJu, E-mail: cchun1130@jnu.ac.kr [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Im, Young Jun, E-mail: imyoungjun@jnu.ac.kr [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

    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.

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

    Science.gov (United States)

    Neuvonen, Maarit; Ahola, Tero

    2009-01-01

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

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

    Science.gov (United States)

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

    2015-09-14

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

  19. Yes-associated protein and p53-binding protein-2 interact through their WW and SH3 domains.

    Science.gov (United States)

    Espanel, X; Sudol, M

    2001-04-27

    To understand the role of the Yes-associated protein (YAP), binding partners of its WW1 domain were isolated by a yeast two-hybrid screen. One of the interacting proteins was identified as p53-binding protein-2 (p53BP-2). YAP and p53BP-2 interacted in vitro and in vivo using their WW1 and SH3 domains, respectively. The YAP WW1 domain bound to the YPPPPY motif of p53BP-2, whereas the p53BP-2 SH3 domain interacted with the VPMRLR sequence of YAP, which is different from other known SH3 domain-binding motifs. By mutagenesis, we showed that this unusual SH3 domain interaction was due to the presence of three consecutive tryptophans located within the betaC strand of the SH3 domain. A point mutation within this triplet, W976R, restored the binding selectivity to the general consensus sequence for SH3 domains, the PXXP motif. A constitutively active form of c-Yes was observed to decrease the binding affinity between YAP and p53BP-2 using chloramphenicol acetyltransferase/enzyme-linked immunosorbent assay, whereas the overexpression of c-Yes did not modify this interaction. Since overexpression of an activated form of c-Yes resulted in tyrosine phosphorylation of p53BP-2, we propose that the p53BP-2 phosphorylation, possibly in the WW1 domain-binding motif, might negatively regulate the YAP.p53BP-2 complex.

  20. Characterization of a novel prokaryotic GDP dissociation inhibitor domain from the G protein coupled membrane protein FeoB.

    Science.gov (United States)

    Eng, Edward T; Jalilian, Amir R; Spasov, Krasimir A; Unger, Vinzenz M

    2008-01-25

    The FeoB family of membrane embedded G proteins are involved with high affinity Fe(II) uptake in prokaryotes. Here, we report that FeoB harbors a novel GDP dissociation inhibitor-like domain that specifically stabilizes GDP-binding through an interaction with the switch I region of the G protein. We show that the stabilization of GDP binding is conserved between species despite a high degree of sequence variability in their guanine nucleotide dissociation inhibitor (GDI)-like domains, and demonstrate that the presence of the membrane embedded domain increases GDP-binding affinity roughly 150-fold over the level accomplished by action of the GDI-like domain alone. To our knowledge, this is the first example for a prokaryotic GDI, targeting a bacterial G protein-coupled membrane process. Our findings suggest that Fe(II) uptake in bacteria involves a G protein regulatory pathway reminiscent of signaling mechanisms found in higher-order organisms.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  2. An expanded view of the protein folding landscape of PDZ domains

    DEFF Research Database (Denmark)

    Hultqvist, Greta; Pedersen, Søren W; Chi, Celestine N.;

    2012-01-01

    Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting...... for the folding reaction under different conditions, as seen for the PDZ protein domain family. We have here analyzed the folding kinetics of two PDZ domains and found that a previously undetected third transition state is rate limiting under conditions that stabilize the native state relative to the denatured...

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Tom G Richardson

    Full Text Available 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.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.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.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.

  5. Protein complex prediction via improved verification methods using constrained domain-domain matching.

    Science.gov (United States)

    Zhao, Yang; Hayashida, Morihiro; Nacher, Jose C; Nagamochi, Hiroshi; Akutsu, Tatsuya

    2012-01-01

    Identification of protein complexes within protein-protein interaction networks is one of the important objectives in functional genomics. Ozawa et al. proposed a verification method of protein complexes by introducing a structural constraint. In this paper, we propose an improved integer programming-based method based on the idea that a candidate complex should not be divided into many small complexes, and combination methods with maximal components and extreme sets. The results of computational experiments suggest that our methods outperform the method by Ozawa et al. We prove that the verification problems are NP-hard, which justifies the use of integer programming.

  6. Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.

    Directory of Open Access Journals (Sweden)

    Tobias Haase

    Full Text Available BACKGROUND: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC Förster resonance energy transfer (FRET was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC beta1 and alpha subunits. METHODOLOGY/PRINCIPAL FINDINGS: Cyan fluorescent protein (CFP was used as FRET donor and yellow fluorescent protein (YFP as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged alpha subunits with the carboxy-terminally tagged beta1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the alpha subunits is close to the carboxy-terminus of the beta1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. CONCLUSIONS/SIGNIFICANCE: Based on the ability of an amino-terminal fragment of the beta1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alphacatbetacat, Winger and Marletta (Biochemistry 2005, 44:4083-90 have proposed a direct interaction of the amino-terminal region of beta1 with the catalytic domains. In support of such a concept of "trans" regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed "fluorescent-conjoined" sGC's by fusion of the alpha amino-terminus to the beta1 carboxy-terminus leading to a

  7. Npw38, a novel nuclear protein possessing a WW domain capable of activating basal transcription.

    Science.gov (United States)

    Komuro, A; Saeki, M; Kato, S

    1999-05-01

    We have found a novel cDNA encoding a 265 amino acid protein possessing a WW domain in our full-length cDNA bank. The WW domain was sandwiched between an acidic region and an acidic-basic amino acid repetitive region. In vitro transcription/translation of the cDNA produced a 38 kDa product that was also found in the cell lysate by western blot analysis. Thus this protein is named the nuclear protein containing a WW domain with a molecular mass of 38 kDa, Npw38. Immunofluorescence studies and expression of a fusion protein to a green fluorescent protein revealed that this protein is localized in the nucleus. Npw38 was shown to be capable of binding to a poly(rG) resin. Interestingly, the WW domain of Npw38 was found to function as a transcriptional activator in CHO cells using the GAL4 DNA-binding fusion system. Furthermore, the WW domains of human YAP and Pin1 were demonstrated to have a similar transcription-promoting activity. Combined mutation of the conserved first and second Trp residues and a hydrophobic triplet of TyrTyrTrp in the WW domain of Npw38 abolished the transcription-promoting activity, but single mutations of these sites did not. These results suggest that some WW domains potentially possess transcription-promoting activity in mammalian cells.

  8. An update on the LIM and SH3 domain protein 1 (LASP1): a versatile structural, signaling, and biomarker protein

    OpenAIRE

    2014-01-01

    The gene encoding the LIM and SH3 domain protein (LASP1) was cloned two decades ago from a cDNA library of breast cancer metastases. As the first protein of a class comprising one N-terminal LIM and one C-terminal SH3 domain, LASP1 founded a new LIM-protein subfamily of the nebulin group. Since its discovery LASP1 proved to be an extremely versatile protein because of its exceptional structure allowing interaction with various binding partners, its ubiquitous expression in normal tissues, alb...

  9. The CBS domain protein MJ0729 of Methanocaldococcus jannaschii binds DNA.

    Science.gov (United States)

    Aguado-Llera, David; Oyenarte, Iker; Martínez-Cruz, Luis Alfonso; Neira, José L

    2010-11-05

    The cystathionine beta-synthase (CBS) domains function as regulatory motifs in several proteins. Elucidating how CBS domains exactly work is relevant because several genetic human diseases have been associated with mutations in those motifs. Here, we show, for the first time, that a CBS domain binds calf-thymus DNA and E-boxes recognized by transcription factors. We have carried out the DNA-binding characterization of the CBS domain protein MJ0729 from Methanocaldococcus jannaschii by biochemical and spectroscopic techniques. Binding induces conformational changes in the protein, and involves the sole tryptophan residue. The apparent dissociation constant for the E-boxes is ∼10 μM. These results suggest that CBS domains might interact with DNA. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Heintz, Udo; Meinhart, Anton; Winkler, Andreas, E-mail: andreas.winkler@mpimf-heidelberg.mpg.de [Max Planck Institute for Medical Research, Heidelberg (Germany)

    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

  11. CATHEDRAL: a fast and effective algorithm to predict folds and domain boundaries from multidomain protein structures.

    Directory of Open Access Journals (Sweden)

    Oliver C Redfern

    2007-11-01

    Full Text Available 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

  12. 3DSwap: Curated knowledgebase of proteins involved in 3D domain swapping

    KAUST Repository

    Shameer, Khader

    2011-09-29

    Three-dimensional domain swapping is a unique protein structural phenomenon where two or more protein chains in a protein oligomer share a common structural segment between individual chains. This phenomenon is observed in an array of protein structures in oligomeric conformation. Protein structures in swapped conformations perform diverse functional roles and are also associated with deposition diseases in humans. We have performed in-depth literature curation and structural bioinformatics analyses to develop an integrated knowledgebase of proteins involved in 3D domain swapping. The hallmark of 3D domain swapping is the presence of distinct structural segments such as the hinge and swapped regions. We have curated the literature to delineate the boundaries of these regions. In addition, we have defined several new concepts like \\'secondary major interface\\' to represent the interface properties arising as a result of 3D domain swapping, and a new quantitative measure for the \\'extent of swapping\\' in structures. The catalog of proteins reported in 3DSwap knowledgebase has been generated using an integrated structural bioinformatics workflow of database searches, literature curation, by structure visualization and sequence-structure-function analyses. The current version of the 3DSwap knowledgebase reports 293 protein structures, the analysis of such a compendium of protein structures will further the understanding molecular factors driving 3D domain swapping. The Author(s) 2011.

  13. The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin β1 endocytosis.

    Science.gov (United States)

    Teckchandani, Anjali; Mulkearns, Erin E; Randolph, Timothy W; Toida, Natalie; Cooper, Jonathan A

    2012-08-01

    Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin β1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain-binding sites. This mutant localized to clathrin structures with integrin β1, AP2, and reduced amounts of Eps15. Of interest, although integrin β1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin β1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin β1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.

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

    Science.gov (United States)

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

    2014-02-21

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

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

    Science.gov (United States)

    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.

  16. Point 5 - civil engineering : surface site, SX, pit, buildings, PX

    CERN Multimedia

    2000-01-01

    > de 00 :00 à 13 :15 le 06 janvier 2000 - vues extérieures du montage du bâtiment SX, du chantier, du puit PX dont le pourtour est congelé- installation des tubes de congélation du sol- bâtiments de la Cryogénie, avec mur anti-bruit- fond des puits creusement et évacuation des déblais > de 13 :17 à 17 :38 le 10 février 2000 - avancement du montage du SX - vues générales du chantier en surface > de 13 :40 à 24 :00 le 10 février 2000 - bâtiment SX vide, fin d’installation des ponts roulants - ferraillage de la dalle d’un bâtiment

  17. MATRIX EQUATION AXB = E WITH PX = sXP CONSTRAINT

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The matrix equation AXB = E with the constraint PX = sXP is considered, where P is a given Hermitian matrix satisfying P2 = I and s = ±1. By an eigenvalue decomposition of P, the constrained problem can be equivalently transformed to a well-known unconstrained problem of matrix equation whose coefficient matrices contain the corresponding eigenvector, and hence the constrained problem can be solved in terms of the eigenvectors of P. A simple and eigenvector-free formula of the general solutions to the constrained problem by generalized inverses of the coefficient matrices A and B is presented.Moreover, a similar problem of the matrix equation with generalized constraint is discussed.

  18. OPUS-Dom: applying the folding-based method VECFOLD to determine protein domain boundaries.

    Science.gov (United States)

    Wu, Yinghao; Dousis, Athanasios D; Chen, Mingzhi; Li, Jialin; Ma, Jianpeng

    2009-01-30

    In this article, we present a de novo method for predicting protein domain boundaries, called OPUS-Dom. The core of the method is a novel coarse-grained folding method, VECFOLD, which constructs low-resolution structural models from a target sequence by folding a chain of vectors representing the predicted secondary-structure elements. OPUS-Dom generates a large ensemble of folded structure decoys by VECFOLD and labels the domain boundaries of each decoy by a domain parsing algorithm. Consensus domain boundaries are then derived from the statistical distribution of the putative boundaries and three empirical sequence-based domain profiles. OPUS-Dom generally outperformed several state-of-the-art domain prediction algorithms over various benchmark protein sets. Even though each VECFOLD-generated structure contains large errors, collectively these structures provide a more robust delineation of domain boundaries. The success of OPUS-Dom suggests that the arrangement of protein domains is more a consequence of limited coordination patterns per domain arising from tertiary packing of secondary-structure segments, rather than sequence-specific constraints.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

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

  20. The 10 kDa domain of human erythrocyte protein 4.1 binds the Plasmodium falciparum EBA-181 protein

    Directory of Open Access Journals (Sweden)

    Coetzer Theresa L

    2006-11-01

    Full Text Available Abstract Background Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181 is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise molecular interaction between the two proteins. Methods 4.1R structural domains (30, 16, 10 and 22 kDa domain and the 4.1R binding region in EBA-181 were synthesized in specific Escherichia coli strains as recombinant proteins and purified using magnetic bead technology. Recombinant proteins were subsequently used in blot-overlay and histidine pull-down assays to determine the binding domain in 4.1R. Results Blot overlay and histidine pull-down experiments revealed specific interaction between the 10 kDa domain of 4.1R and EBA-181. Binding was concentration dependent as well as saturable and was abolished by heat denaturation of 4.1R. Conclusion The interaction of EBA-181 with the highly conserved 10 kDa domain of 4.1R provides new insight into the molecular mechanisms utilized by P. falciparum during erythrocyte entry. The results highlight the potential multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite's life cycle.

  1. Reassessing Domain Architecture Evolution of Metazoan Proteins: The Contribution of Different Evolutionary Mechanisms

    Directory of Open Access Journals (Sweden)

    Laszlo Patthy

    2011-08-01

    Full Text Available In the accompanying papers we have shown that sequence errors of public databases and confusion of paralogs and epaktologs (proteins that are related only through the independent acquisition of the same domain types significantly distort the picture that emerges from comparison of the domain architecture (DA of multidomain Metazoan proteins since they introduce a strong bias in favor of terminal over internal DA change. The issue of whether terminal or internal DA changes occur with greater probability has very important implications for the DA evolution of multidomain proteins since gene fusion can add domains only at terminal positions, whereas domain-shuffling is capable of inserting domains both at internal and terminal positions. As a corollary, overestimation of terminal DA changes may be misinterpreted as evidence for a dominant role of gene fusion in DA evolution. In this manuscript we show that in several recent studies of DA evolution of Metazoa the authors used databases that are significantly contaminated with incomplete, abnormal and mispredicted sequences (e.g., UniProtKB/TrEMBL, EnsEMBL and/or the authors failed to separate paralogs and epaktologs, explaining why these studies concluded that the major mechanism for gains of new domains in metazoan proteins is gene fusion. In contrast with the latter conclusion, our studies on high quality orthologous and paralogous Swiss-Prot sequences confirm that shuffling of mobile domains had a major role in the evolution of multidomain proteins of Metazoa and especially those formed in early vertebrates.

  2. Molecular evolution of Cide family proteins: Novel domain formation in early vertebrates and the subsequent divergence

    Directory of Open Access Journals (Sweden)

    Sun Zhirong

    2008-05-01

    Full Text Available Abstract Background Cide family proteins including Cidea, Cideb and Cidec/Fsp27, contain an N-terminal CIDE-N domain that shares sequence similarity to the N-terminal CAD domain (NCD of DNA fragmentation factors Dffa/Dff45/ICAD and Dffb/Dff40/CAD, and a unique C-terminal CIDE-C domain. We have previously shown that Cide proteins are newly emerged regulators closely associated with the development of metabolic diseases such as obesity, diabetes and liver steatosis. They modulate many metabolic processes such as lipolysis, thermogenesis and TAG storage in brown adipose tissue (BAT and white adipose tissue (WAT, as well as fatty acid oxidation and lipogenesis in the liver. Results To understand the evolutionary process of Cide proteins and provide insight into the role of Cide proteins as potential metabolic regulators in various species, we searched various databases and performed comparative genomic analysis to study the sequence conservation, genomic structure, and phylogenetic tree of the CIDE-N and CIDE-C domains of Cide proteins. As a result, we identified signature sequences for the N-terminal region of Dffa, Dffb and Cide proteins and CIDE-C domain of Cide proteins, and observed that sequences homologous to CIDE-N domain displays a wide phylogenetic distribution in species ranging from lower organisms such as hydra (Hydra vulgaris and sea anemone (Nematostella vectensis to mammals, whereas the CIDE-C domain exists only in vertebrates. Further analysis of their genomic structures showed that although evolution of the ancestral CIDE-N domain had undergone different intron insertions to various positions in the domain among invertebrates, the genomic structure of Cide family in vertebrates is stable with conserved intron phase. Conclusion Based on our analysis, we speculate that in early vertebrates CIDE-N domain was evolved from the duplication of NCD of Dffa. The CIDE-N domain somehow acquired the CIDE-C domain that was formed around the

  3. Direct interaction of the N-terminal domain of ribosomal protein S1 with protein S2 in Escherichia coli.

    Science.gov (United States)

    Byrgazov, Konstantin; Manoharadas, Salim; Kaberdina, Anna C; Vesper, Oliver; Moll, Isabella

    2012-01-01

    Despite of the high resolution structure available for the E. coli ribosome, hitherto the structure and localization of the essential ribosomal protein S1 on the 30 S subunit still remains to be elucidated. It was previously reported that protein S1 binds to the ribosome via protein-protein interaction at the two N-terminal domains. Moreover, protein S2 was shown to be required for binding of protein S1 to the ribosome. Here, we present evidence that the N-terminal domain of S1 (amino acids 1-106; S1(106)) is necessary and sufficient for the interaction with protein S2 as well as for ribosome binding. We show that over production of protein S1(106) affects E. coli growth by displacing native protein S1 from its binding pocket on the ribosome. In addition, our data reveal that the coiled-coil domain of protein S2 (S2α(2)) is sufficient to allow protein S1 to bind to the ribosome. Taken together, these data uncover the crucial elements required for the S1/S2 interaction, which is pivotal for translation initiation on canonical mRNAs in gram-negative bacteria. The results are discussed in terms of a model wherein the S1/S2 interaction surface could represent a possible target to modulate the selectivity of the translational machinery and thereby alter the translational program under distinct conditions.

  4. NMR assignments for the insertion domain of bacteriophage Sf6 coat protein.

    Science.gov (United States)

    Tripler, Therese N; Teschke, Carolyn M; Alexandrescu, Andrei T

    2017-04-01

    The P22 bacteriophage group is a subgroup of the λ phage supercluster, comprised of the three major sequence types Sf6, P22, and CUS-3, based on their capsid proteins. Our goal is to investigate the extent to which structure-function relationships are conserved for the viral coat proteins and I-domains in this subgroup. Sf6 is a phage that infects the human pathogen Shigella flexneri. The coat protein of Sf6 assembles into a procapsid, which further undergoes maturation during DNA packaging into an infectious virion. The Sf6 coat protein contains a genetically inserted domain, termed the I-domain, similar to the ones present in the P22 and CUS-3 coat proteins. Based on the P22 example, I-domains play important functional roles in capsid assembly, stability, viability, and size-determination. Here we report the (1)H, (15)N, and (13)C chemical shift assignments for the I-domain of the Sf6 phage coat protein. Chemical shift-based secondary structure prediction and hydrogen-bond patterns from a long-range HNCO experiment indicate that the Sf6 I-domain adopts a 6-stranded β-barrel fold like those of P22 and CUS-3 but with important differences, including the absence of the D-loop that is critical for capsid assembly and the addition of a novel disordered loop region.

  5. Characterization of a novel protein-binding module--the WW domain.

    Science.gov (United States)

    Sudol, M; Chen, H I; Bougeret, C; Einbond, A; Bork, P

    1995-08-01

    We have identified, characterized and cloned human, mouse and chicken cDNA of a novel protein that binds to the Src homology domain 3 (SH3) of the Yes proto-oncogene product. We subsequently named it YAP for Yes-associated protein. Analysis of the YAP sequence revealed a protein module that was found in various structural, regulatory and signaling molecules. Because one of the prominent features of this sequence motif is the presence of two conserved tryptophans (W), we named it the WW domain. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP which we named WBP-1 and WBP-2. Peptide sequence comparison between the two partial clones revealed a homologous proline-rich region. Binding assays and site-specific mutagenesis have shown that the proline-rich motif binds with relatively high affinity and specificity to the WW domain of YAP, with a preliminary consensus that is different from the SH3-binding PXXP motif. This suggests that the WW domain has a role in mediating protein-protein interactions via proline-rich regions, similar but distinct from Src homology 3 (SH3) domains. Based on this finding, we hypothesize that additional protein modules exist and that they could be isolated using proline-rich peptides as functional probes.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ananth Prakash

    Full Text Available 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.

  8. Molecular evolution of Cide family proteins: Novel domain formation in early vertebrates and the subsequent divergence

    OpenAIRE

    2008-01-01

    Abstract Background Cide family proteins including Cidea, Cideb and Cidec/Fsp27, contain an N-terminal CIDE-N domain that shares sequence similarity to the N-terminal CAD domain (NCD) of DNA fragmentation factors Dffa/Dff45/ICAD and Dffb/Dff40/CAD, and a unique C-terminal CIDE-C domain. We have previously shown that Cide proteins are newly emerged regulators closely associated with the development of metabolic diseases such as obesity, diabetes and liver steatosis. They modulate many metaboli...

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

    Science.gov (United States)

    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.

  10. Ab initio and homology based prediction of protein domains by recursive neural networks

    Directory of Open Access Journals (Sweden)

    Mooney Catherine

    2009-06-01

    Full Text Available Abstract Background Proteins, especially larger ones, are often composed of individual evolutionary units, domains, which have their own function and structural fold. Predicting domains is an important intermediate step in protein analyses, including the prediction of protein structures. Results We describe novel systems for the prediction of protein domain boundaries powered by Recursive Neural Networks. The systems rely on a combination of primary sequence and evolutionary information, predictions of structural features such as secondary structure, solvent accessibility and residue contact maps, and structural templates, both annotated for domains (from the SCOP dataset and unannotated (from the PDB. We gauge the contribution of contact maps, and PDB and SCOP templates independently and for different ranges of template quality. We find that accurately predicted contact maps are informative for the prediction of domain boundaries, while the same is not true for contact maps predicted ab initio. We also find that gap information from PDB templates is informative, but, not surprisingly, less than SCOP annotations. We test both systems trained on templates of all qualities, and systems trained only on templates of marginal similarity to the query (less than 25% sequence identity. While the first batch of systems produces near perfect predictions in the presence of fair to good templates, the second batch outperforms or match ab initio predictors down to essentially any level of template quality. We test all systems in 5-fold cross-validation on a large non-redundant set of multi-domain and single domain proteins. The final predictors are state-of-the-art, with a template-less prediction boundary recall of 50.8% (precision 38.7% within ± 20 residues and a single domain recall of 80.3% (precision 78.1%. The SCOP-based predictors achieve a boundary recall of 74% (precision 77.1% again within ± 20 residues, and classify single domain proteins as

  11. Evolution of SET-domain protein families in the unicellular and multicellular Ascomycota fungi

    Directory of Open Access Journals (Sweden)

    Moriyama Etsuko N

    2008-07-01

    Full Text Available Abstract Background The evolution of multicellularity is accompanied by the occurrence of differentiated tissues, of organismal developmental programs, and of mechanisms keeping the balance between proliferation and differentiation. Initially, the SET-domain proteins were associated exclusively with regulation of developmental genes in metazoa. However, finding of SET-domain genes in the unicellular yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe suggested that SET-domain proteins regulate a much broader variety of biological programs. Intuitively, it is expected that the numbers, types, and biochemical specificity of SET-domain proteins of multicellular versus unicellular forms would reflect the differences in their biology. However, comparisons across the unicellular and multicellular domains of life are complicated by the lack of knowledge of the ancestral SET-domain genes. Even within the crown group, different biological systems might use the epigenetic 'code' differently, adapting it to organism-specific needs. Simplifying the model, we undertook a systematic phylogenetic analysis of one monophyletic fungal group (Ascomycetes containing unicellular yeasts, Saccharomycotina (hemiascomycetes, and a filamentous fungal group, Pezizomycotina (euascomycetes. Results Systematic analysis of the SET-domain genes across an entire eukaryotic phylum has outlined clear distinctions in the SET-domain gene collections in the unicellular and in the multicellular (filamentous relatives; diversification of SET-domain gene families has increased further with the expansion and elaboration of multicellularity in animal and plant systems. We found several ascomycota-specific SET-domain gene groups; each was unique to either Saccharomycotina or Pezizomycotina fungi. Our analysis revealed that the numbers and types of SET-domain genes in the Saccharomycotina did not reflect the habitats, pathogenicity, mechanisms of sexuality, or the ability to

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

    Science.gov (United States)

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

    2015-06-01

    Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein-albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

  13. Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

    DEFF Research Database (Denmark)

    Pedersen, Søren W; Moran, Griffin E; Sereikaité, Vita

    2016-01-01

    drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C-terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys......PDZ domains are ubiquitous small protein domains that are mediators of numerous protein-protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling-transduction complexes. In recent years, PDZ domains have emerged as novel and exciting...

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

    Science.gov (United States)

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

    2012-09-01

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

  15. A Novel Method for Prediction of Protein Domain Using Distance-Based Maximal Entropy

    Institute of Scientific and Technical Information of China (English)

    Shu-xue Zou; Yan-xin Huang; Yan Wang; Chun-guang Zhou

    2008-01-01

    Detecting the boundaries of protein domains is an important and challenging task in both experimental and computational structural biology. In this paper, a promising method for detecting the domain structure of a protein from sequence information alone is presented. The method is based on analyzing multiple sequence alignments derived from a database search. Multiple measures are defined to quantify the domain information content of each position along the sequence. Then they are combined into a single predictor using support vector machine. What is more important, the domain detection is first taken as an imbalanted data learning problem. A novel undersampling method is proposed on distance-based maximal entropy in the feature space of Support Vector Machine (SVM). The overall precision is about 80%. Simulation results demonstrate that the method can help not only in predicting the complete 3D structure of a protein but also in the machine learning system on general imbalanced datasets.

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Science.gov (United States)

    Mannakee, Brian K; Gutenkunst, Ryan N

    2016-07-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Brian K Mannakee

    2016-07-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2014-10-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Evelyn Walenta

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

  2. Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases

    OpenAIRE

    1996-01-01

    GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchor...

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

    Science.gov (United States)

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

    2017-07-19

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

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

    Science.gov (United States)

    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.

  5. N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

    Directory of Open Access Journals (Sweden)

    Etai Jacob

    2013-04-01

    Full Text Available Computational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain.

  6. Fitting hidden Markov models of protein domains to a target species: application to Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Terrapon Nicolas

    2012-05-01

    Full Text Available Abstract Background Hidden Markov Models (HMMs are a powerful tool for protein domain identification. The Pfam database notably provides a large collection of HMMs which are widely used for the annotation of proteins in new sequenced organisms. In Pfam, each domain family is represented by a curated multiple sequence alignment from which a profile HMM is built. In spite of their high specificity, HMMs may lack sensitivity when searching for domains in divergent organisms. This is particularly the case for species with a biased amino-acid composition, such as P. falciparum, the main causal agent of human malaria. In this context, fitting HMMs to the specificities of the target proteome can help identify additional domains. Results Using P. falciparum as an example, we compare approaches that have been proposed for this problem, and present two alternative methods. Because previous attempts strongly rely on known domain occurrences in the target species or its close relatives, they mainly improve the detection of domains which belong to already identified families. Our methods learn global correction rules that adjust amino-acid distributions associated with the match states of HMMs. These rules are applied to all match states of the whole HMM library, thus enabling the detection of domains from previously absent families. Additionally, we propose a procedure to estimate the proportion of false positives among the newly discovered domains. Starting with the Pfam standard library, we build several new libraries with the different HMM-fitting approaches. These libraries are first used to detect new domain occurrences with low E-values. Second, by applying the Co-Occurrence Domain Discovery (CODD procedure we have recently proposed, the libraries are further used to identify likely occurrences among potential domains with higher E-values. Conclusion We show that the new approaches allow identification of several domain families previously absent in

  7. Insights into common functional domains of tospovirus NSm proteins

    Science.gov (United States)

    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...

  8. Domains of Bacillus thuringiensis crystal proteins involved in insecticidal activity

    NARCIS (Netherlands)

    Bosch, H.J.; Schipper, B.; Kleij, van der H.; Maagd, de R.A.; Stiekema, W.J.

    1994-01-01

    The expected increase in application of Bacillus thuringiensis (Bt) in crop protection makes it necessary to anticipate the development of Bt-resistant insects. To safeguard the long-term use of Bt-based insecticides, we studied the mode of action of Bt crystal proteins. CryIA(b), CryIC and CryIE ar

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

    Directory of Open Access Journals (Sweden)

    Žárský Viktor

    2008-04-01

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

  10. Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis

    Directory of Open Access Journals (Sweden)

    Schlegel Brigitte

    2004-03-01

    Full Text Available Abstract Background High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination. Results 88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D 1H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains were quickly identified as being well folded and suitable for structural analysis. Conclusion The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D 1H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics.

  11. Novel insights into the function of the conserved domain of the CAP superfamily of proteins

    NARCIS (Netherlands)

    Olrichs, N.K.|info:eu-repo/dai/nl/304837571; Helms, J.B.|info:eu-repo/dai/nl/080626742

    2016-01-01

    Members of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) superfamily are found in a remarkable variety of biological species. The presence of a highly conserved CAP domain defines the CAP family members, which in many cases is linked to other functional

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

    Science.gov (United States)

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

    2015-07-23

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

  13. DCD – a novel plant specific domain in proteins involved in development and programmed cell death

    Directory of Open Access Journals (Sweden)

    Doerks Tobias

    2005-07-01

    Full Text Available Abstract Background Recognition of microbial pathogens by plants triggers the hypersensitive reaction, a common form of programmed cell death in plants. These dying cells generate signals that activate the plant immune system and alarm the neighboring cells as well as the whole plant to activate defense responses to limit the spread of the pathogen. The molecular mechanisms behind the hypersensitive reaction are largely unknown except for the recognition process of pathogens. We delineate the NRP-gene in soybean, which is specifically induced during this programmed cell death and contains a novel protein domain, which is commonly found in different plant proteins. Results The sequence analysis of the protein, encoded by the NRP-gene from soybean, led to the identification of a novel domain, which we named DCD, because it is found in plant proteins involved in development and cell death. The domain is shared by several proteins in the Arabidopsis and the rice genomes, which otherwise show a different protein architecture. Biological studies indicate a role of these proteins in phytohormone response, embryo development and programmed cell by pathogens or ozone. Conclusion It is tempting to speculate, that the DCD domain mediates signaling in plant development and programmed cell death and could thus be used to identify interacting proteins to gain further molecular insights into these processes.

  14. Atrophin-1, the DRPLA gene product, interacts with two families of WW domain-containing proteins.

    Science.gov (United States)

    Wood, J D; Yuan, J; Margolis, R L; Colomer, V; Duan, K; Kushi, J; Kaminsky, Z; Kleiderlein, J J; Sharp, A H; Ross, C A

    1998-06-01

    Atrophin-1 contains a polyglutamine repeat, expansion of which is responsible for dentatorubral and pallidoluysian atrophy (DRPLA). The normal function of atrophin-1 is unknown. We have identified five atrophin-1 interacting proteins (AIPs) which bind to atrophin-1 in the vicinity of the polyglutamine tract using the yeast two-hybrid system. Four of the interactions were confirmed using in vitro binding assays. All five interactors contained multiple WW domains. Two are novel. The AIPs can be divided into two distinct classes. AIP1 and AIP3/WWP3 are MAGUK-like multidomain proteins containing a number of protein-protein interaction modules, namely a guanylate kinase-like region, two WW domains, and multiple PDZ domains. AIP2/WWP2, AIP4, and AIP5/WWP1 are highly homologous, each having four WW domains and a HECT domain characteristic of ubiquitin ligases. These interactors are similar to recently isolated huntingtin-interacting proteins, suggesting possible commonality of function between two proteins responsible for very similar diseases.

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

    Directory of Open Access Journals (Sweden)

    Defne Surujon

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  17. Sirt1 carboxyl-domain is an ATP-repressible domain that is transferrable to other proteins

    Science.gov (United States)

    Kang, Hyeog; Oka, Shinichi; Lee, Duck-Yeon; Park, Junhong; Aponte, Angel M.; Jung, Young-Sang; Bitterman, Jacob; Zhai, Peiyong; He, Yi; Kooshapur, Hamed; Ghirlando, Rodolfo; Tjandra, Nico; Lee, Sean B.; Kim, Myung K.; Sadoshima, Junichi; Chung, Jay H.

    2017-01-01

    Sirt1 is an NAD+-dependent protein deacetylase that regulates many physiological functions, including stress resistance, adipogenesis, cell senescence and energy production. Sirt1 can be activated by energy deprivation, but the mechanism is poorly understood. Here, we report that Sirt1 is negatively regulated by ATP, which binds to the C-terminal domain (CTD) of Sirt1. ATP suppresses Sirt1 activity by impairing the CTD's ability to bind to the deacetylase domain as well as its ability to function as the substrate recruitment site. ATP, but not NAD+, causes a conformational shift to a less compact structure. Mutations that prevent ATP binding increase Sirt1's ability to promote stress resistance and inhibit adipogenesis under high-ATP conditions. Interestingly, the CTD can be attached to other proteins, thereby converting them into energy-regulated proteins. These discoveries provide insight into how extreme energy deprivation can impact Sirt1 activity and underscore the complex nature of Sirt1 structure and regulation. PMID:28504272

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

    Science.gov (United States)

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

    2014-09-02

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Freiburger, Lee, E-mail: lee.freiburger@tum.de; Sonntag, Miriam, E-mail: miriam.sonntag@mytum.de; Hennig, Janosch, E-mail: janosch.hennig@helmholtz-muenchen.de [Helmholtz Zentrum München, Institute of Structural Biology (Germany); Li, Jian, E-mail: lijianzhongbei@163.com [Chinese Academy of Sciences, Tianjin Institute of Industrial Biotechnology (China); Zou, Peijian, E-mail: peijian.zou@helmholtz-muenchen.de; Sattler, Michael, E-mail: sattler@helmholtz-muenchen.de [Helmholtz Zentrum München, Institute of Structural Biology (Germany)

    2015-09-15

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

  20. Increased Concentration of Polyvalent Phospholipids in the Adsorption Domain of a Charged Protein

    CERN Document Server

    Haleva, E; Diamant, H; Haleva, Emir; Ben-Tal, Nir; Diamant, Haim

    2004-01-01

    We studied the adsorption of a charged protein onto an oppositely charged membrane, composed of mobile phospholipids of differing valence, using a statistical-thermodynamical approach. A two-block model was employed, one block corresponding to the protein-affected region on the membrane, referred to as the adsorption domain, and the other to the unaffected remainder of the membrane. We calculated the protein-induced lipid rearrangement in the adsorption domain as arising from the interplay between the electrostatic interactions in the system and the mixing entropy of the lipids. Equating the electrochemical potentials of the lipids in the two blocks yields an expression for the relations among the various lipid fractions in the adsorption domain, indicating a sensitive (exponential) dependence on lipid valence. This expression is a result of the two-block picture but does not depend on further details of the protein-membrane interaction. We subsequently calculated the lipid fractions themselves using the Pois...

  1. Inhibition of Tcf3 binding by I-mfa domain proteins.

    Science.gov (United States)

    Snider, L; Thirlwell, H; Miller, J R; Moon, R T; Groudine, M; Tapscott, S J

    2001-03-01

    We have determined that I-mfa, an inhibitor of several basic helix-loop-helix (bHLH) proteins, and XIC, a Xenopus ortholog of human I-mf domain-containing protein that shares a highly conserved cysteine-rich C-terminal domain with I-mfa, inhibit the activity and DNA binding of the HMG box transcription factor XTcf3. Ectopic expression of I-mfa or XIC in early Xenopus embryos inhibited dorsal axis specification, the expression of the Tcf3/beta-catenin-regulated genes siamois and Xnr3, and the ability of beta-catenin to activate reporter constructs driven by Lef/Tcf binding sites. I-mfa domain proteins can regulate both the Wnt signaling pathway and a subset of bHLH proteins, possibly coordinating the activities of these two critical developmental pathways.

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

    Science.gov (United States)

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

    2015-09-01

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

  3. Integrating folding kinetics and protein function: biphasic kinetics and dual binding specificity in a WW domain.

    Science.gov (United States)

    Karanicolas, John; Brooks, Charles L

    2004-03-09

    Because of the association of beta-sheet formation with the initiation and propagation of amyloid diseases, model systems have been sought to further our understanding of this process. WW domains have been proposed as one such model system. Whereas the folding of the WW domains from human Yes-associated protein (YAP) and Pin have been shown to obey single-exponential kinetics, the folding of the WW domain from formin-binding protein (FBP) 28 has been shown to proceed via biphasic kinetics. From an analysis of free-energy landscapes from atomic-level molecular dynamics simulations, the biphasic folding kinetics observed in the FBP WW domain may be traced to the ability of this WW domain to adopt two slightly different forms of packing in its hydrophobic core. This conformational change is propagated along the peptide backbone and affects the position of a tryptophan residue shown in other WW domains to play a key role in binding. The WW domains of Pin and YAP do not support more than one type of packing each, leading to monophasic folding kinetics. The ability of the FBP WW domain to assume two different types of packing may, in turn, explain the capacity of this WW domain to bind two classes of ligand, a property that is not shared by other WW domains. These findings lead to the hypothesis that lability with respect to conformations separated by an observable barrier as a requirement for function is incompatible with the ability of a protein to fold via single-exponential kinetics.

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

    Directory of Open Access Journals (Sweden)

    Sofiya Fedosyuk

    2016-12-01

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

  5. Structure and function of the regulatory HRDC domain from human Bloom syndrome protein.

    Science.gov (United States)

    Kim, Young Mee; Choi, Byong-Seok

    2010-11-01

    The helicase and RNaseD C-terminal (HRDC) domain, conserved among members of the RecQ helicase family, regulates helicase activity by virtue of variations in its surface residues. The HRDC domain of Bloom syndrome protein (BLM) is known as a critical determinant of the dissolution function of double Holliday junctions by the BLM-Topoisomerase IIIα complex. In this study, we determined the solution structure of the human BLM HRDC domain and characterized its DNA-binding activity. The BLM HRDC domain consists of five α-helices with a hydrophobic 3(10)-helical loop between helices 1 and 2 and an extended acidic surface comprising residues in helices 3-5. The BLM HRDC domain preferentially binds to ssDNA, though with a markedly low binding affinity (K(d) ∼100 μM). NMR chemical shift perturbation studies suggested that the critical DNA-binding residues of the BLM HRDC domain are located in the hydrophobic loop and the N-terminus of helix 2. Interestingly, the isolated BLM HRDC domain had quite different DNA-binding modes between ssDNA and Holliday junctions in electrophoretic mobility shift assay experiments. Based on its surface charge separation and DNA-binding properties, we suggest that the HRDC domain of BLM may be adapted for a unique function among RecQ helicases--that of bridging protein and DNA interactions.

  6. PAS domain of the deduced Org35 protein mediates the interaction with NifA

    Institute of Scientific and Technical Information of China (English)

    TU Ran; CUI Yanhua; CHEN Sanfeng; LI Jilun

    2006-01-01

    NifA in Azospirillum brasilense plays a key role in regulating the synthesis of nitrogenase in response to ammonia and oxygen available. Recently,our laboratory has identified four clones, whose gene prodcuts interact with NifA, from A. brasilense Sp7genomic libraries by using the yeast two-hybrid system with NifA as bait. We are interested in clone S35,one of the four clones, because it contains a PAS-domain coding region. The entire open reading frame (ORF) for the PAS domain-containing protein was isolated and designated as org35 here. org35gene is 2211-bp long and encodes a protein of 736aa with a predicted molecular weight of about 78.4 kD.The predicted amino acid sequence of org35 has similarity to some two-component sensor kinase/response regulator hybrids of bacteria. Structural analyses showed that Org35 comprises at least three discrete conserved domains: the N-terminal PAS, the central histidine protein kinase (HPK) and the C-terminal response regulator (RR). The PAS domain of the deduced Org35 protein was found to interact directly with NifA, but the central HPK and the C-terminal RR domains of Org35 were not. These results indicated that interaction between NifA and Org35 was mediated by PAS domain.

  7. HMM-FRAME: accurate protein domain classification for metagenomic sequences containing frameshift errors

    Directory of Open Access Journals (Sweden)

    Sun Yanni

    2011-05-01

    Full Text Available Abstract Background Protein domain classification is an important step in metagenomic annotation. The state-of-the-art method for protein domain classification is profile HMM-based alignment. However, the relatively high rates of insertions and deletions in homopolymer regions of pyrosequencing reads create frameshifts, causing conventional profile HMM alignment tools to generate alignments with marginal scores. This makes error-containing gene fragments unclassifiable with conventional tools. Thus, there is a need for an accurate domain classification tool that can detect and correct sequencing errors. Results We introduce HMM-FRAME, a protein domain classification tool based on an augmented Viterbi algorithm that can incorporate error models from different sequencing platforms. HMM-FRAME corrects sequencing errors and classifies putative gene fragments into domain families. It achieved high error detection sensitivity and specificity in a data set with annotated errors. We applied HMM-FRAME in Targeted Metagenomics and a published metagenomic data set. The results showed that our tool can correct frameshifts in error-containing sequences, generate much longer alignments with significantly smaller E-values, and classify more sequences into their native families. Conclusions HMM-FRAME provides a complementary protein domain classification tool to conventional profile HMM-based methods for data sets containing frameshifts. Its current implementation is best used for small-scale metagenomic data sets. The source code of HMM-FRAME can be downloaded at http://www.cse.msu.edu/~zhangy72/hmmframe/ and at https://sourceforge.net/projects/hmm-frame/.

  8. Bioinformatic identification of genes encoding C1q-domain containing proteins in zebrafish

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    C1q is the first subcomponent of classical pathway in the complement system and a major link between innate and acquired immunities. The globular (gC1q) domain similar with C1q was also found in many non-complement C1q-domain-containing (C1qDC) proteins which have similar crystal structure to that of the multifunctional tumor necrosis factor (TNF) ligand family, and also have diverse functions. In this study, we identified a total of 52 independent gene sequences encoding C1q-domain-containing proteins through comprehensive searches of zebrafish genome, cDNA and EST databases. In comparison to 31 orthologous genes in human and different numbers in other species, a significant selective pressure was suggested during vertebrate evolution. Domain organization of C1q-domain-containing (C1qDC) proteins mainly includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 11 highly conserved residues within the C1q domain, among which 2 are invariant within the zebrafish gene set. A more extensive database searches also revealed homologous C1qDC proteins in other vertebrates, invertebrates and even bacterium, but no homologous sequences for encoding C1qDC proteins were found in many species that have a more recent evolutionary history with zebrafish. Therefore, further studies on C1q-domain-containing genes among different species will help us understand evolutionary mechanism of innate and acquired immunities.

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

    Science.gov (United States)

    Walkup, Ward G; Kennedy, Mary B

    2014-06-01

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

  10. Structural Rigidity of Paranemic (PX) and Juxtapose (JX) DNA Nanostructures

    CERN Document Server

    Santosh, Mogurampelly; 10.1016/j.bpj.2011.08.007

    2011-01-01

    Crossover motifs are integral components for designing DNA based nanostructures and nanomechanical devices due to their enhanced rigidity compared to the normal B-DNA. Although the structural rigidity of the double helix B-DNA has been investigated extensively using both experimental and theoretical tools, to date there is no quantitative information about structural rigidity and the mechanical strength of parallel crossover DNA motifs. We have used fully atomistic molecular dynamics simulations in explicit solvent to get the force-extension curve of parallel DNA nanostructures to characterize their mechanical rigidity. In the presence of mono-valent Na+ ions, we find that the stretch modulus (\\gamma_1) of the paranemic crossover (PX) and its topo-isomer JX DNA structure is significantly higher (~ 30%) compared to normal B-DNA of the same sequence and length. However, this is in contrast to the original expectation that these motifs are almost twice rigid compared to the double-stranded B-DNA. When the DNA mo...

  11. Multicanonical Molecular Dynamics Simulations of the N-terminal Domain of Protein L9

    Science.gov (United States)

    Yaşar, Fatih; Jiang, Ping; Hansmann, Ulrich H. E.

    2014-01-01

    We describe multicanonical molecular dynamic simulations of the N-terminal domain of the protein L9. Analyzing free energy landscapes and thermal ordering, we propose a possible folding mechanism for the protein. By comparing our results with that of molecular dynamics runs of the protein at constant temperature, we find that multicanonical molecular dynamics leads to orders of magnitude higher sampling of folding transitions. PMID:25253918

  12. Cellular prion protein expression is not regulated by the Alzheimer's amyloid precursor protein intracellular domain.

    Directory of Open Access Journals (Sweden)

    Victoria Lewis

    Full Text Available There is increasing evidence of molecular and cellular links between Alzheimer's disease (AD and prion diseases. The cellular prion protein, PrP(C, modulates the post-translational processing of the AD amyloid precursor protein (APP, through its inhibition of the β-secretase BACE1, and oligomers of amyloid-β bind to PrP(C which may mediate amyloid-β neurotoxicity. In addition, the APP intracellular domain (AICD, which acts as a transcriptional regulator, has been reported to control the expression of PrP(C. Through the use of transgenic mice, cell culture models and manipulation of APP expression and processing, this study aimed to clarify the role of AICD in regulating PrP(C. Over-expression of the three major isoforms of human APP (APP(695, APP(751 and APP(770 in cultured neuronal and non-neuronal cells had no effect on the level of endogenous PrP(C. Furthermore, analysis of brain tissue from transgenic mice over-expressing either wild type or familial AD associated mutant human APP revealed unaltered PrP(C levels. Knockdown of endogenous APP expression in cells by siRNA or inhibition of γ-secretase activity also had no effect on PrP(C levels. Overall, we did not detect any significant difference in the expression of PrP(C in any of the cell or animal-based paradigms considered, indicating that the control of cellular PrP(C levels by AICD is not as straightforward as previously suggested.

  13. On the Role of Internal Water on Protein Thermal Stability: the Case of Homologous G-domains

    OpenAIRE

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

    2014-01-01

    In this work we address the question whether the enhanced stability of thermophilic proteins has a direct connection with internal hydration. Our model systems are two homologues G-domains of different stability: the mesophilic G domain of the Elongation-Factor thermal unstable protein from E. coli and the hyperthermophilic G domain of the EF-α protein from S. solfataricus. Using molecular dynamics simulation at the microsecond time-scale we show that both proteins host water molecules in int...

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

    Energy Technology Data Exchange (ETDEWEB)

    Saito, Yuriko [Molecular Imaging Center, National Institute of Radiological Sciences (Japan); Graduate School of Pharmaceutical Sciences, Chiba University (Japan); Furukawa, Takako, E-mail: tfuru@nirs.go.jp [Molecular Imaging Center, National Institute of Radiological Sciences (Japan); Biomedical Imaging Research Center, University of Fukui (Japan); Arano, Yasushi [Graduate School of Pharmaceutical Sciences, Chiba University (Japan); Fujibayashi, Yasuhisa [Molecular Imaging Center, National Institute of Radiological Sciences (Japan); Biomedical Imaging Research Center, University of Fukui (Japan); Saga, Tsuneo [Molecular Imaging Center, National Institute of Radiological Sciences (Japan)

    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.

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

    Science.gov (United States)

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

    2012-03-01

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

  16. The delta domain of the HK97 major capsid protein is essential for assembly.

    Science.gov (United States)

    Oh, Bonnie; Moyer, Crystal L; Hendrix, Roger W; Duda, Robert L

    2014-05-01

    The 102 residue N-terminal extension of the HK97 major capsid protein, the delta domain, is normally present during the assembly of immature HK97 procapsids, but it is removed during maturation like well-known internal scaffolding proteins of other tailed phages and herpesviruses. The delta domain also shares other unusual properties usually found in other viral and phage scaffolding proteins, including its location on the inside of the capsid, a high predicted and measured α-helical content, and an additional prediction for the ability to form parallel coiled-coils. Viral scaffolding proteins are essential for capsid assembly and phage viability, so we tested whether the HK97 delta domain was essential for capsid assembly. We studied the effects of deleting all or parts of the delta domain on capsid assembly and on complementation of capsid-protein-defective phage, and our results demonstrate that the delta domain is required for HK97 capsid assembly. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Hierarchical involvement of various GGDEF domain proteins in rdar morphotype development of Salmonella enterica serovar Typhimurium.

    Science.gov (United States)

    Kader, Abdul; Simm, Roger; Gerstel, Ulrich; Morr, Michael; Römling, Ute

    2006-05-01

    GGDEF and EAL domain proteins are involved in the turnover of the novel secondary messenger cyclic-di(3'-->5')-guanylic acid (c-di-GMP) in many bacteria. In this work the role of the 12 GGDEF domain proteins encoded by the Salmonella enterica serovar Typhimurium (S. Typhimurium) chromosome in rdar morphotype development was investigated. Previously, it was shown that the GGDEF domain protein AdrA activated the biosynthesis of cellulose by production of c-di-GMP. Enhancement of the c-di-GMP levels by overexpression of the GGDEF domain protein AdrA did lead to the activation of curli fimbriae biosynthesis through the elevated expression of CsgD and CsgA. Although knock-out of the chromosomal copy of adrA influenced CsgA expression, CsgD expression was not altered, although more than half of the total cellular c-di-GMP was produced by AdrA at 16 h of growth. On the other hand, chromosomally encoded GGDEF-EAL domain proteins STM2123 and STM3388 were required to additively activate CsgD expression on a transcriptional and post-transcriptional level. Enhanced c-di-GMP levels did overcome temperature regulation of rdar morphotype expression by activation of curli fimbriae as well as cellulose biosynthesis through CsgD expression. Thus in the regulatory cascade leading to rdar morphotype expression c-di-GMP activates several subsequent steps in the network.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    Juliana Bernardes

    2016-07-01

    Full Text Available 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

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

    Science.gov (United States)

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

    2013-09-09

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

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

    Directory of Open Access Journals (Sweden)

    Wenlin An

    Full Text Available 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.

  2. Energetic Calculations to Decipher pH-Dependent Oligomerization and Domain Swapping of Proteins.

    Science.gov (United States)

    Shingate, Prashant; Warwicker, Jim; Sowdhamini, Ramanathan

    2015-01-01

    Domain swapping mechanism is a specialised mode of oligomerization of proteins in which part of a protein is exchanged in a non-covalent manner between constituent subunits. This mechanism is highly affected by several physiological conditions. Here, we present a detailed analysis ofthe effect of pH on different regions of the domain swapped oligomer by considering examples which are known to be sensitive to pH in transiting from monomeric to domain-swapped dimeric form. The energetic calculations were performed using a specialized method which considers changes in pH and subsequent changes in the interactions between subunits. This analysis provides definitive hints about the pH-dependence switch from monomer to domain-swapped oligomer and the steps that may be involved in the swapping mechanism.

  3. Energetic Calculations to Decipher pH-Dependent Oligomerization and Domain Swapping of Proteins.

    Directory of Open Access Journals (Sweden)

    Prashant Shingate

    Full Text Available Domain swapping mechanism is a specialised mode of oligomerization of proteins in which part of a protein is exchanged in a non-covalent manner between constituent subunits. This mechanism is highly affected by several physiological conditions. Here, we present a detailed analysis ofthe effect of pH on different regions of the domain swapped oligomer by considering examples which are known to be sensitive to pH in transiting from monomeric to domain-swapped dimeric form. The energetic calculations were performed using a specialized method which considers changes in pH and subsequent changes in the interactions between subunits. This analysis provides definitive hints about the pH-dependence switch from monomer to domain-swapped oligomer and the steps that may be involved in the swapping mechanism.

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

    Science.gov (United States)

    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.

  5. Structure of the Bro1 domain protein BROX and functional analyses of the ALIX Bro1 domain in HIV-1 budding.

    Directory of Open Access Journals (Sweden)

    Qianting Zhai

    Full Text Available BACKGROUND: 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(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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

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

    Science.gov (United States)

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

    2010-07-16

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

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

    Science.gov (United States)

    Han, Weidong; Li, Xiaolei; Fu, Xiaobing

    2011-01-01

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

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

    Science.gov (United States)

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

    2003-03-03

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

  9. Understanding the PxS Aspect of Within-Person Variation: A Variance Partitioning Approach

    Directory of Open Access Journals (Sweden)

    Brian eLakey

    2016-01-01

    Full Text Available This article reviews a variance partitioning approach to within-person variation based on Generalizability (G Theory and the Social Relations Model (SRM. The approach conceptualizes an important part of within-person variation as Person x Situation (PxS interactions: differences among persons in their profiles of responses across the same situations. The approach provided the first quantitative method for capturing within-person variation and demonstrated very large PxS effects for a wide range of constructs. These include anxiety, five-factor personality traits, perceived social support, leadership, and task performance. Although PxS effects are commonly very large, conceptual and analytic obstacles have thwarted consistent progress. For example, how does one develop a psychological, versus purely statistical, understanding of PxS effects? How does one forecast future behavior when the criterion is a PxS effect? How can understanding PxS effects contribute to psychological theory? This review describes potential solutions to these and other problems developed in the course of conducting research on the PxS aspect of social support. Additional problems that need resolution are identified.

  10. The myofibrillar protein, projectin, is highly conserved across insect evolution except for its PEVK domain.

    Science.gov (United States)

    Ayme-Southgate, Agnes J; Southgate, Richard J; Philipp, Richard A; Sotka, Erik E; Kramp, Catherine

    2008-12-01

    All striated muscles respond to stretch by a delayed increase in tension. This physiological response, known as stretch activation, is, however, predominantly found in vertebrate cardiac muscle and insect asynchronous flight muscles. Stretch activation relies on an elastic third filament system composed of giant proteins known as titin in vertebrates or kettin and projectin in insects. The projectin insect protein functions jointly as a "scaffold and ruler" system during myofibril assembly and as an elastic protein during stretch activation. An evolutionary analysis of the projectin molecule could potentially provide insight into how distinct protein regions may have evolved in response to different evolutionary constraints. We mined candidate genes in representative insect species from Hemiptera to Diptera, from published and novel genome sequence data, and carried out a detailed molecular and phylogenetic analysis. The general domain organization of projectin is highly conserved, as are the protein sequences of its two repeated regions-the immunoglobulin type C and fibronectin type III domains. The conservation in structure and sequence is consistent with the proposed function of projectin as a scaffold and ruler. In contrast, the amino acid sequences of the elastic PEVK domains are noticeably divergent, although their length and overall unusual amino acid makeup are conserved. These patterns suggest that the PEVK region working as an unstructured domain can still maintain its dynamic, and even its three-dimensional, properties, without the need for strict amino acid conservation. Phylogenetic analysis of the projectin proteins also supports a reclassification of the Hymenoptera in relation to Diptera and Coleoptera.

  11. Sequential unfolding of the two-domain protein Pseudomonas stutzeri cytochrome c(4)

    DEFF Research Database (Denmark)

    Andersen, Niels Højmark; Jensen, Thomas Jon; Nørgaard, Allan

    2002-01-01

    F stutzeri cytochrome c. is a di-haem protein, composed of two globular domains each with His-Met coordinated haem. and a hydrogen bond network between the domains. The domain foldings are highly symmetric but with specific differences including structural differences of ligand coordination......, and different spin states of the oxidised haem groups. We have studied unfolding of oxidised P. stutzeri cyt c(4) induced thermally and by chemical denaturants Horse heart cyt c was a reference molecule. Isothermal unfolding induced by guanidinium chloride and acid was followed by Soret. alpha/beta. and 701-nm...

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

    Energy Technology Data Exchange (ETDEWEB)

    Cook, William J.; Senkovich, Olga; Chattopadhyay, Debasish (UAB)

    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. Association Rules Mining Based on SVM and Its Application in Simulated Moving Bed PX Adsorption Process%基于支持向量基的关联规则挖掘及其在模拟移动床PX吸附分离过程中的应用

    Institute of Scientific and Technical Information of China (English)

    张英; 苏宏业; 褚健

    2005-01-01

    In this paper, a novel data mining method is introduced to solve the multi-objective optimization problems of process industry. A hyperrectangle association rule mining (HARM) algorithm based on support vector machines (SVMs) is proposed. Hyperrectangles rules are constructed on the base of prototypes and support vectors (SVs) under some heuristic limitations. The proposed algorithm is applied to a simulated moving bed (SMB) paraxylene (PX) adsorption process. The relationships between the key process variables and some objective variables such as purity, recovery rate of PX are obtained. Using existing domain knowledge about PX adsorption process, most of the obtained association rules can be explained.

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

    Science.gov (United States)

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

    2014-02-01

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

  15. Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation.

    Science.gov (United States)

    Hao, Yu-Jun; Song, Qing-Xin; Chen, Hao-Wei; Zou, Hong-Feng; Wei, Wei; Kang, Xu-Sheng; Ma, Biao; Zhang, Wan-Ke; Zhang, Jin-Song; Chen, Shou-Yi

    2010-10-01

    Plant-specific transcription factor NAC proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. In the NAC family, some members function as transcription activators while others act as repressors. In the present study we found that though the full-length GmNAC20 from soybean did not have transcriptional activation activity, the carboxy-terminal activation domain of GmNAC20 had high transcriptional activation activity in the yeast assay system. Deletion experiments revealed an active repression domain with 35 amino acids, named NARD (NAC Repression Domain), in the d subdomain of NAC DNA-binding domain. NARD can reduce the transcriptional activation ability of diverse transcription factors when fused to either the amino-terminal or the carboxy-terminal of the transcription factors. NARD-like sequences are also present in other NAC family members and they are functional repression domain when fused to VP16 in plant protoplast assay system. Mutation analysis of conserved amino acid residues in NARD showed that the hydrophobic LVFY motif may partially contribute to the repression function. It is hypothesized that the interactions between the repression domain NARD and the carboxy-terminal activation domain may finally determine the ability of NAC family proteins to regulate downstream gene expressions.

  16. 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.

    Science.gov (United States)

    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. © 2015 Wiley Periodicals, Inc.

  17. Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.

    Science.gov (United States)

    de Juan-Franco, Elena; Caruz, Antonio; Pedrajas, J R; Lechuga, Laura M

    2013-04-07

    We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.

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

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    Chumporn Soowannayan

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

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

    Science.gov (United States)

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

    2016-01-04

    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. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Peng; Li, Jingzhi; Sha, Bingdong

    2016-11-29

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

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

    DEFF Research Database (Denmark)

    Blaise, Mickael; Alsarraf, Husam Mohammad Ali Baker; Wong, Jaslyn

    2012-01-01

    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...

  2. Structure of the single-strand annealing domain of human RAD52 protein

    OpenAIRE

    Singleton, Martin R.; Wentzell, Lois M.; Liu, Yilun; West, Stephen C.; Wigley, Dale B.

    2002-01-01

    In eukaryotic cells, RAD52 protein plays a central role in genetic recombination and DNA repair by (i) promoting the annealing of complementary single-stranded DNA and (ii) stimulation of the RAD51 recombinase. The single-strand annealing domain resides in the N-terminal region of the protein and is highly conserved, whereas the nonconserved RAD51-interaction domain is located in the C-terminal region. An N-terminal fragment of human RAD52 (residues 1–209) has been purified to homogeneity and...

  3. Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions.

    Science.gov (United States)

    Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E; Levesque, Brié; Pedersen, Stine B; Bartels, Lina; Wapenaar, Hannah; Ye, Fei; Zhang, Mingjie; Bowen, Mark E; Strømgaard, Kristian

    2017-09-15

    The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

  4. On Diophantine equation px4 - ( p-1) y2 =z4%关于丢番图方程px4-(p-1)y2=z4

    Institute of Scientific and Technical Information of China (English)

    姜信君; 佟瑞洲

    2012-01-01

    对任意的奇素数p,还没有找到给出丢番图方程px4(p1)y2=z4的全部正整数解的统一的初等方法,目前只解决了某类特殊的奇素数p的求解问题,例如王洪昌等人完全解决了p-1=Q2;或2Q2;或qQ2,2|Q,q≡3(mod4)为奇素数,Q为正整数的情形.认为对某类特殊的奇素数p求解丢番图方程px4-(p-1)y2=z4,目的是对任意的奇素数p,寻找给出丢番图方程px4-(p-1)y2=z4的全部正整数解的统一解法,当p=2q+1,q≡5(mod8),p,q为奇素数时,利用初等方法把方程px4-(p- 1)y2=z4化为方程x2 +my2=z2,从而给出方程px4-(p-1)y2=z4的全部正整数解;当q为任意正整数时,上述解法仍然适用,因此对任意给定的奇素数p,实际上已经给出了丢番图方程px4-(p-1)y2=z4的全部正整数解的统一解法.%When p is an arbitrary odd prime number, there is no unified primary method of all positive integer solutions to the Diophantine equation px4 (p- l)y2 = z4, but only the solution of some certain types of special odd primes number p.such as Wang Hongchang etc have completely resolved p-l = Q2:Or 2Q2 :orqQ2,2|Q, q≡3(modk) with q is an odd prime number and Q is a positive integer. People solve the Diophantine equation px4 - (p-l)y2 = z4 with some special types of odd prime number p to find a unified approach of all positive integer solutions to Diophantine equation px4 -(p-1) y2=z4. When p=2q+l,q≡5(mod8) ,p,q is an odd prime number, we put equation px4 - (p-l)y2 = z4 into equation χ =z2 by the elementary methods, by which we give the all solution to Diophantine equation px4-(p-l)y2=z4. When q is an arbitrary positive integer, the above solution is still applicable. In fact, we have given a unified method of all positive integer solutions to Diophantine equations px4 - (p- l)y2 = z4, when p is an arbitrary given odd prime number.

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

    NARCIS (Netherlands)

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

    1991-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hai Fang

    2014-10-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  8. Sequence diversity in the A domain of Staphylococcus aureus fibronectin-binding protein A

    Directory of Open Access Journals (Sweden)

    Speziale Pietro

    2008-05-01

    Full Text Available Abstract Background Fibronectin-binding protein A (FnBPA mediates adhesion of Staphylococcus aureus to fibronectin, fibrinogen and elastin. We previously reported that S. aureus strain P1 encodes an FnBPA protein where the fibrinogen/elastin-binding domain (A domain is substantially divergent in amino acid sequence from the archetypal FnBPA of S. aureus NCTC8325, and that these variations created differences in antigenicity. In this study strains from multilocus sequence types (MLST that spanned the genetic diversity of S.aureus were examined to determine the extent of FnBPA A domain variation within the S. aureus population and its effect on ligand binding and immuno-crossreactivity. Results Seven different isotype forms (I – VII of the FnBPA A domain were identified which were between 66 to 76% identical in amino acid sequence in any pair-wise alignment. The fnbA allelic variants in strains of different multilocus sequence type were identified by DNA hybridization using probes specific for sequences encoding the highly divergent N3 sub-domain of different isotypes. Several isotypes were not restricted to specific clones or clonal complexes but were more widely distributed. It is highly likely that certain fnbA genes have been transferred horizontally. Residues lining the putative ligand-binding trench were conserved, which is consistent with the ability of each A domain isotype to bind immobilized fibrinogen and elastin by the dock-latch-lock mechanism. Variant amino acid residues were mapped on a three-dimensional model of the FnBPA A domain and were predicted to be surface-exposed. Polyclonal antibodies raised against the recombinant isotype I A domain bound that protein with a 4 – 7 fold higher apparent affinity compared to the A domains of isotypes II – VII, while some monoclonal antibodies generated against the isotype I A domain showed reduced or no binding to the other isotypes. Conclusion The FnBPA A domain occurs in at least 7

  9. Thermal stability of pepsin: A predictive thermodynamic model of a multi-domain protein

    Directory of Open Access Journals (Sweden)

    Ali Asghar Rastegari

    2017-03-01

    Full Text Available Pepsin is generally used in the preparation of F(ab2 fragments from antibodies. The antibodies that are one of the largest and fastest growing categories of bio- pharmaceutical candidates. Differential scanning calorimetric is principally suitable method to follow the energetics of a multi-domain, fragment to perform a more exhaustive description of the thermodynamics in an associating system. The thermodynamical models of analysis include the construction of a simultaneous fitting of a theoretical expression. The expression depending on the equilibrium unfolding data from multimeric proteins that have a two-state monomer. The aim of the present study is considering the DSC data in connection with pepsin going through reversible thermal denaturation. Afterwards, we calculate the homology modeling identification of pepsin in complex multi-domain families with varied domain architectures. In order to analyze the DSC data, the thermal denaturation of multimer proteins were considered, the “two independent two-state sequential transitions with domains dissociation model” was introduced by using of the effective ΔG concept. The reversible unfolding of the protein description was followed by the two-state transition quantities which is a slower irreversible process of aggregation. The protein unfolding is best described by two non-ideal transitions, suggesting the presence of unfolding intermediates. These evaluations are also applicable for high throughput investigation of protein stability.

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

    Science.gov (United States)

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

    2008-04-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

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

    Science.gov (United States)

    Lorent, Joseph Helmuth; Levental, Ilya

    2015-11-01

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

  13. Domain-Swapped Dimers of Intracellular Lipid-Binding Proteins: Evidence for Ordered Folding Intermediates.

    Science.gov (United States)

    Assar, Zahra; Nossoni, Zahra; Wang, Wenjing; Santos, Elizabeth M; Kramer, Kevin; McCornack, Colin; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H

    2016-09-06

    Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2014-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  18. A preliminary study on functional domains of small heat shock protein Hsp16.3

    Institute of Scientific and Technical Information of China (English)

    CHANG Yong; LI Xuemei; RAO Zihe

    2004-01-01

    Hsp16.3, the small heat shock protein (sHSP) from Mycobacterium tuberculosis, was originally identified as an immunodominant antigen,which possesses three functional domains typical of sHSP family, namely the N-terminal hydrophobic region, α-crystallin domain and a short non-conserved C-terminal extension.To further understand the functional assignment of these independent regions, the three functional domains of Hsp16.3 were defined and the two N- or C-terminal truncated Hsp16.3 remnants were successfully cloned, expressed and purified.In the far and near circular dichroism analysis, the results showed that these remnants expressed similar secondary and tertiary structures to that of wild-type protein.During the reassembly of wild-type nonamer, the C-terminal truncated remnant could interact with the wild-type protein to form hetero-oligomers.When trypsin is used to digest the wild-type Hsp16.3, its α-crystallin domain could resist such degradation.Taken together, these results indicate that the stable secondary and tertiary structures of Hsp16.3 are mainly kept by its α-crystallin domain.

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

    Science.gov (United States)

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

    2009-12-01

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

  20. Reversible Conformational Changes of PsbO Protein Detected by Terahertz Time-Domain Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    CHEN Hua; CHEN Gui-Ying; LI Shu-Qin; WANG Li

    2009-01-01

    We used a terahertz time-domain spectroscope (THz-TDS) to detect the reversible conformations2 changes of PsbO protein induced by N-bromosuccinimide and Guanidine Hydrochloride.The veracity and sensitivity are confirmed by the fluorescence emission spectra.The results demonstrate that THz-TDS has both advantages and disadvantages in monitoring the denaturation process of proteins,which is important in applying THz-TDS technique to studying biomolecules.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Ralf eEnz

    2012-04-01

    Full Text Available 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.

  3. Structure-function analysis of the NB-ARC domain of plant disease resistance proteins.

    Science.gov (United States)

    van Ooijen, Gerben; Mayr, Gabriele; Kasiem, Mobien M A; Albrecht, Mario; Cornelissen, Ben J C; Takken, Frank L W

    2008-01-01

    Resistance (R) proteins in plants are involved in pathogen recognition and subsequent activation of innate immune responses. Most resistance proteins contain a central nucleotide-binding domain. This so-called NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. The NB-ARC domain is a functional ATPase domain, and its nucleotide-binding state is proposed to regulate activity of the R protein. A highly conserved methionine-histidine-aspartate (MHD) motif is present at the carboxy-terminus of ARC2. An extensive mutational analysis of the MHD motif in the R proteins I-2 and Mi-1 is reported. Several novel autoactivating mutations of the MHD invariant histidine and conserved aspartate were identified. The combination of MHD mutants with autoactivating hydrolysis mutants in the NB subdomain showed that the autoactivation phenotypes are not additive. This finding indicates an important regulatory role for the MHD motif in the control of R protein activity. To explain these observations, a three-dimensional model of the NB-ARC domain of I-2 was built, based on the APAF-1 template structure. The model was used to identify residues important for I-2 function. Substitution of the selected residues resulted in the expected distinct phenotypes. Based on the model, it is proposed that the MHD motif fulfils the same function as the sensor II motif found in AAA+ proteins (ATPases associated with diverse cellular activities)-co-ordination of the nucleotide and control of subdomain interactions. The presented 3D model provides a framework for the formulation of hypotheses on how mutations in the NB-ARC exert their effects.

  4. A tetravalent dengue vaccine containing a mix of domain III-P64k and domain III-capsid proteins induces a protective response in mice.

    Science.gov (United States)

    Izquierdo, Alienys; García, Angélica; Lazo, Laura; Gil, Lázaro; Marcos, Ernesto; Alvarez, Mayling; Valdés, Iris; Hermida, Lisset; Guillén, Gerardo; Guzmán, María G

    2014-10-01

    Recombinant fusion proteins containing domain III of the dengue virus envelope protein fused to the P64k protein from Neisseria meningitidis and domain III of dengue virus type 2 (D2) fused to the capsid protein of this serotype were immunogenic and conferred protection in mice against lethal challenge, as reported previously. Combining the domain III-P64k recombinant proteins of dengue virus types 1, 3 and 4 (D1, D3, and D4) with the domain III-capsid protein from D2, we obtained a novel tetravalent formulation containing different antigens. Here, the IgG and neutralizing antibody response, the cellular immune response, and the protective capacity against lethal challenge in mice immunized with this tetravalent formulation were evaluated. The neutralizing antibody response obtained against D1, D2 and D3, together with the high levels of IFNγ secretion induced after stimulation with the four dengue serotypes, supports the strategy of using a new tetravalent formulation containing domain III of the envelope protein fused to the capsid protein of each dengue virus serotype.

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

    Science.gov (United States)

    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.

  6. A fluorescent cassette-based strategy for engineering multiple domain fusion proteins

    Directory of Open Access Journals (Sweden)

    Khorchid Ahmad

    2003-07-01

    Full Text Available Abstract Background The engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all. Results Therefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence. Conclusions Finally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.

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

    Science.gov (United States)

    Zhang, Yanfeng; Gao, Xiaoli; Michael Garavito, R

    2011-04-22

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

  8. Structural and functional analysis of the S-layer protein crystallisation domain of Lactobacillus acidophilus ATCC 4356 : evidence for protein : protein interaction of two subdomains

    NARCIS (Netherlands)

    Smit, E.; Jager, D.; Martinez, B.; Tielen, F.J.; Pouwels, P.H.

    2002-01-01

    The structure of the crystallisation domain, SAN, of the S A-protein of Lactobacillus acidophilus ATCC 4356 was analysed by insertion and deletion mutagenesis, and by proteolytic treatment. Mutant S A-protein synthesised in Escherichia coli with 7-13 amino acid insertions near the N terminus or with

  9. Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

    Institute of Scientific and Technical Information of China (English)

    Jian-lin Dou; Tao Jing; Jing-jing Fan; Zhi-ming Yuan

    2011-01-01

    A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection.Due to its ubiquitous ability to invade host cells,Salmonella typhimurium might be a good candidate for displaying viral antigens.We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S.typhimurium BRD509 using the ice nucleation protein.The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated.The results showed that display motifs in the protein can target integral foreign protein on the surface of S.typhimurium BRD509.Moreover,recombinant strains with surface displayed viral proteins retained their invasiveness,suggesting that the recombinant S.typhimurium can be used as live vaccine vector for eliciting complete immunogenicity.The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.

  10. Multiple protein domains mediate interaction between Bcl10 and MALT1.

    Science.gov (United States)

    Langel, Felicia D; Jain, Nidhi A; Rossman, Jeremy S; Kingeter, Lara M; Kashyap, Anuj K; Schaefer, Brian C

    2008-11-21

    Bcl10 and MALT1 are essential mediators of NF-kappaB activation in response to the triggering of a diverse array of transmembrane receptors, including antigen receptors. Additionally, both proteins are translocation targets in MALT lymphoma. Thus, a detailed understanding of the interaction between these mediators is of considerable biological importance. Previous studies have indicated that a 13-amino acid region downstream of the Bcl10 caspase recruitment domain (CARD) is responsible for interacting with the immunoglobulin-like domains of MALT1. We now provide evidence that the death domain of MALT1 and the CARD of Bcl10 also contribute to Bcl10-MALT1 interactions. Although a direct interaction between the MALT1 death domain and Bcl10 cannot be detected via immunoprecipitation, FRET data strongly suggest that the death domain of MALT1 contributes significantly to the association between Bcl10 and MALT1 in T cells in vivo. Furthermore, analysis of point mutants of conserved residues of Bcl10 shows that the Bcl10 CARD is essential for interaction with the MALT1 N terminus. Mutations that disrupt proper folding of the Bcl10 CARD strongly impair Bcl10-MALT1 interactions. Molecular modeling and functional analyses of Bcl10 point mutants suggest that residues Asp(80) and Glu(84) of helix 5 of the Bcl10 CARD directly contact MALT1. Together, these data demonstrate that the association between Bcl10 and MALT1 involves a complex interaction between multiple protein domains. Moreover, the Bcl10-MALT1 interaction is the second reported example of interactions between a CARD and a non-CARD protein region, which suggests that many signaling cascades may utilize CARD interactions with non-CARD domains.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fernandes, Humberto; Franklin, Edward; Khan, Amir R. (Trinity)

    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.

  12. Viral proteins acquired from a host converge to simplified domain architectures.

    Directory of Open Access Journals (Sweden)

    Nadav Rappoport

    2012-02-01

    Full Text Available The infection cycle of viruses creates many opportunities for the exchange of genetic material with the host. Many viruses integrate their sequences into the genome of their host for replication. These processes may lead to the virus acquisition of host sequences. Such sequences are prone to accumulation of mutations and deletions. However, in rare instances, sequences acquired from a host become beneficial for the virus. We searched for unexpected sequence similarity among the 900,000 viral proteins and all proteins from cellular organisms. Here, we focus on viruses that infect metazoa. The high-conservation analysis yielded 187 instances of highly similar viral-host sequences. Only a small number of them represent viruses that hijacked host sequences. The low-conservation sequence analysis utilizes the Pfam family collection. About 5% of the 12,000 statistical models archived in Pfam are composed of viral-metazoan proteins. In about half of Pfam families, we provide indirect support for the directionality from the host to the virus. The other families are either wrongly annotated or reflect an extensive sequence exchange between the viruses and their hosts. In about 75% of cross-taxa Pfam families, the viral proteins are significantly shorter than their metazoan counterparts. The tendency for shorter viral proteins relative to their related host proteins accounts for the acquisition of only a fragment of the host gene, the elimination of an internal domain and shortening of the linkers between domains. We conclude that, along viral evolution, the host-originated sequences accommodate simplified domain compositions. We postulate that the trimmed proteins act by interfering with the fundamental function of the host including intracellular signaling, post-translational modification, protein-protein interaction networks and cellular trafficking. We compiled a collection of hijacked protein sequences. These sequences are attractive targets for

  13. The WW domain protein Kibra acts upstream of Hippo in Drosophila

    DEFF Research Database (Denmark)

    Baumgartner, Roland; Poernbacher, Ingrid; Buser, Nathalie;

    2010-01-01

    inactivating the transcriptional coactivator Yorkie is well established, much less is known about the upstream events that regulate Hippo signaling activity. The FERM domain proteins Expanded and Merlin appear to represent two different signaling branches that feed into the Hippo pathway. Signaling...

  14. A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein

    KAUST Repository

    Smirnova, Ekaterina

    2016-08-22

    Background: CASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Shoseyov, Oded (Karmey Yosef, IL)

    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.

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

    Science.gov (United States)

    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.

  17. 无限畅快 丽台Winfast PX6800 GS TDH显卡

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    不久前,丽台科技(Leadtek),发表了玩家级3D绘图显示卡-WinFast PX6800 GS。WinFast PX6800 GS的出现将效能以及兼容性推向另一个顶点,支持Microsoft Dierct X 9.0c Shader Model 3.0与OpenGL.2.0。

  18. Molecular cloning and partial characterization of a plant VAP33 homologue with a major sperm protein domain

    NARCIS (Netherlands)

    Laurent, F.; Labesse, G.; Wit, de P.

    2000-01-01

    In a search for proteins interacting with the resistance protein Cf9 from tomato, a new cDNA was cloned and characterized. Protein sequence database searches suggested that the 120 residue-N terminal domain of the encoded protein (named VAP27) is highly similar to the VAP33 protein family from anima

  19. Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1.

    Science.gov (United States)

    Meza-Carmen, Victor; Pacheco-Rodriguez, Gustavo; Kang, Gi Soo; Kato, Jiro; Donati, Chiara; Zhang, Chun-Yi; Vichi, Alessandro; Payne, D Michael; El-Chemaly, Souheil; Stylianou, Mario; Moss, Joel; Vaughan, Martha

    2011-06-28

    ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa protein containing a functional ADP-ribosylation factor (GTP hydrolase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase domains. ARD1 activation by the guanine nucleotide-exchange factor cytohesin-1 was known. GTPase and E3 ligase activities of ARD1 suggest roles in protein transport and turnover. To explore this hypothesis, we used mouse embryo fibroblasts (MEFs) from ARD1-/- mice stably transfected with plasmids for inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady-state levels in cells by autoubiquitination. Based on reported growth factor receptor-cytohesin interactions, EGF receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p = 0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGF-β receptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its autoubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.

  20. Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

    Science.gov (United States)

    Filikov, Anton V.; James, Thomas L.

    1998-05-01

    A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

  1. Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs.

    Science.gov (United States)

    Fox, Philip D; Haberkorn, Christopher J; Weigel, Aubrey V; Higgins, Jenny L; Akin, Elizabeth J; Kennedy, Matthew J; Krapf, Diego; Tamkun, Michael M

    2013-09-01

    In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

  2. Acquired Thermotolerance and Heat Shock Proteins in Thermophiles from the Three Phylogenetic Domains

    DEFF Research Database (Denmark)

    Trent, Jonathan D.; Gabrielsen, Mette; Jensen, Bo

    1994-01-01

    at 70 degrees C and heat shocked at 88 degrees C for 60 min showed thermotolerance at 95 degrees C, and Thermomyces lanuginosus grown at 50 degrees C and heat shocked at 55 degrees C for 60 min showed thermotolerance at 58 degrees C. Determinations of protein synthesis during heat shock revealed...... differences in the dominant heat shock proteins for each species. For B. caldolyticus, a 70-kDa protein dominated while for S. shibatae, a 55-kDa protein dominated and for T. lanuginosus, 31- to 33-kDa proteins dominated. Reagents that disrupted normal protein synthesis during heat shock prevented......Thermophilic organisms from each of the three phylogenetic domains (Bacteria, Archaea, and Eucarya) acquired thermotolerance after heat shock. Bacillus caldolyticus grown at 60 degrees C and heat shocked at 69 degrees C for 10 min showed thermotolerance at 74 degrees C, Sulfolobus shibatae grown...

  3. Yeast Two-Hybrid Screening for Proteins that Interact with the Extracellular Domain of Amyloid Precursor Protein.

    Science.gov (United States)

    Yu, You; Li, Yinan; Zhang, Yan

    2016-04-01

    Alzheimer's disease (AD) is a neurodegenerative disorder in which amyloid β plaques are a pathological characteristic. Little is known about the physiological functions of amyloid β precursor protein (APP). Based on its structure as a type I transmembrane protein, it has been proposed that APP might be a receptor, but so far, no ligand has been reported. In the present study, 9 proteins binding to the extracellular domain of APP were identified using a yeast two-hybrid system. After confirming the interactions in the mammalian system, mutated PLP1, members of the FLRT protein family, and KCTD16 were shown to interact with APP. These proteins have been reported to be involved in Pelizaeus-Merzbacher disease (PMD) and axon guidance. Therefore, our results shed light on the mechanisms of physiological function of APP in AD, PMD, and axon guidance.

  4. The Evolutionary History of MAPL (Mitochondria-Associated Protein Ligase) and Other Eukaryotic BAM/GIDE Domain Proteins.

    Science.gov (United States)

    Wideman, Jeremy G; Moore, Blake P

    2015-01-01

    MAPL (mitochondria-associated protein ligase, also called MULAN/GIDE/MUL1) is a multifunctional mitochondrial outer membrane protein found in human cells that contains a unique BAM (beside a membrane) domain and a C-terminal RING-finger domain. MAPL has been implicated in several processes that occur in animal cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis. Previous studies demonstrated that the BAM domain is present in diverse organisms in which most of these processes do not occur, including plants, archaea, and bacteria. Thus the conserved function of MAPL and its BAM domain remains an open question. In order to gain insight into its conserved function, we investigated the evolutionary origins of MAPL by searching for homologues in predicted proteomes of diverse eukaryotes. We show that MAPL proteins with a conserved BAM-RING architecture are present in most animals, protists closely related to animals, a single species of fungus, and several multicellular plants and related green algae. Phylogenetic analysis demonstrated that eukaryotic MAPL proteins originate from a common ancestor and not from independent horizontal gene transfers from bacteria. We also determined that two independent duplications of MAPL occurred, one at the base of multicellular plants and another at the base of vertebrates. Although no other eukaryote genome examined contained a verifiable MAPL orthologue, BAM domain-containing proteins were identified in the protists Bigelowiella natans and Ectocarpus siliculosis. Phylogenetic analyses demonstrated that these proteins are more closely related to prokaryotic BAM proteins and therefore likely arose from independent horizontal gene transfers from bacteria. We conclude that MAPL proteins with BAM-RING architectures have been present in the holozoan and viridiplantae lineages since their very beginnings. Our work paves

  5. The Evolutionary History of MAPL (Mitochondria-Associated Protein Ligase and Other Eukaryotic BAM/GIDE Domain Proteins.

    Directory of Open Access Journals (Sweden)

    Jeremy G Wideman

    Full Text Available MAPL (mitochondria-associated protein ligase, also called MULAN/GIDE/MUL1 is a multifunctional mitochondrial outer membrane protein found in human cells that contains a unique BAM (beside a membrane domain and a C-terminal RING-finger domain. MAPL has been implicated in several processes that occur in animal cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis. Previous studies demonstrated that the BAM domain is present in diverse organisms in which most of these processes do not occur, including plants, archaea, and bacteria. Thus the conserved function of MAPL and its BAM domain remains an open question. In order to gain insight into its conserved function, we investigated the evolutionary origins of MAPL by searching for homologues in predicted proteomes of diverse eukaryotes. We show that MAPL proteins with a conserved BAM-RING architecture are present in most animals, protists closely related to animals, a single species of fungus, and several multicellular plants and related green algae. Phylogenetic analysis demonstrated that eukaryotic MAPL proteins originate from a common ancestor and not from independent horizontal gene transfers from bacteria. We also determined that two independent duplications of MAPL occurred, one at the base of multicellular plants and another at the base of vertebrates. Although no other eukaryote genome examined contained a verifiable MAPL orthologue, BAM domain-containing proteins were identified in the protists Bigelowiella natans and Ectocarpus siliculosis. Phylogenetic analyses demonstrated that these proteins are more closely related to prokaryotic BAM proteins and therefore likely arose from independent horizontal gene transfers from bacteria. We conclude that MAPL proteins with BAM-RING architectures have been present in the holozoan and viridiplantae lineages since their very beginnings

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Pratibha; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.in

    2015-08-15

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

  8. The human YB-1 cold shock domain : structural, dynamical and binding properties of the central nucleic acid binding domain of the human Y-box protein YB-1, a transcription and translation regulating protein

    NARCIS (Netherlands)

    Kloks, Cathelijne Petra Anne Marie

    2003-01-01

    Y-box proteins are a highly conserved group of proteins present in bacteria, plants and animals. They are essential in regulating transcription and translation and the coupling between theses two processes. Their central domain, the so-called cold shock domain (CSD), is responsible for the nucleic a

  9. The human YB-1 cold shock domain : structural, dynamical and binding properties of the central nucleic acid binding domain of the human Y-box protein YB-1, a transcription and translation regulating protein

    NARCIS (Netherlands)

    Kloks, Cathelijne Petra Anne Marie

    2003-01-01

    Y-box proteins are a highly conserved group of proteins present in bacteria, plants and animals. They are essential in regulating transcription and translation and the coupling between theses two processes. Their central domain, the so-called cold shock domain (CSD), is responsible for the nucleic

  10. MADS on the move : a study on MADS domain protein function and movement during floral development in Arabidopsis thaliana

    NARCIS (Netherlands)

    Urbanus, S.L.

    2010-01-01

    In this thesis we investigated the behaviour of fluorescently-tagged MADS domain proteins during floral development in the model plant Arabidopsis thaliana, and explored the importance of intercellular transport via plasmodesmata for MADS domain transcription factor functioning. The MADS domain tran

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic...... origin. These observations prompted our search for additional EH-containing proteins in mammalian cells. Using an EH domain-specific probe derived from the eps15 cDNA, we cloned and characterized a cDNA encoding an EH-containing protein with overall similarity to Eps15; we designated this protein Eps15r...

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa [Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Yang, Jun-Mo [Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul 135-710 (Korea, Republic of); Kang, Jong-Sun, E-mail: kangj01@skku.edu [Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Hahn, Myong-Joon, E-mail: hahnmj@skku.edu [Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of)

    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.

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

    Science.gov (United States)

    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.

  14. Conformational diversity in the TPR domain-mediated interaction of protein phosphatase 5 with Hsp90.

    Science.gov (United States)

    Cliff, Matthew J; Harris, Richard; Barford, David; Ladbury, John E; Williams, Mark A

    2006-03-01

    Protein phosphatase 5 (Ppp5) is one of several proteins that bind to the Hsp90 chaperone via a tetratricopeptide repeat (TPR) domain. We report the solution structure of a complex of the TPR domain of Ppp5 with the C-terminal pentapeptide of Hsp90. This structure has the "two-carboxylate clamp" mechanism of peptide binding first seen in the Hop-TPR domain complexes with Hsp90 and Hsp70 peptides. However, NMR data reveal that the Ppp5 clamp is highly dynamic, and that there are multiple modes of peptide binding and mobility throughout the complex. Although this interaction is of very high affinity, relatively few persistent contacts are found between the peptide and the Ppp5-TPR domain, thus explaining its promiscuity in binding both Hsp70 and Hsp90 in vivo. We consider the possible implications of this dynamic structure for the mechanism of relief of autoinhibition in Ppp5 and for the mechanisms of TPR-mediated recognition of Hsp90 by other proteins.

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

    Science.gov (United States)

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

    2015-05-18

    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.

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

    Science.gov (United States)

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

    2012-09-01

    YAP (Yes-associated protein) is a potent oncogene and a major effector of the mammalian Hippo tumor suppressor pathway. In this review, our emphasis is on the structural basis of how YAP recognizes its various cellular partners. In particular, we discuss the role of LATS kinase and AMOTL1 junction protein, two key cellular partners of YAP that bind to its WW domain, in mediating cytoplasmic localization of YAP and thereby playing a key role in the regulation of its transcriptional activity. Importantly, the crystal structure of an amino-terminal domain of YAP in complex with the carboxy-terminal domain of TEAD transcription factor was only recently solved at atomic resolution, while the structure of WW domain of YAP in complex with a peptide containing the PPxY motif has been available for more than a decade. We discuss how such structural information may be exploited for the rational development of novel anti-cancer therapeutics harboring greater efficacy coupled with low toxicity. We also embark on a brief discussion of how recent in silico studies led to identification of the cardiac glycoside digitoxin as a potential modulator of WW domain-ligand interactions. Conversely, dobutamine was identified in a screen of known drugs as a compound that promotes cytoplasmic localization of YAP, thereby resulting in growth suppressing activity. Finally, we discuss how a recent study on the dynamics of WW domain folding on a biologically critical time scale may provide a tool to generate repertoires of WW domain variants for regulation of the Hippo pathway toward desired, non-oncogenic outputs. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. The Bromodomain and Extra-Terminal Domain (BET Family: Functional Anatomy of BET Paralogous Proteins

    Directory of Open Access Journals (Sweden)

    Yasushi Taniguchi

    2016-11-01

    Full Text Available The Bromodomain and Extra-Terminal Domain (BET family of proteins is characterized by the presence of two tandem bromodomains and an extra-terminal domain. The mammalian BET family of proteins comprises BRD2, BRD3, BRD4, and BRDT, which are encoded by paralogous genes that may have been generated by repeated duplication of an ancestral gene during evolution. Bromodomains that can specifically bind acetylated lysine residues in histones serve as chromatin-targeting modules that decipher the histone acetylation code. BET proteins play a crucial role in regulating gene transcription through epigenetic interactions between bromodomains and acetylated histones during cellular proliferation and differentiation processes. On the other hand, BET proteins have been reported to mediate latent viral infection in host cells and be involved in oncogenesis. Human BRD4 is involved in multiple processes of the DNA virus life cycle, including viral replication, genome maintenance, and gene transcription through interaction with viral proteins. Aberrant BRD4 expression contributes to carcinogenesis by mediating hyperacetylation of the chromatin containing the cell proliferation-promoting genes. BET bromodomain blockade using small-molecule inhibitors gives rise to selective repression of the transcriptional network driven by c-MYC These inhibitors are expected to be potential therapeutic drugs for a wide range of cancers. This review presents an overview of the basic roles of BET proteins and highlights the pathological functions of BET and the recent developments in cancer therapy targeting BET proteins in animal models.

  18. PRGL:A cell wall proline-rich protein containning GASA domain in Gerbera hybrida

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    PRPs (proline-rich proteins) are a group of cell wall proteins characterized by their proline and hy- droproline-rich repetitive peptides. The expression of PRPs in plants is stimulated by wounding and environmental stress. GASA (gibberellic acid stimulated in Arabidopsis) proteins are small peptides sharing a 60 amino acid conserved C-terminal domain containing twelve invariant cysteine residues. Most of GASAs reported are localized to apoplasm or cell wall and their expression was regulated by gibberellins (GAs). It has been reported that, in French bean, these two proteins encoding by two distinct genes formed a two-component chitin-receptor involved in plant-pathogen interactions when plant was infected. We cloned a full-length cDNA of PRGL (proline-rich GASA-like) gene which encodes a protein containing both PRP and GASA-like domains. It is demonstrated that PRGL is a new protein with characteristics of PRP and GASA by analyzing its protein structure and gene expression.

  19. Functional analysis of schistosomes EF-hand domain-containing tegument proteins

    Institute of Scientific and Technical Information of China (English)

    YU FuDong; KANG Bin; LI YuanYuan; LI YiXue

    2007-01-01

    Schistosomes cause schistosomiasis disease which severely threatens human health. Little is known about the functions of EF-hand domain containing schistosomes tegument proteins other than as antigens. More possible functions of these tegument proteins were investigated with in silico analyses including protein-protein functional interaction, site-specific variation and glycosylation modification. The analysis results suggested that schistosomes could actively modulate host immune responses for its own favor through functional interactions with host proteins with immunomodulatory function, and passively regulate host immune responses through sequence variation under positive selection and glycosylating the recognition sites of host immune attack. In addition, the analysis of the C-terminal domain of these tegument proteins indicated that they could assist schistosomes in escaping host immune attacks through inhibiting chemotaxis and non-complement fixing antibody (IgG4) responses. In summary, our results suggested that these tegument antigen proteins could assist schistosomes in escaping and modulating host immune responses for self-protection during the process of host-para- site interaction.

  20. Angiogenesis-associated crosstalk between collagens, CXC chemokines, and thrombospondin domain-containing proteins.

    Science.gov (United States)

    Rivera, Corban G; Bader, Joel S; Popel, Aleksander S

    2011-08-01

    Excessive vascularization is a hallmark of many diseases including cancer, rheumatoid arthritis, diabetic nephropathy, pathologic obesity, age-related macular degeneration, and asthma. Compounds that inhibit angiogenesis represent potential therapeutics for many diseases. Karagiannis and Popel [Proc. Natl. Acad. Sci. USA 105(37):13775-13780, 2008] used a bioinformatics approach to identify more than 100 peptides with sequence homology to known angiogenesis inhibitors. The peptides could be grouped into families by the conserved domain of the proteins they were derived from. The families included type IV collagen fibrils, CXC chemokine ligands, and type I thrombospondin domain-containing proteins. The relationships between these families have received relatively little attention. To investigate these relationships, we approached the problem by placing the families of proteins in the context of the human interactome including >120,000 physical interactions among proteins, genes, and transcripts. We built on a graph theoretic approach to identify proteins that may represent conduits of crosstalk between protein families. We validated these findings by statistical analysis and analysis of a time series gene expression data set taken during angiogenesis. We identified six proteins at the center of the angiogenesis-associated network including three syndecans, MMP9, CD44, and versican. These findings shed light on the complex signaling networks that govern angiogenesis phenomena.

  1. A novel PAN/apple domain-containing protein from Toxoplasma gondii: characterization and receptor identification.

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    Haiyan Gong

    Full Text Available Toxoplasma gondii is an intracellular parasite that invades nucleated cells, causing toxoplasmosis in humans and animals worldwide. The extremely wide range of hosts susceptible to T. gondii is thought to be the result of interactions between T. gondii ligands and receptors on its target cells. In this study, a host cell-binding protein from T. gondii was characterized, and one of its receptors was identified. P104 (GenBank Access. No. CAJ20677 is 991 amino acids in length, containing a putative 26 amino acid signal peptide and 10 PAN/apple domains, and shows low homology to other identified PAN/apple domain-containing molecules. A 104-kDa host cell-binding protein was detected in the T. gondii lysate. Immunofluorescence assays detected P104 at the apical end of extracellular T. gondii. An Fc-fusion protein of the P104 N-terminus, which contains two PAN/apple domains, showed strong affinity for the mammalian and insect cells evaluated. This binding was not related to protein-protein or protein-lipid interactions, but to a protein-glycosaminoglycan (GAG interaction. Chondroitin sulfate (CS, a kind of GAG, was shown to be involved in adhesion of the Fc-P104 N-terminus fusion protein to host cells. These results suggest that P104, expressed at the apical end of the extracellular parasite, may function as a ligand in the attachment of T. gondii to CS or other receptors on the host cell, facilitating invasion by the parasite.

  2. LRR conservation mapping to predict functional sites within protein leucine-rich repeat domains.

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    Laura Helft

    Full Text Available 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.

  3. The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain.

    Science.gov (United States)

    Minakshi, Rinki; Padhan, Kartika; Rehman, Safikur; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2014-10-13

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a positive stranded RNA virus with ∼30kb genome. Among all open reading frames (orfs) of this virus, the orf3a is the largest, and encodes a protein of 274 amino acids, named as 3a protein. Sequence analysis suggests that the orf3a aligned to one calcium pump present in Plasmodium falciparum and the enzyme glutamine synthetase found in Leptospira interrogans. This sequence similarity was found to be limited only to amino acid residues 209-264 which form the cytoplasmic domain of the orf3a. Furthermore, this region was predicted to be involved in the calcium binding. Owing to this hypothesis, we were driven to establish its calcium binding property in vitro. Here, we expressed and purified the cytoplasmic domain of the 3a protein, called Cyto3a, as a recombinant His-tagged protein in the E. coli. The calcium binding nature was established by performing various staining methods such as ruthenium red and stains-all. (45)Ca overlay method was also done to further support the data. Since the 3a protein forms ion channels, we were interested to see any conformational changes occurring in the Cyot3a upon calcium binding, using fluorescence spectroscopy and circular dichroism. These studies clearly indicate a significant change in the conformation of the Cyto3a protein after binding with calcium. Our results strongly suggest that the cytoplasmic domain of the 3a protein of SARS-CoV binds calcium in vitro, causing a change in protein conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Targeting diverse protein-protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold.

    Science.gov (United States)

    Checco, James W; Kreitler, Dale F; Thomas, Nicole C; Belair, David G; Rettko, Nicholas J; Murphy, William L; Forest, Katrina T; Gellman, Samuel H

    2015-04-14

    Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues ("α/β-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF165-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain-mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

  5. Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases.

    Science.gov (United States)

    Ghai, Rajesh; Mobli, Mehdi; Norwood, Suzanne J; Bugarcic, Andrea; Teasdale, Rohan D; King, Glenn F; Collins, Brett M

    2011-05-10

    Following endocytosis, the fates of receptors, channels, and other transmembrane proteins are decided via specific endosomal sorting pathways, including recycling to the cell surface for continued activity. Two distinct phox-homology (PX)-domain-containing proteins, sorting nexin (SNX) 17 and SNX27, are critical regulators of recycling from endosomes to the cell surface. In this study we demonstrate that SNX17, SNX27, and SNX31 all possess a novel 4.1/ezrin/radixin/moesin (FERM)-like domain. SNX17 has been shown to bind to Asn-Pro-Xaa-Tyr (NPxY) sequences in the cytoplasmic tails of cargo such as LDL receptors and the amyloid precursor protein, and we find that both SNX17 and SNX27 display similar affinities for NPxY sorting motifs, suggesting conserved functions in endosomal recycling. Furthermore, we show for the first time that all three proteins are able to bind the Ras GTPase through their FERM-like domains. These interactions place the PX-FERM-like proteins at a hub of endosomal sorting and signaling processes. Studies of the SNX17 PX domain coupled with cellular localization experiments reveal the mechanistic basis for endosomal localization of the PX-FERM-like proteins, and structures of SNX17 and SNX27 determined by small angle X-ray scattering show that they adopt non-self-assembling, modular structures in solution. In summary, this work defines a novel family of proteins that participate in a network of interactions that will impact on both endosomal protein trafficking and compartment specific Ras signaling cascades.

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

    Science.gov (United States)

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

    1996-01-01

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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Gustavo Caetano-Anollés

    2011-11-01

    Full Text Available 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

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    containing the ZAC-C2 domain bind anionic phospholipids non-specifically, with some variance in Ca2+ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1-174) for phosphatidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact...... zinc finger motif, but proteins containing only the zinc finger domain (residues 1-105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence...

  10. The Role of the Coat Protein A-Domain in P22 Bacteriophage Maturation

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    David S. Morris

    2014-07-01

    Full Text Available Bacteriophage P22 has long been considered a hallmark model for virus assembly and maturation. Repurposing of P22 and other similar virus structures for nanotechnology and nanomedicine has reinvigorated the need to further understand the protein-protein interactions that allow for the assembly, as well as the conformational shifts required for maturation. In this work, gp5, the major coat structural protein of P22, has been manipulated in order to examine the mutational effects on procapsid stability and maturation. Insertions to the P22 coat protein A-domain, while widely permissive of procapsid assembly, destabilize the interactions necessary for virus maturation and potentially allow for the tunable adjustment of procapsid stability. Future manipulation of this region of the coat protein subunit can potentially be used to alter the stability of the capsid for controllable disassembly.

  11. Expression of LIM-domain binding protein (ldb) genes during zebrafish embryogenesis.

    Science.gov (United States)

    Toyama, R; Kobayashi, M; Tomita, T; Dawid, I B

    1998-02-01

    LIM homeodomain proteins are developmental regulators whose functions depend on synergism with LIM domain binding proteins (Ldb proteins). We have isolated four members of the ldb gene family from the zebrafish, Danio rerio. Ldb1, Ldb2 and Ldb3 share 95%, 73% and 62% amino acid identity with mouse Ldb1, respectively. In overlay assays, Ldb proteins bind LIM homeodomain proteins and LMO1, but not zyxin or MLP. Whole mount in situ hybridization showed that zebrafish ldb1 is expressed ubiquitously from gastrulation onward. Ldb2 is ubiquitous at gastrulation, and later is found in many but not all tissues, especially the anterior central nervous system (CNS) and vasculature. Ldb3 mRNA was expressed primarily in the anterior CNS.

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

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    László Patthy

    2011-07-01

    Full Text Available 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

  13. Cellulose affinity purification of fusion proteins tagged with fungal family 1 cellulose-binding domain.

    Science.gov (United States)

    Sugimoto, Naohisa; Igarashi, Kiyohiko; Samejima, Masahiro

    2012-04-01

    N- or C-terminal fusions of red-fluorescent protein (RFP) with various fungal cellulose-binding domains (CBDs) belonging to carbohydrate binding module (CBM) family 1 were expressed in a Pichia pastoris expression system, and the resulting fusion proteins were used to examine the feasibility of large-scale affinity purification of CBD-tagged proteins on cellulose columns. We found that RFP fused with CBD from Trichoderma reesei CBHI (CBD(Tr)(CBHI)) was expressed at up to 1.2g/l in the culture filtrate, which could be directly injected into the cellulose column. The fusion protein was tightly adsorbed on the cellulose column in the presence of a sufficient amount of ammonium sulfate and was efficiently eluted with pure water. Bovine serum albumin (BSA) was not captured under these conditions, whereas both BSA and the fusion protein were adsorbed on a phenyl column, indicating that the cellulose column can be used for the purification of not only hydrophilic proteins but also for hydrophobic proteins. Recovery of various fusion proteins exceeded 80%. Our results indicate that protein purification by expression of a target protein as a fusion with a fungal family 1 CBD tag in a yeast expression system, followed by affinity purification on a cellulose column, is simple, effective and easily scalable.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chengliang [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Zhang, Qiongdi [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Hang, Tianrong [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Tao, Yue [Shanghai Children’s Medical Center, 1678 Dongfang Road, Pudong, Shanghai 200120, People’s Republic of (China); Ma, Xukai [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Wu, Minhao; Zhang, Xuan, E-mail: xuanzbin@ustc.edu.cn; Zang, Jianye, E-mail: xuanzbin@ustc.edu.cn [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China)

    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.

  15. Crystal structure of the actin binding domain of the cyclase-associated protein.

    Science.gov (United States)

    Dodatko, Tetyana; Fedorov, Alexander A; Grynberg, Marcin; Patskovsky, Yury; Rozwarski, Denise A; Jaroszewski, Lukasz; Aronoff-Spencer, Eliah; Kondraskina, Elena; Irving, Tom; Godzik, Adam; Almo, Steven C

    2004-08-24

    Cyclase-associated protein (CAP or Srv2p) is a modular actin monomer binding protein that directly regulates filament dynamics and has been implicated in a number of complex developmental and morphological processes, including mRNA localization and the establishment of cell polarity. The crystal structure of the C-terminal dimerization and actin monomer binding domain (C-CAP) reveals a highly unusual dimer, composed of monomers possessing six coils of right-handed beta-helix flanked by antiparallel beta-strands. Domain swapping, involving the last two strands of each monomer, results in the formation of an extended dimer with an extensive interface. This structural and biochemical characterization provides new insights into the organization and potential mechanistic properties of the multiprotein assemblies that integrate dynamic actin processes into the overall physiology of the cell. An unanticipated finding is that the unique tertiary structure of the C-CAP monomer provides a structural model for a wide range of molecules, including RP2 and cofactor C, proteins involved in X-linked retinitis pigmentosa and tubulin maturation, respectively, as well as several uncharacterized proteins that exhibit very diverse domain organizations. Thus, the unusual right-handed beta-helical fold present in C-CAP appears to support a wide range of biological functions.

  16. The chromosome axis controls meiotic events through a hierarchical assembly of HORMA domain proteins.

    Science.gov (United States)

    Kim, Yumi; Rosenberg, Scott C; Kugel, Christine L; Kostow, Nora; Rog, Ofer; Davydov, Vitaliy; Su, Tiffany Y; Dernburg, Abby F; Corbett, Kevin D

    2014-11-24

    Proteins of the HORMA domain family play central, but poorly understood, roles in chromosome organization and dynamics during meiosis. In Caenorhabditis elegans, four such proteins (HIM-3, HTP-1, HTP-2, and HTP-3) have distinct but overlapping functions. Through combined biochemical, structural, and in vivo analysis, we find that these proteins form hierarchical complexes through binding of their HORMA domains to cognate peptides within their partners' C-terminal tails, analogous to the "safety belt" binding mechanism of Mad2. These interactions are critical for recruitment of HIM-3, HTP-1, and HTP-2 to chromosome axes. HTP-3, in addition to recruiting the other HORMA domain proteins to the axis, plays an independent role in sister chromatid cohesion and double-strand break formation. Finally, we find that mammalian HORMAD1 binds a motif found both at its own C terminus and at that of HORMAD2, indicating that this mode of intermolecular association is a conserved feature of meiotic chromosome structure in eukaryotes. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Tetratricopeptide repeat domain 9A is an interacting protein for tropomyosin Tm5NM-1

    Directory of Open Access Journals (Sweden)

    Ho Gay

    2008-08-01

    Full Text Available Abstract Background Tetratricopeptide repeat domain 9A (TTC9A protein is a recently identified protein which contains three tetratricopeptide repeats (TPRs on its C-terminus. In our previous studies, we have shown that TTC9A was a hormonally-regulated gene in breast cancer cells. In this study, we found that TTC9A was over-expressed in breast cancer tissues compared with the adjacent controls (P Methods Breast samples from 25 patients including the malignant breast tissues and the adjacent normal tissues were processed for Southern blot analysis. Yeast-two-hybrid assay, GST pull-down assay and co-immunoprecipitation were used to identify and verify the interaction between TTC9A and other proteins. Results Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins. The interaction between TTC9A and Tm5NM-1 was further confirmed by GST pull-down assay and co-immunoprecipitation in mammalian cells. TTC9A domains required for the interaction were also characterized in this study. The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role. Conclusion Since the primary function of tropomyosin is to stabilize actin filament, its interaction with TTC9A may play a role in cell shape and motility. In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading. We speculate that TTC9A acts as a chaperone protein to facilitate the function of tropomyosins in stabilizing microfilament and it may play a role in cancer cell invasion and metastasis.

  18. Predicting the impact of alternative splicing on plant MADS domain protein function.

    Directory of Open Access Journals (Sweden)

    Edouard I Severing

    Full Text Available Several genome-wide studies demonstrated that alternative splicing (AS significantly increases the transcriptome complexity in plants. However, the impact of AS on the functional diversity of proteins is difficult to assess using genome-wide approaches. The availability of detailed sequence annotations for specific genes and gene families allows for a more detailed assessment of the potential effect of AS on their function. One example is the plant MADS-domain transcription factor family, members of which interact to form protein complexes that function in transcription regulation. Here, we perform an in silico analysis of the potential impact of AS on the protein-protein interaction capabilities of MIKC-type MADS-domain proteins. We first confirmed the expression of transcript isoforms resulting from predicted AS events. Expressed transcript isoforms were considered functional if they were likely to be translated and if their corresponding AS events either had an effect on predicted dimerisation motifs or occurred in regions known to be involved in multimeric complex formation, or otherwise, if their effect was conserved in different species. Nine out of twelve MIKC MADS-box genes predicted to produce multiple protein isoforms harbored putative functional AS events according to those criteria. AS events with conserved effects were only found at the borders of or within the K-box domain. We illustrate how AS can contribute to the evolution of interaction networks through an example of selective inclusion of a recently evolved interaction motif in the MADS AFFECTING FLOWERING1-3 (MAF1-3 subclade. Furthermore, we demonstrate the potential effect of an AS event in SHORT VEGETATIVE PHASE (SVP, resulting in the deletion of a short sequence stretch including a predicted interaction motif, by overexpression of the fully spliced and the alternatively spliced SVP transcripts. For most of the AS events we were able to formulate hypotheses about the

  19. Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1.

    Science.gov (United States)

    Dobriyal, Neha; Tripathi, Prerna; Sarkar, Susrita; Tak, Yogesh; Verma, Amit K; Sahi, Chandan

    2017-03-06

    J proteins are obligate co-chaperones of Hsp70s. Via their signature J domain, all J proteins interact with their partner Hsp70s and stimulate their weak ATPase activity, which is vital for Hsp70 functions. The dependency of J proteins on their J domain is such that mutations in critical amino acids in the J domain often results into a null phenotype for a particular J protein. Here, we show that the J domain of Djp1, a cytosolic J protein important for peroxisomal protein import in Saccharomyces cerevisiae, is partially dispensable. A complete deletion of Djp1 J domain resulted into only partial loss in peroxisomal protein import function. Instead, the C-terminal domain of Djp1 was found to be essential for proper localization of the peroxisomal targeted GFP-PTS1. Furthermore, we show that Caj1, another cytosolic J protein, also has some role in peroxisomal protein import. Caj1 was found to be partially redundant with Djp1 as cells lacking both Djp1 and Caj1 resulted into a much more severe defect in GFP-PTS1 localization. Based on these results, we propose that dispensability of J domains could be attributed to genetic redundancy between different J proteins sharing common structural topology and cellular localization.

  20. Recombinant MafA protein containing its own protein transduction domain stimulates insulin gene expression in IEC-6 cells.

    Science.gov (United States)

    Lu, Jun; Luo, Hailan; Wu, Huiling; Lan, Michael S; Tan, Jianming; Lu, Daru

    2011-07-18

    MafA, a basic leucine zipper (bZIP) transcription factor, functions as a potent activator of insulin gene transcription in β-cell. In this paper, we aimed to investigate whether the entire MafA protein has the self-delivery activity, and that the arginine- and lysine-rich sequence in MafA bZIP domain is an efficient protein transduction domain (PTD). Entire MafA protein internalization was evaluated with Western blot and immunofluorescence. The distribution of the PTD-EGFP (enhanced green fluorescence protein) was examined by fluorescent microscope observation. Luciferase reporter assay was used to investigate the effect of the transduced MafA protein on insulin 2 promoter activity. Additionally, we conducted RT-PCR to detect the expression of insulin mRNA in MafA treated IEC-6 cells. The arginine- and lysine-rich peptide of MafA serves as a novel PTD. PTD-EGFP can permeate into various cell types including Min6 (a β-cell line), and transduce in a dose- and time-dependent manner. The cellular uptake of MafA PTD can be completely blocked by heparin, whereas cytochalasin D and amiloride were partially effective in blocking the PTD-EGFP protein entry. Transduced intact MafA protein behaves in the same way as the endogenous MafA, stimulating the transcription of insulin promoter and further inducing insulin expression in treated non-β-cell line (IEC-6). These results indicate that the MafA PTD could serve as a therapeutic delivery vehicle, and further suggest that MafA protein transduction could be a valuable strategy for enhancing insulin gene transcription without requiring gene transfer technology. Copyright © 2011. Published by Elsevier Inc.

  1. Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family

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    Liban, Tyler J.; Medina, Edgar M.; Tripathi, Sarvind; Sengupta, Satyaki; Henry, R. William; Buchler, Nicolas E.; Rubin, Seth M. (UCSC); (Duke); (MSU)

    2017-04-24

    The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD–CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein–E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

  2. NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain

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    Adrian Velazquez-Campoy

    2013-06-01

    Full Text Available The nonstructural protein 3 (NS3 from the hepatitis C virus (HCV is responsible for processing the non-structural region of the viral precursor polyprotein in infected hepatic cells. NS3 protease activity, located at the N-terminal domain, is a zinc-dependent serine protease. A zinc ion, required for the hydrolytic activity, has been considered as a structural metal ion essential for the structural integrity of the protein. In addition, NS3 interacts with another cofactor, NS4A, an accessory viral protein that induces a conformational change enhancing the hydrolytic activity. Biophysical studies on the isolated protease domain, whose behavior is similar to that of the full-length protein (e.g., catalytic activity, allosteric mechanism and susceptibility to inhibitors, suggest that a considerable global conformational change in the protein is coupled to zinc binding. Zinc binding to NS3 protease can be considered as a folding event, an extreme case of induced-fit binding. Therefore, NS3 protease is an intrinsically (partially disordered protein with a complex conformational landscape due to its inherent plasticity and to the interaction with its different effectors. Here we summarize the results from a detailed biophysical characterization of this enzyme and present new experimental data.

  3. Expression, Purification, and Functional Characterization of Atypical Xenocin, Its Immunity Protein, and Their Domains from Xenorhabdus nematophila

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    Jitendra Singh Rathore

    2013-01-01

    Full Text Available Xenorhabdus nematophila, a gram-negative bacterium belonging to the family Enterobacteriaceae is a natural symbiont of a soil nematode from the family Steinernematidae. In this study cloning, expression, and purification of broad range iron regulated multidomain bacteriocin called xenocin from X. nematophila (66 kDa, encoded by xcinA gene and its multidomain immunity protein (42 kDa, encoded by ximB gene have been done. xcinA-ximB (N′ terminal 270 bp, translocation, and translocation-receptor domain of xcinA, ximB, and its hemolysin domain were cloned, expressed, and purified by single step Ni-NTA chromatography under native conditions. In the functional characterization, neutralization of xcinA toxicity by immunity domain of ximB gene was determined by endogenous assay. Exogenous toxic assays results showed that only the purified recombinant xenocin-immunity domain (10 kDa protein complex had toxic activity. Atypical cognate immunity protein (42 kDa of xenocin was fusion of immunity domain (10 kDa and hemolysin domain (32 kDa. In silico analysis of immunity protein revealed its similarity with hemolysin and purine NTPase like proteins. Hemolytic activity was not observed in immunity protein or in its various domains; however, full-length immunity protein lacking Walker motif showed ATPase activity. Finally, using circular dichroism performed secondary structural analyses of all the recombinant proteins/protein complexes.

  4. The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles.

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    Christopher M Ziegler

    2016-03-01

    Full Text Available 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.

  5. A single whey acidic protein domain containing protein (SWD) inhibits bacteria invasion and dissemination in shrimp Marsupenaeus japonicus.

    Science.gov (United States)

    Jiang, Hai-Shan; Sun, Chen; Wang, Tong; Zhao, Xiao-Fan; Wang, Jin-Xing

    2013-08-01

    The single whey acidic protein (WAP) domain containing proteins (SWDs) in crustacean belong to type III crustins and have antiprotease activities and/or antimicrobial activities. Their functions in vivo in crustacean immunity need to be clarify. In this study, a new single WAP domain containing protein (SWD) was obtained from Marsupenaeus japonicus, designated as MjSWD. The full-length cDNA of MjSWD was 522 bp.The open reading frame of MjSWD encoded a protein of 79 amino acids, with a 24 amino acid signal peptide and a WAP domain. Tissue distribution analysis revealed that MjSWD transcripts were generally expressed in all the tested tissues, including hemocytes, heart, hepatopancreas, gill, stomach and intestine. The time course expression of MjSWD was analyzed by quantitative real time PCR, and the results exhibited that MjSWD was upregulated after bacteria (Vibrio anguillarum, Staphylococcus aureus) and white spot syndrome virus (WSSV) challenge in gills and stomach of the shrimp. The purified recombinant protein of MjSWD could bind to several Gram-negative and Gram-positive bacteria though binding to microbial polysaccharides (peptidoglycan). MjSWD could inhibit the activity of Subtilisin A and Proteinase K and bacteria-secreted proteases. The results of natural infection with MjSWD incubated bacteria showed that the inhibition of MjSWD against bacterial secreted proteases was contributed to inhibiting bacteria invasion and dissemination in the shrimp. The MjSWD is, thus, involved in the shrimp antibacterial innate immunity.

  6. Identification of the functional domains of the telomere protein Rap1 in Schizosaccharomyces pombe.

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    Ikumi Fujita

    Full Text Available The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1(+ have revealed that the long N-terminal region (1-456 a.a. [amino acids] of Rap1 (full length: 693 a.a. is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457-693 a.a. containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.

  7. Ephemeral Protein Binding to DNA Shapes Stable Nuclear Bodies and Chromatin Domains.

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    Brackley, Chris A; Liebchen, Benno; Michieletto, Davide; Mouvet, Francois; Cook, Peter R; Marenduzzo, Davide

    2017-03-28

    Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear bodies exchange rapidly with the soluble pool while the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins able to switch between an "on" (binding) and an "off" (nonbinding) state. This system provides a model for any DNA-binding protein that can be posttranslationally modified to change its affinity for DNA (e.g., through phosphorylation). Protein switching is a nonequilibrium process, and it leads to the formation of clusters of self-limiting size, where individual proteins in a cluster exchange with the soluble pool with kinetics similar to those seen in photobleaching experiments. This behavior contrasts sharply with that exhibited by nonswitching proteins, which are permanently in the on-state; when these bind to DNA nonspecifically, they form clusters that grow indefinitely in size. To explain these findings, we propose a mean-field theory from which we obtain a scaling relation between the typical cluster size and the protein switching rate. Protein switching also reshapes intrachromatin contacts to give networks resembling those seen in topologically associating domains, as switching markedly favors local (short-range) contacts over distant ones. Our results point to posttranslational modification of chromatin-bridging proteins as a generic mechanism driving the self-assembly of highly dynamic, nonequilibrium, protein clusters with the properties of nuclear bodies.

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

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    Ghiselli Giancarlo

    2005-01-01

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

  9. Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins.

    Science.gov (United States)

    Kirubakaran, Palani; Pfeiferová, Lucie; Boušová, Kristýna; Bednarova, Lucie; Obšilová, Veronika; Vondrášek, Jiří

    2016-10-01

    Artificial multidomain proteins with enhanced structural and functional properties can be utilized in a broad spectrum of applications. The design of chimeric fusion proteins utilizing protein domains or one-domain miniproteins as building blocks is an important advancement for the creation of new biomolecules for biotechnology and medical applications. However, computational studies to describe in detail the dynamics and geometry properties of two-domain constructs made from structurally and functionally different proteins are lacking. Here, we tested an in silico design strategy using all-atom explicit solvent molecular dynamics simulations. The well-characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ), along with 5 artificial domains and 2 types of molecular linkers, were selected to construct chimeric two-domain molecules. The influence of the artificial domains on the structure and dynamics of the PDZ3 and SH3 domains was determined using a range of analyses. We conclude that the artificial domains can function as allosteric modulators of the PDZ3 and SH3 domains. Proteins 2016; 84:1358-1374. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Solution structure of the HRDC domain of human Bloom syndrome protein BLM.

    Science.gov (United States)

    Sato, Akiko; Mishima, Masaki; Nagai, Aki; Kim, Sun-Yong; Ito, Yutaka; Hakoshima, Toshio; Jee, Jun-Goo; Kitano, Ken

    2010-10-01

    Bloom syndrome is a rare genetic disorder characterized by severe growth retardation and cancer predisposition. The disease is caused by a loss of function of the Bloom syndrome protein (BLM), a member of the RecQ family of DNA helicases. Here we report on the first 3D structure of a BLM fragment, a solution structure of the C-terminal helicase-and-ribonuclease D-C-terminal (HRDC) domain from human BLM. The structure reveals unique features of BLM HRDC that are distinct from the HRDC domain of Werner syndrome protein. In particular, BLM HRDC retains many acidic residues exposed to the solvent, which makes the domain surface extensively electronegative. Consistent with this, fluorescence polarization assays showed an inability of isolated BLM HRDC to interact with DNA substrates. Analyses employing ultracentrifugation, gel-filtration, CD spectroscopy and dynamic light scattering showed that the BLM HRDC domain exists as a stable monomer in solution. The results show that BLM HRDC is a compact, robust and acidic motif which may play a distinct role apart from DNA binding.

  11. Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

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    Wilbur, Jeremy D., E-mail: jwilbur@msg.ucsf.edu [Graduate Program in Biophysics, University of California, San Francisco, California 94143 (United States); Hwang, Peter K. [Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143 (United States); Brodsky, Frances M. [The G. W. Hooper Foundation, Departments of Microbiology and Immunology and of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94143 (United States); Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 (United States); Fletterick, Robert J. [Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143 (United States); Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 (United States); Graduate Program in Biophysics, University of California, San Francisco, California 94143 (United States)

    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. Strategy to target the substrate binding site of SET domain protein methyltransferases.

    Science.gov (United States)

    Nguyen, Kong T; Li, Fengling; Poda, Gennadiy; Smil, David; Vedadi, Masoud; Schapira, Matthieu

    2013-03-25

    Protein methyltransferases (PMTs) are a novel gene family of therapeutic relevance involved in chromatin-mediated signaling and other biological mechanisms. Most PMTs are organized around the structurally conserved SET domain that catalyzes the methylation of a substrate lysine. A few potent chemical inhibitors compete with the protein substrate, and all are anchored in the channel recruiting the methyl-accepting lysine. We propose a novel strategy to design focused chemical libraries targeting the substrate binding site, where a limited number of warheads each occupying the lysine-channel of multiple enzymes would be decorated by different substituents. A variety of sequence and structure-based approaches used to analyze the diversity of the lysine channel of SET domain PMTs support the relevance of this strategy. We show that chemical fragments derived from published inhibitors are valid warheads that can be used in the design of novel focused libraries targeting other PMTs.

  13. Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery.

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    Jan Mani

    Full Text Available Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs. GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are

  14. The Arabidopsis PLAT domain protein1 is critically involved in abiotic stress tolerance.

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    Tae Kyung Hyun

    Full Text Available Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.

  15. The Arabidopsis PLAT domain protein1 is critically involved in abiotic stress tolerance.

    Science.gov (United States)

    Hyun, Tae Kyung; van der Graaff, Eric; Albacete, Alfonso; Eom, Seung Hee; Großkinsky, Dominik K; Böhm, Hannah; Janschek, Ursula; Rim, Yeonggil; Ali, Walid Wahid; Kim, Soo Young; Roitsch, Thomas

    2014-01-01

    Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.

  16. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    Energy Technology Data Exchange (ETDEWEB)

    Lee, A L [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    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.

  17. Viroporin potential of the lentivirus lytic peptide (LLP domains of the HIV-1 gp41 protein

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    Garry Robert F

    2007-11-01

    Full Text Available Abstract 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.

  18. The HhH domain of the human DNA repair protein XPF forms stable homodimers.

    Science.gov (United States)

    Das, Devashish; Tripsianes, Konstantinos; Jaspers, Nicolaas G J; Hoeijmakers, Jan H J; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2008-03-01

    The human XPF-ERCC1 protein complex plays an essential role in nucleotide excision repair by catalysing positioned nicking of a DNA strand at the 5' side of the damage. We have recently solved the structure of the heterodimeric complex of the C-terminal domains of XPF and ERCC1 (Tripsianes et al., Structure 2005;13:1849-1858). We found that this complex comprises a pseudo twofold symmetry axis and that the helix-hairpin-helix motif of ERCC1 is required for DNA binding, whereas the corresponding domain of XPF is functioning as a scaffold for complex formation with ERCC1. Despite the functional importance of heterodimerization, the C-terminal domain of XPF can also form homodimers in vitro. We here compare the stabilities of homodimeric and heterodimeric complexes of the C-terminal domains of XPF and ERCC1. The higher stability of the XPF HhH complexes under various experimental conditions, determined using CD and NMR spectroscopy and mass spectrometry, is well explained by the structural differences that exist between the HhH domains of the two complexes. The XPF HhH homodimer has a larger interaction interface, aromatic stacking interactions, and additional hydrogen bond contacts as compared to the XPF/ERCC1 HhH complex, which accounts for its higher stability.

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

    OpenAIRE

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

    2015-01-01

    CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-induced mutations to the 5’ exons of candidate genes 1–5 , but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR mutagenesis to exons encoding functional protein domains. This generates a higher propor...

  20. On-Chip Manipulation of Protein-Coated Magnetic Beads via Domain-Wall Conduits

    DEFF Research Database (Denmark)

    Donolato, Marco; Vavassori, Paolo; Gobbi, Marco;

    2010-01-01

    Geometrically constrained magnetic domain walls (DWs) in magnetic nanowires can be manipulated at the nanometer scale. The inhomogeneous magnetic stray field generated by a DW can capture a magnetic nanoparticle in solution. On-chip nanomanipulation of individual magnetic beads coated with proteins...... is demonstrated through the motion of geometrically constrained DWs in specially designed magnetic nanoconduits fully integrated in a lab-on-a-chip platform....

  1. Solution structure and peptide binding of the PTB domain from the AIDA1 postsynaptic signaling scaffolding protein.

    Directory of Open Access Journals (Sweden)

    Ekaterina Smirnova

    Full Text Available AIDA1 links persistent chemical signaling events occurring at the neuronal synapse with global changes in gene expression. Consistent with its role as a scaffolding protein, AIDA1 is composed of several protein-protein interaction domains. Here we report the NMR structure of the carboxy terminally located phosphotyrosine binding domain (PTB that is common to all AIDA1 splice variants. A comprehensive survey of peptides identified a consensus sequence around an NxxY motif that is shared by a number of related neuronal signaling proteins. Using peptide arrays and fluorescence based assays, we determined that the AIDA1 PTB domain binds amyloid protein precursor (APP in a similar manner to the X11/Mint PTB domain, albeit at reduced affinity (∼10 µM that may allow AIDA1 to effectively sample APP, as well as other protein partners in a variety of cellular contexts.

  2. SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

    Directory of Open Access Journals (Sweden)

    Shchelkunov Sergei N

    2010-10-01

    Full Text Available Abstract Background Variola virus (VARV the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

  3. Protein folding of the SAP domain, a naturally occurring two-helix bundle.

    Science.gov (United States)

    Dodson, Charlotte A; Arbely, Eyal

    2015-07-01

    The SAP domain from the Saccharomyces cerevisiae Tho1 protein is comprised of just two helices and a hydrophobic core and is one of the smallest proteins whose folding has been characterised. Φ-value analysis revealed that Tho1 SAP folds through a transition state where helix 1 is the most extensively formed element of secondary structure and flickering native-like core contacts from Leu35 are also present. The contacts that contribute most to native state stability of Tho1 SAP are not formed in the transition state.

  4. Degradation of LIM domain-binding protein three during processing of Spanish dry-cured ham

    OpenAIRE

    2014-01-01

    Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9 months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-9...

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

    OpenAIRE

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

    2012-01-01

    YAP (Yes-associated protein) is a potent oncogene and a major effector of the mammalian Hippo tumor suppressor pathway. In this review, our emphasis is on the structural basis of how YAP recognizes its various cellular partners. In particular, we discuss the role of LATS kinase and AMOTL1 junction protein, two key cellular partners of YAP that bind to its WW domain, in mediating cytoplasmic localization of YAP and thereby playing a key role in the regulation of its transcriptional activity. I...

  6. HD-GYP domain proteins regulate biofilm formation and virulence in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Ryan, Robert P.; Lucey, Jean; O'Donovan, Karen

    2009-01-01

    2572 had a negative influence on swarming that was cryptic and was revealed only after removal of an uncharacterized C-terminal domain. Mutation of PA4108, PA4781 and PA2572 had distinct effects on biofilm formation and architecture of P. aeruginosa. All three proteins contributed to virulence of P...... residues (YN-GYP). Here we have investigated the role of these proteins in biofilm formation, virulence factor synthesis and virulence of P. aeruginosa. Mutation of PA4108 and PA4781 led to an increase in the level of cyclic-di-GMP in P. aeruginosa, consistent with the predicted activity of the encoded...

  7. RNA-binding Domain of the Key Structural Protein P7 for the Rice dwarf virus Particle Assembly

    Institute of Scientific and Technical Information of China (English)

    Bo-Xiong ZHONG; Yan-Wei SHEN; Toshihiro OMURA

    2005-01-01

    The Rice dwarf virus (RDV) P7 structural protein is the key protein in the RDV particle assembly. The P7 protein was digested partially or completely by Staphylococcus aureus V8 protease and/or Pseudomonas fragi Asp-N protease. The molecular mass and the N-terminal amino acid sequence of the polypeptide fragments of the P7 protein were determined by SDS-PAGE and the Edman degradation method,respectively. Then the polypeptides were located in the deduced amino acid sequence of the RDV P7 protein based on the nucleotide sequence information, with the knowledge of the specific cleavage sites of the Staphylococcus aureus V8 and Pseudomonasfragi Asp-N protease, and the two RNA-binding domains in the P7 protein were identified. Domain 1 was located in the residue 128-249 containing 122 amino acids and domain 2 was located in the residue 325-355 containing 31 amino acids. Thus, these two domains may play an important role in the virus particle assembly by contributing to the packaging of viral dsRNAs inside the particles. The two domains may be novel RNA-binding domains, because no amino acid sequences highly similar to the conservative sequences of known dsRNA-binding domains reported so far. The similarity between the motif of domain 1 and the motif of the DNA-binding protein suggests that the DNA-binding activity of the RDV P7 protein may be due to this sequence. The similarity between the motif of domain 1 and the motif of the RNA polymerase domain suggests that the P7 protein may also play a role in RNA synthesis,besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  9. Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis

    Institute of Scientific and Technical Information of China (English)

    Fangfang Li; Fanping Meng; Quanxin Jin; Changyuan Sun; Yingxin Li; Honghua Li; Songzhu Jin

    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 pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia 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 speciifc immunosuppressive therapy of myasthenia gravis.

  10. Arabidopsis RADICAL-INDUCED CELL DEATH1 belongs to the WWE protein-protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses.

    Science.gov (United States)

    Ahlfors, Reetta; Lång, Saara; Overmyer, Kirk; Jaspers, Pinja; Brosché, Mikael; Tauriainen, Airi; Kollist, Hannes; Tuominen, Hannele; Belles-Boix, Enric; Piippo, Mirva; Inzé, Dirk; Palva, E Tapio; Kangasjärvi, Jaakko

    2004-07-01

    Experiments with several Arabidopsis thaliana mutants have revealed a web of interactions between hormonal signaling. Here, we show that the Arabidopsis mutant radical-induced cell death1 (rcd1), although hypersensitive to apoplastic superoxide and ozone, is more resistant to chloroplastic superoxide formation, exhibits reduced sensitivity to abscisic acid, ethylene, and methyl jasmonate, and has altered expression of several hormonally regulated genes. Furthermore, rcd1 has higher stomatal conductance than the wild type. The rcd1-1 mutation was mapped to the gene At1g32230 where it disrupts an intron splice site resulting in a truncated protein. RCD1 belongs to the (ADP-ribosyl)transferase domain-containing subfamily of the WWE protein-protein interaction domain protein family. The results suggest that RCD1 could act as an integrative node in hormonal signaling and in the regulation of several stress-responsive genes.

  11. Characterization and Evolution of the Cell Cycle-Associated Mob Domain-Containing Proteins in Eukaryotes

    Directory of Open Access Journals (Sweden)

    Nicola Vitulo

    2007-01-01

    Full Text Available 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, confi rming the universal distribution of this protein family and suggesting its prominent biological function. The phylogenetic analysis reveals fi ve 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.

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

    Directory of Open Access Journals (Sweden)

    Asita Elengoe

    2015-01-01

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

  13. I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions

    Energy Technology Data Exchange (ETDEWEB)

    Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Yoshimitsu, Makoto; Hachiman, Miho [Division of Hematology and Immunology, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Ikeda, Masanori [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

    2015-12-15

    The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner. Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability. - Highlights: • I-mfa domain proteins, HIC and I-mfa, specifically interact with HTLV-1 Tax. • HIC and I-mfa repress the Tax-dependent transactivation of HTLV-1 LTR. • HIC represses the Tax-dependent transactivation of NF-κΒ. • HIC decreases the nuclear distribution of Tax. • HIC stimulates the proteasomal degradation of Tax.

  14. Structure of the RecQ C-terminal domain of human Bloom syndrome protein.

    Science.gov (United States)

    Kim, Sun-Yong; Hakoshima, Toshio; Kitano, Ken

    2013-11-21

    Bloom syndrome is a rare genetic disorder characterized by genomic instability and cancer predisposition. The disease is caused by mutations of the Bloom syndrome protein (BLM). Here we report the crystal structure of a RecQ C-terminal (RQC) domain from human BLM. The structure reveals three novel features of BLM RQC which distinguish it from the previous structures of the Werner syndrome protein (WRN) and RECQ1. First, BLM RQC lacks an aromatic residue at the tip of the β-wing, a key element of the RecQ-family helicases used for DNA-strand separation. Second, a BLM-specific insertion between the N-terminal helices exhibits a looping-out structure that extends at right angles to the β-wing. Deletion mutagenesis of this insertion interfered with binding to Holliday junction. Third, the C-terminal region of BLM RQC adopts an extended structure running along the domain surface, which may facilitate the spatial positioning of an HRDC domain in the full-length protein.

  15. A Variable Light Domain Fluorogen Activating Protein Homodimerizes To Activate Dimethylindole Red

    Energy Technology Data Exchange (ETDEWEB)

    Senutovitch, Nina; Stanfield, Robyn L.; Bhattacharyya, Shantanu; Rule, Gordon S.; Wilson, Ian A.; Armitage, Bruce A.; Waggoner, Alan S.; Berget, Peter B. (Scripps); (CM)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-15

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

  17. SRA-domain proteins required for DRM2-mediated de novo DNA methylation.

    Directory of Open Access Journals (Sweden)

    Lianna M Johnson

    2008-11-01

    Full Text Available De novo DNA methylation and the maintenance of DNA methylation in asymmetrical sequence contexts is catalyzed by homologous proteins in plants (DRM2 and animals (DNMT3a/b. In plants, targeting of DRM2 depends on small interfering RNAs (siRNAs, although the molecular details are still unclear. Here, we show that two SRA-domain proteins (SUVH9 and SUVH2 are also essential for DRM2-mediated de novo and maintenance DNA methylation in Arabidopsis thaliana. At some loci, SUVH9 and SUVH2 act redundantly, while at other loci only SUVH2 is required, and this locus specificity correlates with the differing DNA-binding affinity of the SRA domains within SUVH9 and SUVH2. Specifically, SUVH9 preferentially binds methylated asymmetric sites, while SUVH2 preferentially binds methylated CG sites. The suvh9 and suvh2 mutations do not eliminate siRNAs, suggesting a role for SUVH9 and SUVH2 late in the RNA-directed DNA methylation pathway. With these new results, it is clear that SRA-domain proteins are involved in each of the three pathways leading to DNA methylation in Arabidopsis.

  18. Functional characterisation of the WW minimal domain for delivering therapeutic proteins by adenovirus dodecahedron.

    Directory of Open Access Journals (Sweden)

    Ana Villegas-Méndez

    Full Text Available Protein transduction offers a great therapeutic potential by efficient delivery of biologically active cargo into cells. The Adenovirus Dd (Dodecahedron has recently been shown to deliver proteins fused to the tandem WW(2-3-4 structural domains from the E3 ubiquitin ligase Nedd4. In this study, we conclusively show that Dd is able to efficiently deliver cargo inside living cells, which mainly localize in fast moving endocytic vesicles, supporting active transport along the cytoskeleton. We further improve this delivery system by expressing a panel of 13 WW-GFP mutant forms to characterize their binding properties towards Dd. We identified the domain WW(3 and its mutant form WW(3_10_13 to be sufficient for optimal binding to Dd. We greatly minimise the interacting WW modules from 20 to 6 kDa without compromising its efficient delivery by Dd. Using these minimal WW domains fused to the tumor suppressor p53 protein, we show efficient cellular uptake and distribution into cancer cells, leading to specific induction of apoptosis in these cells. Taken together, these findings represent a step further towards the development of a Dd-based delivery system for future therapeutic application.

  19. Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

    Directory of Open Access Journals (Sweden)

    Alessandra Pasquo

    Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

  20. StAR-related lipid transfer domain protein 5 binds primary bile acids[S

    Science.gov (United States)

    Létourneau, Danny; Lorin, Aurélien; Lefebvre, Andrée; Frappier, Vincent; Gaudreault, Francis; Najmanovich, Rafael; Lavigne, Pierre; LeHoux, Jean-Guy

    2012-01-01

    Steroidogenic acute regulatory-related lipid transfer (START) domain proteins are involved in the nonvesicular intracellular transport of lipids and sterols. The STARD1 (STARD1 and STARD3) and STARD4 subfamilies (STARD4–6) have an internal cavity large enough to accommodate sterols. To provide a deeper understanding on the structural biology of this domain, the binding of sterols to STARD5, a member of the STARD4 subfamily, was monitored. The SAR by NMR [1H-15N heteronuclear single-quantum coherence (HSQC)] approach, complemented by circular dichroism (CD) and isothermal titration calorimetry (ITC), was used. Titration of STARD5 with cholic (CA) and chenodeoxycholic acid (CDCA), ligands of the farnesoid X receptor (FXR), leads to drastic perturbation of the 1H-15N HSQC spectra and the identification of the residues in contact with those ligands. The most perturbed residues in presence of ligands are lining the internal cavity of the protein. Ka values of 1.8·10−4 M−1 and 6.3·104 M−1 were measured for CA and CDCA, respectively. This is the first report of a START domain protein in complex with a sterol ligand. Our original findings indicate that STARD5 may be involved in the transport of bile acids rather than cholesterol. PMID:23018617

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

    Science.gov (United States)

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

    2017-08-25

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

  2. Overexpression of AtGRDP2, a novel glycine-rich domain protein, accelerates plant growth and improves stress tolerance

    OpenAIRE

    Maria Azucena Ortega-Amaro; Aida Araceli Rodriguez-Hernandez; Margarita eRodriguez-Kessler; Eloisa eHernandez-Lucero; Sergio eRosales Mendoza; Alejandro eIbañez-Salazar; Pablo eDelgado; Juan Francisco Jimenez Bremont

    2015-01-01

    Proteins with glycine-rich signatures have been reported in a wide variety of organisms including plants, mammalians, fungi, and bacteria. Plant glycine-rich protein genes exhibit developmental and tissue-specific expression patterns. Herein, we present the characterization of the AtGRDP2 gene using Arabidopsis null and knockdown mutants and, Arabidopsis and lettuce over-expression lines. AtGRDP2 encodes a short glycine-rich domain protein, containing a DUF1399 domain and a putative RNA recog...

  3. The complex regulation of HIC (Human I-mfa domain containing protein) expression.

    Science.gov (United States)

    Reiss-Sklan, Ella; Levitzki, Alexander; Naveh-Many, Tally

    2009-07-07

    Human I-mfa domain containing protein (HIC) differentially regulates transcription from viral promoters. HIC affects the Wnt pathway, the JNK/SAPK pathway and the activity of positive transcription elongation factor-b (P-TEFb). Studies exploring HIC function in mammalian cells used ectopically expressed HIC due to undetected endogenous HIC protein. HIC mRNA contains exceptionally long 5' and 3' untranslated regions (UTRs) compared to the average length of mRNA UTRs. Here we show that HIC protein is subject to strict repression at multiple levels. The HIC mRNA UTRs reduce the expression of HIC or of a reporter protein: The HIC 3'-UTR decreases both HIC and reporter mRNA levels, whereas upstream open reading frames located in the 5'-UTR repress the translation of HIC or of the reporter protein. In addition, ectopically expressed HIC protein is degraded by the proteasome, with a half-life of approximately 1 h, suggesting that upon activation, HIC expression in cells may be transient. The strict regulation of HIC expression at the levels of mRNA stability, translation efficiency and protein stability suggests that expression of the HIC protein and its involvement in the various pathways is required only under specific cellular conditions.

  4. The complex regulation of HIC (Human I-mfa domain containing protein expression.

    Directory of Open Access Journals (Sweden)

    Ella Reiss-Sklan

    Full Text Available Human I-mfa domain containing protein (HIC differentially regulates transcription from viral promoters. HIC affects the Wnt pathway, the JNK/SAPK pathway and the activity of positive transcription elongation factor-b (P-TEFb. Studies exploring HIC function in mammalian cells used ectopically expressed HIC due to undetected endogenous HIC protein. HIC mRNA contains exceptionally long 5' and 3' untranslated regions (UTRs compared to the average length of mRNA UTRs. Here we show that HIC protein is subject to strict repression at multiple levels. The HIC mRNA UTRs reduce the expression of HIC or of a reporter protein: The HIC 3'-UTR decreases both HIC and reporter mRNA levels, whereas upstream open reading frames located in the 5'-UTR repress the translation of HIC or of the reporter protein. In addition, ectopically expressed HIC protein is degraded by the proteasome, with a half-life of approximately 1 h, suggesting that upon activation, HIC expression in cells may be transient. The strict regulation of HIC expression at the levels of mRNA stability, translation efficiency and protein stability suggests that expression of the HIC protein and its involvement in the various pathways is required only under specific cellular conditions.

  5. HMMCAS: a web tool for the identification and domain annotations of Cas proteins.

    Science.gov (United States)

    Chai, Guoshi; Yu, Min; Jiang, Lixu; Duan, Yaocong; Huang, Jian

    2017-02-07

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immune systems are discovered in many bacteria and most archaea. These systems are encoded by cas (CRISPR-associated) operons that have an extremely diverse architecture. The most crucial step in the depiction of cas operons composition is the identification of cas genes or Cas proteins. With the continuous increase of the newly sequenced archaeal and bacterial genomes, the recognition of new Cas proteins is becoming possible, which not only provides candidates for novel genome editing tools but also helps to understand the prokaryotic immune system better. Here we describe HMMCAS, a web service for the detection of CRISPR-associated structural and functional domains in protein sequences. HMMCAS uses hmmscan similarity search algorithm in HMMER3.1 to provide a fast, interactive service based on a comprehensive collection of hidden Markov models of Cas protein family. It can accurately identify the Cas proteins including those fusion proteins, for example the Cas1-Cas4 fusion protein in Candidatus Chloracidobacterium thermophilum B (Cab. thermophilum B). HMMCAS can also find putative cas operon and determine which type it belongs to. HMMCAS is freely available at http://i.uestc.edu.cn/hmmcas.

  6. Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila.

    Science.gov (United States)

    Nakano, Y; Nystedt, S; Shivdasani, A A; Strutt, H; Thomas, C; Ingham, P W

    2004-06-01

    The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. Here, we identify domains essential for Smo activity and investigate the sub-cellular distribution of the wild type protein in vivo. We find that deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins we show that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. We further demonstrate that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction.

  7. Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

    Science.gov (United States)

    Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

    2009-02-01

    Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

  8. Identification of routing determinants in the cytosolic domain of a secretory granule-associated integral membrane protein.

    Science.gov (United States)

    Milgram, S L; Mains, R E; Eipper, B A

    1996-07-19

    We have investigated the trafficking of integral membrane peptidylglycine alpha-amidating monooxygenase (PAM) in the neuroendocrine AtT-20 cell line. This bifunctional enzyme has two domains which together catalyze the COOH-terminal alpha-amidation of peptidylglycine substrates yielding amidated products stored in secretory granules. As soluble proteins, both catalytic domains were independently targeted to secretory granules. In contrast, membrane PAM was largely localized to the trans-Golgi network (TGN). Upon truncation of its cytoplasmic COOH-terminal domain, membrane PAM was less efficiently cleaved by secretory granule enzymes and accumulated on the plasma membrane. When transferred to the lumenal domain of the interleukin 2 receptor alpha-chain (Tac protein), the cytoplasmic domain of PAM caused rerouting of Tac from the surface to the TGN and supported internalization of Tac antibody from the plasma membrane. To define sequences in the cytoplasmic domain of integral membrane PAM involved in its trafficking, we expressed PAM proteins containing truncations, deletions, or point mutations in the COOH-terminal cytoplasmic domain. PAM proteins were not retained in the TGN when half of the cytoplasmic domain was deleted; such proteins accumulated on the plasma membrane, were not efficiently internalized, and were cleaved to generate a bifunctional PAM protein that was not stored in secretory granules. A tyrosine-based internalization motif was identified, which was not required for efficient cleavage of full-length integral membrane PAM by secretory granule enzymes. Deletion of an 18-amino acid domain surrounding this Tyr residue both diminished cleavage of membrane PAM by secretory granule enzymes and eliminated internalization of PAM from the plasma membrane. The cytoplasmic domain is responsible for retaining membrane PAM in the TGN and for retrieving membrane PAM from the cell surface, while the lumenal catalytic domains of PAM appear to be responsible for

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

    Directory of Open Access Journals (Sweden)

    Katrina F Tjhung

    2015-04-01

    Full Text Available 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 charged FLAG sequence, which is known to be post-translationally excised from pIII 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..

  10. Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins.

    Science.gov (United States)

    Shenkarev, Zakhar O; Lyukmanova, Ekaterina N; Butenko, Ivan O; Petrovskaya, Lada E; Paramonov, Alexander S; Shulepko, Mikhail A; Nekrasova, Oksana V; Kirpichnikov, Mikhail P; Arseniev, Alexander S

    2013-02-01

    Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.

  11. Characterization of the domains of zRICH, a protein induced during optic nerve regeneration in zebrafish.

    Science.gov (United States)

    Challa, Madhavi; Chapa, Gloria R; Govindaraju, Suman; González-García, Maribel; Ballestero, Rafael P

    2006-07-19

    Teleost fish show a remarkable capability of nerve regeneration in their CNS, while injuries to axon fibers in the CNS of mammals result in degeneration and loss of function. Understanding this difference has biomedical consequences to humans. Both extrinsic factors from the neuronal environment and intrinsic neuronal factors seem to play a role in successful nerve regeneration. Among the intrinsic factors, a number of proteins termed axonal growth associated proteins (GAPs) are strongly induced during axon regeneration. RICH proteins are axonal GAPs that show homology to mammalian myelin marker proteins termed CNPases. Sequence analysis distinguishes three domains in these proteins. In this report, mutant versions of zebrafish RICH proteins were generated to study the roles of the domains of the protein at biochemical and cellular levels. The central CNPase homology domain was sufficient for catalytic activity. The amino terminal acidic domain causes the anomalous electrophoretic migration observed for RICH proteins. The small C-terminal domain bears an isoprenylation motif and is necessary for the interaction of zRICH with cellular membranes. At the cellular level, expression of wild-type zRICH protein in PC12 cells did not induce neurite generation. Additionally, neither the expression of wild-type zRICH nor the expression of mutant versions of the protein interfered with the levels of differentiation of PC12 cells induced by nerve growth factor, suggesting that, at least in this model of neuronal differentiation, zRICH proteins do not participate in the process of generation of neurites.

  12. Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

    Directory of Open Access Journals (Sweden)

    Gonzalo P Solis

    Full Text Available Analyses of cultured cells and transgenic mice expressing prion protein (PrP deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1 the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2 the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and

  13. Crystal structure of the GTPase-activating protein-related domain from IQGAP1.

    Science.gov (United States)

    Kurella, Vinodh B; Richard, Jessica M; Parke, Courtney L; Lecour, Louis F; Bellamy, Henry D; Worthylake, David K

    2009-05-29

    IQGAP1 is a 190-kDa molecular scaffold containing several domains required for interaction with numerous proteins. One domain is homologous to Ras GTPase-activating protein (GAP) domains. However, instead of accelerating hydrolysis of bound GTP on Ras IQGAP1, using its GAP-related domain (GRD) binds to Cdc42 and Rac1 and stabilizes their GTP-bound states. We report here the crystal structure of the isolated IQGAP1 GRD. Despite low sequence conservation, the overall structure of the GRD is very similar to the GAP domains from p120 RasGAP, neurofibromin, and SynGAP. However, instead of the catalytic "arginine finger" seen in functional Ras GAPs, the GRD has a conserved threonine residue. GRD residues 1099-1129 have no structural equivalent in RasGAP and are seen to form an extension at one end of the molecule. Because the sequence of these residues is highly conserved, this region likely confers a functionality particular to IQGAP family GRDs. We have used isothermal titration calorimetry to demonstrate that the isolated GRD binds to active Cdc42. Assuming a mode of interaction similar to that displayed in the Ras-RasGAP complex, we created an energy-minimized model of Cdc42.GTP bound to the GRD. Residues of the GRD that contact Cdc42 map to the surface of the GRD that displays the highest level of sequence conservation. The model indicates that steric clash between threonine 1046 with the phosphate-binding loop and other subtle changes would likely disrupt the proper geometry required for GTP hydrolysis.

  14. Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

    Directory of Open Access Journals (Sweden)

    Matej Skočaj

    Full Text Available Ostreolysin A (OlyA is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II-Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1-positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-04-22

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

  16. Crystal structure of the extracellular domain of human myelin protein zero

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S.; Brunzelle, Joseph S.; Kovari, Iulia A.; Sohi, Jasloveleen; Kamholz, John; Kovari, Ladislau C. (WSU-MED); (NWU)

    2012-03-27

    Charcot-Marie-Tooth disease (CMT), a hereditary motor and sensory neuropathy, is the most common genetic neuropathy with an incidence of 1 in 2600. Several forms of CMT have been identified arising from different genomic abnormalities such as CMT1 including CMT1A, CMT1B, and CMTX. CMT1 with associated peripheral nervous system (PNS) demyelination, the most frequent diagnosis, demonstrates slowed nerve conduction velocities and segmental demyelination upon nerve biopsy. One of its subtypes, CMT1A, presents a 1.5-Mb duplication in the p11-p12 region of the human chromosome 17 which encodes peripheral myelin protein 22 (PMP22). CMT1B, a less common form, arises from the mutations in the myelin protein zero (MPZ) gene on chromosome 1, region q22-q23, which encodes the major structural component of the peripheral myelin. A rare type of CMT1 has been found recently and is caused by point mutations in early growth response gene 2 (EGR2), encoding a zinc finger transcription factor in Schwann cells. In addition, CMTX, an X-linked form of CMT, arises from a mutation in the connexin-32 gene. Myelin protein zero, associated with CMT1B, is a transmembrane protein of 219 amino acid residues. Human MPZ consists of three domains: 125 residues constitute the glycosylated immunoglobulin-like extracellular domain; 27 residues span the membrane; and 67 residues comprise the highly basic intracellular domain. MPZ makes up approximately 50% of the protein content of myelin, and is expressed predominantly in Schwann cells, the myelinating cell of the PNS. Myelin protein zero, a homophilic adhesion molecule, is a member of the immunoglobulin super-family and is essential for normal myelin structure and function. In addition, MPZ knockout mice displayed abnormal myelin that severely affects the myelination pathway, and overexpression of MPZ causes congenital hypomyelination of peripheral nerves. Myelin protein zero mutations account for {approx}5% of patients with CMT. To date, over 125

  17. The TriTryp Phosphatome: analysis of the protein phosphatase catalytic domains

    Directory of Open Access Journals (Sweden)

    Huxley-Jones Julie

    2007-11-01

    Full Text Available Abstract Background The genomes of the three parasitic protozoa Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the main subject of this study. These parasites are responsible for devastating human diseases known as Chagas disease, African sleeping sickness and cutaneous Leishmaniasis, respectively, that affect millions of people in the developing world. The prevalence of these neglected diseases results from a combination of poverty, inadequate prevention and difficult treatment. Protein phosphorylation is an important mechanism of controlling the development of these kinetoplastids. With the aim to further our knowledge of the biology of these organisms we present a characterisation of the phosphatase complement (phosphatome of the three parasites. Results An ontology-based scan of the three genomes was used to identify 86 phosphatase catalytic domains in T. cruzi, 78 in T. brucei, and 88 in L. major. We found interesting differences with other eukaryotic genomes, such as the low proportion of tyrosine phosphatases and the expansion of the serine/threonine phosphatase family. Additionally, a large number of atypical protein phosphatases were identified in these species, representing more than one third of the total phosphatase complement. Most of the atypical phosphatases belong to the dual-specificity phosphatase (DSP family and show considerable divergence from classic DSPs in both the domain organisation and sequence features. Conclusion The analysis of the phosphatome of the three kinetoplastids indicates that they possess orthologues to many of the phosphatases reported in other eukaryotes, including humans. However, novel domain architectures and unusual combinations of accessory domains, suggest distinct functional roles for several of the kinetoplastid phosphatases, which await further experimental exploration. These distinct traits may be exploited in the selection of suitable new targets for drug development to prevent

  18. Mutagenesis of bacteriophage IKe major coat protein transmembrane domain: role of an interfacial proline residue.

    Science.gov (United States)

    Williams, K A; Deber, C M

    1993-10-15

    The transmembrane (TM) domain of the 53-residue major coat protein of the M13-related bacteriophage IKe (residues 24-42: LISQTWPVVTTVVVAGVLI) has been subjected to randomized mutagenesis to probe the conformation and stability of the TM domain, as well as the effect of structurally-important residues such as proline. TM mutants were obtained by the Eckstein method of site-directed mutagenesis using the IKe genome as template so as to eliminate the need for subcloning. Over 40 single- and double-site viable mutants of bacteriophage IKe were isolated. Every residue in the TM segment, except the highly conserved Trp29, could be mutated to at least one other residue; polar and charged mutations occurred in the TM segment adjacent to the N-terminal domain (residues 24-28), while non-polar substitutions predominated in the C-terminal portion (residues 30-42). The Pro30 locus tolerated four mutations-Ala, Gly, Cys, and Ser- which represent the four side chains of least volume. Mutant coat proteins obtained directly from the phage in milligram quantities were studied by circular dichroism spectroscopy and SDS-PAGE gels. Wild type IKe coat protein solubilized in sodium deoxycholate micelles was found to occur as an alpha-helical, monomeric species which is stable at 95 degrees C, whereas the mutant Pro30-->Gly undergoes an irreversible conformational transition at ca. 90 degrees C to an aggregated beta-sheet structure. The result that Pro30 stabilizes the TM helix in the micellar membrane suggests a sterically-restricted location for the wild type Pro pyrrolidine side chain in the bulky Trp-Pro-Val triad, where it may be positioned to direct the initiation of the subsequent TM core domain helix.

  19. Virus-Induced Chaperone-Enriched (VICE domains function as nuclear protein quality control centers during HSV-1 infection.

    Directory of Open Access Journals (Sweden)

    Christine M Livingston

    2009-10-01

    Full Text Available Virus-Induced Chaperone-Enriched (VICE domains form adjacent to nuclear viral replication compartments (RC during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70, the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42 degrees C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.

  20. Ensemble models of proteins and protein domains based on distance distribution restraints.

    Science.gov (United States)

    Jeschke, Gunnar

    2016-04-01

    Conformational ensembles of intrinsically disordered peptide chains are not fully determined by experimental observations. Uncertainty due to lack of experimental restraints and due to intrinsic disorder can be distinguished if distance distributions restraints are available. Such restraints can be obtained from pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy applied to pairs of spin labels. Here, we introduce a Monte Carlo approach for generating conformational ensembles that are consistent with a set of distance distribution restraints, backbone dihedral angle statistics in known protein structures, and optionally, secondary structure propensities or membrane immersion depths. The approach is tested with simulated restraints for a terminal and an internal loop and for a protein with 69 residues by using sets of sparse restraints for underlying well-defined conformations and for published ensembles of a premolten globule-like and a coil-like intrinsically disordered protein.

  1. Exploring Symmetry as an Avenue to the Computational Design of Large Protein Domains

    Energy Technology Data Exchange (ETDEWEB)

    Fortenberry, Carie; Bowman, Elizabeth Anne; Proffitt, Will; Dorr, Brent; Combs, Steven; Harp, Joel; Mizoue, Laura; Meiler, Jens (Vanderbilt)

    2012-03-15

    It has been demonstrated previously that symmetric, homodimeric proteins are energetically favored, which explains their abundance in nature. It has been proposed that such symmetric homodimers underwent gene duplication and fusion to evolve into protein topologies that have a symmetric arrangement of secondary structure elements - 'symmetric superfolds'. Here, the ROSETTA protein design software was used to computationally engineer a perfectly symmetric variant of imidazole glycerol phosphate synthase and its corresponding symmetric homodimer. The new protein, termed FLR, adopts the symmetric ({beta}{alpha}){sub 8} TIM-barrel superfold. The protein is soluble and monomeric and exhibits two-fold symmetry not only in the arrangement of secondary structure elements but also in sequence and at atomic detail, as verified by crystallography. When cut in half, FLR dimerizes readily to form the symmetric homodimer. The successful computational design of FLR demonstrates progress in our understanding of the underlying principles of protein stability and presents an attractive strategy for the in silico construction of larger protein domains from smaller pieces.

  2. Protein CTC from Aquifex aeolicus possesses a full-sized 5S rRNA-binding domain.

    Science.gov (United States)

    Korobeinikova, A V; Shestakov, S A; Korepanov, A P; Garber, M B; Gongadze, G M

    2009-03-01

    Ribosomal 5S RNA is the only identified target for proteins of the CTC family. All known proteins of this family, except for CTC from Aquifex aeolicus, contain a full-sized 5S rRNA-binding domain. In the present study a mistake in the published A. aeolicus genome is corrected. It has been demonstrated that the ctc gene of this organism encodes the protein with a full-length 5S rRNA-binding domain. This protein binds specifically to the bacterial 5S rRNA. Thereby, our data show that CTC A. aeolicus is not an exception from the other known CTC proteins.

  3. Prediction of Certain Well-Characterized Domains of Known Functions within the PE and PPE Proteins of Mycobacteria.

    Science.gov (United States)

    Sultana, Rafiya; Tanneeru, Karunakar; Kumar, Ashwin B R; Guruprasad, Lalitha

    2016-01-01

    The PE and PPE protein family are unique to mycobacteria. Though the complete genome sequences for over 500 M. tuberculosis strains and mycobacterial species are available, few PE and PPE proteins have been structurally and functionally characterized. We have therefore used bioinformatics tools to characterize the structure and function of these proteins. We selected representative members of the PE and PPE protein family by phylogeny analysis and using structure-based sequence annotation identified ten well-characterized protein domains of known function. Some of these domains were observed to be common to all mycobacterial species and some were species specific.

  4. The PX-EM algorithm for fast stable fitting of Henderson's mixed model

    Directory of Open Access Journals (Sweden)

    Van Dyk David A

    2000-03-01

    Full Text Available Abstract This paper presents procedures for implementing the PX-EM algorithm of Liu, Rubin and Wu to compute REML estimates of variance covariance components in Henderson's linear mixed models. The class of models considered encompasses several correlated random factors having the same vector length e.g., as in random regression models for longitudinal data analysis and in sire-maternal grandsire models for genetic evaluation. Numerical examples are presented to illustrate the procedures. Much better results in terms of convergence characteristics (number of iterations and time required for convergence are obtained for PX-EM relative to the basic EM algorithm in the random regression.

  5. Tracing the origin of functional and conserved domains in the human proteome: implications for protein evolution at the modular level.

    Science.gov (United States)

    Pal, Lipika R; Guda, Chittibabu

    2006-11-07

    The functional repertoire of the human proteome is an incremental collection of functions accomplished by protein domains evolved along the Homo sapiens lineage. Therefore, knowledge on the origin of these functionalities provides a better understanding of the domain and protein evolution in human. The lack of proper comprehension about such origin has impelled us to study the evolutionary origin of human proteome in a unique way as detailed in this study. This study reports a unique approach for understanding the evolution of human proteome by tracing the origin of its constituting domains hierarchically, along the Homo sapiens lineage. The uniqueness of this method lies in subtractive searching of functional and conserved domains in the human proteome resulting in higher efficiency of detecting their origins. From these analyses the nature of protein evolution and trends in domain evolution can be observed in the context of the entire human proteome data. The method adopted here also helps delineate the degree of divergence of functional families occurred during the course of evolution. This approach to trace the evolutionary origin of functional domains in the human proteome facilitates better understanding of their functional versatility as well as provides insights into the functionality of hypothetical proteins present in the human proteome. This work elucidates the origin of functional and conserved domains in human proteins, their distribution along the Homo sapiens lineage, occurrence frequency of different domain combinations and proteome-wide patterns of their distribution, providing insights into the evolutionary solution to the increased complexity of the human proteome.

  6. WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN

    Energy Technology Data Exchange (ETDEWEB)

    Rangaraj, K.; Cooper, P.K.; Trego, K.S.

    2009-01-01

    The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG) and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C-terminal region of

  7. Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p.

    Science.gov (United States)

    Koyama, Masako; Hirano, Hidemi; Shirai, Natsuki; Matsuura, Yoshiyuki

    2017-08-08

    Xpo1p (yeast CRM1) is the major nuclear export receptor that carries a plethora of proteins and ribonucleoproteins from the nucleus to cytoplasm. The passage of the Xpo1p nuclear export complex through nuclear pore complexes (NPCs) is facilitated by interactions with nucleoporins (Nups) containing extensive repeats of phenylalanine-glycine (so-called FG repeats), although the precise role of each Nup in the nuclear export reaction remains incompletely understood. Here we report structural and biochemical characterization of the interactions between the Xpo1p nuclear export complex and the FG repeats of Nup42p, a nucleoporin localized at the cytoplasmic face of yeast NPCs and has characteristic SxFG/PxFG sequence repeat motif. The crystal structure of Xpo1p-PKI-Nup42p-Gsp1p-GTP complex identified three binding sites for the SxFG/PxFG repeats on HEAT repeats 14-20 of Xpo1p. Mutational analyses of Nup42p showed that the conserved serines and prolines in the SxFG/PxFG repeats contribute to Xpo1p-Nup42p binding. Our structural and biochemical data suggest that SxFG/PxFG-Nups such as Nup42p and Nup159p at the cytoplasmic face of NPCs provide high-affinity docking sites for the Xpo1p nuclear export complex in the terminal stage of NPC passage and that subsequent disassembly of the nuclear export complex facilitates recycling of free Xpo1p back to the nucleus. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  8. Dimerization of the transmembrane domain of amyloid precursor proteins and familial Alzheimer's disease mutants

    Directory of Open Access Journals (Sweden)

    Fraser Paul E

    2008-01-01

    Full Text Available Abstract Background Amyloid precursor protein (APP is enzymatically cleaved by γ-secretase to form two peptide products, either Aβ40 or the more neurotoxic Aβ42. The Aβ42/40 ratio is increased in many cases of familial Alzheimer's disease (FAD. The transmembrane domain (TM of APP contains the known dimerization motif GXXXA. We have investigated the dimerization of both wild type and FAD mutant APP transmembrane domains. Results Using synthetic peptides derived from the APP-TM domain, we show that this segment is capable of forming stable transmembrane dimers. A model of a dimeric APP-TM domain reveals a putative dimerization interface, and interestingly, majority of FAD mutations in APP are localized to this interface region. We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers. Conclusion The dissociation constants are correlated to both the Aβ42/Aβ40 ratio and the mean age of disease onset in AD patients. We also show that these TM-peptides reduce Aβ production and Aβ42/Aβ40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and γ-secretase components, potentially revealing a new class of γ-secretase inhibitors.

  9. A Conserved Myc Protein Domain, MBIV, Regulates DNA Binding, Apoptosis, Transformation, and G2 Arrest†

    Science.gov (United States)

    Cowling, Victoria H.; Chandriani, Sanjay; Whitfield, Michael L.; Cole, Michael D.

    2006-01-01

    The myc family of oncogenes is well conserved throughout evolution. Here we present the characterization of a domain conserved in c-, N-, and L-Myc from fish to humans, N-Myc317-337, designated Myc box IV (MBIV). A deletion of this domain leads to a defect in Myc-induced apoptosis and in some transformation assays but not in cell proliferation. Unlike other Myc mutants, MycΔMBIV is not a simple loss-of-function mutant because it is hyperactive for G2 arrest in primary cells. Microarray analysis of genes regulated by N-MycΔMBIV reveals that it is weakened for transactivation and repression but not nearly as defective as N-MycΔMBII. Although the mutated region is not part of the previously defined DNA binding domain, we find that N-MycΔMBIV has a significantly lower affinity for DNA than the wild-type protein in vitro. Furthermore, chromatin immunoprecipitation shows reduced binding of N-MycΔMBIV to some target genes in vivo, which correlates with the defect in transactivation. Thus, this conserved domain has an unexpected role in Myc DNA binding activity. These data also provide a novel separation of Myc functions linked to the modulation of DNA binding activity. PMID:16705173

  10. Regulation and action of the bacterial enhancer-binding protein AAA+ domains

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Nixon, B. Tracy (Penn)

    2008-08-04

    Bacterial EBPs (enhancer-binding proteins) play crucial roles in regulating cellular responses to environmental changes, in part by providing efficient control over {sigma}{sup 54}-dependent gene transcription. The AAA+ (ATPase associated with various cellular activites) domain of the EBPs, when assembled into a ring, uses energy from ATP binding, hydrolysis and product release to remodel the {sigma}{sup 54}-RNAP (RNA polymerase) holoenzyme so that it can transition from closed to open form at promoter DNA. The assembly, and hence activity, of these ATPases are regulated by many different signal transduction mechanisms. Recent advances in solution scattering techniques, when combined with high-resolution structures and biochemical data, have enabled us to obtain mechanistic insights into the regulation and action of a subset of these {sigma}{sup 54} activators: those whose assembly into ring form is controlled by two-component signal transduction. We review (i) experimental considerations of applying the SAXS (small-angle X-ray scattering)/WAXS (wide-angle X-ray scattering) technique, (ii) distinct regulation mechanisms of the AAA+ domains of three EBPs by similar two-component signal transduction receiver domains, and (iii) major conformational changes and correlated {sigma}{sup 54}-binding activity of an isolated EBP AAA+ domain in the ATP hydrolysis cycle.

  11. Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain.

    Science.gov (United States)

    Sheng, Ren; Jung, Da-Jung; Silkov, Antonina; Kim, Hyunjin; Singaram, Indira; Wang, Zhi-Gang; Xin, Yao; Kim, Eui; Park, Mi-Jeong; Thiagarajan-Rosenkranz, Pallavi; Smrt, Sean; Honig, Barry; Baek, Kwanghee; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

    2016-08-19

    Lymphocyte-specific protein-tyrosine kinase (Lck) plays an essential role in T cell receptor (TCR) signaling and T cell development, but its activation mechanism is not fully understood. To explore the possibility that plasma membrane (PM) lipids control TCR signaling activities of Lck, we measured the membrane binding properties of its regulatory Src homology 2 (SH2) and Src homology 3 domains. The Lck SH2 domain binds anionic PM lipids with high affinity but with low specificity. Electrostatic potential calculation, NMR analysis, and mutational studies identified the lipid-binding site of the Lck SH2 domain that includes surface-exposed basic, aromatic, and hydrophobic residues but not the phospho-Tyr binding pocket. Mutation of lipid binding residues greatly reduced the interaction of Lck with the ζ chain in the activated TCR signaling complex and its overall TCR signaling activities. These results suggest that PM lipids, including phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, modulate interaction of Lck with its binding partners in the TCR signaling complex and its TCR signaling activities in a spatiotemporally specific manner via its SH2 domain. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Synthetic protein scaffolds based on peptide motifs and cognate adaptor domains for improving metabolic productivity

    Directory of Open Access Journals (Sweden)

    Anselm H.C. Horn

    2015-11-01

    Full Text Available The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.

  13. The Myb-domain protein ULTRAPETALA1 INTERACTING FACTOR 1 controls floral meristem activities in Arabidopsis.

    Science.gov (United States)

    Moreau, Fanny; Thévenon, Emmanuel; Blanvillain, Robert; Lopez-Vidriero, Irene; Franco-Zorrilla, Jose Manuel; Dumas, Renaud; Parcy, François; Morel, Patrice; Trehin, Christophe; Carles, Cristel C

    2016-04-01

    Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression. © 2016. Published by The Company of Biologists Ltd.

  14. Structural, evolutionary and functional analysis of the NAC domain protein family in Eucalyptus.

    Science.gov (United States)

    Hussey, Steven G; Saïdi, Mohammed N; Hefer, Charles A; Myburg, Alexander A; Grima-Pettenati, Jacqueline

    2015-06-01

    NAC domain transcription factors regulate many developmental processes and stress responses in plants and vary widely in number and family structure. We analysed the characteristics and evolution of the NAC gene family of Eucalyptus grandis, a fast-growing forest tree in the rosid order Myrtales. NAC domain genes identified in the E. grandis genome were subjected to amino acid sequence, phylogenetic and motif analyses. Transcript abundance in developing tissues and abiotic stress conditions in E. grandis and E. globulus was quantified using RNA-seq and reverse transcription quantitative PCR (RT-qPCR). One hundred and eighty-nine E. grandis NAC (EgrNAC) proteins, arranged into 22 subfamilies, are extensively duplicated in subfamilies associated with stress response. Most EgrNAC genes form tandem duplicate arrays that frequently carry signatures of purifying selection. Sixteen amino acid motifs were identified in EgrNAC proteins, eight of which are enriched in, or unique to, Eucalyptus. New candidates for the regulation of normal and tension wood development and cold responses were identified. This first description of a Myrtales NAC domain family reveals an unique history of tandem duplication in stress-related subfamilies that has likely contributed to the adaptation of eucalypts to the challenging Australian environment. Several new candidates for the regulation of stress, wood formation and tree-specific development are reported.

  15. Identification of Domains for Efficient Notch Signaling Activity in Immobilized Notch Ligand Proteins.

    Science.gov (United States)

    Liu, Ledi; Wada, Hiroe; Matsubara, Natsuki; Hozumi, Katsuto; Itoh, Motoyuki

    2017-04-01

    Notch is a critical signaling pathway that controls cell fate and tissue homeostasis, but the functional characterization of Notch ligand domains that activate Notch receptors remains incomplete. Here, we established a method for immobilizing Notch ligand proteins onto beads to measure time-dependent Notch activity after the addition of Notch ligand-coated beads. A comparison between activities by the Notch ligand found on the cell surface to that of the ligand immobilized on beads showed that immobilized Notch ligand protein produces comparable signal activity during the first 10 h. Follow-up truncation studies showed that the N-terminal epidermal growth factor (EGF) repeat three region of delta like canonical Notch ligand 4 (DLL4) or jagged 1 (JAG1) is the minimum region for activating Notch signaling, and the DLL4 EGF repeat three domain may have a role in activation through a mechanism other than by increasing binding affinity. In addition, we found that reconstruction of the DLL4 delta and OSM-11 (DOS) motif (N257P) resulted in an increase in both binding affinity and signaling activity, which suggests that the role of the DOS motif is conserved among Notch ligands. Furthermore, active DLL4 protein on beads promoted T cell differentiation or inhibited B cell differentiation in vitro, whereas JAG1 proteins on beads did not have any effect. Taken together, our findings provide unambiguous evidence for the role of different Notch ligands and their domains in Notch signal activation, and may be potential tools for controlling Notch signaling activation. J. Cell. Biochem. 118: 785-796, 2017. © 2016 Wiley Periodicals, Inc.

  16. Domain a' of protein disulfide isomerase plays key role in inhibiting alpha-synuclein fibril formation.

    Science.gov (United States)

    Cheng, Han; Wang, Lei; Wang, Chih-chen

    2010-07-01

    alpha-Synuclein (alpha Syn) is the main component of Lewy bodies formed in midbrain dopaminergic neurons which is a pathological characteristic of Parkinson's disease. It has been recently showed to induce endoplasmic reticulum (ER) stress and impair ER functions. However, the mechanism of how ER responds to alpha Syn toxicity is poorly understood. In the present study, we found that protein disulfide isomerase (PDI), a stress protein abundant in ER, effectively inhibits alpha Syn fibril formation in vitro. In PDI molecule with a structure of abb'xa'c, domain a' was found to be essential and sufficient for PDI to inhibit alpha Syn fibril formation. PDI was further found to be more avid for binding with intermediate species formed during alpha Syn fibril formation, and the binding was more intensive in the later lag phase. Our results provide new insight into the role of PDI in protecting ER from the deleterious effects of misfolded protein accumulation in many neurodegenerative diseases.

  17. Origin and Status of Homologous Proteins of Biomineralization (Biosilicification in the Taxonomy of Phylogenetic Domains

    Directory of Open Access Journals (Sweden)

    Igor E. Pamirsky

    2013-01-01

    Full Text Available The taxonomic affiliation (in the systematisation of viruses, and biological domains of known peptides and proteins of biomineralization (silicateins, silaffins, silacidins and silicase and their primary structure homologues were analyzed (methods in silico; using Uniprot database. The total number of known peptides and proteins of biosilicification was counted. The data of the quantitative distribution of the detected homologues found in nature are presented. The similarity of the primary structures of silaffins, silacidins, silicateins, silicase, and their homologues was 21–94%, 45–98%, 39–50%, and 28–40%, respectively. These homologues are found in many organisms, from the Protista to the higher plants and animals, including humans, as well as in bacteria and extracellular agents, and they perform a variety of biological functions, such as biologically controlled mineralisation. The provisional classification of these biomineralization proteins is presented. The interrelation of the origin of the first organic polymers and biomineralization is discussed.

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

    Science.gov (United States)

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

    2017-01-01

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

  19. Directed evolution of human heavy chain variable domain (VH) using in vivo protein fitness filter.

    Science.gov (United States)

    Kim, Dong-Sik; Song, Hyung-Nam; Nam, Hyo Jung; Kim, Sung-Geun; Park, Young-Seoub; Park, Jae-Chan; Woo, Eui-Jeon; Lim, Hyung-Kwon

    2014-01-01

    Human immunoglobulin heavy chain variable domains (VH) are promising scaffolds for antigen binding. However, VH is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the VH domain, we performed in vivo protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of E. coli. After screening a human germ-line VH library, 95% of the VH proteins obtained were identified as VH3 family members; one VH protein, MG2x1, stood out among separate clones expressing individual VH variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in VH. Comparison of the crystal structures of the VH variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the VH interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered VH acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the VH variant as a scaffold was qualified as most of VH clones selected randomly were expressed as soluble form in E. coli regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected VH conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display.

  20. The intact Kunitz domain protects the amyloid precursor protein from being processed by matriptase-2.

    Science.gov (United States)

    Beckmann, Anna-Madeleine; Glebov, Konstantin; Walter, Jochen; Merkel, Olaf; Mangold, Martin; Schmidt, Frederike; Becker-Pauly, Christoph; Gütschow, Michael; Stirnberg, Marit

    2016-08-01

    Proteolytic processing of the amyloid precursor protein (APP) leads to amyloid-β (Aβ) peptides. So far, the mechanism of APP processing is insufficiently characterized at the molecular level. Whereas the knowledge of Aβ generation by several proteases has been expanded, the contribution of the Kunitz-type protease inhibitor domain (KPI) present in two major APP isoforms to the complex proteolytic processing of APP is poorly understood. In this study, we have identified KPI-containing APP as a very potent, slow-binding inhibitor for the membrane-bound proteolytic regulator of iron homeostasis matriptase-2 by forming stable complexes with its target protease in HEK cells. Inhibition and complex formation depend on the intact KPI domain. By inhibiting matriptase-2, KPI-containing APP is protected from matriptase-2-mediated proteolysis within the Aβ region, thus preventing the generation of N-terminally truncated Aβ.

  1. Assembly of the intrinsic factor domains and oligomerization of the protein in the presence of cobalamin

    DEFF Research Database (Denmark)

    Fedosov, Sergey N; Fedosova, Natalya U; Berglund, Lars;

    2004-01-01

    oligomerized. A mixture of two fragments IF(30) + IF(20) and Cbl produced a firm complex, IF(30+20).Cbl, which could not associate to dimers. In contrast to IF(30+20).Cbl, the saturated full-length monomers IF(50).Cbl dimerized with K(d) approximately 1 microM. We suggest a two-domain organization of the full......-length protein, where two distant units, IF(30) and IF(20), can be assembled only by Cbl. They are connected by a protease-sensitive link, whose native structure is likely to be important for dimerization. However, linkage between two domains is not compulsory for Cbl binding. Advantages of the two...

  2. Birth and death of protein domains: A simple model of evolution explains power law behavior

    Directory of Open Access Journals (Sweden)

    Berezovskaya Faina S

    2002-10-01

    Full Text Available Abstract Background Power distributions appear in numerous biological, physical and other contexts, which appear to be fundamentally different. In biology, power laws have been claimed to describe the distributions of the connections of enzymes and metabolites in metabolic networks, the number of interactions partners of a given protein, the number of members in paralogous families, and other quantities. In network analysis, power laws imply evolution of the network with preferential attachment, i.e. a greater likelihood of nodes being added to pre-existing hubs. Exploration of different types of evolutionary models in an attempt to determine which of them lead to power law distributions has the potential of revealing non-trivial aspects of genome evolution. Results A simple model of evolution of the domain composition of proteomes was developed, with the following elementary processes: i domain birth (duplication with divergence, ii death (inactivation and/or deletion, and iii innovation (emergence from non-coding or non-globular sequences or acquisition via horizontal gene transfer. This formalism can be described as a birth, death and innovation model (BDIM. The formulas for equilibrium frequencies of domain families of different size and the total number of families at equilibrium are derived for a general BDIM. All asymptotics of equilibrium frequencies of domain families possible for the given type of models are found and their appearance depending on model parameters is investigated. It is proved that the power law asymptotics appears if, and only if, the model is balanced, i.e. domain duplication and deletion rates are asymptotically equal up to the second order. It is further proved that any power asymptotic with the degree not equal to -1 can appear only if the hypothesis of independence of the duplication/deletion rates on the size of a domain family is rejected. Specific cases of BDIMs, namely simple, linear, polynomial and rational

  3. Thermal denaturation of protein studied by terahertz time-domain spectroscopy

    Science.gov (United States)

    Fu, Xiuhua; Li, Xiangjun; Liu, Jianjun; Du, Yong; Hong, Zhi

    2012-12-01

    In this study, the absorption spectra of native or thermal protein were measured in 0.2-1.4THz using terahertz time-domain spectroscopy (THz-TDS) system at room temperature, their absorption spectra and the refractive spectra were obtained. Experimental results indicate that protein both has strong absorption but their characteristics were not distinct in the THz region, and the absorption decreased during thermal denatured state. In order to prove protein had been denatured, we used Differential scanning calorimeter (DSC) measured their denatured temperature, from their DSC heating traces, collagen Td=101℃, Bovine serum albumin Td=97℃. While we also combined the Fourier transform infrared spectrometer (FTIR) to investigate their secondary and tertiary structure before and after denatuation, but the results did not have the distinct changes. We turned the absorption spectra and the refractive spectra to the dielectric spectra, and used the one-stage Debye model simulated the terahertz dielectric spectra of protein before and after denaturation. This research proved that the terahertz spectrum technology is feasible in testing protein that were affected by temperature or other factors which can provide theoretical foundation in the further study about the THz spectrum of protein and peptide temperature stability.

  4. Isolation of Camelid Single-Domain Antibodies Against Native Proteins Using Recombinant Multivalent Peptide Ligands.

    Science.gov (United States)

    Alturki, Norah A; Henry, Kevin A; MacKenzie, C Roger; Arbabi-Ghahroudi, Mehdi

    2015-01-01

    Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (1) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (2) expression, purification, and characterization of the multivalent peptide-VT ligands; (3) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (4) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield custom peptide-VT ligands that appear to maintain the antigenic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest.

  5. Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-binding context.

    Science.gov (United States)

    Viganò, M A; Di Rocco, G; Zappavigna, V; Mavilio, F

    1998-11-01

    Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

  6. A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement

    Directory of Open Access Journals (Sweden)

    Finan Christopher L

    2005-03-01

    Full Text Available Abstract Background In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by the production of a secreted growth factor. This autocrine resuscitation-promoting factor (Rpf is the founder member of a family of proteins found throughout and confined to the actinobacteria (high G + C Gram-positive bacteria. The aim of this work was to search for and characterise a cognate gene family in the firmicutes (low G + C Gram-positive bacteria and obtain information about how they may control bacterial growth and resuscitation. Results In silico analysis of the accessory domains of the Rpf proteins permitted their classification into several subfamilies. The RpfB subfamily is related to a group of firmicute proteins of unknown function, represented by YabE of Bacillus subtilis. The actinobacterial RpfB and firmicute YabE proteins have very similar domain structures and genomic contexts, except that in YabE, the actinobacterial Rpf domain is replaced by another domain, which we have called Sps. Although totally unrelated in both sequence and secondary structure, the Rpf and Sps domains fulfil the same function. We propose that these proteins have undergone "non-orthologous domain displacement", a phenomenon akin to "non-orthologous gene displacement" that has been described previously. Proteins containing the Sps domain are widely distributed throughout the firmicutes and they too fall into a number of distinct subfamilies. Comparative analysis of the accessory domains in the Rpf and Sps proteins, together with their weak similarity to lytic transglycosylases, provide clear evidence that they are muralytic enzymes. Conclusions The results indicate that the firmicute Sps proteins and the actinobacterial Rpf proteins are cognate and that they control bacterial culturability via enzymatic modification of the bacterial cell envelope.

  7. Single TRAM domain RNA-binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii.

    Science.gov (United States)

    Taha; Siddiqui, K S; Campanaro, S; Najnin, T; Deshpande, N; Williams, T J; Aldrich-Wright, J; Wilkins, M; Curmi, P M G; Cavicchioli, R

    2016-09-01

    TRAM domain proteins present in Archaea and Bacteria have a β-barrel shape with anti-parallel β-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins.

  8. The First Residue of the PWWP Motif Modulates HATH Domain Binding, Stability, and Protein-Protein Interaction.

    Science.gov (United States)

    Hung, Yi-Lin; Lee, Hsia-Ju; Jiang, Ingjye; Lin, Shang-Chi; Lo, Wei-Cheng; Lin, Yi-Jan; Sue, Shih-Che

    2015-07-01

    Hepatoma-derived growth factor (hHDGF) and HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic structural motif, namely the PWWP motif. The HATH domain has attracted attention because of its ability to bind with heparin/heparan sulfate, DNA, and methylated histone peptide. Depending on the sequence of the PWWP motif, HRP HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro) forms. A-type HATH is highly unstable and tends to precipitate in solution. We replaced the Pro residue in P-type HATHHDGF with Ala and evaluated the influence on structure, dynamics, and ligand binding. Nuclear magnetic resonance (NMR) hydrogen/deuterium exchange and circular dichroism (CD) measurements revealed reduced stability. Analysis of NMR backbone (15)N relaxations (R1, R2, and nuclear Overhauser effect) revealed additional backbone dynamics in the interface between the β-barrel and the C-terminal helix bundle. The β1-β2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction through the loop. A-type HATH, therefore, shows a stronger tendency to aggregate when binding with heparin and DNA oligomers. This study defines the role of the first residue of the PWWP motif in modulating HATH domain stability and oligomer formation in binding.

  9. The Copper Metabolism MURR1 Domain protein 1 (COMMD1) modulates the aggregation of misfolded protein species in a client-specific manner

    NARCIS (Netherlands)

    W.I.M. Vonk (Willianne I.); V. Kakkar (Vaishali); P. Bartuzi (Paulina); D. Jaarsma (Dick); R. Berger (Ruud); M.A. Hofker (Marten); L.W.J. Klomp (Leo W.); C. Wijmenga (Cisca); H. Kampinga (Harm); B. van de Sluis (Bart)

    2014-01-01

    textabstractThe Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-tr

  10. The Copper Metabolism MURR1 Domain Protein 1 (COMMD1) Modulates the Aggregation of Misfolded Protein Species in a Client-Specific Manner

    NARCIS (Netherlands)

    Vonk, Willianne I. M.; Kakkar, Vaishali; Bartuzi, Paulina; Jaarsma, Dick; Berger, Ruud; Hofker, Marten H.; Klomp, Leo W. J.; Wijmenga, Cisca; Kampinga, Harm H.; van de Sluis, Bart

    2014-01-01

    The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-kappa B and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transport

  11. Membrane-spanning domain of bovine foamy virus transmembrane protein having cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    MA Yonggang; YU Hong; WANG Jinzhong; CHEN Qimin; GENG Yunqi

    2006-01-01

    Foamy viruses (FVs) have broad cellular tropism infecting vertebrates from fish to human being,which indicates that Env protein has a high capability for membrane fusion.Conservative features in all FV transmembrane (TM) proteins include a region of hydrophobic domain called membrane-spanning domain (MSD),which contains several stretches of hydrophobic amino acids.To investigate whether these features were associated with the cytotoxicity effect of TM on Escherichia coli,a series of mutants were constructed and expressed in the E.coli BL21 (DE3) using pET-32a (+) as expressing vector.The results showed that only TM3 without MSD was expressed in E.coli,whereas the other two containing full or part of the MSD (TM1 and TM2) could not be expressed.Furthermore,the bacterial amount and living bacteria analysis revealed that the cytotoxicity of TM was dependent on its MSD,especially on the stretches of hydrophobic amino acids.Western blotting analysis showed that TM3 protein was purified with affinity purification.

  12. Arabidopsis mRNA secondary structure correlates with protein function and domains

    Science.gov (United States)

    Vandivier, Lee E.; Li, Fan; Zheng, Qi; Willmann, Matthew R.; Chen, Ying; Gregory, Brian D.

    2013-01-01

    RNAs fold into intricate structures that are determined by specific base pairing interactions encoded within their primary sequences. Recently, a number of transcriptome-wide studies have suggested that RNA secondary structure is a potent cis-acting regulator of numerous post-transcriptional processes in viruses and eukaryotes. However, the need for experimentally-based structure determination methods has not been well addressed. Here, we show that the regulatory significance of Arabidopsis RNA secondary structure is revealed specifically through high-throughput, sequencing-based, structure mapping data, not by computational prediction. Additionally, we find that transcripts with similar levels of secondary structure in their UTRs (5' or 3') or CDS tend to encode proteins with coherent functions. Finally, we reveal that portions of mRNAs encoding predicted protein domains are significantly more structured than those specifying inter-domain regions. In total, our findings show the utility of high-throughput, sequencing-based, structure-mapping approaches and suggest that mRNA folding regulates protein maturation and function. PMID:23603972

  13. Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice.

    Science.gov (United States)

    Froese, Alexander; Breher, Stephanie S; Waldeyer, Christoph; Schindler, Roland F R; Nikolaev, Viacheslav O; Rinné, Susanne; Wischmeyer, Erhard; Schlueter, Jan; Becher, Jan; Simrick, Subreena; Vauti, Franz; Kuhtz, Juliane; Meister, Patrick; Kreissl, Sonja; Torlopp, Angela; Liebig, Sonja K; Laakmann, Sandra; Müller, Thomas D; Neumann, Joachim; Stieber, Juliane; Ludwig, Andreas; Maier, Sebastian K; Decher, Niels; Arnold, Hans-Henning; Kirchhof, Paulus; Fabritz, Larissa; Brand, Thomas

    2012-03-01

    Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention.

  14. De novo designed protein transduction domain mimics from simple synthetic polymers.

    Science.gov (United States)

    Tezgel, A Özgül; Telfer, Janice C; Tew, Gregory N

    2011-08-08

    Protein transduction domains (PTDs) that readily transverse cellular membranes are of great interest and are attractive tools for the intracellular delivery of bioactive molecules. Learning to program synthetic polymers and oligomers with the appropriate chemical information to capture adequately the biological activity of proteins is critical to our improved understanding of how these natural molecules work. In addition, the versatility of these synthetic mimics provides the opportunity to discover analogs with superior properties compared with their native sequences. Here we report the first detailed structure-activity relationship of a new PTD family of polymers based on a completely abiotic backbone. The synthetic approach easily allows doubling the density of guanidine functional groups, which increases the transduction efficiency of the sequences. Cellular uptake studies on three different cell lines (HEK 293T, CHO, and Jurkat T cells) confirm that these synthetic analogs are highly efficient novel protein transduction domain mimics (PTDMs), which are more effective than TAT(49-57) and nonaarginine (R9) and also highlight the usefulness of polymer chemistry at the chemistry-biology interface.

  15. A novel DNA-binding domain in the Shrunken initiator-binding protein (IBP1).

    Science.gov (United States)

    Lugert, T; Werr, W

    1994-06-01

    South-western screening of lambda gt11 expression library with a fragment of the Shrunken promoter containing the initiator element resulted in cloning of a novel maize gene. The encoded initiator-binding protein (IBP1) interacts at the transcription start site of the Shrunken promoter. Analysis of the 680 amino acid (aa) long polypeptide revealed a novel bipartite DNA-binding domain at the carboxyl terminus. In its amino-terminal part, it is weakly related to Myb R-repeats but the following basic region is also essential for DNA binding. A region of similarity to the conserved 2.1 and 2.2 motifs in bacterial sigma-factors is located close to the IBP1 amino terminus. Two putative nuclear localization signals are compatible with the presence of antigenically related polypeptides in nuclear protein extracts. The IBP1 gene was mapped to the long arm of chromosome 9 (9L095); a second highly related gene IBP2 is located on the short arm of chromosome 1 (1S014). Both genes encode proteins sharing 93% similarity and are transcribed with similar activity in different plant organs. A small 82 nucleotide intron in the IBP2 transcript is found unspliced to a variable degree in different tissues. Translation of this incompletely processed transcript would result in a truncated amino-terminal polypeptide lacking the DNA-binding domain.

  16. cDNA cloning and characterization of the carboxylesterase pxCCE016b from the diamondback moth, Plutella xylostella L.

    Institute of Scientific and Technical Information of China (English)

    HU Zhen-di; FENG Xia; LIN Qing-sheng; CHEN Huan-yu; LI Zhen-yu; YIN Fei; LIANG Pei; GAO Xi-wu

    2016-01-01

    Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a ful-length cDNA of carboxylesterasepxCCE016b was cloned and exogenously expressed inEscherichia coliat the ifrst time, which contained a 1693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of thepxCCE016b was signiifcantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore,pxCCE016b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfuly with prokaryotic cel. Western blot analysis showed that it was coded by pxCCE016b. Al the above ifndings provide important information for further functional study, although we are uncertainty

  17. Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Bradley R.; Drake, Eric J.; Shi, Ce; Aldrich, Courtney C.; Gulick, Andrew M.

    2016-09-05

    Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.

  18. Size and shape of the repetitive domain of high molecular weight wheat gluten proteins. 1. Small angle neutron scattering

    NARCIS (Netherlands)

    Egelhaaf, SU; van Swieten, E; Bosma, T; de Boef, E; van Dijk, AA; Robillard, GT; Egelhaaf, Stefan U.

    2003-01-01

    The solution structure of the central repetitive domain of high molecular weight (HMW) wheat gluten proteins has been investigated for a range of concentrations and temperatures using mainly small-angle neutron scattering. A representative part of the repetitive domain (dBl) was studied as well as a

  19. Partial analysis of the central domain of the Bacillus sphaericus JG-A12 S-layer protein

    Energy Technology Data Exchange (ETDEWEB)

    Schnorpfeil, M.; Raff, J.; Flemming, K.; Selenska-Pobell, S.

    2002-05-01

    310 amino acids from the central domain of the B. sphaericus JG-A12 S-layer were analyzed. In contrast to the N-terminal domain of this protein, which possesses a unique structure, the part studied in this work shares a significant identity with the corresponding parts of several other B. sphaericus S-layers. (orig.)

  20. Photochemical Reactions of the LOV and LOV-Linker Domains of the Blue Light Sensor Protein YtvA

    NARCIS (Netherlands)

    Choi, S.; Nakasone, Y.; Hellingwerf, K.J.; Terazima, M.

    2016-01-01

    YtvA is a blue light sensor protein composed of an N-terminal LOV (light-oxygen-voltage) domain, a linker helix, and the C-terminal sulfate transporter and anti-sigma factor antagonist domain. YtvA is believed to act as a positive regulator for light and salt stress responses by regulating the

  1. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Andrew Loyd [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the δ-Al-ε 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 βαβ-βαβ pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel β-sheet. In addition 15N T1, T2, and 15N/1H 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 1H, 13C, and 15N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and 15N 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.

  2. A Novel Signal Transduction Protein: Combination of Solute Binding and Tandem PAS-like Sensor Domains in One Polypeptide Chain

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Rui; Wilton, R; Cuff, M. E.; Endres, M.; Babnigg, G.; Edirisinghe, J. N.; Henry, C. S.; Schiffer, M.; Pokkuluri, P. R.

    2017-04-01

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

  3. Tracing the origin of functional and conserved domains in the human proteome: implications for protein evolution at the modular level

    OpenAIRE

    Guda Chittibabu; Pal Lipika R

    2006-01-01

    Abstract Background The functional repertoire of the human proteome is an incremental collection of functions accomplished by protein domains evolved along the Homo sapiens lineage. Therefore, knowledge on the origin of these functionalities provides a better understanding of the domain and protein evolution in human. The lack of proper comprehension about such origin has impelled us to study the evolutionary origin of human proteome in a unique way as detailed in this study. Results This stu...

  4. Polarization of cumulative protons in the reaction. gamma. A r arrow pX

    Energy Technology Data Exchange (ETDEWEB)

    Avakyan, R.O.; Avetisyan, A.E.; Arestakesyan, G.A.; Bartikyan, M.V.; Garibyan, Y.A.; Grigoryan, A.E.; Eganov, V.S.; Karapetyan, A.P.; Karapetyan, M.P.; Keropyan, I.A.; and others

    1989-02-01

    The polarization of cumulative protons has been measured in the reaction {gamma}{ital A}{r arrow}{ital pX} in the proton-energy region 170--270 MeV for C and Sn nuclei in bremsstrahlung beams produced by 4.5- and 1.5-GeV electrons. The measured polarization value is close to zero.

  5. INFERENCE ON P(X < Y FOR EXPONENTIATED FAMILY OF DISTRIBUTIONS

    Directory of Open Access Journals (Sweden)

    Sudhansu S. Maiti

    2011-04-01

    Full Text Available Inference on R = P(X < Y has been considered when X and Y belong to independent exponentiated family of distributions. Maximum Likelihood Estimator (MLE, Uniformly Minimum Variance Unbiased Estimator (UMVUE and Bayes Estimator of R has been derived and compared through simulation study. Exact and approximate confidence intervals and Bayesian credible intervals have also been derived.

  6. WW domain binding protein-2, an E6-associated protein interacting protein, acts as a coactivator of estrogen and progesterone receptors.

    Science.gov (United States)

    Dhananjayan, Sarath C; Ramamoorthy, Sivapriya; Khan, Obaid Y; Ismail, Ayesha; Sun, Jun; Slingerland, Joyce; O'Malley, Bert W; Nawaz, Zafar

    2006-10-01

    WW domain binding protein-2 (WBP-2) was cloned as an E6-associated protein interacting protein, and its role in steroid hormone receptors functions was investigated. We show that WBP-2 specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER), whereas it did not have any significant effect on the androgen receptor, glucocorticoid receptor, or the activation functions of p53 and VP-16. Depletion of endogenous WBP-2 with small interfering RNAs indicated that WBP-2 was required for the proper functioning of PR and ER. We also demonstrated that WBP-2 contains an intrinsic activation domain. Moreover, chromatin immunoprecipitation assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. Mutational analysis suggests that one of three polyproline (PY) motifs of WBP-2 is essential for its coactivation and intrinsic activation functions. We show that WBP-2 and E6-associated protein each enhance PR function, and their effect on PR action are additive when coexpressed, suggesting a common signaling pathway. In this study, we also demonstrate that the WBP-2 binding protein, Yes kinase-associated protein (YAP) enhances PR transactivation, but YAP's coactivation function is absolutely dependent on WBP-2. Taken together, our data establish the role of WBP-2 and YAP as coactivators for ER and PR transactivation pathways.

  7. Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

    Science.gov (United States)

    Lila, T; Drubin, D G

    1997-02-01

    In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions.

  8. Developmental defects in mutants of the PsbP domain protein 5 in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Johnna L Roose

    Full Text Available Plants contain an extensive family of PsbP-related proteins termed PsbP-like (PPL and PsbP domain (PPD proteins, which are localized to the thylakoid lumen. The founding member of this family, PsbP, is an established component of the Photosystem II (PS II enzyme, and the PPL proteins have also been functionally linked to other photosynthetic processes. However, the functions of the remaining seven PPD proteins are unknown. To elucidate the function of the PPD5 protein (At5g11450 in Arabidopsis, we have characterized a mutant T-DNA insertion line (SALK_061118 as well as several RNAi lines designed to suppress the expression of this gene. The functions of the photosynthetic electron transfer reactions are largely unaltered in the ppd5 mutants, except for a modest though significant decrease in NADPH dehydrogenase (NDH activity. Interestingly, these mutants show striking plant developmental and morphological defects. Relative to the wild-type Col-0 plants, the ppd5 mutants exhibit both increased lateral root branching and defects associated with axillary bud formation. These defects include the formation of additional rosettes originating from axils at the base of the plant as well as aerial rosettes formed at the axils of the first few nodes of the shoot. The root-branching phenotype is chemically complemented by treatment with the synthetic strigolactone, GR24. We propose that the developmental defects observed in the ppd5 mutants are related to a deficiency in strigolactone biosynthesis.

  9. Conserved TRAM Domain Functions as an Archaeal Cold Shock Protein via RNA Chaperone Activity

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    2017-08-01

    Full Text Available Cold shock proteins (Csps enable organisms to acclimate to and survive in cold environments and the bacterial CspA family exerts the cold protection via its RNA chaperone activity. However, most Archaea do not contain orthologs to the bacterial csp. TRAM, a conserved domain among RNA modification proteins ubiquitously distributed in organisms, occurs as an individual protein in most archaeal phyla and has a structural similarity to Csp proteins, yet its biological functions remain unknown. Through physiological and biochemical studies on four TRAM proteins from a cold adaptive archaeon Methanolobus psychrophilus R15, this work demonstrated that TRAM is an archaeal Csp and exhibits RNA chaperone activity. Three TRAM encoding genes (Mpsy_0643, Mpsy_3043, and Mpsy_3066 exhibited remarkable cold-shock induced transcription and were preferentially translated at lower temperature (18°C, while the fourth (Mpsy_2002 was constitutively expressed. They were all able to complement the cspABGE mutant of Escherichia coli BX04 that does not grow in cold temperatures and showed transcriptional antitermination. TRAM3066 (gene product of Mpsy_3066 and TRAM2002 (gene product of Mpsy_2002 displayed sequence-non-specific RNA but not DNA binding activity, and TRAM3066 assisted RNases in degradation of structured RNA, thus validating the RNA chaperone activity of TRAMs. Given the chaperone activity, TRAM is predicted to function beyond a Csp.

  10. Single-stranded DNA-binding proteins: multiple domains for multiple functions.

    Science.gov (United States)

    Dickey, Thayne H; Altschuler, Sarah E; Wuttke, Deborah S

    2013-07-01

    The recognition of single-stranded DNA (ssDNA) is integral to myriad cellular functions. In eukaryotes, ssDNA is present stably at the ends of chromosomes and at some promoter elements. Furthermore, it is formed transiently by several cellular processes including telomere synthesis, transcription, and DNA replication, recombination, and repair. To coordinate these diverse activities, a variety of proteins have evolved to bind ssDNA in a manner specific to their function. Here, we review the recognition of ssDNA through the analysis of high-resolution structures of proteins in complex with ssDNA. This functionally diverse set of proteins arises from a limited set of structural motifs that can be modified and arranged to achieve distinct activities, including a range of ligand specificities. We also investigate the ways in which these domains interact in the context of large multidomain proteins/complexes. These comparisons reveal the structural features that define the range of functions exhibited by these proteins.

  11. Thermodynamic Stabilization of the Folded Domain of Prion Protein Inhibits Prion Infection in Vivo

    Directory of Open Access Journals (Sweden)

    Qingzhong Kong

    2013-07-01

    Full Text Available Prion diseases, or transmissible spongiform encephalopathies (TSEs, are associated with the conformational conversion of the cellular prion protein, PrPC, into a protease-resistant form, PrPSc. Here, we show that mutation-induced thermodynamic stabilization of the folded, α-helical domain of PrPC has a dramatic inhibitory effect on the conformational conversion of prion protein in vitro, as well as on the propagation of TSE disease in vivo. Transgenic mice expressing a human prion protein variant with increased thermodynamic stability were found to be much more resistant to infection with the TSE agent than those expressing wild-type human prion protein, in both the primary passage and three subsequent subpassages. These findings not only provide a line of evidence in support of the protein-only model of TSEs but also yield insight into the molecular nature of the PrPC→PrPSc conformational transition, and they suggest an approach to the treatment of prion diseases.

  12. Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E. coli

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and are involved in signal transduction. Uncontrolled signaling from receptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamma tory responses and diseases such as cancer and atherosclerosis. Thus, inhibitors that block the activity of tyrosine kinases or the signaling pathways of PTKs activation could be assumed as the potential candidate for drug development. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK gene was amplified by PCR us ing the cDNA of abl from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tagged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5α and was induced to express PTK protein. The expression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reached peak level about 40% of the host total pro tein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PTK fusion pro tein presented higher activity for tyrosine phosphorylation.

  13. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

    Science.gov (United States)

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J.

    2015-04-01

    The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane.

  14. Protein-protein and protein-lipid interactions in domain-assembly : Lessons from giant unilamellar vesicles

    NARCIS (Netherlands)

    Kahya, Nicoletta

    2010-01-01

    Giant Unilamellar Vesicles (GUVs) provide a key model membrane system to study lipid-lipid and lipid-protein interactions, which are relevant to vital cellular processes, by (single-molecule) optical microscopy. Here, we review the work on reconstitution techniques for membrane proteins and other pr

  15. A noncanonical PWI domain in the N-terminal helicase-associated region of the spliceosomal Brr2 protein.

    Science.gov (United States)

    Absmeier, Eva; Rosenberger, Leonie; Apelt, Luise; Becke, Christian; Santos, Karine F; Stelzl, Ulrich; Wahl, Markus C

    2015-04-01

    The spliceosomal RNA helicase Brr2 is required for the assembly of a catalytically active spliceosome on a messenger RNA precursor. Brr2 exhibits an unusual organization with tandem helicase units, each comprising dual RecA-like domains and a Sec63 homology unit, preceded by a more than 400-residue N-terminal helicase-associated region. Whereas recent crystal structures have provided insights into the molecular architecture and regulation of the Brr2 helicase region, little is known about the structural organization and function of its N-terminal part. Here, a near-atomic resolution crystal structure of a PWI-like domain that resides in the N-terminal region of Chaetomium thermophilum Brr2 is presented. CD spectroscopic studies suggested that this domain is conserved in the yeast and human Brr2 orthologues. Although canonical PWI domains act as low-specificity nucleic acid-binding domains, no significant affinity of the unusual PWI domain of Brr2 for a broad spectrum of DNAs and RNAs was detected in band-shift assays. Consistently, the C. thermophilum Brr2 PWI-like domain, in the conformation seen in the present crystal structure, lacks an expanded positively charged surface patch as observed in at least one canonical, nucleic acid-binding PWI domain. Instead, in a comprehensive yeast two-hybrid screen against human spliceosomal proteins, fragments of the N-terminal region of human Brr2 were found to interact with several other spliceosomal proteins. At least one of these interactions, with the Prp19 complex protein SPF27, depended on the presence of the PWI-like domain. The results suggest that the N-terminal region of Brr2 serves as a versatile protein-protein interaction platform in the spliceosome and that some interactions require or are reinforced by the PWI-like domain.

  16. Implementation of innovative pulsed xenon ultraviolet (PX-UV) environmental cleaning in an acute care hospital.

    Science.gov (United States)

    Fornwalt, Lori; Riddell, Brad

    2014-01-01

    It is widely acknowledged that the hospital environment is an important reservoir for many of the pathogenic microbes associated with health care-associated infections (HAIs). Environmental cleaning plays an important role in the prevention and containment of HAIs, in patient safety, and the overall experience of health care facilities. New technologies, such as pulsed xenon ultraviolet (PX-UV) light systems are an innovative development for enhanced cleaning and decontamination of hospital environments. A portable PX-UV disinfection device delivers pulsed UV light to destroy microbial pathogens and spores, and can be used in conjunction with manual environmental cleaning. In addition, this technology facilitates thorough disinfection of hospital rooms in 10-15 minutes. The current study was conducted to evaluate whether the introduction of the PX-UV device had a positive impact on patient satisfaction. Satisfaction was measured using the Hospital Consumer Assessment of Healthcare Providers and Systems (HCAHPS) survey. In 2011, prior to the introduction of the PX-UV system, patient HCAHPS scores for cleanliness averaged 75.75%. In the first full quarter after enhanced cleaning of the facility was introduced, this improved to 83%. Overall scores for the hospital rose from 76% (first quarter, 2011) to 87.6% (fourth quarter, 2012). As a result of this improvement, the hospital received 1% of at-risk reimbursement from the inpatient prospective payment system as well as additional funding. Cleanliness of the hospital environment is one of the questions included in the HCAHPS survey and one measure of patient satisfaction. After the introduction of the PX-UV system, the score for cleanliness and the overall rating of the hospital rose from below the fiftieth to the ninety-ninth percentile. This improvement in the patient experience was associated with financial benefits to the hospital.

  17. Evolutionary stabilization of the gene-3-protein of phage fd reveals the principles that govern the thermodynamic stability of two-domain proteins.

    Science.gov (United States)

    Martin, Andreas; Schmid, Franz X

    2003-05-09

    The gene-3-protein (G3P) of filamentous phage is essential for their propagation. It consists of three domains. The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor. Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability. In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P. Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains. The method is limited by the proteolytic stability of G3P itself. We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections. Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C. The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined. In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step. N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken. The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains. These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and

  18. The BAR Domain Protein PICK1 Controls Vesicle Number and Size in Adrenal Chromaffin Cells

    DEFF Research Database (Denmark)

    da Silva Pinheiro, Paulo César; Jansen, Anna M; de Wit, Heidi

    2014-01-01

    Protein Interacting with C Kinase 1 (PICK1) is a Bin/Amphiphysin/Rvs (BAR) domain protein involved in AMPA receptor trafficking. Here, we identify a selective role for PICK1 in the biogenesis of large, dense core vesicles (LDCVs) in mouse chromaffin cells. PICK1 colocalized with syntaxin-6......, a marker for immature granules. In chromaffin cells isolated from a PICK1 knockout (KO) mouse the amount of exocytosis was reduced, while release kinetics and Ca(2+) sensitivity were unaffected. Vesicle-fusion events had a reduced frequency and released lower amounts of transmitter per vesicle (i.......e., reduced quantal size). This was paralleled by a reduction in the mean single-vesicle capacitance, estimated by averaging time-locked capacitance traces. EM confirmed that LDCVs were fewer and of markedly reduced size in the PICK1 KO, demonstrating that all phenotypes can be explained by reductions...

  19. The Arabidopsis PLAT domain protein1 is critically involved in abiotic stress tolerance

    DEFF Research Database (Denmark)

    Hyun, Tae Kyung; van der Graaff, Eric; Albacete, Alfonso

    2014-01-01

    and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination....... Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located...... of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions...

  20. Degradation of LIM domain-binding protein three during processing of Spanish dry-cured ham.

    Science.gov (United States)

    Gallego, Marta; Mora, Leticia; Fraser, Paul D; Aristoy, María-Concepción; Toldrá, Fidel

    2014-04-15

    Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9 months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-90) providing evidence for the complexity and variability of proteolytic reactions throughout the whole process of dry-curing. Methionine oxidation has been observed in several peptides by the end of the process. The potential of some of the identified peptides to be used as biomarkers of dry-cured ham processing has also been considered.

  1. SAS-1 is a C2 domain protein critical for centriole integrity in C. elegans.

    Science.gov (United States)

    von Tobel, Lukas; Mikeladze-Dvali, Tamara; Delattre, Marie; Balestra, Fernando R; Blanchoud, Simon; Finger, Susanne; Knott, Graham; Müller-Reichert, Thomas; Gönczy, Pierre

    2014-11-01

    Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD) syndrome.

  2. SAS-1 is a C2 domain protein critical for centriole integrity in C. elegans.

    Directory of Open Access Journals (Sweden)

    Lukas von Tobel

    2014-11-01

    Full Text Available Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD syndrome.

  3. InterPro in 2017—beyond <