Latest Articles Include:
- Jonathan Widom 1955–2011
- Nat Struct Mol Biol 18(9):965-966 (2011)
Article preview View full access options Nature Structural & Molecular Biology | Obituary Jonathan Widom 1955–2011 * Jeffrey Hayes1Journal name:Nature Structural & Molecular BiologyVolume: 18,Pages:965–966Year published:(2011)DOI:doi:10.1038/nsmb.2132Published online06 September 2011 Rick Gaber This past month, many of us in the biochemistry and structural biology communities were shocked and saddened to learn of the untimely death of Jon Widom at the too-early age of 55. Jon was a leading scientist working at the forefront of understanding how genomes are organized into arrays of nucleosomes and higher-order chromatin structures. His research spanned the biology and physical chemistry of macromolecular interactions, especially as applied to DNA mechanics and DNA-histone complexes, and he published numerous seminal works. Jeffrey Hayes Jon Widom was trained as a chemist, receiving a BA from Cornell University in 1977. He pursued doctoral research with Robert L. (Buzz) Baldwin at Stanford University, where he focused on cation-induced condensation of DNA as a model for understanding compaction of phage DNA into toroidal conformations1. This work introduced Jon to the physical chemistry and mechanics of DNA and, perhaps more apropos of his life's work, to the problem of fitting a genome's worth of DNA into a functionally confining space such as a phage capsid or a eukaryotic nucleus2. Upon receiving his PhD in biochemistry in 1982, Jon ventured to the famed Laboratory of Molecular Biology at the Medical Research Council Laboratory in Cambridge, England, to work with Nobel laureate Sir Aaron Klug on the structure of eukaryotic chromatin. Specifically, Jon applied low-angle X-ray scattering and other techniques to study the conformation and nucleosome packaging arrangements in the so-called 30-nm-diameter chro! matin fiber, providing evidence for the proposed solenoid model of the fiber3. Of special note was Jon's seminal study of cation-induced chromatin compaction, published as a single-author paper from his postdoctoral work, which set the stage for a multitude of modern analyses of salt-dependent folding and compaction of model chromatin systems4. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Structural & Molecular Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Affiliations * Jeffrey Hayes is at the University of Rochester Medical Center, Rochester, New York, USA. Author Details * Jeffrey Hayes Search for this author in: * NPG journals * PubMed * Google Scholar - A new twist in actin filament nucleation
- Nat Struct Mol Biol 18(9):967-969 (2011)
Article preview View full access options Nature Structural & Molecular Biology | News and Views A new twist in actin filament nucleation * Marie-France Carlier1Journal name:Nature Structural & Molecular BiologyVolume: 18,Pages:967–969Year published:(2011)DOI:doi:10.1038/nsmb.2130Published online06 September 2011 One of the most versatile regulators of actin assembly, the WASP homology 2 (WH2) domain, reveals previously unknown facets by combining with a newly discovered actin-nucleating dimeric structure in the effector protein VopL from Vibrio parahaemolyticus. Figures at a glance * Figure 1: Summary of the structural organization and nucleating activities of the various VopL constructs. * Figure 2: Possible models for the nucleation activity of VopL. () The VCD-organized hexameric nucleus stabilized by the WH2 array of VopL1, 2. Consecutive binding of actin subunits to the opposing WH2 domains on each protomer leads to a hexamer in which constraints imposed by the binding of the amphipathic helix of each WH2 domain impose actin-actin longitudinal bonds that are incompatible with the helical structure of the filament18, 19 (tilted orientation of the actin subunits). Formation of lateral interactions between the two opposing strands induces the isomerization of the hexamer of WH2-bound actins into a helical hexameric nucleus, with concomitant dissociation from VopL. () VopL facilitates spontaneous nucleation. (1) Spontaneous nucleation of actin alone through formation of a longitudinal dimer and helical trimer20. (2) VCD-catalyzed nucleation. The binding of G-actin () to VCD reduces the electronegativity of actin in the VCD–A complex and allows easier formation of longitudinal dimers VCD–A2 and lateral association of a! ctin leading to VCD–A3, enhancing the formation of A3 nuclei. (3) W-VCD does not nucleate as well as VCD. The amphipathic helix of each W domain bound to the barbed face of actin makes an abortive complex2. (4) Nucleation catalyzed by W2-VCD (not shown) and W3-VCD. The first and second WH2 domains bind a G-actin with higher affinity than the third one2 and bring it into longitudinal association with the VCD-bound first actin, facilitating the transient formation of a higher amount of W3-VCD–A3 than that shown in , leading to a higher stationary amount of A3 nuclei. This model is similar to one that accounts for nucleation of actin filaments by Cobl17. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Structural & Molecular Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Marie-France Carlier is in Dynamique du Cytosquelette et Motilité Cellulaire, UPR 3089, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Marie-France Carlier Author Details * Marie-France Carlier Contact Marie-France Carlier Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling
- Nat Struct Mol Biol 18(9):971-976 (2011)
Nature Structural & Molecular Biology | Article The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling * Daniela Ungureanu1 * Jinhua Wu2, 6 * Tuija Pekkala1 * Yashavanthi Niranjan1 * Clifford Young3, 6 * Ole N Jensen3 * Chong-Feng Xu2, 6 * Thomas A Neubert2 * Radek C Skoda4 * Stevan R Hubbard2 * Olli Silvennoinen1, 5 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:971–976Year published:(2011)DOI:doi:10.1038/nsmb.2099Received17 January 2011Accepted14 June 2011Published online14 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased JAK2 activity, contributing to myeloproliferative neoplasms (MPNs). Here we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2: Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling. Notably, different MPN mutations abrogated JH2 activity in cells, and in MPN (V617F) patient cells phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized mechanism to control basal activity and signaling of JAK2. View full text Figures at a glance * Figure 1: Identification of JAK2 JH2 catalytic activity in vitro. () In vitro kinase assay with purified JAK2 GST-JH2 with [32P-γ]ATP in the absence or presence of divalent cations. () Time-course kinase assay with purified JAK2 GST-JH2 in the presence of [γ-32P]ATP or unlabeled ATP. () Autoradiography of kinase assay (30 min) using purified JAK2 JH2 and JH1 domain and [γ-32P]ATP, in the absence or presence of cations. Coomassie staining shows the protein levels of JH1 and JH2. * Figure 2: Identification of phosphorylated residues in JAK2 JH2. () Chromatogram of JAK2 JH2 purification showing the peaks from anion-exchange chromatography. () Coomassie staining of a native gel of JH2-peak1 and JH2-peak2 proteins. () Coomassie staining of a native gel of purified JH2-peak1 and JH2-peak2 after a kinase reaction. () MS-MS spectra of the phosphorylated residues in a JAK2 JH2-peak2 4-h kinase assay. Left, JH2-peak2 is stoichiometrically phosphorylated at Ser523. Right, JH2-peak2 is partially phosphorylated at Tyr570. * Figure 3: Analysis of JAK2 JH2 autophosphorylation and ATP-binding activity. () Time-course kinase assay of purified JH2-peak1 and JH2-peak2. () Time-course kinase assay of purified JH2 S523A and Y570F mutants compared to JH2-peak2. () Fluorescence measurement of an ATP-binding assay of JAK2 JH2-peak2. () Kd measurement of mant-ATP binding to JH2-peak2. Graph shows mean ± s.d. of three independent experiments. * Figure 4: Analysis of JAK2 signaling in mammalian cells. () Phosphorylation of wild-type JAK2 (WT) and its mutants in JAK2-deficient γ2A cells. Hemagglutinin (HA)-tagged JAK2 proteins were immunoprecipitated with anti-HA antibody, and JAK2 phosphorylation is shown by western blotting. Anti-HA western blots show protein levels for each independent experiment. () Phosphorylation of JAK2 JH2 in γ2A cells. () As in , but with the K882D mutant that abrogates JH1 activity. (,) Phosphorylation of STAT1 in response to IFN-γ stimulation, and phosphorylation of STAT5 in response to Epo stimulation in γ2A cells. () Effect of JAK2 K581A mutation on STAT1 transcription activation using an IFN–γ—dependent GAS luciferase reporter. Graph shows mean ± s.d. of three independent experiments (P < 0.05). () Effect of the JAK2 K581A mutation on STAT5 transcription activation using an SPI-Luc2 luciferase reporter. The basal wild-type JAK2 activity was set to 1 for all experiments. Graph shows mean ± s.d. of six independent experiments (P < 0.! 05). * Figure 5: Phosphorylation of different JAK2 MPN mutants. () Phosphorylation of wild-type JAK2 (WT) and MPN mutants in JAK2-deficient γ2A cells. () Phosphorylation of JAK2 JH2 in γ2A cells. Author information * Abstract * Author information * Supplementary information Affiliations * Institute of Biomedical Technology, University of Tampere, Tampere, Finland. * Daniela Ungureanu, * Tuija Pekkala, * Yashavanthi Niranjan & * Olli Silvennoinen * Structural Biology Program, Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, New York, USA. * Jinhua Wu, * Chong-Feng Xu, * Thomas A Neubert & * Stevan R Hubbard * Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. * Clifford Young & * Ole N Jensen * Department of Biomedicine, University Hospital Basel, Basel, Switzerland. * Radek C Skoda * Tampere University Hospital, Tampere, Finland. * Olli Silvennoinen * Present addresses: Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA (J.W.); Analytical Development, Biogen Idec, Cambridge, Massachusetts, USA (C.‐F.X.); Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark (C.Y.). * Jinhua Wu, * Clifford Young & * Chong-Feng Xu Contributions D.U. performed the experiments and wrote the paper. O.S. and S.R.H. designed the experiments and wrote the paper. J.W. performed the in vitro experiments with recombinant proteins. T.P. and Y.N. performed the mutagenesis experiments in mammalian cells. C.Y., O.N.J., T.A.N. and C.-F.X. performed the experiments for MS analysis. R.C.S. performed the experiments for clinical sample analysis. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Olli Silvennoinen Author Details * Daniela Ungureanu Search for this author in: * NPG journals * PubMed * Google Scholar * Jinhua Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Tuija Pekkala Search for this author in: * NPG journals * PubMed * Google Scholar * Yashavanthi Niranjan Search for this author in: * NPG journals * PubMed * Google Scholar * Clifford Young Search for this author in: * NPG journals * PubMed * Google Scholar * Ole N Jensen Search for this author in: * NPG journals * PubMed * Google Scholar * Chong-Feng Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas A Neubert Search for this author in: * NPG journals * PubMed * Google Scholar * Radek C Skoda Search for this author in: * NPG journals * PubMed * Google Scholar * Stevan R Hubbard Search for this author in: * NPG journals * PubMed * Google Scholar * Olli Silvennoinen Contact Olli Silvennoinen Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (14M) Supplementary Figures 1–10 and Supplementary Methods Additional data Entities in this article * Interferon gamma IFNG Homo sapiens * View in UniProt * View in Entrez Gene * Thrombopoietin THPO Homo sapiens * View in UniProt * View in Entrez Gene * Prolactin PRL Homo sapiens * View in UniProt * View in Entrez Gene * Somatotropin GH1 Homo sapiens * View in UniProt * View in Entrez Gene * Tyrosine-protein kinase JAK3 JAK3 Homo sapiens * View in UniProt * View in Entrez Gene * Tyrosine-protein kinase JAK1 JAK1 Homo sapiens * View in UniProt * View in Entrez Gene * Erythropoietin EPO Homo sapiens * View in UniProt * View in Entrez Gene * Non-receptor tyrosine-protein kinase TYK2 TYK2 Homo sapiens * View in UniProt * View in Entrez Gene * Interleukin-3 IL3 Homo sapiens * View in UniProt * View in Entrez Gene * Signal transducer and activator of transcription 1-alpha/beta STAT1 Homo sapiens * View in UniProt * View in Entrez Gene * STE20-related kinase adapter protein alpha STRADA Homo sapiens * View in UniProt * View in Entrez Gene * Granulocyte-macrophage colony-stimulating factor CSF2 Homo sapiens * View in UniProt * View in Entrez Gene * Leptin LEP Homo sapiens * View in UniProt * View in Entrez Gene * Interleukin-5 IL5 Homo sapiens * View in UniProt * View in Entrez Gene * Tyrosine-protein kinase JAK2 JAK2 Homo sapiens * View in UniProt * View in Entrez Gene * Peripheral plasma membrane protein CASK CASK Homo sapiens * View in UniProt * View in Entrez Gene * Inactive serine/threonine-protein kinase VRK3 VRK3 Homo sapiens * View in UniProt * View in Entrez Gene * Signal transducer and activator of transcription 5A STAT5A Homo sapiens * View in UniProt * View in Entrez Gene * Erythropoietin receptor EPOR Homo sapiens * View in UniProt * View in Entrez Gene * Receptor tyrosine-protein kinase erbB-3 ERBB3 Homo sapiens * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase WNK1 WNK1 Homo sapiens * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase haspin GSG2 Homo sapiens * View in UniProt * View in Entrez Gene - Splicing enhances recruitment of methyltransferase HYPB/Setd2 and methylation of histone H3 Lys36
- Nat Struct Mol Biol 18(9):977-983 (2011)
Nature Structural & Molecular Biology | Article Splicing enhances recruitment of methyltransferase HYPB/Setd2 and methylation of histone H3 Lys36 * Sérgio Fernandes de Almeida1 * Ana Rita Grosso1 * Frederic Koch2 * Romain Fenouil2 * Sílvia Carvalho1 * Jorge Andrade1 * Helena Levezinho1 * Marta Gut3 * Dirk Eick4 * Ivo Gut3 * Jean-Christophe Andrau2 * Pierre Ferrier2 * Maria Carmo-Fonseca1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:977–983Year published:(2011)DOI:doi:10.1038/nsmb.2123Received11 April 2011Accepted13 July 2011Published online26 July 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Several lines of recent evidence support a role for chromatin in splicing regulation. Here, we show that splicing can also contribute to histone modification, which implies bidirectional communication between epigenetic mechanisms and RNA processing. Genome-wide analysis of histone methylation in human cell lines and mouse primary T cells reveals that intron-containing genes are preferentially marked with histone H3 Lys36 trimethylation (H3K36me3) relative to intronless genes. In intron-containing genes, H3K36me3 marking is proportional to transcriptional activity, whereas in intronless genes, H3K36me3 is always detected at much lower levels. Furthermore, splicing inhibition impairs recruitment of H3K36 methyltransferase HYPB (also known as Setd2) and reduces H3K36me3, whereas splicing activation has the opposite effect. Moreover, the increase of H3K36me3 correlates with the length of the first intron, consistent with the view that splicing enhances H3 methylation. We propos! e that splicing is mechanistically coupled to recruitment of HYPB/Setd2 to elongating RNA polymerase II. View full text Figures at a glance * Figure 1: Patterns of H3K36 trimethylation in intron-containing and intronless genes. () H3K36me3 ChIP-seq average profiles across intron-containing (dashed lines) and intronless genes (solid lines), grouped according to expression level (red, high; green, medium; blue, low). Human H1299 cells: for intron-containing genes, the profiles correspond to 634 genes with high, 593 genes with medium and 436 genes with low level of expression; for intronless genes, the profiles correspond to 11 genes with high, 16 genes with medium and 62 genes with low level of expression. Mouse CD4+ CD8+ T cells: for intron-containing genes, the profiles correspond to 917 genes with high, 920 genes with medium and 872 genes with low level of expression; for intronless genes, the profiles correspond to 16 genes with high, 13 genes with medium and 63 genes with low expression level. The x axis extends from the TSS to the 3′ end of the genes and is scaled along the gene bodies in 100 equally sized bins (introns not depicted). () H3K36me3 ChIP-seq average profiles for intron-containin! g and intronless genes with equal average lengths and equal high-expression levels. The x axis is scaled as in . () ChIP-seq average profiles for indicated chromatin marks on genes containing a second exon that is either excluded or included in the mRNA, as assessed from RNA-seq reads of the same cells. Exons and introns represented in 20 and 40 equally sized bins, respectively. Box plots represent distribution of expression levels for each gene set. () Same as in , but alternatively or constitutively spliced exons were defined according to the University of California, Santa Cruz (UCSC) Genome Browser database. () H3K36me3 ChIP-seq average profiles at single nucleosome resolution across intron-containing and intronless genes grouped according to expression level. The graph on the left depicts all genes. The graph on the right depicts genes with equal average lengths and equal high-expression levels. Graphs show mean and s.e.m. (). Asterisks denote P < 0.005. Student's t-te! st. * Figure 2: In an intronless gene, H3K36me3 remains low irrespective of transcriptional activation. () Results of experiments with RNA from undifferentiated (T0) or differentiated (T48) MEL cells that was reverse transcribed with random primers and PCR amplified using primer pairs that detect U2AF1 pre-mRNA and ZRSR1 RNA. The amount of PCR product was estimated by qRT-PCR and normalized to the levels of GAPDH. () Results of ChIP assays to compare H3K36me3 in the intronless ZRSR1 gene and the intron-containing U2AF1 gene using MEL cells that were either uninduced (T0) or induced to undergo erythroid differentiation for 48 h (T48). ChIP signals for H3K36me3 () and H3K9Ac () were normalized to total H3. Histograms and graphs depict mean and s.d. from at least three independent experiments. The schematic diagrams of the regions amplified by each primer set are represented below each graph. Blue boxes represent exons; IG, intergenic region; Pr, promoter region. * Figure 3: H3K36me3 is highly dynamic in intron-containing genes. Results from experiments with HeLa cells after transcription inhibition with DRB for the indicated times. ChIP signals obtained with H3K36me3 antibody are normalized to total histone H3 (), and ChIP signals obtained with antibody N20 against Pol II () are represented as percentage of the input. Schematic diagrams represent the regions amplified by each primer set for the indicated genes. An intergenic region (IG) was amplified as a control. Exons are indicated as blue boxes. All graphs depict mean and s.d. from at least three independent experiments. * Figure 4: Splicing inhibition reduces H3K36me3 and HYPB/Setd2 recruitment in intron-containing genes. () Western blot analysis of HeLa cell lysates prepared 48 h after transfection with siRNAs against luciferase (GL2) and SAP130. The blot was probed with the indicated antibodies. Molecular weight markers are shown on the left. () Results from experiments with RNA from HeLa cells that were either transfected with the indicated siRNAs or treated with meayamycin. qRT-PCR was performed to measure the amount of total RNA present in whole-cell lysates or chromatin fractions and the amount of unspliced RNA from the indicated genes. The amount of PCR product estimated by qRT-PCR was normalized to the levels of U6 small nucleolar RNA (snRNA). Results are shown as the fold change over GL2- or DMSO-treated cells. () ChIP assays were carried out for the indicated genes. Signals obtained with H3K36me3 antibody are normalized to total histone H3 (), and signals obtained with anti-HYPB/Setd2 antibody are represented as percentage of the input (). Schematic diagrams represent the regions am! plified by each primer set. An intergenic region (IG) was amplified as a control. Exons are indicated as blue boxes. Histograms and graphs depict mean and s.d. from at least three independent experiments. * Figure 5: Exon inclusion by alternative splicing increases HYPB/Setd2 recruitment and H3K36me3. () Schematic representation of the CD44 gene, indicating constitutively and alternatively spliced exons. () HeLa cells were treated overnight with 40 ng ml−1 PMA and inclusion of exons v5 and v10 was quantified by qRT-PCR. The histogram shows the ratio of the values obtained before and after PMA treatment, normalized to total CD44 transcripts obtained with primers for CD44 exon 1 mRNA. Error bars represent the s.d. from three independent experiments. () ChIP assays were done using primers for CD44 exons 1, 2, 3, v5, v10, 16, 17, 18 and a control intergenic region (IG). HeLa cells were either mock treated (−), treated with PMA overnight or simultaneously treated with PMA and 100 μM DRB for 2 h. Signals obtained with antibodies directed to Pol II (N20) and HYPB/Setd2 are represented as percentage of the input, and signals obtained with the antibody to H3K36me3 are normalized to total histone H3. Schematic diagrams represent the regions amplified by each primer set, with e! xons indicated in boxes. The graphs depict mean and s.d. from at least three independent experiments. * Figure 6: H3K36me3 does not mirror Ser2P Pol II occupancy. () ChIP-seq average profiles for initiating Pol II (Ser5P), elongating Pol II (Ser2P) and H3K36me3 along highly and medium expressed intron-containing genes. Panel () depicts average profiles for H3K36me3 and Ser2P Pol II along genes that contain the second exon located within 1,000 ± 30 (red), 1,500 ± 30 (green) or 2,400 ± 30 (blue) nucleotides from the TSS. () Splicing enhances H3K36me3 through HYPB/Setd2 recruitment. HYPB/Setd2 binds to the hyperphosphorylated CTD of Pol II and methylates the nucleosomes reassembled behind the elongating polymerase. As Pol II transcribes through the 3′ splice site, allowing for spliceosome assembly on the pre-mRNA, recruitment of HYPB/Setd2 is enhanced, resulting in higher levels of H3K36me3. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * GSE30902 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. * Sérgio Fernandes de Almeida, * Ana Rita Grosso, * Sílvia Carvalho, * Jorge Andrade, * Helena Levezinho & * Maria Carmo-Fonseca * Centre d'Immunologie de Marseille-Luminy, Centre National de la Recherche Scientifique (CNRS) UMR6102, Institut National de la Santé et de la Recherche Médicale (INSERM), U631, Université de la Méditerranée, Marseille, France. * Frederic Koch, * Romain Fenouil, * Jean-Christophe Andrau & * Pierre Ferrier * Centre Nacional D'Anàlisi Genòmica, Parc Cientific de Barcelona, Barcelona, Spain. * Marta Gut & * Ivo Gut * Department of Molecular Epigenetics, Helmholtz Center Munich, Center of Integrated Protein Science, Munich, Germany. * Dirk Eick Contributions S.F.deA. and M.C.-F. conceived the project and designed the experiments. S.F.deA., S.C., J.A., H.L. conducted and analyzed the wet-lab experiments. F.K., I.G., J.-C.A. and P.F. conceived the framework of the ChIP-seq studies. F.K. and J.-C.A. designed the ChIP-seq experiments. D.E. produced and provided the Ser2P and Ser5P Pol II antibodies. R.F. and F.K. carried out the bioinformatics preprocessing of ChIP-seq data. ChIP-seq and RNA-seq preprocessing materials were prepared by F.K.. M.G. and I.G. conducted all ChIP-seq– and RNA-sequencing experiments. A.R.G carried out the bioinformatic analysis of microarray, ChIP-seq and RNA-seq data. S.F.deA., A.R.G. and M.C.-F. wrote the manuscript. All authors reviewed the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Maria Carmo-Fonseca or * Jean-Christophe Andrau or * Pierre Ferrier Author Details * Sérgio Fernandes de Almeida Search for this author in: * NPG journals * PubMed * Google Scholar * Ana Rita Grosso Search for this author in: * NPG journals * PubMed * Google Scholar * Frederic Koch Search for this author in: * NPG journals * PubMed * Google Scholar * Romain Fenouil Search for this author in: * NPG journals * PubMed * Google Scholar * Sílvia Carvalho Search for this author in: * NPG journals * PubMed * Google Scholar * Jorge Andrade Search for this author in: * NPG journals * PubMed * Google Scholar * Helena Levezinho Search for this author in: * NPG journals * PubMed * Google Scholar * Marta Gut Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk Eick Search for this author in: * NPG journals * PubMed * Google Scholar * Ivo Gut Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-Christophe Andrau Contact Jean-Christophe Andrau Search for this author in: * NPG journals * PubMed * Google Scholar * Pierre Ferrier Contact Pierre Ferrier Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Carmo-Fonseca Contact Maria Carmo-Fonseca Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3.2M) Supplementary Figures 1–6, Supplementary Tables 1 and 2 and Supplementary Methods Additional data Entities in this article * Splicing factor U2AF 35 kDa subunit U2af1 Mus musculus * View in UniProt * View in Entrez Gene * U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit-related protein 1 Zrsr1 Mus musculus * View in UniProt * View in Entrez Gene * Proliferating cell nuclear antigen PCNA Homo sapiens * View in UniProt * View in Entrez Gene * CD44 antigen CD44 Homo sapiens * View in UniProt * View in Entrez Gene * T-cell surface glycoprotein CD4 Cd4 Mus musculus * View in UniProt * View in Entrez Gene * Histone-lysine N-methyltransferase, H3 lysine-36 specific SET2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * T-cell surface glycoprotein CD4 CD4 Homo sapiens * View in UniProt * View in Entrez Gene * TATA-box-binding protein TBP Homo sapiens * View in UniProt * View in Entrez Gene * Receptor-type tyrosine-protein phosphatase C PTPRC Homo sapiens * View in UniProt * View in Entrez Gene * Splicing factor 3B subunit 3 SF3B3 Homo sapiens * View in UniProt * View in Entrez Gene * Cyclin-dependent kinase 2 CDK2 Homo sapiens * View in UniProt * View in Entrez Gene * Histone-lysine N-methyltransferase SETD2 SETD2 Homo sapiens * View in UniProt * View in Entrez Gene * Protein yippee-like 5 YPEL5 Homo sapiens * View in UniProt * View in Entrez Gene - Simultaneous visualization of the extracellular and cytoplasmic domains of the epidermal growth factor receptor
- Nat Struct Mol Biol 18(9):984-989 (2011)
Nature Structural & Molecular Biology | Article Simultaneous visualization of the extracellular and cytoplasmic domains of the epidermal growth factor receptor * Li-Zhi Mi1 * Chafen Lu1 * Zongli Li2 * Noritaka Nishida3 * Thomas Walz2 * Timothy A Springer1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:984–989Year published:(2011)DOI:doi:10.1038/nsmb.2092Received02 February 2011Accepted25 May 2011Published online07 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg To our knowledge, no structural study to date has characterized, in an intact receptor, the coupling of conformational change in extracellular domains through a single-pass transmembrane domain to conformational change in cytoplasmic domains. Here we examine such coupling, and its unexpected complexity, using nearly full-length epidermal growth factor receptor (EGFR) and negative-stain EM. The liganded, dimeric EGFR ectodomain can couple both to putatively active, asymmetrically associated kinase dimers and to putatively inactive, symmetrically associated kinase dimers and monomers. Inhibitors that stabilize the active or inactive conformation of the kinase active site, as well as mutations in the kinase dimer interface and a juxtamembrane phosphorylation site, shift the equilibrium among the three kinase association states. This coupling of one conformation of an activated receptor ectodomain to multiple kinase-domain arrangements reveals previously unanticipated complexity! in transmembrane signaling and facilitates regulation of receptor function in the juxtamembrane and cytoplasmic environments. View full text Figures at a glance * Figure 1: Unliganded EGFR adopts a monomeric tethered conformation. () Superose 6 gel filtration chromatograms of EGFR with the indicated pretreatments. D, dimer; M, monomer. In preparations with EGF, the amount of monomer present varied in independent preparations with the same mutation or drug, and thus variations in monomer content in the profiles in are not meaningful. () Representative EM class averages of unliganded EGF receptors. () The ectodomain after masking. () Bottom, the best cross-correlating projections from the unliganded ectodomain crystal structure. Top, enlarged ribbon diagrams in same orientation. () The best correlating projections with a monomer from the dimeric, liganded ectodomain crystal structure. Cross-correlation coefficients are shown below the projections. Scale bars, 10 nm. * Figure 2: The liganded EGF receptor ectodomain can link to multiple kinase-domain dimerization states. (,) Representative EM class averages of EGF-bound receptors with asymmetric () or symmetric () kinase-domain dimers, with masked regions and cross-correlations for each average shown below in –. (,) Class averages after masking of all but the ectodomain. (,) Best-correlating ectodomain projections. Enlarged ribbon diagrams are shown in upper parts of ,. (,) Class averages after masking of all but the kinase domain. (–) Best-correlating asymmetric kinase domain (,) and symmetric kinase domain (,) crystal structure projections. Cross-correlation scores are shown below projections. Scale bars,10 nm. (,) Positions and two-dimensional orientations of ectodomain and kinase-domain crystal structures, determined by cross-correlation with masked class averages in ,,,. Structures are shown as ribbon diagrams enlarged relative to the class averages while maintaining spatial relationships. The better-correlating asymmetric kinase dimer is shown in , and the better-correlating symmet! ric kinase dimer in . () Schematic diagram of the asymmetric kinase dimer in which one kinase (green) activates the other (orange). The position of Val924 is marked with a star. Modified from reference 6. * Figure 3: Mutational disruption of the asymmetric kinase dimer. (,) Representative class averages of EGFR V924R mutant in complex with EGF with unassociated kinase monomers () or symmetric kinase dimers (). (,) Positions and two-dimensional orientations of ectodomain and kinase-domain crystal structures as in Figure 2m,n, determined using masked class averages in ,. (,) Representative class averages of EGFR T669D S671D mutant in complex with EGF with unassociated kinase monomers () or symmetric kinase dimers (). (,) Positions and two-dimensional orientations of ectodomain and kinase-domain crystal structures as in Figure 2m,n, determined using masked class averages from , as described in Figure 2. Scale bars, 10 nm. * Figure 4: Kinase inhibitors gefitinib and PD168393 cooperate with EGF in promoting the asymmetric kinase dimer. (,) Representative class averages of EGFR in complex with EGF and gefitinib or PD168393. Representative asymmetric dimeric (1), symmetric dimeric (2) and unassociated kinase domains (3) are shown. (,) The kinase domain after masking. (–) Best-correlating projections with asymmetric (,) and symmetric dimers (,) from kinase crystal structure. Cross-correlation scores are shown below each projection. Scale bars, 10 nm. (,) Positions and two-dimensional orientations of ectodomain and kinase-domain crystal structures determined using masked class averages as in Figure 2. () Fraction of asymmetric-like kinase dimers, symmetric-like kinase dimers and kinase monomers in the presence of EGF and indicated inhibitors or mutations, calculated using all class averages with well-resolved kinase domains and the number of particles in each class average. WT, wild-type. * Figure 5: Effect of the inhibitors lapatinib and HKI-272 on kinase-domain dimerization. (,) Representative class averages of EGFR in complex with EGF and lapatinib as symmetric kinase dimers () or unassociated kinase monomers (). () Top, representative class averages of EGFR in complex with EGF and HKI-272. Scale bars, 10 nm. Bottom, positions and two-dimensional orientations of ectodomain and kinase-domain crystal structures, as determined using masked class averages as in Figure 2. Author information * Abstract * Author information * Supplementary information Affiliations * Immune Disease Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA. * Li-Zhi Mi, * Chafen Lu & * Timothy A Springer * Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA. * Zongli Li & * Thomas Walz * Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan. * Noritaka Nishida Contributions L.-Z.M. prepared constructs, designed and carried out experiments, and wrote the manuscript. C.L. prepared constructs, designed and carried out experiments, and discussed the writeup. N.N. performed early EM experiments. Z.L. trained N.N. and L.-Z.M., and maintained and supervised the EM facility. T.W. discussed and supervised EM experiments and strategy, wrote the manuscript, and helped respond to referees. T.A.S. designed the overall experimental approach, supervised experiments, wrote the manuscript and responded to referees. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Timothy A Springer Author Details * Li-Zhi Mi Search for this author in: * NPG journals * PubMed * Google Scholar * Chafen Lu Search for this author in: * NPG journals * PubMed * Google Scholar * Zongli Li Search for this author in: * NPG journals * PubMed * Google Scholar * Noritaka Nishida Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Walz Search for this author in: * NPG journals * PubMed * Google Scholar * Timothy A Springer Contact Timothy A Springer Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (11M) Supplementary Figures 1–12 Additional data Entities in this article * Epidermal growth factor receptor EGFR Homo sapiens * View in UniProt * View in Entrez Gene * Pro-epidermal growth factor EGF Homo sapiens * View in UniProt * View in Entrez Gene * Receptor tyrosine-protein kinase erbB-3 ERBB3 Homo sapiens * View in UniProt * View in Entrez Gene * ERBB receptor feedback inhibitor 1 ERRFI1 Homo sapiens * View in UniProt * View in Entrez Gene - Structure-function studies of nucleocytoplasmic transport of retroviral genomic RNA by mRNA export factor TAP
- Nat Struct Mol Biol 18(9):990-998 (2011)
Nature Structural & Molecular Biology | Article Structure-function studies of nucleocytoplasmic transport of retroviral genomic RNA by mRNA export factor TAP * Marianna Teplova1 * Lara Wohlbold2 * Nyan W Khin1 * Elisa Izaurralde2 * Dinshaw J Patel1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:990–998Year published:(2011)DOI:doi:10.1038/nsmb.2094Received02 December 2010Accepted01 June 2011Published online07 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg mRNA export is mediated by the TAP–p15 heterodimer, which belongs to the family of NTF2-like export receptors. TAP–p15 heterodimers also bind to the constitutive transport element (CTE) present in simian type D retroviral RNAs, and they mediate the export of viral unspliced RNAs to the host cytoplasm. We have solved the crystal structure of the RNA recognition and leucine-rich repeat motifs of TAP bound to one symmetrical half of the CTE RNA. L-shaped conformations of protein and RNA are involved in a mutual molecular embrace on complex formation. We have monitored the impact of structure-guided mutations on binding affinities in vitro and transport assays in vivo. Our studies define the principles by which CTE RNA subverts the mRNA export receptor TAP, thereby facilitating the nuclear export of viral genomic RNAs, and, more generally, provide insights on cargo RNA recognition by mRNA export receptors. View full text Figures at a glance * Figure 1: Structure of TAP-NTD bound to the hCTE RNA. () Domain architecture of full-length TAP and TAP-NTD construct (amino acids 96–362) consisting of the RRM (green) and LRR (blue) domains used for crystallization of the complex. () The full-length CTE RNA, consisting of two large internal loops whose sequences are related by two-fold symmetry (indicated by thick and thin curved arrows). () Sequence of one symmetrical half of CTE (half CTE, designated hCTE). Residues 1–62 of hCTE correspond to the half of the molecule that contains the hairpin loop (top half) of the full-length CTE in , with the hairpin loop replaced by a stable GAAA RNA loop (red) in the construct used for crystallization. The bases shown in red are different from the corresponding bases in the wild-type CTE sequence and were incorporated to facilitate efficient in vitro transcription (5′-GG), to incorporate a hammerhead ribozyme cleavage site (UC-3′) and to improve crystal quality (GAAA loop with GC closing pair). The residues are numbered from 1 t! o 62. (,) Two alternative views of the crystal structure of the 1:1 complex of TAP-NTD bound to hCTE. The RRM domain is shown in green, the LRR domain in blue and the RNA in wheat. Flipped-out RNA residues A13, G14 and A15 are shown in yellow. The N and C termini of TAP-NTD and the 5′ and 3′ ends of the RNA are labeled. * Figure 2: Fold of the internal loop and bulged bases in the hCTE–TAP complex. () The sequence and numbering of the internal loop and bulged bases of hCTE, with dashed red lines indicating the formation of four noncanonical pairs on complex formation. () A stereo pair of the structure of the zippered-up internal loop of hCTE in the complex. The RNA is shown in wheat, and flipped out A13, G14 and A15 are shown in yellow. The core of the fold contains four stacked pairs, with junctional U11•G50 and G18-C43 pairs aligned at right angles to each other. (–) Pairing alignments of noncanonical pairs in the zippered-up internal loop of the complex. The noncanonical pairing alignments are U11•G50 (), A12•A48 (), C16•C49 () and A17•G47 (). (,) Conformation of the AA bulges in the upper (A39-A40) and lower (A54-A55) stems of the hCTE RNA in the complex. The A39-A40 bulge in the upper stem of the complex is shown in , with A39 flipped out; A40 is stacked in the duplex and forms an A40•(G21•U38) triple. The A54-A55 bulge in the lower stem of the com! plex is shown in , with both A54 and A55 flipped out but mutually stacked on each other. * Figure 3: Key interactions between hCTE RNA and the TAP-NTD in the complex. () Schematic highlighting intermolecular hydrogen bond and hydrophobic protein-RNA contacts in the complex. hCTE RNA residues involved in base-specific and sugar phosphate–specific recognition are represented as shaded and red-outlined boxes, respectively. Amino acid residues of RRM (green) and LRR (blue) involved in hydrogen bonding and hydrophobic or stacking interactions are shown by solid and dashed arrows, respectively. () Intermolecular contacts between residues of hCTE RNA (A20, A31-U34 and G50-G51) and the RRM domain of TAP-NTD in the complex. () Intermolecular contacts between residues of hCTE RNA (A13-G14 and A54-A55) and the LRR domain of TAP-NTD in the complex. (–) Intermolecular hydrogen bonds involving flipped-out A13, which is inserted between Arg276 and Arg233 (), flipped-out G14, which is positioned on the surface patch involving Tyr278, Arg279 and Lys304 (), and flipped out A15, which is inserted between Glu151 and the sugar of G51 (). * Figure 4: In vitro ITC, and direct and competitive filter-binding data for TAP-NTD and hCTE RNA mutants. (,) ITC () and nitrocellulose filter–binding () curves for complex formation between TAP-NTD, and its mutants, with hCTE RNA. Wild-type TAP-NTD (WT) is represented by red circles, the dual R233A R276A mutant by orange diamonds, the triple Y278A R279A K304A mutant by blue pentagons, the single E151A mutant by green squares and the dual R128E R158E mutant by black triangles. () Summary of the binding constants measured from ITC and filter-binding assays. (–) Competitive nitrocellulose filter–binding assay curves for complex formation between TAP-NTD and hCTE RNA mutants. Curves for wild-type hCTE with residue A13 (red circles), and the G13 (blue triangles) and C13 (green squares) mutants, are shown in . Curves for wild-type hCTE with residue G14 (red circles), and the A14 (blue triangles) and C14 (green squares) mutants, are shown in . Curves for wild-type hCTE with residue A15 (in red circles) and the G15 (blue triangles) and C15 (green squares) mutants are shown in . (! ) Summary of the binding constants from competitive filter binding assays. * Figure 5: In vivo RNA export assay. () Schematic representation of the pCMV128-RLuc-CTE reporter and the corresponding control without CTE. SD, splicing donor site; SA, splicing acceptor site. () Human cells were transfected with a mixture of three plasmids: one expressing pCMV128-RLuc-CTE reporter or a control reporter lacking the CTE, another expressing firefly luciferase (FLuc), and a third expressing TAP (wild type or one of the mutants). A plasmid expressing p15 was included in the transfection mixtures as indicated. RLuc activity was normalized to that of the FLuc transfection control and set to 1 in the absence of exogenous TAP. Mean values ± s.d. are shown. () Human cells were transfected with the pCMV128-RLuc-CTE reporter carrying wild-type CTE or a mutant. Plasmids expressing TAP and p15 were co-transfected as indicated. A plasmid expressing FLuc served as a transfection control. Luciferase activity was analyzed as described in . () Human cells were transfected with a mixture of three plasmids: one ! expressing the pCMV128-RLuc reporter lacking the CTE, another expressing FLuc, and a third expressing TAP (wild type or mutant). A plasmid expressing p15 was included in the transfection mixtures. RLuc and FLuc activities were measured and analyzed as described in . * Figure 6: Stoichiometry of TAP-NTD binding to CTE RNA. () Electrophoretic mobility gel-shift data for binding of TAP-NTD to full-length CTE establishing 2:1 stoichiometry of the complex. The positions of the free CTE and of the TAP-NTD–CTE complexes are indicated on the right. TAP-NTD to CTE molar ratios are listed above the lanes. CTE is fully bound at 2:1 molar ratio for TAP-NTD/CTE. () Electrophoretic mobility gel-shift data for binding of TAP-NTD to hCTE establishing 1:1 stoichiometry of the complex. MW, molecular weight. () Gel-filtration profiles (above) monitoring the interaction between TAP-NTD (blue) and full-length CTE (red), and a calibration curve for an analytical gel-filtration column shown with molecular mass standards (below). The mixture of TAP-NTD and CTE at a 2:1 protein/RNA ratio (green) migrates as a single peak corresponding to a higher-molecular-weight fraction. The elution volumes of TAP-NTD (blue triangle), CTE (red triangle) and TAP-NTD + CTE complex (green diamond) are denoted on the calibration curv! e. () Nitrocellulose filter binding curve for the complex between TAP-NTD and full-length CTE. The apparent Kd measured by nonlinear least-squares fit according to equation (1) in the Online Methods is listed, ± fitting error. () Stoichiometry of TAP-NTD binding to CTE measured by the filter binding assay. The total RNA concentration used in the equilibrations is listed and is 16-fold greater than the Kd of TAP-NTD for CTE as determined by direct titration (). The data are compared to theoretical saturation curves for 1:1, 2:1 and 4:1 protein/RNA stoichiometry. The 2:1 curve most closely approximates the data, establishing that two copies of TAP-NTD interact with a single CTE molecule. The plots in and represent mean ± s.d. for two independent measurements. * Figure 7: Packing of two molecules of complex (TAP-NTD bound to hCTE) in the crystallographic asymmetric unit and a model of the 2:1 complex of TAP-NTD bound to full-length CTE. () The alignment of two hCTEs in the crystallographic asymmetric unit of the complex. The bases in red reflect differences from the wild-type CTE sequence. () The orientation of two molecules of complex (TAP-NTD bound to hCTE RNA) in the crystallographic asymmetric unit. () The full-length CTE RNA consisting of two large internal loops, whose sequences are related by two-fold symmetry (indicated by thick and thin curved arrows). () Model of the complex of full-length CTE with two TAP-NTD molecules bound to two CTE internal loops. The model was generated based on the structure of the two molecules of complex (TAP-NTD bound to hCTE) in the crystallographic asymmetric unit. The missing parts of the full-length CTE were modeled using idealized A-form RNA duplexes and RNA structural elements from previously determined structures (see Supplementary Methods for details) followed by rounds of idealization of geometric parameters with REFMAC45 and Coot (http://biop.ox.ac.uk/coot/) pr! ograms. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3RW7 * 3RW6 * 3RW7 * 3RW6 Referenced accessions Protein Data Bank * 1FO1 * 1FO1 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Marianna Teplova, * Nyan W Khin & * Dinshaw J Patel * Max Planck Institute for Developmental Biology, Tübingen, Germany. * Lara Wohlbold & * Elisa Izaurralde Contributions Constructs design, protein and RNA preparation and purification, crystallization of complex and its structure determination and in vitro binding assays were undertaken by M.T. with the assistance of N.W.K. under the supervision of D.J.P. The in vivo transport assays were performed by L.W. under the supervision of E.I. The paper was written by M.T., D.J.P. and E.I. with input from the other authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Dinshaw J Patel Author Details * Marianna Teplova Search for this author in: * NPG journals * PubMed * Google Scholar * Lara Wohlbold Search for this author in: * NPG journals * PubMed * Google Scholar * Nyan W Khin Search for this author in: * NPG journals * PubMed * Google Scholar * Elisa Izaurralde Search for this author in: * NPG journals * PubMed * Google Scholar * Dinshaw J Patel Contact Dinshaw J Patel Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (717 KB) Supplementary Figures 1–6 and Supplementary Methods Additional data Entities in this article * U2 small nuclear ribonucleoprotein A' SNRPA1 Homo sapiens * View in UniProt * View in Entrez Gene * Toll-like receptor 3 TLR3 Homo sapiens * View in UniProt * View in Entrez Gene * Lupus La protein SSB Homo sapiens * View in UniProt * View in Entrez Gene * Nuclear RNA export factor 1 NXF1 Homo sapiens * View in UniProt * View in Entrez Gene * THO complex subunit 4 THOC4 Homo sapiens * View in UniProt * View in Entrez Gene * Exportin-5 XPO5 Homo sapiens * View in UniProt * View in Entrez Gene * RNA, U2 small nuclear 1 RNU2-1 Homo sapiens * View in Entrez Gene * U2 small nuclear ribonucleoprotein B'' SNRPB2 Homo sapiens * View in UniProt * View in Entrez Gene * Nuclear transport factor 2 NUTF2 Homo sapiens * View in UniProt * View in Entrez Gene * NTF2-related export protein 1 NXT1 Homo sapiens * View in UniProt * View in Entrez Gene * Exportin-1 XPO1 Homo sapiens * View in UniProt * View in Entrez Gene * GTP-binding nuclear protein Ran RAN Homo sapiens * View in UniProt * View in Entrez Gene - An autoinhibitory helix in the C-terminal region of phospholipase C-β mediates Gαq activation
- Nat Struct Mol Biol 18(9):999-1005 (2011)
Nature Structural & Molecular Biology | Article An autoinhibitory helix in the C-terminal region of phospholipase C-β mediates Gαq activation * Angeline M Lyon1 * Valerie M Tesmer1 * Vishan D Dhamsania1 * David M Thal1 * Joanne Gutierrez2 * Shoaib Chowdhury2 * Krishna C Suddala3 * John K Northup2 * John J G Tesmer1, 4 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:999–1005Year published:(2011)DOI:doi:10.1038/nsmb.2095Received12 February 2011Accepted01 June 2011Published online07 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The enzyme phospholipase C-β (PLCβ) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the Gq family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCβ3 considerably increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCβ. View full text Figures at a glance * Figure 1: Primary and tertiary structures of PLCβ family members and comparison of cephalopod PLC21 with the Gαq–PLCβ3 complex. () Primary structure of human PLCβ3. PLCβ3 truncations used in this paper are indicated below the diagram. Numbers above the diagram correspond to amino acid positions at domain boundaries. () Crystal structure of LPLC21. LPLC21 crystallized as a dimer with pseudo two-fold symmetry. Domains are colored as in . The Hα2′-Hα3 hairpin from the proximal CTR is shown in cyan, and the catalytic Ca2+ is shown as a black sphere. Disordered loops are drawn as dashed lines, with the exception of the connection between the C2 domain and the beginning of Hα2′, which is ambiguous in the dimer interface. The C terminus of the C2 domain and start of Hα2′ are marked with pink and blue asterisks, respectively. N- and C-terminal ends of the protein fragment resolved in the crystal structure are labeled N and C, respectively. () Crystal structure of SPLC21. Domains are colored as in . () Crystal structure of the Gαq–PLCβ3 complex (PDB 3OHM)23. Hα1 and Hα2, which form the prima! ry Gαq binding site, are shown in dark blue. Residues corresponding to Hα2′ in the PLC21 structures are shown in cyan. Activated Gαq is shown in light gray, with GDP and AlF4− colored red and Mg2+ colored black. * Figure 2: Interactions of Hα2′ with the catalytic core. () The SPLC21 Hα2′ helix docks in a conserved cleft formed between the TIM barrel and C2 domains, in close proximity to the active site and the X-Y linker. The shorter Hα3 helix forms a hairpin interaction with Hα2′ stabilized by hydrophobic interactions. Domains are colored as in Figure 1a. () Specific interactions of SPLC21 Hα2′ with the catalytic core. Side chains that make large contributions to the binding interface are shown as sticks with carbons colored according to their respective domains and nitrogens colored blue. () The Hα2′–catalytic core interaction is recapitulated in a crystal contact of the Gαq–PLCβ3 structure (PDB 3OHM)23. Domains are colored as in Figure 1d. The subunit of Gαq shown is in complex with a different catalytic core in the crystal lattice. () Specific interactions between Hα2′ and the catalytic core in human PLCβ3. Residues analogous to those of SPLC21 shown in are drawn as sticks, and site-directed mutations created in! this study to perturb the interface are indicated. The SPLC21 Hα2′ helix is continuous, whereas Hα2′ in human PLCβ3 is kinked at Ala877, as if to optimize the interactions of the Leu879 side chain, which is smaller than that of the corresponding Phe804 residue in SPLC21. * Figure 3: Functional studies of PLCβ3 variants. () The proximal CTR stabilizes the catalytic core. Thermo Fluor assays were used to measure the melting point of three PLCβ3 variants by monitoring the change in fluorescence of 1-anilinonaphthalene-8-sulfonic acid (ANS). Representative curves are shown for PLCβ3 (circles), PLCβ3-Δ892 (squares) and PLCβ3-Δ847 (triangles). PLCβ3-Δ847 is 5–7 °C less stable (left-shifted) than PLCβ3 or PLCβ3-Δ892 (see Supplementary Table 1). AU, arbitrary units. () Comparison of the basal activity of PLCβ3 variants. Deletion of the proximal CTR in PLCβ3-Δ847 increases basal activity relative to PLCβ3-Δ892. The higher basal activity of PLCβ3 reflects the contribution of more distal regions of the CTR to maximal activity. Activity was measured by counting free [3H]-IP3 released from liposomes containing [3H]-PIP2 at 30 °C in the presence of ~200 nM free Ca2+ at four to five time points. The data shown represent at least four individual experiments conducted in duplicate ± s.! e.m. () Mutation of PLCβ3 at positions that contribute to the Hα2′–catalytic core interface substantially increase basal activity, indicating that this interaction is involved in autoinhibition. The data shown represents at least four individual experiments conducted in duplicate ± s.e.m. () Distal regions of the PLCβ3 CTR enhance binding to Gαq. FCPIA was used to quantify the ability of PLCβ3 truncations to displace Alexa Fluor 488 (AF488)-labeled PLCβ3-Δ892 (R872A L876A L879A triple mutant) from biotinylated, AlF4−-activated Gαi/q bound to streptavidin beads. Representative curves for PLCβ3 (circles), PLCβ3-Δ892 (squares) and PLCβ3-Δ847 (triangles) are shown. See Table 3 for measured inhibition constants. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3QR0 * 3QR1 * 3QR0 * 3QR1 Referenced accessions Protein Data Bank * 2ZKM * 3OHM * 2ZKM * 3OHM Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, USA. * Angeline M Lyon, * Valerie M Tesmer, * Vishan D Dhamsania, * David M Thal & * John J G Tesmer * Laboratory of Cell Biology, National Institute on Deafness and Other Communication Disorders, US National Institutes of Health, Rockville, Maryland, USA. * Joanne Gutierrez, * Shoaib Chowdhury & * John K Northup * Department of Biophysics, University of Michigan, Ann Arbor, Michigan, USA. * Krishna C Suddala * Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA. * John J G Tesmer Contributions A.M.L., V.M.T., J.K.N. and J.J.G.T. designed the overall experimental approach. J.G., S.C. and J.K.N. purified LPLC21 and SPLC21, and cloned and sequenced cDNA encoding SPLC21. K.C.S. crystallized LPLC21. A.M.L. crystallized SPLC21 and determined the crystal structures of LPLC21 and SPLC21. A.M.L. and V.M.T. cloned, expressed and purified human PLCβ3 variants. V.M.T. cloned, expressed and purified Gαq. A.M.L. did all activity-based assays. D.M.T. helped design and, together with V.D.D., conducted Thermo Fluor and FCPIA assays. A.M.L., V.M.T. and J.J.G.T. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * John J G Tesmer or * John K Northup Author Details * Angeline M Lyon Search for this author in: * NPG journals * PubMed * Google Scholar * Valerie M Tesmer Search for this author in: * NPG journals * PubMed * Google Scholar * Vishan D Dhamsania Search for this author in: * NPG journals * PubMed * Google Scholar * David M Thal Search for this author in: * NPG journals * PubMed * Google Scholar * Joanne Gutierrez Search for this author in: * NPG journals * PubMed * Google Scholar * Shoaib Chowdhury Search for this author in: * NPG journals * PubMed * Google Scholar * Krishna C Suddala Search for this author in: * NPG journals * PubMed * Google Scholar * John K Northup Contact John K Northup Search for this author in: * NPG journals * PubMed * Google Scholar * John J G Tesmer Contact John J G Tesmer Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Methods Additional data Entities in this article * Guanine nucleotide-binding protein G(q) subunit alpha Gnaq Mus musculus * View in UniProt * View in Entrez Gene * Phospholipase C Sepia officinalis * View in UniProt * 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase classes I and II Plc21C Drosophila melanogaster * View in UniProt * View in Entrez Gene * Synembryn-A Ric8 Mus musculus * View in UniProt * View in Entrez Gene * 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-2 PLCB2 Homo sapiens * View in UniProt * View in Entrez Gene * 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-3 PLCB3 Homo sapiens * View in UniProt * View in Entrez Gene * Phospholipase C Loligo pealeii * View in UniProt * 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase norpA Drosophila melanogaster * View in UniProt * View in Entrez Gene * Ras-related C3 botulinum toxin substrate 1 RAC1 Homo sapiens * View in UniProt * View in Entrez Gene - Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition
- Nat Struct Mol Biol 18(9):1006-1014 (2011)
Nature Structural & Molecular Biology | Article Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition * Elena Khazina1 * Vincent Truffault1 * Regina Büttner1 * Steffen Schmidt1 * Murray Coles2 * Oliver Weichenrieder1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1006–1014Year published:(2011)DOI:doi:10.1038/nsmb.2097Received03 January 2011Accepted02 June 2011Published online07 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The LINE-1 (L1) retrotransposon emerges as a major source of human interindividual genetic variation, with important implications for evolution and disease. L1 retrotransposition is poorly understood at the molecular level, and the mechanistic details and evolutionary origin of the L1-encoded L1ORF1 protein (L1ORF1p) are particularly obscure. Here three crystal structures of trimeric L1ORF1p and NMR solution structures of individual domains reveal a sophisticated and highly structured, yet remarkably flexible, RNA-packaging protein. It trimerizes via an N-terminal, ion-containing coiled coil that serves as scaffold for the flexible attachment of the central RRM and the C-terminal CTD domains. The structures explain the specificity for single-stranded RNA substrates, and a mutational analysis indicates that the precise control of domain flexibility is critical for retrotransposition. Although the evolutionary origin of L1ORF1p remains unclear, our data reveal previously undet! ected structural and functional parallels to viral proteins. View full text Figures at a glance * Figure 1: Domain structure of human L1ORF1p. () Domain boundaries. The crystallized part is colored gray (coiled coil), red (RRM domain) and blue (CTD), with hinges in lime. Yellow circles, ion-binding heptad repeats; cyan, heptads with an RhxxhE trimerization motif39. () NMR ensemble for the RRM domain with the disordered loop L(β2-β3) highlighted in yellow (Pro204–Thr213) and with the conserved salt bridges shown as sticks. () NMR ensemble for the CTD domain with the hinge at Trp264 highlighted in lime and with important side chains (Tyr282 and cis-proline Pro283) from the conserved α-hairpin shown as sticks. () Trimerization of L1ORF1p and details of the coiled coil (crystal form I). Heptad repeats are numbered I–VI and have alternating gray and blue-white colors, with central chloride ions shown as yellow spheres. Positions within heptads are generally numbered a–g, where a and d form hydrophobic layers in the core of the coiled coil. Left: externally stabilizing hydrogen bonds (including RhxxhE trimerizat! ion motifs39 in heptads II and V) are shown as dotted lines, with the corresponding residues as cyan sticks. Center: side chains of the hydrophobic core (positions a and d of each heptad) are shown as sticks with ion-coordinating residues in green. Right: conserved and functionally important side chains on the surface of the coiled coil are shown as magenta sticks. * Figure 2: Crystal structure and domain organization of the human L1ORF1p trimer. () Overview of the trimer (crystal form I) with the coiled coil in gray, the RRM domains in red and the CTD domains in blue. Central chloride ions are shown as yellow spheres. Side view (left) and top view (right). () Comparison of the three monomers with the interdomain linkers in lime and with the corresponding hinges marked by green arrows (side view). Residues Arg261 and Tyr282 are shown as black sticks to illustrate the different CTD orientations. (,) Asymmetric interfaces of the RRM domains (, top view) and details of the hinges (, side view). Interdomain hydrogen bonds are drawn as dashed lines, with the corresponding interface residues as sticks. The black triangle indicates the pseudo—three-fold axis of the coiled coil. Helix α3N is drawn as a simple loop for clarity. * Figure 3: Flexibility of the L1ORF1p trimer. () Comparison of the three crystal forms, cfI (left), cfII (center) and cfIII (right), colored according to monomers. Circles highlight the different CTD domain orientations, and residues Arg261 and Tyr282 of monomer C are shown as black sticks. (–) Flexibility of the CTD. The CTD is shown in the 'resting' (; cfI, monomer B), 'lifted' (; cfIII, monomer B) and 'twisted' (; cfI, monomer C) position. Selected side chains are shown as sticks and colored magenta if found important for retrotransposition, green if found irrelevant and yellow if not tested. Residues marked with asterisks have been found to be important for nucleic acid binding. * Figure 4: Nucleic acid–binding properties of the L1ORF1p trimer. () Electrostatic potential mapped onto the molecular surface of crystal form III with the superimposed NMR ensembles for the loops L(β2-β3) as yellow tubes. Potentials are contoured from −15 kT/e (red) to +15 kT/e (blue). Left, front view; right, back view. (–) Size-exclusion chromatography was done with various nucleic acid substrates (red lines) in the absence (dashed lines) or presence (solid lines) of L1ORF1p trimers (blue lines, dashed in the absence of nucleic acid substrate). Elution volumes of the complexes and free components are indicated by arrows and dotted gray lines. Starting concentrations for trimer and nucleic acid were, respectively, 25 and 45 μM (), 25 and 27 μM (), 25 and 45 μM (), 45 and 20 μM () and 25 and 45 μM (). Concentrations for were 50 μM of each preformed complex. * Figure 5: Mutational analysis of L1ORF1p. () RNA binding analyzed by size-exclusion chromatography. Binding of single-stranded RNA (27U RNA, red lines) was tested for variants of L1ORF1p (blue lines, dashed in the absence of nucleic acid substrate). Elution volumes of the complexes and free components are indicated by arrows and dotted gray lines. Apparent concentrations are calculated from the relative absorption properties of the components. Starting concentrations for trimer and nucleic acid were 25 and 45 μM, respectively. () Retrotransposition activities for variants of L1ORF1p. Activity was scored relative to the wild-type protein (bars with values below), using an active-site mutant of the L1ORF2p reverse transcriptase (D702A) as a negative control. R261K is also inactive16, 29 and was not determined here (n.d.). Error bars are s.d. from three independent experiments. * Figure 6: Structural parallels to viral proteins. () Bilobal architecture and basic RNA-binding groove of viral nucleoproteins. The ribbon representations (left) of L1ORF1p (crystal form III, monomer C) and of the nucleoprotein (NP) from the influenza A virus (PDB-ID 2iqh)35 reveal two RNA-binding lobes (red and blue) with a positively charged RNA-binding cleft in between (right; electrostatic surface potential contoured between −15 kT/e, red and + 15 kT/e, blue). () Trimeric coiled coils of viral membrane fusion proteins. Post-fusion structures of HTLV-1 gp21 (PDB-ID 1mg1)40, HIV-1 gp41 (PDB-ID 1env)41 and influenza A hemagglutinin (PDB-ID 1qu1)42 reveal variable 'insertion domains' (red) and C-terminal sequences (blue) that are attached to the surface of the coiled coil, as well as central chloride ions (yellow spheres, gp21) and an RhxxhE trimerization motif39 (cyan sticks, gp41). Yellow helices indicate regions in hemagglutinin undergoing a coil-to-helix transition upon membrane fusion. The dotted line provides a refe! rence for a structural alignment. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2yko * 2ykp * 2ykq * 2ldy * 2yko * 2ykp * 2ykq * 2ldy Referenced accessions Biological Magnetic Resonance Data Bank * 17686 Protein Data Bank * 2wpq * 2w7a * 2iqh * 1mg1 * 1env * 1qu1 * 2wpq * 2w7a * 2iqh * 1mg1 * 1env * 1qu1 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany. * Elena Khazina, * Vincent Truffault, * Regina Büttner, * Steffen Schmidt & * Oliver Weichenrieder * Department of Protein Evolution, Max Planck Institute for Developmental Biology, Tübingen, Germany. * Murray Coles Contributions E.K. is the leading author, designed, performed and analyzed experiments, established cell culture, determined the crystal structures and wrote the manuscript. V.T. and M.C. recorded and analyzed NMR spectra, determined and analyzed NMR structures, and contributed to the manuscript. R.B. established cell culture, and performed and analyzed experiments. S.S. analyzed data and contributed to the manuscript. O.W. designed research, analyzed experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Oliver Weichenrieder Author Details * Elena Khazina Search for this author in: * NPG journals * PubMed * Google Scholar * Vincent Truffault Search for this author in: * NPG journals * PubMed * Google Scholar * Regina Büttner Search for this author in: * NPG journals * PubMed * Google Scholar * Steffen Schmidt Search for this author in: * NPG journals * PubMed * Google Scholar * Murray Coles Search for this author in: * NPG journals * PubMed * Google Scholar * Oliver Weichenrieder Contact Oliver Weichenrieder Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information Movies * Supplementary Movie 1 (13M) Flexibility and domain movements in the L1ORF1p trimer. While rotating once around the pseudo–three-fold axis of the trimer, the movie smoothly interpolates between the structural conformations observed in the three crystal forms (cfI, cfII and cfIII). Morphing transitions are from cfI via cfII to cfII and directly back to cfI. This is repeated once. The coiled coil is colored in gray, the RRM domains are in red and the CTD domains are in blue. Interdomain linkers are in lime and the central chloride ions are shown as yellow spheres. PDF files * Supplementary Text and Figures (10M) Supplementary Figures 1–5 and Supplementary Methods Additional data Entities in this article * Hemagglutinin HA Influenza A virus (strain A/Puerto Rico/8/1934 H1N1) * View in UniProt * View in Entrez Gene * Envelope glycoprotein gp62 Human T-cell leukemia virus 1 (strain Japan MT-2 subtype A) * View in UniProt * Envelope glycoprotein gp160 env Human immunodeficiency virus type 1 group M subtype B (isolate HXB2) * View in UniProt * View in Entrez Gene - Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation
- Nat Struct Mol Biol 18(9):1015-1019 (2011)
Nature Structural & Molecular Biology | Article Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation * Xuefeng Chen1 * Hengyao Niu2 * Woo-Hyun Chung1 * Zhu Zhu1 * Alma Papusha1 * Eun Yong Shim3 * Sang Eun Lee3 * Patrick Sung2 * Grzegorz Ira1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1015–1019Year published:(2011)DOI:doi:10.1038/nsmb.2105Received20 October 2010Accepted13 June 2011Published online14 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg DNA recombination pathways are regulated by the cell cycle to coordinate with replication. Cyclin-dependent kinase (Cdk1) promotes efficient 5′ strand resection at DNA double-strand breaks (DSBs), the initial step of homologous recombination and damage checkpoint activation. The Mre11–Rad50–Xrs2 complex with Sae2 initiates resection, whereas two nucleases, Exo1 and Dna2, and the DNA helicase–topoisomerase complex Sgs1–Top3–Rmi1 generate longer ssDNA at DSBs. Using Saccharomyces cerevisiae, we provide evidence for Cdk1-dependent phosphorylation of the resection nuclease Dna2 at Thr4, Ser17 and Ser237 that stimulates its recruitment to DSBs, resection and subsequent Mec1-dependent phosphorylation. Poorly recruited dna2T4A S17A S237A and dna2ΔN248 mutant proteins promote resection only in the presence of Exo1, suggesting cross-talk between Dna2- and Exo1-dependent resection pathways. View full text Figures at a glance * Figure 1: Cdk1 regulates long-range double-strand break resection by Dna2. () Analysis of initial and extensive resection in mutants with either active or inactive Cdk1 kinase. The Southern blots corresponding to these experiments are presented in Supplementary Figure 1. Error bars correspond to s.d. of three independent experiments. () Southern blot analysis of initial resection in mutants. Smearing and additional bands below the HO cut band typical for mutants that lack extensive resection are indicated by asterisks. () Analysis of resection in cells where Cdk1 activity is blocked at 4 h after break induction, when all cells have initiated resection. () Recruitment of Dna2 to DSBs was monitored in indicated mutants by ChIP. Error bars correspond to s.d. of three independent experiments. IP, normalized fold increase in DNA amount compared to time '0' in immunoprecipitated samples. * Figure 2: Dna2 is phosphorylated by Cdk1 and Mec1. () Western blot analysis of Dna2-9 × Myc phosphorylation in cdk1-as1 with or without Cdk1 inhibitor and in checkpoint-deficient cells in response to a single DSB. () In vitro phosphorylation of wild-type Dna2 or dna2 mutant proteins lacking single or multiple Cdk1 phosphorylation consensus sites. () Western blot analysis of Dna2 phosphorylation in wild-type cells and indicated dna2 mutant cells. * Figure 3: Dna2 phosphorylation by Cdk1 stimulates resection. () Analysis of 5′ strand resection in dna2Δ cells complemented with plasmids carrying either wild-type or a mutant DNA2 allele. Error bars correspond to s.d. of three independent experiments. The Southern blots corresponding to these experiments are presented in Supplementary Figure 3. () Analysis of resection in exo1Δ dna2Δ cells complemented with plasmids carrying either wild-type or a mutant DNA2 allele. () Analysis of sensitivity to methyl methanesulfonate (MMS) and camptothecin in the indicated mutants. * Figure 4: Dna2 phosphorylation by Cdk1 is needed for its recruitment to double-strand breaks. () Analysis of DSB recruitment of GFP-tagged wild-type Dna2 and indicated mutant dna2 proteins. () Analysis of recruitment of Flag-tagged wild-type Dna2 and indicated mutant dna2 proteins to DSB ends by ChIP using primers specific for sequences located 1 kb upstream of the DSB. Error bars correspond to s.d. of three independent experiments. IP, normalized fold increase in DNA amount compared to time '0' in immunoprecipitated samples. () Analysis of recruitment of GFP-tagged phosphomimic dna2S17D S237D protein to DSB ends in Cdk1- and Ku-deficient cells. Arrows indicate position of weak Dna2-GFP loci. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, USA. * Xuefeng Chen, * Woo-Hyun Chung, * Zhu Zhu, * Alma Papusha & * Grzegorz Ira * Department of Molecular Biophysics & Biochemistry, Yale University School of Medicine, New Haven, Connecticut, USA. * Hengyao Niu & * Patrick Sung * Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA. * Eun Yong Shim & * Sang Eun Lee Contributions X.C. constructed most of the strains and plasmids, analyzed Dna2 phosphorylation in cells, carried out fluorescence microscopy and ChIP experiments. W.-H.C. and A.P. analyzed resection in mutants, and H.N. purified proteins and conducted all the in vitro experiments. Z.Z. constructed the initial plasmids carrying dna2 mutant alleles. X.C., H.N., P.S. and G.I. designed the experiments, analyzed the data and wrote the manuscript. E.Y.S. and S.E.L. did the ChIP assay for Sgs1-13 × Myc. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Patrick Sung or * Grzegorz Ira Author Details * Xuefeng Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Hengyao Niu Search for this author in: * NPG journals * PubMed * Google Scholar * Woo-Hyun Chung Search for this author in: * NPG journals * PubMed * Google Scholar * Zhu Zhu Search for this author in: * NPG journals * PubMed * Google Scholar * Alma Papusha Search for this author in: * NPG journals * PubMed * Google Scholar * Eun Yong Shim Search for this author in: * NPG journals * PubMed * Google Scholar * Sang Eun Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick Sung Contact Patrick Sung Search for this author in: * NPG journals * PubMed * Google Scholar * Grzegorz Ira Contact Grzegorz Ira Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods Additional data Entities in this article * DNA damage response protein kinase DUN1 DUN1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA repair protein RAD50 RAD50 Homo sapiens * View in UniProt * View in Entrez Gene * Double-strand break repair protein MRE11A MRE11A Homo sapiens * View in UniProt * View in Entrez Gene * DNA endonuclease ctp1 ctp1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Nibrin NBN Homo sapiens * View in UniProt * View in Entrez Gene * ATP-dependent DNA helicase II subunit 2 YKU80 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * ATP-dependent DNA helicase II subunit 1 YKU70 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA repair and recombination protein PIF1 PIF1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Pantothenate transporter FEN2 FEN2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase CHK1 CHK1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Ribonucleotide reductase inhibitor protein SML1 SML1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Exodeoxyribonuclease 1 EXO1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA endonuclease SAE2 SAE2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA topoisomerase 3 TOP3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * ATP-dependent helicase SGS1 SGS1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Double-strand break repair protein MRE11 MRE11 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA replication ATP-dependent helicase DNA2 DNA2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA repair protein XRS2 XRS2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA repair protein RAD50 RAD50 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * ATP-dependent DNA helicase SRS2 SRS2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase MEC1 MEC1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * RecQ-mediated genome instability protein 1 RMI1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Cyclin-dependent kinase 1 CDC28 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Breast cancer type 2 susceptibility protein BRCA2 Homo sapiens * View in UniProt * View in Entrez Gene * DNA repair protein rhp9 crb2 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * DNA repair protein RAD9 RAD9 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase RAD53 RAD53 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Bud site selection protein 5 BUD5 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Probable DNA-binding protein SNT1 SNT1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA endonuclease RBBP8 RBBP8 Homo sapiens * View in UniProt * View in Entrez Gene * Meiosis-specific protein SPO11 SPO11 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA repair protein rhp51 rhp51 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Transcription-associated protein 1 TRA1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA repair protein RAD51 homolog 1 RAD51 Homo sapiens * View in UniProt * View in Entrez Gene * Replication factor A protein 2 RFA2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Bloom syndrome, RecQ helicase-like BLM Homo sapiens * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase TEL1 TEL1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * G2/mitotic-specific cyclin-2 CLB2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene - A universal pathway for kinesin stepping
- Nat Struct Mol Biol 18(9):1020-1027 (2011)
Nature Structural & Molecular Biology | Article A universal pathway for kinesin stepping * Bason E Clancy1 * William M Behnke-Parks2 * Johan O L Andreasson3 * Steven S Rosenfeld4 * Steven M Block1, 5 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1020–1027Year published:(2011)DOI:doi:10.1038/nsmb.2104Received30 March 2011Accepted14 June 2011Published online14 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation i! s also responsible for ensuring gating in kinesin. View full text Figures at a glance * Figure 1: Single-molecule records and backstepping velocity for Kin6AA. () Representative traces obtained under constant force at indicated loads, at 2 mM ATP. Light traces are unfiltered; darker traces are median-filtered. The records show clear 8-nm forward and backward steps. () Double-log plot of backstepping velocity versus ATP concentration under −7 pN load (black dots; mean ± s.e.m.; N = 21−62). The solid line (red) shows the global fit to all single-molecule data for the model described in the text. Inset, histogram of the backward step size at a −7 pN load for all ATP concentrations (mean ± s.d., 8.12 ± 1.72 nm; N = 1,906) and Gaussian fit (red line), centered at 7.97 nm. * Figure 2: Bidirectionality of Kin6AA as a function of load and ATP concentration. Solid lines (red) show the global fits to all single-molecule data for the model described in the text. () Velocity, v (mean ± s.e.m.) versus force for 2 mM ATP (N = 25−164) and 10 μM ATP (N = 18−74). Stall occurs where the fit data cross the horizontal dashed gray line at v = 0. () Step ratio, SR (mean ± s.e.m.) versus force at 2 mM ATP (N = 264−3,331), 10 μM ATP (N = 235−1,412) and 2 μM ATP (N = 90−368). Stall occurs where the fit data cross the horizontal dashed gray line at SR = 1. () Reciprocal randomness, r−1 (mean ± s.e.m.). Note that the fit data cross r−1 = 0 at the stall forces for the data in . * Figure 3: Model for stepping by kinesin dimers, showing forward-stepping, backward-stepping and futile-hydrolysis pathways. See text. () Left, the numbers assigned to each of the five states. The molecular configurations of the kinesin dimer on the microtubule thought to correspond to each of the states are illustrated, along with any nucleotides bound. No particular docking state of the neck-linker is implied in this diagram. Kinesin heads are color-coded (red, blue). Starting from state (1) (middle row), forward steps are accomplished by ascending the diagram, and backward steps by descending. () Reaction diagram for the model, showing the transition rates among states. Load- and ATP-dependent transitions are indicated. Three main pathways are shaded: forward stepping (yellow), backward stepping (orange) and futile hydrolysis (light green). Largely irreversible transitions between states that produce ±8-nm displacements are shown as green arrows. In this minimal model, fast transitions occurring in rapid succession were combined to generate composite states in several instances. Note: the tran! sition from state (4) to state (3) in the futile-hydrolysis pathway involves ATP binding to the rear head, but, unlike in the stepping pathways, heads do not swap positions and no step is taken. * Figure 4: Fluorescence data for Kin6AA and KinWT with TMR probes attached to both neck-linkers. (,) Steady-state TMR fluorescence emission spectra for KinWT () and Kin6AA (), which monitor neck-linker separation under the following conditions: microtubules plus 2 mM AMP-PNP (red), microtubules plus apyrase to remove nucleotides (black, dashed) and 2 mM ADP without microtubules (blue). The large signal increase in the absence of nucleotide (apyrase present) for Kin6AA is consistent with neck-linker separation. In the inset cartoons, approximate locations of the TMR probes (at position 333) are indicated (yellow circles), as well as the neck-linker inserts (blue lines). (,) Pre–steady-state TMR kinetic records for KinWT and Kin6AA. TMR-labeled motors complexed to a five-fold excess of microtubules and treated with apyrase were mixed with 2 mM ATP. The initial increase in fluorescence seen for KinWT is absent for Kin6AA, indicating that before mixing with ATP, both heads of Kin6AA are bound to the microtubule, and consequently the neck-linkers of this mutant are separat! ed. * Figure 5: Binding of 2′dmT to Kin6AA. () A complex of Kin6AA and microtubules was preformed and mixed in a stopped-flow apparatus with 2′dmT (Supplementary Methods). The resulting fluorescence signal (red) consisted of three sequential phases: a first phase of increasing fluorescence, a lag phase, and a second phase of increasing fluorescence. Fitting this signal required three exponential terms (black curve). Two terms corresponding to the phases of increasing fluorescence were associated with rate constants of 81.5 ± 21.0 s−1 and 3.0 ± 0.1 s−1. The third term was associated with a low-amplitude decreasing phase, consistent with a lag, and with a rate constant of 55.6 ± 24.8 s−1. The same experiment in the absence of microtubules (gray) produced a fluorescence increase with a single exponential phase and a rate constant of 45.0 ± 1.0 s−1. Inset, fractional amplitude of the first phase versus 2′dmT concentration. () Rate constant for the first phase of fluorescence increase versus 2′dmT concent! ration. Data (black dots; mean ± s.e.m.) were fit to a hyperbola (red curve) that extrapolates to 61 ± 12 s−1 at zero 2′dmT and is associated with a second-order rate constant of 2.6 ± 0.3 μM−1 s−1. Inset, rate constant for the second phase of fluorescence versus 2′dmT concentration, which averages 3.0 ± 0.4 s1 (red line). () Rate of initial decay of TMR fluorescence as a function of ATP concentration, compared to the rate of the lag phase in . Rates of the TMR fluorescence decay (red dots; mean ± s.e.m.; N = 18−35) were fit to a rectangular hyperbola (black curve); the asymptotic rate at saturating ATP was 90 ± 4 s−1. The y intercept of the fit at 11 ± 5 s−1 is interpreted as the rate at which a head rebinds to the microtubule. Blue squares represent rate constant for the lag phase versus 2′dmT concentration (mean ± s.e.m.; N = 10−20). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Biology, Stanford University, Stanford, California, USA. * Bason E Clancy & * Steven M Block * Department of Biology, Columbia University, New York, New York, USA. * William M Behnke-Parks * Department of Physics, Stanford University, Stanford, California, USA. * Johan O L Andreasson * Department of Neurology, Columbia University, New York, New York, USA. * Steven S Rosenfeld * Department of Applied Physics, Stanford University, Stanford, California, USA. * Steven M Block Contributions B.E.C., W.M.B.-P. and J.O.L.A. designed and carried out experiments, collected and analyzed data, and cowrote the paper. B.E.C. and J.O.L.A. carried out modeling and fits. S.M.B. and S.S.R. helped to design experiments and analyze results, and cowrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Steven M Block Author Details * Bason E Clancy Search for this author in: * NPG journals * PubMed * Google Scholar * William M Behnke-Parks Search for this author in: * NPG journals * PubMed * Google Scholar * Johan O L Andreasson Search for this author in: * NPG journals * PubMed * Google Scholar * Steven S Rosenfeld Search for this author in: * NPG journals * PubMed * Google Scholar * Steven M Block Contact Steven M Block Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (508K) Supplementary Figures 1–6 and Supplementary Methods Additional data Entities in this article * Kinesin-1 heavy chain KIF5B Homo sapiens * View in UniProt * View in Entrez Gene - An unusual dimeric structure and assembly for TLR4 regulator RP105–MD-1
- Nat Struct Mol Biol 18(9):1028-1035 (2011)
Nature Structural & Molecular Biology | Article An unusual dimeric structure and assembly for TLR4 regulator RP105–MD-1 * Sung-il Yoon1 * Minsun Hong1 * Ian A Wilson1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1028–1035Year published:(2011)DOI:doi:10.1038/nsmb.2106Received24 December 2010Accepted14 June 2011Published online21 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg RP105–MD-1 modulates the TLR4–MD-2-mediated, innate immune response against bacterial lipopolysaccharide (LPS). The crystal structure of the bovine 1:1 RP105–MD-1 complex bound to a putative endogenous lipid at 2.9 Å resolution shares a similar overall architecture to its homolog TLR4–MD-2 but assembles into an unusual 2:2 homodimer that differs from any other known TLR-ligand assembly. The homodimer is assembled in a head-to-head orientation that juxtaposes the N-terminal leucine-rich repeats (LRRs) of the two RP105 chains, rather than the usual tail-to-tail configuration of C-terminal LRRs in ligand-activated TLR dimers, such as TLR1–TRL2, TLR2–TLR6, TLR3–TLR3 and TLR4–TLR4. Another unusual interaction is mediated by an RP105-specific asparagine-linked glycan, which wedges MD-1 into the co-receptor binding concavity on RP105. This unique mode of assembly represents a new paradigm for TLR complexes and suggests a molecular mechanism for regulating LPS respo! nses. View full text Figures at a glance * Figure 1: Overall architecture of the 2:2 sRP105–MD-1 complex. The 1:1 sRP105–MD-1 building block that corresponds to unliganded 1:1 sTLR4–MD-2 complex is designated by sRP105a–MD-1a (yellow and red; translucent molecular surface with underlying ribbon representation) or sRP105b–MD-1b (blue and gray; ribbon only). The 1:1 complex formation is driven by the primary binding interface between RP105 and MD-1. Two copies of the 1:1 complex associate in a symmetrical manner in a head-to-head mode through a unique dimerization interface. RP105 Asn402- and Asn451-linked glycans that are well ordered in the sRP105a–MD-1a or sRP105b–MD-1b interface region are shown in magenta and cyan stick models, respectively. The two views are shown parallel to the cell surface (above) and from the top looking down onto the cell surface (below). * Figure 2: sRP105 and MD-1 structures as observed in the 2:2 complex. (,) The sRP105 () and MD-1 () structures are represented by yellow ribbon diagrams, and the components of the structure that form each binding interface are colored as indicated in the figure. RP105 residues are labeled in brown. The asparagine-linked glycan at sRP105 Asn402, which is involved in primary interface C, is shown in magenta sticks and the nearby glycan at Asn451 is colored orange. Disulfide bonds are shown in black sticks. sRP105 is divided into three subdomains as indicated by dashed lines that correspond to those designated in previous work in TLR4 as the N-terminal, central and C-terminal domains10. * Figure 3: The sRP105–MD-1 primary interaction. () Overall view of the 1:1 sRP105–MD-1 complex. The 1:1 complex is represented by a ribbon diagram (sRP105, yellow; MD-1, gray) and primary interfaces A, B and C are colored in blue, green and magenta, respectively, on the MD-1 surface. sRP105 primary interface residues are shown in stick models (carbon, yellow; oxygen, red; nitrogen, blue). (–) Close-up view of primary interfaces A (), B () and C (). Interfaces A and B correspond to A patch and B patch, respectively, as designated in previous work in TLR4–MD-2 (ref. 5). MD-1 and sRP105 residues in the primary interface are shown in green and yellow ball-and-stick models, respectively, with oxygens in red and nitrogens in blue. The MD-1 interface is color-coded on a surface representation according to sequence conservation among ten MD-1 orthologs from light green (most conserved) to dark blue (less conserved). Broken dotted lines represent hydrogen bonds or salt bridges. RP105 residues are labeled in brown. * Figure 4: sRP105-specific glycan at Asn402. () Asn402-linked glycan (green sticks) is stabilized by interactions with neighboring sRP105 protein residues, as well as with Asn451-linked glycan (cyan sticks). Hydrogen bonds are represented by dashed lines. Electron density for the Asn402-linked glycan is shown in a pale green mesh at a 1.0-σ level in a 2Fo–Fc map. The sRP105 and MD-1 Cα traces are colored in yellow and gray, respectively. Residual electron density observed beyond Man-A and Man-4 sugars of glycan at Asn402 are circled in red in the inset (top right) and suggest that the glycan is Man8-9GlcNAc2. RP105 residues are labeled in brown. () Schematic diagram of an asparagine-linked high mannose glycan, Man9GlcNAc2. The most frequently found asparagine-linked glycan in insect cells, Man3GlcNAc2, is boxed in dashed lines. Asn402-linked glycan (Man6GlcNAc2) that could be modeled well in the sRP105 structure is enclosed by solid lines. Glycan residues that make contacts with MD-1 are colored in red. The standar! d nomenclature of each sugar moiety is shown in parentheses. * Figure 5: The unique sRP105–MD-1 homodimerization interaction for assembly of the 2:2 complex. () Overall view of the 2:2 sRP105–MD-1 complex. 1:1 sRP105a–MD-1a and sRP105b–MD-1b complexes are shown in a surface representation (yellow sRP105a, gray MD-1a) and in thin coils (blue sRP105b, green MD-1b), respectively. The dimerization interface is colored in red (interfaces α and α′) and cyan (interface β) on the surface representation of the sRP105a–MD-1a. () Close-up view of dimerization interface α. MD-1a and sRP105b residues are shown with green and orange carbons (red oxygens, blue nitrogens) in ball-and-stick models, respectively. The MD-1a interface is color-coded on the surface representation by sequence conservation as in Figure 3. Broken dotted lines represent hydrogen bonds or salt bridges. RP105 residues are labeled in brown. () Close-up view of dimerization interface β. The sRP105a and sRP105b residues are shown in green and orange ball-and-stick models, respectively. The RP105a interface is color-coded on the surface representation according ! to sequence conservation in four RP105 orthologs. * Figure 6: Different organization of the 2:2 sRP105–MD-1 and LPS-bound 2:2 sTLR4–MD-2 homodimeric assemblies. (,) The head-to-head homodimer of 2:2 sRP105–MD-1 () and the tail-to-tail homodimer of LPS-bound 2:2 TLR4–MD-2 (PDB 3FXI)10 (). The surface representations of sRP105 and sTLR4 are rainbow-colored from N terminus (blue) to C terminus (red). MD-1 and MD-2 are shown in magenta ribbons. LPS bound to sTLR4–MD-2 is represented by black sticks. (,) Close-up views of 1:1 sRP105a–MD-1a () and 1:1 sTLR4a–MD-2a (). The distinct dimerization interfaces of 2:2 sRP105–MD-1 and TLR4–MD-2 are shown as red and cyan surface representations over the ribbon diagram of their respective 1:1 complexes. For comparison, the similar primary interfaces A and B are shown in gray surface representations. () The unique head-to-head arrangement of sRP105–MD-1 is verified by mouse MD-1 mutants G52D and G71D, which do not permit homodimerization of the sRP105–MD-1 complex. sRP105 coexpressed with an excess of MD-1 WT or mutants was analyzed by gel-filtration chromatography (top), and its f! ractions were resolved by SDS-PAGE (bottom). mAU, milliabsorbance units. * Figure 7: Two possible models for the interaction between RP105–MD-1 and TLR4–MD-2. (,) Binding models 1 () and 2 (). Potential TLR4–MD-2 binding sites on RP105–MD-1 are highlighted by black circles in left panels, and their resulting complexes are shown in right panels. The sRP105–MD-1 (yellow and gray) and sTLR4–MD-2 (cyan and green) structures are shown by surface representations. MD-2 () and MD-1 () cavities that accommodate LPS molecules are represented by red and black triangles, respectively. Currently, we favor model 2. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3RG1 * 3RG1 Referenced accessions Protein Data Bank * 3MTX * 2Z64 * 3FXI * 3MTX * 2Z64 * 3FXI Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA. * Sung-il Yoon, * Minsun Hong & * Ian A Wilson * The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA. * Ian A Wilson Contributions S.Y. and I.A.W. designed experiments. S.Y. and M.H. conducted experiments. S.Y., M.H. and I.A.W. analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ian A Wilson Author Details * Sung-il Yoon Search for this author in: * NPG journals * PubMed * Google Scholar * Minsun Hong Search for this author in: * NPG journals * PubMed * Google Scholar * Ian A Wilson Contact Ian A Wilson Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–7 Additional data Entities in this article * Variable lymphocyte receptor B Eptatretus burgeri * View in UniProt * Lymphocyte antigen 86 LY86 Homo sapiens * View in UniProt * View in Entrez Gene * Lymphocyte antigen 86 Ly86 Mus musculus * View in UniProt * View in Entrez Gene * Toll-like receptor 4 Tlr4 Mus musculus * View in UniProt * View in Entrez Gene * Toll-like receptor 6 Tlr6 Mus musculus * View in UniProt * View in Entrez Gene * Toll-like receptor 2 Tlr2 Mus musculus * View in UniProt * View in Entrez Gene * Lymphocyte antigen 86 LY86 Gallus gallus * View in UniProt * View in Entrez Gene * Toll-like receptor 3 Tlr3 Mus musculus * View in UniProt * View in Entrez Gene * Lymphocyte antigen 86 LY86 Bos taurus * View in UniProt * View in Entrez Gene * CD180 antigen CD180 Bos taurus * View in UniProt * View in Entrez Gene * Toll-like receptor 1 Tlr1 Mus musculus * View in UniProt * View in Entrez Gene * Lymphocyte antigen 96 Ly96 Mus musculus * View in UniProt * View in Entrez Gene - The structural basis of RNA-catalyzed RNA polymerization
- Nat Struct Mol Biol 18(9):1036-1042 (2011)
Nature Structural & Molecular Biology | Article The structural basis of RNA-catalyzed RNA polymerization * David M Shechner1, 2, 3, 4 * David P Bartel1, 2, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1036–1042Year published:(2011)DOI:doi:10.1038/nsmb.2107Received19 January 2011Accepted22 June 2011Published online21 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Early life presumably required polymerase ribozymes capable of replicating RNA. Known polymerase ribozymes best approximating such replicases use as their catalytic engine an RNA-ligase ribozyme originally selected from random RNA sequences. Here we report 3.15-Å crystal structures of this ligase trapped in catalytically viable preligation states, with the 3′-hydroxyl nucleophile positioned for in-line attack on the 5′-triphosphate. Guided by metal- and solvent-mediated interactions, the 5′-triphosphate hooks into the major groove of the adjoining RNA duplex in an unanticipated conformation. Two phosphates and the nucleophile jointly coordinate an active-site metal ion. Atomic mutagenesis experiments demonstrate that active-site nucleobase and hydroxyl groups also participate directly in catalysis, collectively playing a role that in proteinaceous polymerases is performed by a second metal ion. Thus artificial ribozymes can use complex catalytic strategies that differ! markedly from those of analogous biological enzymes. View full text Figures at a glance * Figure 1: The class I ligase ribozyme preligation complex. () Secondary structure of the C47U crystallization construct17, depicted undergoing ligation (curved arrows). The nucleophile, electrophile and leaving group (red), active-site backbone phosphates (yellow bars) and nucleotides added to facilitate crystallization (gray) are indicated. Residue numbering, base-pair geometries and tertiary interactions, as in reference 17. Inset, primer extension catalyzed by a ligase variant resembling the self-ligation construct, except that exogenous GTP replaces G1–A4. () Overview of the Mg2+–Sr2+ preligation structure, peering into the active site. The U1A protein and its cognate loop have been removed from view. The 5′-GTP is shown as sticks. Active-site metal ions are shown as spheres (orange, Mg2+; green, metal-coordinated water). Meshes are simulated-annealing |Fo| − |Fc| OMIT maps calculated without G1 (magenta, contoured at 5σ) or active-site solvent atoms (dark blue, contoured at 4.5σ). () Superposition of Mg2+–Sr2+ preli! gation and product (PDB ID: 3HHN)17 structures. () Superposition of Mg2+–Sr2+ preligation and product structures (sticks and black lines, respectively) near the active site. A structural metal ion and water molecules are shown as orange and green spheres, respectively; active-site solvent atoms have been removed from view. * Figure 2: The class I ligase active site. () The active site, as observed in the Ca2+–Sr2+ preligation structure. Calcium ions and waters (orange and green spheres, respectively), inner-sphere metal interactions (thin sticks) and outer-sphere interactions and hydrogen bonds (black dashes) are indicated. Meshes are simulated-annealing |Fo| − |Fc| omit maps calculated without GTP (magenta, contoured at 5σ) or active-site solvent atoms (navy, contoured at 4.5σ). () The active site, as observed in the Mg2+–Sr2+ preligation structure. As in , except that the orange spheres depict Mg2+ ions. () Reorientation of the 5′-GTP during metal-ion exchange. () Solvent interactions in active site of the Mg2+–Sr2+ preligation complex. Blue numbers indicate the distance, in angstroms, between atomic centers; the error of these measurements is approximated by the mean crystallographic coordinate precision, 0.4 Å. Waters hydrating the Mg2+ bound by G1 and G2 are represented by black lines. () Summary of phosphorothioate in! terference (black, Mgkthio/Mgkoxy) and metal rescue (blue, (Xkthio/Xkoxy)/(thioMgkthio/Mgkoxy) values for oxygens at the active site. Functional groups on the 5′-GTP were interrogated in primer-extension assays, and those at positions 29 and 30 were interrogated in self-ligation assays. Error values are the s.d. from three independent experiments. * Figure 3: Biochemical interrogation of C47. () View of the active site observed in the Mg2+–Sr2+ complex, roughly orthogonal to that in Figure 2b, highlighting interactions between C47, C30 and the 5′-triphosphate. Depiction and coloring are as in Figure 2b, with the thin red line indicating the assumed line of nucleophilic attack. () Self-ligation rates of C47 variants, relative to that of the parental ligase (red bar). The rates of the faster constructs (red and dark blue bars) were measured at pH 6.0. To allow time courses to approach completion within 24 h, the rates of slower constructs (light blue bars) were measured at pH 8.0 and re-normalized assuming log-linear pH dependencies24. Constructs marked with an asterisk were synthesized with a 2′-deoxyribose at position 47, and rates were normalized to account for this modification. The 4-thiouridine variant showed biphasic kinetics in which approximately 10% reacted in a burst phase with a relative rate of 0.23 ± 0.04 (s.d.); shown is the relative rate rele! vant to the majority of the molecules. Error bars, s.d. from three independent experiments. * Figure 4: Catalytic roles of active-site functional groups. () Proton inventories for self-ligation by wild type and the indicated variants. The solid black line indicates the best fit for a single-proton transfer model; the dashed blue line indicates the best fit for a two-proton transfer model28, 37, 38. Error bars, s.d. from three independent experiments. () Relative rates for self-ligation of constructs with the indicated substitutions at the 2′ position of C30, surveyed in combination with the indicated subsitutions at position 47. For each C47 substitution, rates are normalized to that of the C30 2′-OH construct. Error bars, s.d. from three independent experiments. () Schemes summarizing functional interaction between the C30 2′-hydroxyl and the C47 N4, showing the effects of removing these groups (left) or replacing them with inhibitory groups (right). Substitutions, relative to the wild type, are denoted in red. Fractional numbers indicate the decrease in self-ligation rate, kright/kleft or kbottom/ktop, resulting from ! the indicated substitution. At pH 6.0, the observed rate of the unmodified ligase (upper left corner of each scheme) was 0.93 ± 0.009 min−1 (s.d.). * Figure 5: Transition-state stabilization by protein and RNA active sites. () Catalysis by proteinaceous polymerases28. Black dotted lines indicate bonds formed or broken during the transition state. Red arrows indicate the reaction direction during polymerization. () Catalysis by the class I ligase. Gray dashed lines denote hydrogen bonds. Thick lines denote inner-sphere contacts with a single catalytic metal ion; those in blue are observed crystallographically, those in gray are either inferred from biochemical results or are presumed to be water (not shown). Red arrows indicate the reaction direction during ligation or polymerization. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3R1H * 3R1L * 3R1H * 3R1L Referenced accessions Protein Data Bank * 3HHN * 3HHN Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. * David M Shechner & * David P Bartel * Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * David M Shechner & * David P Bartel * Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * David M Shechner & * David P Bartel * Present addresses: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA, and The Broad Institute, Cambridge, Massachusetts, USA. * David M Shechner Contributions D.M.S. and D.P.B. designed the experiments and wrote the manuscript. D.M.S. carried out the experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David P Bartel Author Details * David M Shechner Search for this author in: * NPG journals * PubMed * Google Scholar * David P Bartel Contact David P Bartel Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information Audio files * Supplementary Movie 1 (938K) Simulated conformational changes and catalysis by the Class I ligase. A series of linear morphs between the Ca2+–Sr2+ preligation, Mg2+–Sr2+ preligation and product structures. Atoms are colored as in Figure 2a,b; the red dotted line indicates the proposed line of nucleophilic attack. PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–5, Supplementary Discussion and Supplementary Methods Additional data Entities in this article * U1 small nuclear ribonucleoprotein A SNRPA Homo sapiens * View in UniProt * View in Entrez Gene - Transfer RNA–mediated regulation of ribosome dynamics during protein synthesis
- Nat Struct Mol Biol 18(9):1043-1051 (2011)
Nature Structural & Molecular Biology | Article Transfer RNA–mediated regulation of ribosome dynamics during protein synthesis * Jingyi Fei1, 3 * Arianne C Richard2, 3 * Jonathan E Bronson1, 3 * Ruben L Gonzalez Jr1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1043–1051Year published:(2011)DOI:doi:10.1038/nsmb.2098Received09 December 2010Accepted14 June 2011Published online21 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Translocation of tRNAs through the ribosome during protein synthesis involves large-scale structural rearrangement of the ribosome and ribosome-bound tRNAs that is accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. How the rearrangement of individual tRNA-ribosome interactions influences tRNA movement during translocation, however, remains largely unknown. To address this question, we used single-molecule FRET to characterize the dynamics of ribosomal pretranslocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate of PRE complex rearrangement into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. Notably, our results suggest that the conformational flexibility of the tRNA molecule has a crucial role in directing the structural dynamics of the PRE complex dur! ing translocation. View full text Figures at a glance * Figure 1: Global states model of the PRE complex, L1-L9 labeling strategy and PRE−A complexes. () Cartoon diagram of global states model of PRE complex. The 30S and 50S subunits, tan and lavender, respectively; L1 stalk, dark blue. tRNAs, brown curves; nascent polypeptide, chain of gold spheres. () Labeling strategy for smFRETL1-L9. The 50S subunit is shown from the perspective of the intersubunit space31 (PDB 2J01). L1 stalk consists of 23S rRNA helices 76–78 (pink) and r-protein L1 (dark blue). r-protein L9, cyan. Donor (Cy3) and the acceptor (Cy5) fluorophores are green and red stars on r-proteins L9 and L1, respectively. Image was rendered using PyMol50. () Cartoon diagram of a PRE−A complex. PRE−A complexes are formed using an L1-L9 labeled 50S subunit and carry a deacylated P-site tRNA. * Figure 2: Sample smFRET versus time trajectories and relative occupancies of smFRET trajectory subpopulations. () Sample smFRET versus time trajectories. Three subpopulations of smFRET trajectories were observed. The first subpopulation, SPGS1 (left), shows a stable FRET state centered at 0.56 ± 0.02; the second subpopulation, SPfluct (middle), fluctuates between two FRET states centered at 0.56 ± 0.02 and 0.36 ± 0.01; and the third subpopulation, SPGS2 (right), shows a stable FRET state centered at 0.36 ± 0.01. Representative Cy3 and Cy5 emission intensity versus time trajectories, green and red, respectively (top row). Corresponding smFRET versus time trajectories, calculated using E = ICy5 / (ICy3 + ICy5), where E is the FRET efficiency at each time point and ICy3 and ICy5 are emission intensities of Cy3 and Cy5, respectively, are in blue (bottom row). () Relative occupancies of the three subpopulations of smFRET trajectories. Percentage of smFRET trajectories occupying SPGS1, SPfluct and SPGS2 for each PRE−A complex without (left) and with (right) EF-G(GDPNP). Data are mean! ± s.d. of three independent measurements (see Supplementary Table 1). * Figure 3: Steady-state smFRET measurements of PRE−A complexes carrying wild-type and elongator tRNAs. Surface contour plots of time evolution of population FRET were generated by superimposing individual smFRET versus time trajectories for each PRE−A complex. Contours are plotted from white (lowest population) to red (highest population). n, number of smFRET trajectories used to construct each contour plot. Corresponding one-dimensional FRET histograms plotted along right-hand y-axis of surface contour plots were generated using first 20 time points from all FRET trajectories in each data set. PRE−A complexes without EF-G(GDPNP), top row; corresponding PRE−A complexes with 2 μM EF-G(GDPNP), bottom row. () PRE−AfMet-1; () PRE−AfMet-2; () PRE−APhe; () PRE−ATyr; () PRE−AGlu; () PRE−AVal. * Figure 4: Design of tRNAfMet2 mutants. () Secondary structure diagram for E. colitRNAfMet2. Three structural features of tRNAfMet differentiating it from all elongator tRNAs, red; mutations designed to convert these three structural features to those found in tRNAPhe are listed. () Three-dimensional structure of E. colitRNAfMet2. Three unique structural features of tRNAfMet are colored as in the secondary structure diagram44 (PDB 3CW6). * Figure 5: Steady-state smFRET measurements on PRE−A complexes carrying tRNAfMet2 mutants. Data are presented as in Figure 3. () PRE−AAnti; () PRE−AAcc; () PRE−AD-flip; () PRE−AD-dis; () PRE−AAcc/D-flip. * Figure 6: P-site tRNA-ribosome interactions within the GS1 and GS2 state of a PRE complex and comparative structural analysis of ribosome-free and ribosome-bound tRNAs. () P-site tRNA-ribosome interactions within GS1 and GS2 state of a PRE complex. Quasi-atomic-resolution models for GS1 (left) and GS2 (right) states of a PRE complex were generated by real-space refinement using rigid body fitting of atomic-resolution structures of the E. coli ribosome (PDB 2AVY and 2AW4) and a P site–bound tRNA (PDB 2J00) into the electron density obtained from cryo-EM reconstructions of the GS1 and GS2 states of a PRE complex (provided by J. Frank, H. Gao and X. Aguirrezabala)32. P/P- and P/E-configured tRNAs, pink and purple, respectively. rRNA helices and r-proteins that interact with aminoacyl acceptor stem (top), D stem (middle) and anticodon stem (bottom) of each tRNA are labeled; nucleotide positions of tRNAfMet2 mutations, red. () Comparative structural analysis of ribosome-free and ribosome-bound tRNAs. Ribosome-free tRNAfMet (ref. 44; PDB 3CW6), A/T-configured tRNAThr (ref. 22; PDB 2WRN), P/P-configured tRNAfMet (ref. 31; PDB 2J00), and P/E-conf! igured tRNAfMet (quasi-atomic-resolution model generated by molecular dynamics flexible fitting40, provided by K. Schulten and B. Liu) are cyan, orange, pink and purple, respectively. The four tRNAs were superimposed using the anticodon stem loops (nucleotides 31–39 for the alignment of P/P-, P/E-configured tRNA to the ribosome-free tRNA, and nucleotides 32–38 for the alignment of A/T-configured tRNA to the ribosome-free tRNA) with PyMol50. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 2J01 * 3CW6 * 2AVY * 2AW4 * 2J00 * 2WRN * 2J01 * 3CW6 * 2AVY * 2AW4 * 2J00 * 2WRN Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Chemistry, Columbia University, New York, New York, USA. * Jingyi Fei, * Jonathan E Bronson & * Ruben L Gonzalez Jr * Department of Biological Sciences, Columbia University, New York, New York, USA. * Arianne C Richard * Present addresses: Department of Physics, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA (J.F.); Immunoregulation Section, Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA (A.C.R.); Boston Consulting Group, New York, New York, USA (J.E.B.). * Jingyi Fei, * Arianne C Richard & * Jonathan E Bronson Contributions J.F. and R.L.G. Jr. designed the research; J.F. and A.C.R. carried out the experiments and analyzed the data; J.E.B. helped with the data analysis; J.F., A.C.R. and R.L.G. Jr. wrote the manuscript; all authors approved the final manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ruben L Gonzalez Jr Author Details * Jingyi Fei Search for this author in: * NPG journals * PubMed * Google Scholar * Arianne C Richard Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan E Bronson Search for this author in: * NPG journals * PubMed * Google Scholar * Ruben L Gonzalez Jr Contact Ruben L Gonzalez Jr Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–5, Supplementary Table 1, Supplementary Methods and Supplementary Discussion Additional data Entities in this article * Elongation factor G fusA Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * 50S ribosomal protein L9 rplI Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * 50S ribosomal protein L1 rplA Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * tRNA metY Escherichia coli str. K-12 substr. MG1655 * View in Entrez Gene * tRNA metZ Escherichia coli str. K-12 substr. MG1655 * View in Entrez Gene - Mechanism of ubiquitylation by dimeric RING ligase RNF4
- Nat Struct Mol Biol 18(9):1052-1059 (2011)
Nature Structural & Molecular Biology | Article Mechanism of ubiquitylation by dimeric RING ligase RNF4 * Anna Plechanovová1 * Ellis G Jaffray1 * Stephen A McMahon2 * Kenneth A Johnson2 * Iva Navrátilová3 * James H Naismith2 * Ronald T Hay1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1052–1059Year published:(2011)DOI:doi:10.1038/nsmb.2108Received19 May 2010Accepted24 June 2011Published online21 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight into the mechanism of RING-mediated ubiquitylation, we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model, the 'Ile44 hydrophobic patch' of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING, and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis. View full text Figures at a glance * Figure 1: Structure of dimeric RING domain of RNF4. () Single-turnover substrate-ubiquitylation assay for RNF4. The UbcH5a~Ub thioester was incubated with 125I-labeled 4× SUMO-2 in the presence or absence of RNF4, followed by SDS-PAGE analysis and phosphorimaging. () Quantification of substrate-ubiquitylation reactions shown in . () Ribbon representation of the dimer of the RNF4 RING domain. Zinc ions are shown as gray spheres and zinc-coordinating residues in stick representation. () A close-up view of the dimerization interface of RNF4. Residues at the dimer interface are shown in stick representation. Blue atoms, nitrogen; red, oxygen; yellow, sulfur. () Mutational analysis of dimerization interface residues. Substrate-ubiquitylation activity of dimerization mutants of RNF4 was determined using the assay described for . Data represent mean ± s.d. of duplicate reactions. Top, Coomassie blue–stained SDS-PAGE gel with purified wild-type (WT) and mutant RNF4 proteins (1 μg). () A model of a complex between the RNF4 RING d! omain and UbcH5b. Putative E2-binding residues of RNF4 that were mutated in this study are shown in stick representation. Active site cysteine of UbcH5b (Cys85) is also shown. () Substrate-ubiquitylation activity of E2-binding mutants of RNF4. () A FRET-based in vitro assay to study dimerization of RNF4. ECFP-RNF4 (wild-type) was mixed with YFP or YFP-RING domain of RNF4 (wild-type or mutant) and FRET signal was measured. Data points represent mean ± s.d. of triplicate measurements. * Figure 2: Ubiquitylation by RNF4 can proceed both in cis and in trans. () Ubiquitylation by RNF4 can proceed in trans. Full-length RNF4 (0.55 μM) and the RNF4 RING domain (0.55 μM) were added to substrate-ubiquitylation reactions, and ubiquitylation activity was determined. () Schematic representation of the experiment in . A heterodimer of the RNF4 RING domain and full-length RNF4 with disrupted E2-binding site (M140A R181A) should be active in a substrate-ubiquitylation reaction provided ubiquitylation by RNF4 can proceed in trans. S2, SUMO-2. () Schematic representation of a hypothesis behind the experiment in . If ubiquitylation by RNF4 can proceed in cis, then a heterodimer of full-length RNF4 and the RNF4 RING domain with disrupted E2-binding site should have substrate-ubiquitylation activity. However, this heterodimer should be inactive if ubiquitylation can only proceed in trans. Therefore, addition of an excess of the RING domain with disrupted E2-binding site to wild-type RNF4 should result in inhibition of substrate-ubiquitylation ! activity provided ubiquitylation cannot proceed in cis. () Ubiquitylation by RNF4 can proceed in cis. The RING domain of RNF4 with disrupted E2-binding site (RING M140A R181A) was added to RNF4 WT (0.275 μM) in 2-, 20- or 200-fold molar excess, and substrate-ubiquitylation activity was determined. () Under the conditions used in , substrate-ubiquitylation activity is proportional to concentration of RNF4 in the reaction. In ,,, the data are mean ± s.d. of duplicate reactions. Experiments were performed twice. * Figure 3: RNF4 preferentially binds ubiquitin-loaded E2 and activates the bond between E2 and ubiquitin. () RNF4 induces hydrolysis of E2~Ub linked via an oxyester bond. Phosphorimager scan of SDS-PAGE gels shows time course of UbcH5a C85S~125I-Ub oxyester stability in the presence or absence of RNF4. () UbcH5a C85S~125I-Ub oxyester was incubated with the RNF4 RING domain or full-length RNF4 (wild-type or mutant as indicated), and rates of oxyester hydrolysis were determined. Data are shown as mean ± s.d. of duplicate reactions. The experiment was performed twice. () RNF4 shows higher affinity for ubiquitin-charged E2 than for free E2 in a pull-down experiment. A mixture of UbcH5a N77A C85S, ubiquitin and the UbcH5a N77A C85S~Ub oxyester was incubated with MBP or MBP-RNF4 (wild-type or mutant), followed by purification on amylose beads. WB, western blot. () A model of the RNF4 RING domain in complex with E2~ubiquitin thioester predicts an interaction between ubiquitin and the RING domain. The RING domain is shown in ribbon form, with one monomer in blue and the other monomer i! n cyan. Putative ubiquitin-interacting residues (Tyr193 and Leu152) are shown as spheres. Ubiquitin is shown in pink with the hydrophobic contact surface (Leu8, Ile44 and Val70) in black. The E2 is shown in tan, with the point of thioester attachment shown as a yellow sphere. () Close-up view of the predicted interaction interface between the hydrophobic patch on ubiquitin and residues Tyr193 and Leu152 on the RING domain of RNF4. * Figure 4: The Ile44-centered hydrophobic patch on ubiquitin is required for RNF4-mediated ubiquitylation. () Ubiquitin mutations used in do not affect formation of the UbcH5a~Ub thioester. UbcH5a was incubated with ubiquitin (wild-type or mutant as indicated) in the presence of Ube1 and ATP, followed by nonreducing SDS-PAGE analysis. () Effects of mutations in ubiquitin on substrate-ubiquitylation activity of RNF4. 4× SUMO-2, radiolabeled with iodine-125, was used as substrate. Data are shown as mean ± s.d. of duplicate reactions. The experiment was performed twice. () Mutation I44A in ubiquitin causes a modest defect in E3-independent transfer of ubiquitin to poly(L-lysine). UbcH5a~125I-Ub thioester was incubated with poly(L-lysine), and samples were taken at indicated times. Subsequently, poly(L-lysine) from samples was purified on SP-Sepharose resin, and radioactivity captured on the beads was quantified by γ-counting. Initial rates were determined from the first three time points. Data are shown as mean ± s.d. of duplicate reactions. () The Ile44 patch on ubiquitin is es! sential for RNF4-mediated cleavage of E2~ubiquitin oxyester. The UbcH5a C85S~Ub oxyester was incubated in the presence or absence of RNF4, and reaction progress was analyzed by SDS-PAGE, followed by staining with SYPRO orange. Reaction rates represent mean ± s.d. of duplicate reactions. The experiment was performed three times. () The Ile44 patch on ubiquitin is required for the interaction between ubiquitin-loaded E2 and RNF4. A mixture of UbcH5a C85S, ubiquitin and the UbcH5a C85S~Ub oxyester was briefly incubated with either MBP or MBP-RNF4 immobilized on amylose beads, followed by a quick washing step. Bound material was resolved by SDS-PAGE. WB, western blot. * Figure 5: Tyr193, a residue located at the dimer interface of the RNF4 RING domain, is required for activation of the thioester bond in the E2~ubiquitin thioester. () The ability of RNF4 Y193H to dimerize was assessed using a FRET-based dimerization assay. Unlabeled RNF4 was titrated into a mixture of ECFP-RNF4 and YFP-RING domain, and FRET signal was measured. Data points represent mean ± s.d. of triplicate measurements. () Mutational analysis of the predicted ubiquitin-binding site on the RNF4 RING domain. Several mutations of Tyr193 were generated. Mutations to alanine and leucine disrupted RNF4 dimerization, but mutations to tryptophan and histidine did not. Substrate-ubiquitylation activity of the mutant proteins was determined using the single-turnover assay described in Figure 1a. () Tyr193 in RNF4 is required for efficient hydrolysis of the E2~ubiquitin oxyester. The UbcH5a C85S~Ub oxyester was incubated with RNF4 (wild-type or mutant as indicated) and the rate of oxyester hydrolysis was determined. () Mutation Y193H in RNF4 disrupts binding to the E2~ubiquitin oxyester. A pull-down experiment was performed as in Figure 4e. ()! Ubiquitylation activity of RNF4 Y193H can be rescued by addition of an inactive RNF4 mutant with disrupted E2-binding site (RNF4 M140A R181A). Substrate-ubiquitylation activity was determined as in Figure 1a. Final concentration of RNF4 mutants in the reaction was either 0.55 μM (+) or 1.1 μM (++). In ,,, data represent mean ± s.d. of duplicate reactions. The experiments were performed twice. * Figure 6: A linear fusion of full-length RNF4 and the RNF4 RING domain shows that E2-binding site in one RING domain and Tyr193 in the other RING are both required for ubiquitylation activity. () A model of a linear fusion of full-length RNF4 and the RING domain of RNF4, based on the structure of the RNF4 RING domain reported here. A short linker between the C terminus of full-length RNF4 and the N terminus of the RING domain is shown as a red dashed line. Tyr193 at the dimer interface and residues important for E2 binding (Met140 and Arg181) are shown in stick representation. The linear fusion protein contains one substrate-binding site, two E2-binding sites and two Tyr193 residues, one on each side of the dimerization interface. A schematic representation of the RNF4-RING fusion protein is shown at right. () Substrate-ubiquitylation activity of RNF4-RING linear fusion proteins, with mutations in full-length RNF4 and/or the RNF4 RING domain. Data are shown as mean ± s.d. of duplicate reactions. The experiment was performed twice. Top, SYPRO orange–stained SDS-PAGE gel with purified RNF4-RING fusion proteins (0.6 μg). () Mutation of Tyr193 in the RING domain t! hat does not bind E2 raises KM and lowers kcat. Michaelis-Menten kinetics were determined from reaction rates at various concentrations of UbcH5a~Ub thioester. () Binding of the E2~ubiquitin oxyester to RNF4-RING fusion proteins correlates well with their ubiquitylation activities. A pull-down experiment was performed as in Figure 4e. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2XEU * 2XEU Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, UK. * Anna Plechanovová, * Ellis G Jaffray & * Ronald T Hay * Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, UK. * Stephen A McMahon, * Kenneth A Johnson & * James H Naismith * Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK. * Iva Navrátilová Contributions A.P. purified RNF4 proteins, carried out crystallography, conducted biochemical analysis and interpreted the data. E.G.J. purified recombinant proteins and carried out ubiquitylation assays. S.A.M., K.A.J. and J.H.N. contributed to structural analysis. I.N. contributed to biochemical analysis. A.P., J.H.N. and R.T.H. wrote the paper. R.T.H. conceived the project and contributed to data interpretation. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ronald T Hay Author Details * Anna Plechanovová Search for this author in: * NPG journals * PubMed * Google Scholar * Ellis G Jaffray Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen A McMahon Search for this author in: * NPG journals * PubMed * Google Scholar * Kenneth A Johnson Search for this author in: * NPG journals * PubMed * Google Scholar * Iva Navrátilová Search for this author in: * NPG journals * PubMed * Google Scholar * James H Naismith Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald T Hay Contact Ronald T Hay Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–9 and Supplementary Methods Additional data Entities in this article * Protein Mdm4 MDM4 Homo sapiens * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase Mdm2 MDM2 Homo sapiens * View in UniProt * View in Entrez Gene * Ubiquitin-conjugating enzyme E2 D2 UBE2D2 Homo sapiens * View in UniProt * View in Entrez Gene * Pre-mRNA-splicing factor 19 PRP19 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Protein PML PML Homo sapiens * View in UniProt * View in Entrez Gene * Ubiquitin-conjugating enzyme E2 D1 UBE2D1 Homo sapiens * View in UniProt * View in Entrez Gene * Baculoviral IAP repeat-containing protein 3 BIRC3 Homo sapiens * View in UniProt * View in Entrez Gene * Small ubiquitin-related modifier 2 SUMO2 Homo sapiens * View in UniProt * View in Entrez Gene * TNF receptor-associated factor 6 TRAF6 Homo sapiens * View in UniProt * View in Entrez Gene * BRCA1-associated RING domain protein 1 BARD1 Homo sapiens * View in UniProt * View in Entrez Gene * Breast cancer type 1 susceptibility protein BRCA1 Homo sapiens * View in UniProt * View in Entrez Gene * E3 ubiquitin ligase RNF4 Rnf4 Rattus norvegicus * View in UniProt * View in Entrez Gene * Ubiquitin-conjugating enzyme E2 D3 UBE2D3 Homo sapiens * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase NEDD4-like NEDD4L Homo sapiens * View in UniProt * View in Entrez Gene * Polycomb complex protein BMI-1 Bmi1 Mus musculus * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase RING2 Rnf2 Mus musculus * View in UniProt * View in Entrez Gene * Ubiquitin-like modifier-activating enzyme 1 UBA1 Homo sapiens * View in UniProt * View in Entrez Gene * Ubiquitin-conjugating enzyme E2 R1 CDC34 Homo sapiens * View in UniProt * View in Entrez Gene * Ubiquitin-conjugating enzyme E2 S UBE2S Homo sapiens * View in UniProt * View in Entrez Gene - Mechanism of actin filament nucleation by Vibrio VopL and implications for tandem W domain nucleation
- Nat Struct Mol Biol 18(9):1060-1067 (2011)
Nature Structural & Molecular Biology | Article Mechanism of actin filament nucleation by Vibrio VopL and implications for tandem W domain nucleation * Suk Namgoong1 * Malgorzata Boczkowska1 * Michael J Glista2 * Jonathan D Winkelman2 * Grzegorz Rebowski1 * David R Kovar2, 3 * Roberto Dominguez1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1060–1067Year published:(2011)DOI:doi:10.1038/nsmb.2109Received21 December 2010Accepted30 June 2011Published online28 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand their more complex eukaryotic analogs. We show that the strong actin filament nucleation activity of Vibrio parahaemolyticusVopL depends on its three W domains and on its dimerization through a unique VopL C-terminal domain (VCD). The VCD shows a previously unknown all-helical fold and interacts with the pointed end of the actin nucleus, contributing to the nucleation activity directly and through duplication of the W domain repeat. VopL promotes rapid cycles of filament nucleation and detachment but generally has no effect on elongation. Profilin inhibits VopL-induced nucleation by competing for actin binding to the W domains. Combined, the results suggest that VopL stabilizes a hexameric double-stranded pointed end nucleus. Analysis of hybrid constructs of VopL and the eukaryotic nucleator Spire suggest that Spire may also function as a dimer in cells. View full text Figures at a glance * Figure 1: Nucleation activity of VopL constructs. () VopL constructs used in this study and alignment of W domains illustrating the sites of mutations. FL, full length. () Time course of polymerization of 2 μM Mg-ATP-actin (6% pyrene-labeled) alone or in the presence of 25 nM VopL constructs (color-coded). Polymerization rates are reported as mean and s.e.m. values (number of measurements ≥ 3). AU, arbitrary units. () Effect of VopL construct concentration on the polymerization rate of pyrene–actin (see also Supplementary Fig. 2). (d) Time course of actin polymerization by VopL constructs containing point mutations in the W domains (shown in ). () Polymerization of 1.5 μM Mg-ATP-actin (33% Oregon Green–labeled) alone or in the presence of 0.1 nM VopL constructs, visualized by TIRF microscopy on NEM-myosin II–coated coverslips, including time-lapse micrographs (see Supplementary Videos 1,2,3 and 4) of representative 19.5-μm2 fields (left) and plots of the growth of 20 filament barbed ends over time (right). Circle! s and arrowheads indicate the pointed and barbed ends of representative filaments. The average number of filaments in a 133-μm2 field of view at 300 s, from two independent experiments, is indicated as f μm−2. Errors for elongation rates in are ± s.d. Scale bar is 5 μm. * Figure 2: VCD mediates dimerization and binds to the pointed end of the polymerization nucleus. () SEC-MALS mass measurements of VopL constructs (theoretical masses in parentheses). Supplementary Table 1 lists the masses of other constructs. () Time course of polymerization of 2 μM Mg-ATP-actin alone or induced by 25 nM VopL constructs. AU, arbitrary units. (,) Gel electrophoresis of supernatant (S) and pellet (P) fractions after co-sedimentation at 224,000g of F-actin with VCD or 1W-VCD. () Mass measurements of 1W-VCD and its complex with actin. () TIRF time-lapse micrographs of actin assembly induced by P-3W-VCD immobilized on a coverslip, showing a filament anchored to the coverslip by its pointed end that dissociates between frames at 220 s and 280 s (top) and a filament that remains anchored throughout (bottom) (Supplementary Videos 5 and 6). Circles and arrowheads indicate the pointed and barbed ends of the filaments. Shown on the right are growth plots of 20 filaments, including tethered and non-tethered (blue dashed lines). () TIRF time-lapse micrographs of ac! tin (green) assembly by 3W-VCD bound to Qdots (red). Upper panels show a Qdot that nucleates 14 filaments (arrowheads and numbers, Supplementary Videos 7 and 8). Middle and lower panels show filaments whose pointed (filled circle) or barbed (arrows) ends appear to be bound to a Qdot (Supplementary Videos 9 and 10). () Plots of the growth of filaments, including non-bound, fast-dissociated, pointed end– and barbed end–bound filaments, and two filaments whose barbed ends detach from the Qdot and whose elongation rates decreased during the experiment. Errors in and are ± s.d. Scale bars in and are 5 μm. * Figure 3: Crystal structure of VCD and SAXS structures of VCD and 1W-VCD–actin. () Two perpendicular views of the crystal structure of VCD and definition of domains and secondary structure shown along the sequence of this domain (Supplementary Video 11). Different colors highlight different domains: base (magenta, green), arm (pink), coiled coil (gold). () Experimental X-ray scattering pattern of VCD (brown) and 1W-VCD–actin (blue) as a function of momentum transfer s = 4πsinθ /λ, where 2θ is the scattering angle and λ = 0.103 nm is the wavelength. The inset shows the normalized distance distribution functions computed with GNOM36. (,) Two perpendicular orientations of the average SAXS envelopes of VCD and 1W-VCD–actin (same orientations as for the crystal structure). CC, coiled coil. * Figure 4: Profilin inhibits polymerization induced by VopL. () Polymerization rates of 2 μM Mg-ATP-actin induced by 5-nM VopL constructs (color-coded) or 0.5 μM F-actin seeds (black) as a function of profilin concentration. Errors are ± s.e.m. AU, arbitrary units. () Visualization by TIRF microscopy of the effect of 2.5 μM profilin on the polymerization of 1.5 μM Mg-ATP-actin alone (Supplementary Video 12) or induced by 0.1 nM P-3W-VCD and 3W-VCD (Supplementary Videos 13 and 14) or mutants P-3W-VCDP191E and 3W-VCDP191E (Supplementary Videos 15,16,17 and 18). Plots of the growth of 20 individual filaments are shown on the right. () Comparison of the nucleation activities and elongation rates measured by TIRF with or without profilin. Errors in and are ± s.d. * Figure 5: Role of dimerization and specific sequence of W domains and inter–W domain linkers in nucleation. () Design of hybrid constructs of VopL and D. melanogasterSpire's repeat of four W domains (Spire4W). In construct 3W-sL3, VopL linker 2 was replaced with Spire linker 3. In construct Spire4W–VCD, VopL's VCD was fused C-terminal to the last W domain of Spire, using the LKKT(V) motifs as a reference for fusion. () Time courses of polymerization of 2 μM Mg-ATP-actin induced by two different concentrations of constructs Spire4W, 3W-sL3 and 3W. () Comparison of the time courses of actin polymerization induced by constructs VopL 3W-VCD and Spire4W–VCD and Spire4W at the indicated concentrations. Errors are ± s.e.m. AU, arbitrary units. * Figure 6: Proposed mechanisms of actin nucleation by VopL and Spire. () Electrostatic surface representation (blue, positively charged; red, negatively charged) of the structures of VCD and W-actin (PDB 2D1K)25, illustrating the existing shape and charge complementarity (indicated by red and blue arrows) between the two structures. () Such complementarity and the necessity to connect the C terminus of the third W domain to the N terminus of VCD lead to a model of the complex in which the first actin subunit interacts with both subunits of the VopL dimer (see Supplementary Video 19). The second actin subunit (gray) might bind on the opposite side of VCD according to the actin filament model31. () Model of nucleation by VopL. VCD plays a dual role, contributing directly to the recruitment of actin subunits by binding to the pointed end of the polymerization nucleus, and enabling the formation of a hexameric nucleus by duplication of the W domain repeat. VopL detaches fast after nucleation, probably owing to steric hindrance of the W domains wit! h longitudinal contacts between actin subunits in the filament21. Upon detachment, VopL might carry with it actin subunits that become part of the nucleus in a new round of polymerization. () Spire's activity is also enhanced by dimerization, which in cells is mediated by interaction of Spire's KIND domain with formins15, 33, 34. CC, coiled coil. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3RYL * 3RYL Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Suk Namgoong, * Malgorzata Boczkowska, * Grzegorz Rebowski & * Roberto Dominguez * Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA. * Michael J Glista, * Jonathan D Winkelman & * David R Kovar * Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA. * David R Kovar Contributions S.N. expressed proteins, did the sedimentation, crystallization, structure determination and SAXS experiments, and participated in most of the other experiments; M.B. carried out pyrene actin polymerization assays; G.R. did ITC and SEC-MALS experiments; M.J.G., J.D.W. and D.R.K. conducted and analyzed TIRF experiments; R.D. did the crystallographic studies, supervised the research and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Roberto Dominguez Author Details * Suk Namgoong Search for this author in: * NPG journals * PubMed * Google Scholar * Malgorzata Boczkowska Search for this author in: * NPG journals * PubMed * Google Scholar * Michael J Glista Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan D Winkelman Search for this author in: * NPG journals * PubMed * Google Scholar * Grzegorz Rebowski Search for this author in: * NPG journals * PubMed * Google Scholar * David R Kovar Search for this author in: * NPG journals * PubMed * Google Scholar * Roberto Dominguez Contact Roberto Dominguez Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information Movies * Supplementary Video 1 (537K) Visualization by TIRF microscopy of actin assembly. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin alone (33% Oregon Green-labeled). Video corresponds to Fig. 1e (top panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 2 (463K) Visualization by TIRF microscopy of actin assembly induced by P-3W-VCD.Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct P-3W-VCD. Video corresponds to Fig. 1e (second panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 3 (180K) Visualization by TIRF microscopy of actin assembly induced by 3W-VCD. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct 3W-VCD. Video corresponds to Fig. 1e (third panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 4 (553K) Visualization by TIRF microscopy of actin assembly induced by VCD. Timelapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct VCD. Video corresponds to Fig. 1e (bottom panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 5 (651K) Visualization by TIRF microscopy of actin assembly induced by immobilized P-3W-VCD (corresponding to Fig. 2f top panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) on a coverslip pre-coated with VopL construct P-3W-VCD. Video corresponds to Fig. 2f (top). A yellow dot marks the immobilized pointed end, and a yellow arrow marks the growing barbed end of one of the filaments that was initially anchored to the coverslip by its pointed end, but then dissociated. Images were captured every 10 s for 5 min, and are displayed at 10 frames s−1. Filaments were considered tethered to the coverslip if the position of one of the ends of the filament did not change for at least five frames. * Supplementary Video 6 (266K) Visualization by TIRF microscopy of actin assembly induced by immobilized P-3W-VCD (corresponding to Fig. 2f bottom panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) on a coverslip pre-coated with VopL construct P-3WVCD, corresponding to Fig. 2f (bottom). A yellow dot marks the immobilized pointed end, and a yellow arrow marks the growing barbed end of one of the filaments that remained anchored to the coverslip by its pointed end. Images were captured every 10 s for 5 min, and are displayed at 10 frames s−1. Filaments were considered tethered to the coverslip if the position of one of the ends of the filament did not change for at least five frames. * Supplementary Video 7 (1M) Visualization by TIRF microscopy of actin assembly induced by Qdot-coupled 3W-VCD (corresponding to Fig. 2g top panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM 3W-VCD coupled with Qdot-625 (red). Images were captured every 2 s for 5 min, and are displayed at 10 frames s−1. The movie shows a Qdot that nucleates the assembly of 15 actin filaments (marked with arrowheads and numbered). * Supplementary Video 8 (672K) Visualization by TIRF microscopy of actin assembly induced by Qdot-coupled 3W-VCD (corresponding to Fig. 2g top panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM 3W-VCD coupled with Qdot-625 (red). Images were captured every 2 s for 5 min, and are displayed at 10 frames s−1. The movie shows another example of a Qdot that nucleates the assembly of multiple (8) actin filaments (marked with arrowheads and numbered). * Supplementary Video 9 (1M) Visualization by TIRF microscopy of actin assembly induced by Qdot-coupled 3W-VCD (corresponding to Fig. 2g middle panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM 3W-VCD coupled with Qdot-625 (red). Video corresponds to Fig. 2g. Images were captured every 2 s for 5 min, and are displayed at 10 frames s−1. The movie shows Qdots that nucleate the assembly of actin filaments that remain attached through their pointed ends (marked by circles) after nucleation and whose barbed ends (marked by arrows) elongate freely. * Supplementary Video 10 (2M) Visualization by TIRF microscopy of actin assembly induced by Qdot-coupled 3W-VCD (corresponding to Fig. 2g bottom panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM 3W-VCD coupled with Qdot-625 (red). Images were captured every 2 s for 5 min, and are displayed at 10 frames s−1. The movie shows a Qdot that nucleates the assembly of an actin filament that remains attached through its barbed and displays accelerated barbed end elongation. The filament buckles similarly to filaments elongated from immobilized formin. The arrow and circle indicate the barbed and pointed ends, respectively. * Supplementary Video 11 (5M) Animation of the crystal structure of VCD. The video shows ribbon and surface representations colored according to three different criteria: domain organization (core, magenta and green; arm, pink; coiled coil, gold), electrostatic charge distribution (blue, positively charged to red, negatively charged), and temperature factor (B-factor) values (blue, lowest to red, highest). * Supplementary Video 12 (328K) Visualization by TIRF microscopy of actin assembly in the presence of profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-Actin (33% Oregon Greenlabeled) with 2.5 μM profilin. Video corresponds to Fig. 4b (top panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 13 (512K) Visualization by TIRF microscopy of actin assembly induced by P-3W-VCD in the presence of profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct P-3W-VCD and 2.5 μM profilin. Video corresponds to Fig. 4b (second panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 14 (319K) Visualization by TIRF microscopy of actin assembly induced by 3W-VCD in the presence of profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct 3W-VCD and 2.5 μM profilin. Video corresponds to Fig. 4b (third panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 15 (418K) Visualization by TIRF microscopy of actin assembly induced by P-3WVCDP191E without profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct P-3W-VCDP191E in the absence (control) of profilin. A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 16 (684K) Visualization by TIRF microscopy of actin assembly induced by P-3WVCDP191E in the presence of profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATPactin (33% Oregon Green-labeled) with 0.1 nM VopL construct P-3W-VCDP191E in the presence of 2.5 μM profilin. Video corresponds to Fig. 4b (fourth panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 17 (627K) Visualization by TIRF microscopy of actin assembly induced by 3W-VCDP191E without profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct 3W-VCDP191E in the absence of profilin. A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 18 (668K) Visualization by TIRF microscopy of actin assembly induced by 3W-VCDP191E in the presence of profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct 3W-VCDP191E in the presence of 2.5 μM profilin. Video corresponds to Fig. 4b (fifth panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. * Supplementary Video 19 (6M) Model of nucleation by VopL. Proposed assembly of actin subunits with VCD at the pointed end of the polymerization nucleus. The video shows ribbon and electrostatic surface representations (blue, positively charged to red, negatively charged). The two chains of VCD are colored green and yellow and the first W-actin complex at the pointed end of the polymerization nucleus is colored blue and red. Note the existing shape and charge complementarity between VCD and the first W-actin complex in the proposed model of interaction. A second W-actin complex, positioned with respect to the first one according to the structure of the actin filament, is show (actin, gray; W domain, red). For the second W-actin complex, the distance between the C-terminus of the W domain and the N13 terminus of VCD is ~40 Å. Not shown in this model are 12 amino acids of the flexible linker between the third W domain and VCD, which with rearrangement of the termini would be sufficient to bridge thedistan! ce between the W domain and VCD. PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–7 and Supplementary Tables 1 and 2. Additional data Entities in this article * Neural Wiskott-Aldrich syndrome protein WASL Homo sapiens * View in UniProt * View in Entrez Gene * Translocated actin-recruiting phosphoprotein CT456 Chlamydia trachomatis * View in UniProt * View in Entrez Gene * Protein cappuccino capu Drosophila melanogaster * View in UniProt * View in Entrez Gene * Vasodilator-stimulated phosphoprotein VASP Homo sapiens * View in UniProt * View in Entrez Gene * Protein cordon-bleu Cobl Mus musculus * View in UniProt * View in Entrez Gene * Protein spire spir Drosophila melanogaster * View in UniProt * View in Entrez Gene * Surface cell antigen 2 Rickettsia parkeri * View in UniProt * VopF Vibrio cholerae * View in UniProt * Formin-1 Fmn1 Mus musculus * View in UniProt * View in Entrez Gene * Protein spire homolog 2 SPIRE2 Homo sapiens * View in UniProt * View in Entrez Gene * Protein spire homolog 1 SPIRE1 Homo sapiens * View in UniProt * View in Entrez Gene * Putative type III secretion system effector protein VopL Vibrio parahaemolyticus * View in UniProt * Profilin-1 PFN1 Homo sapiens * View in UniProt * View in Entrez Gene * Protein diaphanous homolog 1 DIAPH1 Homo sapiens * View in UniProt * View in Entrez Gene * Adenomatous polyposis coli protein APC Homo sapiens * View in UniProt * View in Entrez Gene * Formin-2 Fmn2 Mus musculus * View in UniProt * View in Entrez Gene - Mechanism of actin filament nucleation by the bacterial effector VopL
- Nat Struct Mol Biol 18(9):1068-1074 (2011)
Nature Structural & Molecular Biology | Article Mechanism of actin filament nucleation by the bacterial effector VopL * Bingke Yu1, 2 * Hui-Chun Cheng1, 2 * Chad A Brautigam1 * Diana R Tomchick1 * Michael K Rosen1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1068–1074Year published:(2011)DOI:doi:10.1038/nsmb.2110Received21 December 2010Accepted30 June 2011Published online28 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected into eukaryotic host cells. VopL contains three N-terminal Wiskott-Aldrich homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own and requires the VCD for activity. The VCD forms a U-shaped dimer in the crystal, stabilized by a terminal coiled coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data lead to a model in which VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors. View full text Figures at a glance * Figure 1: Both the WH2 motifs and VCD contribute to actin nucleation by VopL. () Domain structures of VopL constructs used in this work. All constructs lack the N-terminal secretion sequence needed for VopL translocation into host cells, whose deletion does not affect activity (not shown). P: Proline-rich motif. WH2: WASP homology 2 domain. VCD: VopL C-terminal domain. GGS: glycine-glycine-serine linker. The half-time (t1/2) of actin assembly for each VopL protein is given by the average and s.d. measured in three experiments. N.A., not available. () Actin assembly assays contained the indicated concentrations of VopL constructs W3-C, W3 or W4. () Actin assembly assays contained 50 nM W3-C, W2-C, W1-C or VCD. AU, arbitrary units. * Figure 2: Dimerization is necessary for actin nucleation by the VopL WH2 motifs. () Size-exclusion chromatography–MALLS analysis of VopL constructs W3-C (red) and VCD (blue). Normalized scattered light in the chromatographic elution (right y axis) is superimposed on the molecular weight distribution (left y axis). () MALLS analysis of VCDΔh. Data plotted as in . () Actin polymerization assays done with 4 μM actin and 50 nM W3-C, W2-C, W1-C, VCD, W3-GST, W2-GST or W1-GST. AU, arbitrary units. * Figure 3: Structure of the VCD dimer. () Ribbon diagram of the structure; monomers are colored blue and green. The N- and C termini of each monomer are indicated. Red dashed line indicates regions not observed in the electron density map for the green monomer (analogous regions not shown in the blue monomer). Boxed regions are enlarged in and , which show detailed views of the dimer interface. Black dashed lines indicate hydrogen bonds. In all panels, side chains of residues discussed in the text are shown as sticks. * Figure 4: The VCD contributes to actin assembly activity by dimerization and contacts to actin. Assays were done with 4 μM actin and 50 nM (,) or 5 nM () of the indicated VopL proteins. () Comparison of W3-C, W3-CΔh and W3. () Comparison of W3-C with the VCD arm mutants, W3-CKRR and W3-CDVP. () Comparison of W3-C with the VCD base mutants, W3-CKYR, W3-CYR and W3-CEDE. AU, arbitrary units. * Figure 5: Increasing the linker between WH2c and the VCD increases actin assembly activity. Assays contained 50 nM of each VopL protein, W3-C, W2-C, W1-C, W2-L-C or W1-L-C. AU, arbitrary units. * Figure 6: Model for a minimal actin nucleus assembled by VopL. VCD is indicated by blue blocks with ribbons representing the coiled coil. Initial actin monomers are indicated by yellow boxes. Actin-bound WH2c motifs are shown as red cylinders. Red lines indicate linkers between WH2c and VCD. Additional actin monomers assembled by WH2a and WH2b would bind to the upper surfaces of the WH2c-bound actins, making additional short- and long-pitch contacts, further stabilizing the assembly. Actins are implicitly shown with barbed ends directed away from the VCD, but our current data do not speak to the orientation of actin monomers in the VopL nucleus. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3SEO * 3SEO Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA. * Bingke Yu, * Hui-Chun Cheng, * Chad A Brautigam, * Diana R Tomchick & * Michael K Rosen * Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA. * Bingke Yu, * Hui-Chun Cheng & * Michael K Rosen Contributions M.K.R. conceived the project; B.Y. did the work; C.A.B. and D.R.T. assisted with X-ray diffraction data collection and structure determination. H.-C.C. assisted with designing and making the VopL constructs; M.K.R. and B.Y. analyzed the data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael K Rosen Author Details * Bingke Yu Search for this author in: * NPG journals * PubMed * Google Scholar * Hui-Chun Cheng Search for this author in: * NPG journals * PubMed * Google Scholar * Chad A Brautigam Search for this author in: * NPG journals * PubMed * Google Scholar * Diana R Tomchick Search for this author in: * NPG journals * PubMed * Google Scholar * Michael K Rosen Contact Michael K Rosen Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (463K) Supplementary Figures 1–8 Additional data Entities in this article * Putative type III secretion system effector protein VopL Vibrio parahaemolyticus * View in UniProt * Translocated actin-recruiting phosphoprotein CT456 Chlamydia trachomatis * View in UniProt * View in Entrez Gene * VopF Vibrio cholerae * View in UniProt * Protein cappuccino capu Drosophila melanogaster * View in UniProt * View in Entrez Gene * Surface cell antigen 2 Rickettsia parkeri * View in UniProt * Actin-related protein 3 ACTR3 Bos taurus * View in UniProt * View in Entrez Gene * Actin-related protein 2 ACTR2 Bos taurus * View in UniProt * View in Entrez Gene * Protein cordon-bleu Cobl Mus musculus * View in UniProt * View in Entrez Gene * Protein spire spir Drosophila melanogaster * View in UniProt * View in Entrez Gene - Biogenic mechanisms and utilization of small RNAs derived from human protein-coding genes
- Nat Struct Mol Biol 18(9):1075-1082 (2011)
Nature Structural & Molecular Biology | Resource Biogenic mechanisms and utilization of small RNAs derived from human protein-coding genes * Eivind Valen1, 5 * Pascal Preker2, 5 * Peter Refsing Andersen2, 5 * Xiaobei Zhao1 * Yun Chen1 * Christine Ender3 * Anne Dueck4 * Gunter Meister3, 4 * Albin Sandelin1 * Torben Heick Jensen2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1075–1082Year published:(2011)DOI:doi:10.1038/nsmb.2091Received22 February 2011Accepted23 May 2011Published online07 August 2011Corrected online21 August 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Efforts to catalog eukaryotic transcripts have uncovered many small RNAs (sRNAs) derived from gene termini and splice sites. Their biogenesis pathways are largely unknown, but a mechanism based on backtracking of RNA polymerase II (RNAPII) has been suggested. By sequencing transcripts 12–100 nucleotides in length from cells depleted of major RNA degradation enzymes and RNAs associated with Argonaute (AGO1/2) effector proteins, we provide mechanistic models for sRNA production. We suggest that neither splice site–associated (SSa) nor transcription start site–associated (TSSa) RNAs arise from RNAPII backtracking. Instead, SSa RNAs are largely degradation products of splicing intermediates, whereas TSSa RNAs probably derive from nascent RNAs protected by stalled RNAPII against nucleolysis. We also reveal new AGO1/2-associated RNAs derived from 3′ ends of introns and from mRNA 3′ UTRs that appear to draw from noncanonical microRNA biogenesis pathways. View full text Figures at a glance * Figure 1: The majority of gene-derived sRNAs are decay products. () Schematic overview of gene-derived sRNAs identified in this study. Red arrows indicate density and directionality of sequenced sRNAs that are overrepresented at TSSs (yellow), 5′ splice sites (SS, pink), 3′ splice sites (green) and 3′ ends (blue), and to a lesser degree within exons (white). () sRNA density in exons versus introns for different sRNA sizes. The y axes show the number of unique sRNAs per million in the indicated library, counting all nucleotides within each tag. The x axes show 3′ and 5′ parts of internal exons as well as intronic regions normalized to the same length. Positions of exon-intron and intron-exon borders are indicated by dashed gray lines. * Figure 2: sRNAs around splice sites. () Density of unique sRNA 5′ ends around exon-intron, exon-exon and intron-exon junctions. The y axes show the number of unique sRNA 5′ ends per million tags of the indicated libraries, counting only the 5′ nucleotide of each RNA. These values are broken up by sizes of the sRNAs (indicated by color) and represented as stacked bar plots. Dashed lines indicate the locations of exon-intron (left), exon-exon (middle) and intron-exon junctions (right). Negative (upstream) and positive (downstream) x axis coordinates are given relative to these locations. Depletion of sRNA 5′ ends just upstream of exon-intron junctions (left) is due to sRNAs partially mapping to the next exon. sRNA 5′ ends positioned just a few nucleotides upstream of the 3′ splice site are due to mapping ambiguities where the 3′ end of the intron is highly similar to the 3′ end of the preceding exon. () Model for the production of SSa RNAs. Top of figure schematically shows sRNAs found in this stu! dy mapping around exon-intron and intron-exon junctions. Exonic 5′ SSa RNAs are suggested to derive from the decay of upstream exons failing to undergo the second step of splicing (left), whereas intronic sRNAs are suggested to constitute intermediates from the decay of spliced-out introns (right). * Figure 3: sRNA density around TSSs is dependent on promoter type and sRNA length. () sRNA density around TSSs of broad and sharp promoters. sRNAs mapping to the sense (+) and antisense (−) strand, with respect to the associated gene, are colored blue and green, respectively. The y axis shows the number of unique sRNA 3′ ends per million tags and promoter. The x axes show their positions relative to the most prominent TSS. () Distribution of total sRNA tag per million counts in the +10 to +50 region relative to the TSS of the indicated libraries and as a function of sRNA lengths. () Distribution of unique TSSa sRNA 3′ ends per million tags and promoters from the HeLa12–20(N) library. Multi- and single-mapping sRNAs are plotted separately as in and broken down by sRNA sizes as indicated. * Figure 4: TSSa RNAs are likely produced by protection of nascent RNA by stalled RNAPII. () Relative positioning of RNAPII and TSSa RNA 3′ ends from sharp promoters harboring one or more sRNA 3′ ends at position +38 relative to the TSS (the peak in Fig. 3a). The y axes show the average number of RNAPII ChIP tags. The x axes show ChIP signal positions, with dashed lines indicating the +38 position. The colored lines indicate how many sRNA 3′ ends at position +38 were required for the promoter to be included in the analysis (see also Supplementary Fig. 6). () Model for the production of TSSa RNAs. Transcriptional stalling may in some cases trigger nucleolytic degradation of the nascent RNA 5′ end extruding from RNAPII (see text for details). () XRN1/2-dependent production of TSSa RNA. The number of sRNAs per million (counting up to 10 unique sRNAs per position; see Methods) is plotted on the y axis as a function of position relative to the TSS (x axes). Plots on the left show densities of RNA 5′ ends from the HeLa30–100 and HeLa30–100(XRN1/2) librari! es, and plots on the right show densities of RNA 3′ ends from the HeLa18–30 and HeLa18–30(XRN1/2) libraries. * Figure 5: Human tailed mirtrons. () sRNAs at intron termini are enriched in AGO1/2 immunoprecipitates. Numbers of unique sRNA 5′ ends from the HeLa18–30(AGO1/2) and the HeLa18–30 libraries are shown relative to the 5′ splice site (left) or 3′ splice site (right) regions. 3′ ends of the latter class coincide with the intron-exon junction. Dashed lines indicate splice junctions. () Size distribution of 3′ SSa sRNAs. The y axes show the total number of sRNAs per million tags in the −18 to −30 and −50 to −80 regions from the indicated libraries. The x axes show sRNA lengths. () University of California, Santa Cruz (UCSC) genome browser view of a putative 5′ tailed mirtron in the EEF1G gene. Only sRNA reads from the HeLa18–30 and HeLa18–30(AGO1/2) libraries are shown. Unique sRNAs are colored according to how many times they were sequenced; see color legend on the left. Shown at the bottom is the exon structure using UCSC gene annotation and the degree of conservation between 28 verte! brate species (Phastcons conservation). The predicted secondary structure with the nucleotides corresponding to the most frequent reads in red boxes is shown to the right. 'BP1' and 'BP2' point to two consensus branch-point sequences. () Genome browser view of a putative 3′ tailed mirtron in the human KHSRP gene. Conventions are as in . () Validation of expression of both arms of the EEF1G-derived mirtron shown in by splinted ligation of HeLa cell total RNA. Bands correspond to sRNA ~22 nt in length (~36 nt ligation products). '3p' and '5p' denote the 3′ arm and 5′ arm of the EEF1G-derived mirtron, respectively. 'No RNA controls' are reactions in which RNA was omitted. * Figure 6: AGO1/2-associating sRNAs located in 3′ UTRs. () Ratio of unique (left) or all mapped (right) sRNAs in 3′ UTRs versus protein-coding exons (CDS) in the indicated libraries. () sRNA size distribution near gene termini. The y axes show the total number of sRNAs from the indicated libraries whose 5′ ends map within the −(35–18) region upstream of the 3′ UTR end, normalized by library size. The x axes show sRNA lengths. () Density of unique sRNAs within 3′ UTRs. The y axes show the number of unique sRNAs normalized by library size. The x axes show the location within the last 50% of annotated unique spliced 3′ UTRs, where all UTRs are normalized to the same length. () Genome browser view of HeLa18–30(AGO1/2) and HeLa18–30(AGO1/2/RRP40) sRNA reads mapping to the RPL5 gene. Unique RNAs are colored (legend on the left) according to how many times they were sequenced. The canonical 'A(A or U)UAAA' polyadenylation signal sequence and the G- and U-rich sequence downstream of the cleavage site (vertical red arrow! ) are boxed. Also shown is the track for experimentally verified (green bar) and predicted (purple bar) polyadenylation sites. () Detection of RPL5-derived sRNAs by splinted ligation in total HeLa- or Ago-immunoprecipitated RNA. The miR-21 band (positive control) corresponds to an sRNA ~22 nt in length (~36 nt ligation product), and the RPL5-derived band corresponds to an sRNA ~24 nt in length (~38 nt ligation product). Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE29116 Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationCorrigendum 21 August 2011In the version of this article initially published online, in Figure 5a, the x-axis tick marks and labels were placed incorrectly; in Figure 5c, there were two extraneous tracks; and in Figure 5d, the y-axis label was missing, a stem in the RNA was incorrectly colored in gray (instead of red) and the sRNA tracks were incorrectly shifted to the left. These errors have been corrected for the print, PDF and HTML versions of this article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Eivind Valen, * Pascal Preker & * Peter Refsing Andersen Affiliations * The Bioinformatics Centre, Department of Biology and the Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark. * Eivind Valen, * Xiaobei Zhao, * Yun Chen & * Albin Sandelin * Centre for mRNP Biogenesis and Metabolism, Department of Molecular Biology, Aarhus University, Denmark. * Pascal Preker, * Peter Refsing Andersen & * Torben Heick Jensen * Laboratory of RNA Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany. * Christine Ender & * Gunter Meister * Department of Biochemistry, University of Regensburg, Regensburg, Germany. * Anne Dueck & * Gunter Meister Contributions E.V., P.P., P.R.A., G.M., A.S. and T.H.J. designed the experiments. P.P., P.R.A., C.E. and A.D. conducted the experiments. E.V., X.Z., Y.C. and A.S. did the bioinformatics analyses. E.V., P.P., P.R.A., G.M., A.S. and T.H.J. evaluated the results. E.V., P.P., P.R.A., X.Z., Y.C., A.S. and T.H.J. produced the figures. E.V., P.P., P.R.A., A.S. and T.H.J. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Albin Sandelin or * Torben Heick Jensen Author Details * Eivind Valen Search for this author in: * NPG journals * PubMed * Google Scholar * Pascal Preker Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Refsing Andersen Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaobei Zhao Search for this author in: * NPG journals * PubMed * Google Scholar * Yun Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Christine Ender Search for this author in: * NPG journals * PubMed * Google Scholar * Anne Dueck Search for this author in: * NPG journals * PubMed * Google Scholar * Gunter Meister Search for this author in: * NPG journals * PubMed * Google Scholar * Albin Sandelin Contact Albin Sandelin Search for this author in: * NPG journals * PubMed * Google Scholar * Torben Heick Jensen Contact Torben Heick Jensen Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (10M) Supplementary Figures 1–9 and Supplementary Tables 1 and 2 Additional data Entities in this article * Drosha drosha Drosophila melanogaster * View in UniProt * View in Entrez Gene * 5'-3' exoribonuclease 2 XRN2 Homo sapiens * View in UniProt * View in Entrez Gene * microRNA 1982 Mir1982 Mus musculus * View in Entrez Gene * Zyxin ZYX Homo sapiens * View in UniProt * View in Entrez Gene * Endoribonuclease Dicer DICER1 Homo sapiens * View in UniProt * View in Entrez Gene * Ribonuclease 3 DROSHA Homo sapiens * View in UniProt * View in Entrez Gene * 5'-3' exoribonuclease 1 XRN1 Homo sapiens * View in UniProt * View in Entrez Gene * Exosome complex component RRP40 EXOSC3 Homo sapiens * View in UniProt * View in Entrez Gene * Elongation factor 1-gamma EEF1G Homo sapiens * View in UniProt * View in Entrez Gene * microRNA 21 MIR21 Homo sapiens * View in Entrez Gene * Ribonuclease 3 Drosha Mus musculus * View in UniProt * View in Entrez Gene * Endoribonuclease Dicer Dicer1 Mus musculus * View in UniProt * View in Entrez Gene * Protein argonaute-1 EIF2C1 Homo sapiens * View in UniProt * View in Entrez Gene * Serine/arginine-rich splicing factor 1 SRSF1 Homo sapiens * View in UniProt * View in Entrez Gene * Far upstream element-binding protein 2 KHSRP Homo sapiens * View in UniProt * View in Entrez Gene * Endoribonuclease Dcr-1 Dcr-1 Drosophila melanogaster * View in UniProt * View in Entrez Gene * 60S ribosomal protein L5 RPL5 Homo sapiens * View in UniProt * View in Entrez Gene * Protein argonaute-2 EIF2C2 Homo sapiens * View in UniProt * View in Entrez Gene * Double-stranded RNA-specific adenosine deaminase ADAR Homo sapiens * View in UniProt * View in Entrez Gene * Heterogeneous nuclear ribonucleoproteins C1/C2 HNRNPC Homo sapiens * View in UniProt * View in Entrez Gene - Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs
- Nat Struct Mol Biol 18(9):1084 (2011)
Nature Structural & Molecular Biology | Corrigendum Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs * Anthony K L Leung * Amanda G Young * Arjun Bhutkar * Grace X Zheng * Andrew D Bosson * Cydney B Nielsen * Phillip A SharpJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:1084Year published:(2011)DOI:doi:10.1038/nsmb0911-1084aPublished online06 September 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Struct. Mol. Biol.18, 237–244 (2011); published online 23 January 2011; corrected after print 12 May 2011 In the version of this article initially published, the blue curve in Figure 2c was mistakenly replaced with a duplicate of that in Figure 2a. The error has been corrected in the HTML and PDF versions of the article. Additional data Author Details * Anthony K L Leung Search for this author in: * NPG journals * PubMed * Google Scholar * Amanda G Young Search for this author in: * NPG journals * PubMed * Google Scholar * Arjun Bhutkar Search for this author in: * NPG journals * PubMed * Google Scholar * Grace X Zheng Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew D Bosson Search for this author in: * NPG journals * PubMed * Google Scholar * Cydney B Nielsen Search for this author in: * NPG journals * PubMed * Google Scholar * Phillip A Sharp Search for this author in: * NPG journals * PubMed * Google Scholar - The resistance of DMC1 D-loops to dissociation may account for the DMC1 requirement in meiosis
- Nat Struct Mol Biol 18(9):1084 (2011)
Nature Structural & Molecular Biology | Corrigendum The resistance of DMC1 D-loops to dissociation may account for the DMC1 requirement in meiosis * Dmitry V Bugreev * Roberto J Pezza * Olga M Mazina * Oleg N Voloshin * R Daniel Camerini-Otero * Alexander V MazinJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:1084Year published:(2011)DOI:doi:10.1038/nsmb0911-1084bPublished online06 September 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Struct. Mol. Biol.18, 56–60 (2011); published online 12 December 2010; corrected after print 9 August 2011 In the version of this article initially published, the legend for Figure 2d,e did not include the source of the data in those panels. These data originally appeared in ref. 20. The error has been corrected in the HTML and PDF versions of the article. Additional data Author Details * Dmitry V Bugreev Search for this author in: * NPG journals * PubMed * Google Scholar * Roberto J Pezza Search for this author in: * NPG journals * PubMed * Google Scholar * Olga M Mazina Search for this author in: * NPG journals * PubMed * Google Scholar * Oleg N Voloshin Search for this author in: * NPG journals * PubMed * Google Scholar * R Daniel Camerini-Otero Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander V Mazin Search for this author in: * NPG journals * PubMed * Google Scholar - ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair
- Nat Struct Mol Biol 18(9):1084 (2011)
Nature Structural & Molecular Biology | Erratum ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair * Gareth J Williams * R Scott Williams * Jessica S Williams * Gabriel Moncalian * Andrew S Arvai * Oliver Limbo * Grant Guenther * Soumita SilDas * Michal Hammel * Paul Russell * John A TainerJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:1084Year published:(2011)DOI:doi:10.1038/nsmb0911-1084cPublished online06 September 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Struct. Mol. Biol.18, 423–431 (2011); published online 27 March 2011; corrected after print 6 April 2011 In the version of this article initially published, the acronym ENIGMA was spelled out incorrectly in the Acknowledgments section. The error has been corrected in the HTML and PDF versions of the article. Additional data Author Details * Gareth J Williams Search for this author in: * NPG journals * PubMed * Google Scholar * R Scott Williams Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica S Williams Search for this author in: * NPG journals * PubMed * Google Scholar * Gabriel Moncalian Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew S Arvai Search for this author in: * NPG journals * PubMed * Google Scholar * Oliver Limbo Search for this author in: * NPG journals * PubMed * Google Scholar * Grant Guenther Search for this author in: * NPG journals * PubMed * Google Scholar * Soumita SilDas Search for this author in: * NPG journals * PubMed * Google Scholar * Michal Hammel Search for this author in: * NPG journals * PubMed * Google Scholar * Paul Russell Search for this author in: * NPG journals * PubMed * Google Scholar * John A Tainer Search for this author in: * NPG journals * PubMed * Google Scholar - Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes
- Nat Struct Mol Biol 18(9):1084 (2011)
Nature Structural & Molecular Biology | Erratum Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes * David Martin * Cristina Pantoja * Ana Fernández Miñán * Christian Valdes-Quezada * Eduardo Moltó * Fuencisla Matesanz * Ozren Bogdanović * Elisa de la Calle-Mustienes * Orlando Domínguez * Leila Taher * Mayra Furlan-Magaril * Antonio Alcina * Susana Cañón * María Fedetz * María A Blasco * Paulo S Pereira * Ivan Ovcharenko * Félix Recillas-Targa * Lluís Montoliu * Miguel Manzanares * Roderic Guigó * Manuel Serrano * Fernando Casares * José Luis Gómez-SkarmetaJournal name:Nature Structural & Molecular BiologyVolume: 18,Page:1084Year published:(2011)DOI:doi:10.1038/nsmb0911-1084dPublished online06 September 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Struct. Mol. Biol.18, 708–714 (2011); published online 22 May 2011; corrected after print 3 June 2011. In the version of this article initially published, the affiliation for authors at the Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Madrid, Spain, was incomplete. The full affiliation is "Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, CSIC, Madrid, Spain." The error has been corrected in the HTML and PDF versions of the article. Additional data Author Details * David Martin Search for this author in: * NPG journals * PubMed * Google Scholar * Cristina Pantoja Search for this author in: * NPG journals * PubMed * Google Scholar * Ana Fernández Miñán Search for this author in: * NPG journals * PubMed * Google Scholar * Christian Valdes-Quezada Search for this author in: * NPG journals * PubMed * Google Scholar * Eduardo Moltó Search for this author in: * NPG journals * PubMed * Google Scholar * Fuencisla Matesanz Search for this author in: * NPG journals * PubMed * Google Scholar * Ozren Bogdanović Search for this author in: * NPG journals * PubMed * Google Scholar * Elisa de la Calle-Mustienes Search for this author in: * NPG journals * PubMed * Google Scholar * Orlando Domínguez Search for this author in: * NPG journals * PubMed * Google Scholar * Leila Taher Search for this author in: * NPG journals * PubMed * Google Scholar * Mayra Furlan-Magaril Search for this author in: * NPG journals * PubMed * Google Scholar * Antonio Alcina Search for this author in: * NPG journals * PubMed * Google Scholar * Susana Cañón Search for this author in: * NPG journals * PubMed * Google Scholar * María Fedetz Search for this author in: * NPG journals * PubMed * Google Scholar * María A Blasco Search for this author in: * NPG journals * PubMed * Google Scholar * Paulo S Pereira Search for this author in: * NPG journals * PubMed * Google Scholar * Ivan Ovcharenko Search for this author in: * NPG journals * PubMed * Google Scholar * Félix Recillas-Targa Search for this author in: * NPG journals * PubMed * Google Scholar * Lluís Montoliu Search for this author in: * NPG journals * PubMed * Google Scholar * Miguel Manzanares Search for this author in: * NPG journals * PubMed * Google Scholar * Roderic Guigó Search for this author in: * NPG journals * PubMed * Google Scholar * Manuel Serrano Search for this author in: * NPG journals * PubMed * Google Scholar * Fernando Casares Search for this author in: * NPG journals * PubMed * Google Scholar * José Luis Gómez-Skarmeta Search for this author in: * NPG journals * PubMed * Google Scholar
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